MICB 421 Manual 2016 V1.82

1
Table of Contents
Acknowledgements
Course Operation and Rules
A.
B.
C.
D.
E.
F.
G.
Course Overview
Course Operation
Purpose
Learning Objectives
Specific Course Resources
Rules
General Expectations
Project 1 Preparation of Competent E. coli

Project 2 - Research Project
Purpose
Timeline
o
o
o
o
o
o
o
o
Week I
Week II
Week III
Week IV and V
Week VI
Week VII through XI
Week XII
Completing the Project
Materials and Equipment
Schedules, Performance and Marks

Schedule
Performance and Marks
Lab Etiquette
Effort and Etiquette
Participation
Lab Notebook
Reflective Weekly Journal
Project Reports
Theory Exams
Grading Rubrics
Weekly Summaries
Competent Cell Preparation
Individual Project Proposal
Team Project Proposal
Team Oral Presentation
Draft Paper
Lab Notebooks
Video Presentation
Question from Team Presentations
Appendix A - Basic Methods

I. Efficient Organization
II. Aseptic Work
A.
B.
C.
D.
Preparation for Aseptic Work

Flaming
Aseptic Transfer by an Inoculator
Aseptic Transfer by Pipette
III. Mixing Samples

IV. Pipettes and Pipetting
A.
B.
C.
D.
Classes of Pipettes
Sizes of Pipettes
Types of Pipettes and Pipetting Aids
Pipetting Procedures
V. Plating and Enumeration

A.
B.
C.
Streak Plating for Isolates

Enumeration
Replica Plating for Multiple Tests
Appendix B - Laboratory Safety

A.
B.
C.
D.
E.
F.
G.
H.
I.
Laboratory Hazards
WHMIS
National Fire Protection Association Diamond Codes
Gloves
Laboratory Techniques
Personal Practices
Chemical Disinfectants
Disposal Procedures
Radiation Safety and Working Rules
Appendix C Units, Graphs, and Calculations

A.
B.
C.
D.
E.
Units and Prefixes

Graphs
Reliability of Results
Growth Calculation
Dilutions and Dilution Schemes
3
F.
Sample Problems with Dilution and Growth Calculations
Appendix D - Technical Theory

I. Centrifugation
A.
B.
General Information
Types of Centrifuges
II. Electrophoresis
III. Enzyme Assays
IV. Growth Media
V. Buffers
VI. Ion Probe Measurement
A.
B.
pH Probes
Other Probes
VII. Nomenclature
A.
B.
VIII.
Strain Names
Genetic Nomenclature
Protein Isolation
A.
B.
C.
D.
Protein Release from the Cell

Stabilization of Released Enzyme Activity
Isolation of the Released Enzyme Activity
Protein Measurement
IX. Radioactivity
A.
B.
C.
D.
E.
General Information
Nomenclature of Radioactive Molecules
Properties of Some Isotopes
Measuring Radioactivity
Incorporation Properties
X. Spectrophotometry
A.
B.
General Information
Problems and Compromises in Spectrophotometer Design
Appendix E Teams and Teamwork
A.
B.
Teams
Choosing Teams
Appendix F Records and Reports

A.
B.
C.
D.
E.
Project Reports
Format Requirements for Project Reports
Participation Reports
Lab Notebook
Oral Presentations
Microbiology 421
Manual for Experimental
Microbiology
Compiled by:
W.D. Ramey
D.C. Oliver
(2015)
Acknowledgements
This lab manual for MICB 421 has been developed by using an assortment of information and ideas
concerning the aims, objectives and results of former MICB 321, MICB 421 and MICB 421 classes. The
manual is intended to be used in conjunction with the MICB 421 Connect website at
https://connect.ubc.ca/webapps/portal/frameset.jsp?tab_tab_group_id=_2_1&url=%2Fwebapps%2Fblackboard%2F
execute%2Flauncher%3Ftype%3DCourse%26id%3D_27433_1%26url%3D
Nick Cheng wrote many of the directions for operating the equipment and took most of the digital images
that are used throughout the website and the manual. Nick Cheng, Chris Gin, Kevin Lin, Kyla Omilusik,
Lando Robillo, Kristen Schurek, Jennifer Sibley, Gaye Sweet, and Alice Wang helped to locate, develop
and test content for the experiments as well as experimental protocols, assays and proofreading. Karen
Smith compiled details and content for the section on laboratory safety and disposal of hazardous
materials in Appendix B. Kristen Schurek compiled the detailed section on the formatting reports in
Appendix F.
Department of Microbiology and Immunology

University of British Columbia
January 2016
William Ramey / David Oliver
Course Operation and Rules

A. Course Overview
Traditional lab courses tend to present students with experimental recipes and supplies, and
then ask students to follow the recipes, look at the results and write a report. This approach
allows each student to have the same learning experience but it limits the range of available
experiments. In addition, even though instructors expect students to understand the work
before attempting an experiment the traditional lab approach allows some students to get
through the work by following the directions rather than understanding the purpose of the
work. In this course teams of students are expected to design experiments and protocols to
test their approach to provided categories of observations rather than just following a
supplied recipe. In some cases the explanations might require a simple modification of a
previous experiment, in other cases the teams might need to invent an entirely new
experimental protocol. In either case, once the experiment has been acceptably designed,
your team will be expected to prepare the supplies, set up equipment, carry out the
experiment, analyze the results and prepare a formal journal style report that describes the
experimental purpose, the methods, the results and the conclusions. The course instructors
will review this report. The report will then be returned to the team to correct any
significant observed problems in style, logic or fact before it is published to the "UBC
eJournal of Experimental Microbiology and Immunology" (JEMI) where it will be available
on-line. Teams may repeat a previous experiment to ensure that the results were correct but
will still be expected to produce and test an original experiment based on the resulting
observations and explanations. The experiments will have some constraints imposed by
time, equipment and costs.
This approach is intended to provide a more complete and realistic research experience. We
also hope that it will be more interesting for you because you will be contributing
knowledge and skills to solve real scientific questions instead of working on recipes chosen
by the instructor.
B. Course Operation
The class will operate as teams of three or four students.
The majority of the course is concerned with designing and carrying out a research project
but each Tuesday there will be a scheduled class meeting. During that part of the class we
will discuss/review different aspects of project design and advanced molecular and
microbiology. Towards the end of term we will also talk about the final report and the
characteristics of the final report. The actual content of these classes will depend on general
needs for the different projects in the class. Individual weekly meetings will be scheduled
during the lab periods on Tuesday and Thursday afternoons to discuss details of their
projects with each team. By the start of the weekly meeting each team member should have
an idea of the work that the team is intending to complete in the following week and how
the team will do that work.
An original copy of the team records must be maintained in a bound lab book whenever the
team is working in the laboratory. This book must include all important laboratory details
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such as the working hypotheses, ideas, data, explanations of observations, and conclusions
as well as significant discarded ideas, relevant references, equipment names and models,
chemical sources and catalogue numbers. The first page of the book must be reserved for an
index that is updated each week. Each page in the book must be numbered and dated as it is
used so that the important pages can be identified in the index.
The final report must be a formal journal article written in the style of the Experimental Journal
of Microbiology and Immunology. This style is adapted from the style used in the American
Society for Microbiology journals such as J.Bacteriol and J.Immunol. Approximate examples
can be seen in recent volumes of the Journal of Experimental Microbiology and Immunology
(JEMI) at:
https://www. jemi.microbiology.ubc.ca
Keep in mind that the ASM style of citing references changed in January 2011. Please use the
current ASM style:
http://jb.asm.org/site/misc/ifora.xhtml
C.Purpose
The purpose of the course is to carry out a research project involving aspects of molecular
biology and microbiology in order to:
1. demonstrate your ability to apply your skills in this area of science
2. expand your background in this area of research
3. learn additional molecular biology skills
D.Learning Objectives
1. To develop your ability to define a research problem.
2. To develop your ability to adapt your knowledge and background to develop protocols
to experimentally solve scientific problems
.
3. To develop your ability to locate information and background for designing an
experimental protocol.
4. To further your understanding of some of the constraints of an experimental protocol or
testable question as well as recognition of the common elements in research protocols.
5. To further your ability to quantitatively analyze data and formulate logical and testable
mechanistic models that account for the observations.
6. To reinforce your ability to maintain scientific records and communicate scientific
results.
7. To further your ability to prepare and present written scientific reports.
8. To further your ability to organize and complete technical projects with finite resources
and time.
9. To further your ability to work in scientific teams.

E. Specific Course Resources
Most of the course material is described in the lab manual. Some resources, inventories and
assignments for the course are located at the MICB 421 Connect site. There will also be
printed copies of the equipment operation, strain inventories and chemical inventories in
room 104 and 110 and 114 Wesbrook. Some of the files in the website can be downloaded
and searched.
F. Rules
1.
The major part of your lab work each week should be planned to fit your scheduled
class time. However, some work will need to be done at other times to allow your
projects to proceed in a timely manner. If you are working outside the scheduled class
time the available lab hours are 9:00 AM to 5:00 PM. Plan to work within those
times.
2.
There should be two people present when you are working in the lab. One of the
people must be the instructor or the teaching assistants if you are starting any work
involving heat, gas, compressed air or hazardous chemicals. If you are counting
colonies or weighing safe ingredients to prepare media or supplies you do not need
supervision but it might be helpful to have supervision in case questions arise as you
proceed with the work. Work that requires long periods of supervision should be
confined to the scheduled lab times.
3.
You must be certified before you use any hazardous or expensive or fragile equipment.
Certification means that the operation of the equipment has been demonstrated to you
by the MICB 421 instructor or teaching assistant. Please have the person that
trained you initial your lab training record. If there is doubt about the operation of any
of this equipment, it is important to clarify your uncertainty before you use the
equipment.
4.
You must be aware of the hazards of any work that you attempt. As part of that
awareness you must consult the MSDS site for every chemical that you attempt to use
and copy down or print out any significant risks and the corresponding first aid
treatments.
5.
You must understand the intent of each step in every protocol that you attempt to use
because you will make the work safer for everyone in the lab if you understand the
intent of each step in each experiment.
6.
Each team must maintain complete experimental records in a single notebook that will
be submitted to the course at the end of the term. You may keep individual record
books but the team record book must be kept up-to-date each week and available at
any time the team is working in the lab.
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G. General Expectations
We expect you to remember all the general features of basic, safe aseptic work that you
have encountered in other classes. These expectations include:
1.
Disinfect the top of your desk with Westosan at the beginning and the end of each lab
day to minimize the chance of spreading contaminants around. Wash your hands with
disinfectant soap before leaving the lab.
2.
Do not eat or drink in the laboratory.
3.
Do not take cultures out of the laboratory without permission.
4.
Inform the instructors if you spill a culture, and then disinfect the area. Use the brush
at the discard cart to sweep up any broken glass or hazardous chemicals and discard
them to the designated waste containers.
5.
Inform the instructors about any accidents such as cuts, burns or abrasions, even if
they seem slight.
6.
Wear a protective lab coat when working in the laboratory and secure your hair so that
it will not swish into any open flames or chemicals involved in your experiment.
Wear gloves and eye protection when the hazards warrant the use of gloves and eye
protection.
7.
Use the lockers outside room 110, 104, and room 5 to store backpacks and other
equipment and materials that are not needed in the lab room. Lockers can be signed
out by providing a deposit that will be returned when the locker key or lock are
returned
8.
Drain tubes containing toxic or corrosive chemicals to the appropriate discards then
rinse the tubes with cold water before they are discarded.
9.
Cultures and other solutions that are contaminated with microbes should be discarded
in closed flasks or tubes so they can be autoclaved before they are dumped out.
Adequate closure is achieved by covering the top of a flask with a cotton bung or a
layer of aluminum foil when the flasks are discarded to the trays in the lab room.
10. Discard standard calibrated pipettes into the tall discard jars at the bench. Discard
disinfected Pasteur pipettes into glass waste.
11. Keep your work space tidy and organized so it is safe and aseptic.
12. Put away your supplies between lab days and return communal chemicals and
other supplies to the appropriate storage places.
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Project 1 Preparation of Competent Esherichia coli
Purpose:
The intent of this project is to provide an opportunity for your team to begin working
independently in the laboratory setting. Your teams goal is to prepare aliquots of competent E.
coli and to determine the transformation efficiency of your preparation. You have course weeks
2, 3, 4 and 5 to plan, execute and document this task. You will need to source protocols, plan
your experimental steps, and prepare the requisite reagents such as growth media, chemical
solutions, and sterile equipment and glassware. You should include appropriate controls to
demonstrate that your experiment worked as expected. You should also think about replicates
when performing transformations in order to be able to report transformation efficiency with
statistical support. You may choose any (non-pathogenic) E. coli strain available in the MICB
421 Ramey strain collection (e.g HB101, DH5, BL21, B). It is recommended that you think
ahead to Project 2 in order to produce competent E. coli that may be useful to your team later in
the course (Note: competent cells stored in cryoprotectant can be stable for years at -80C).
Similarly, you may select any plasmid available in our collection to test transformation efficiency,
however, it may be prudent to choose something relevant to your subsequent research in Project 2.
Instructions:
Your team is responsible for planning, execution, and documentation of your experiment. Lab
notebook guidelines in Appendix F should be followed. The Instructor and TAs will be available
throughout the scheduled lab period to provide guidance, technical demonstrations (e.g making
media and pouring plates), and training on equipment such as the centrifuge, shakers, and
autoclave. We can also introduce you to reference manuals and online sources of protocols.
The sections of the course manual describing proper aseptic technique, bench organization,
pipetting, and safety should be carefully reviewed before beginning your work.
When determining transformation efficiency, perform an experiment that allows you to graph (1)
plasmid concentration versus transformation efficiency, and (2) plasmid concentration vs total
transformants. How do you predict these plots will compare?
Assessment:
Document Project 1 as experiments in your teams lab notebook. Relevant pages of your teams
lab notebook can be submitted to MICB 421 Connect as a word document or as a scanned PDF of
the lab notebook pages (scanning can be done using the photocopier). The deadline for Project 1
submission is 11:59PM on Feb 5th 2016. The submitted notebook pages will be assessed for
formatting and scientific rigor. The results themselves will not specicially be graded, rather
qualities of the report will be evaluated. We will look for an effective title, a well written and
referenced Introduction section, a complete Materials and Methods section, a Results section
containing appropriate observations and interpretations, well presented data with appropriate
12
statistical backing and noted limitations, objective Conclusion(s), and a feasible Future Directions
section.
Project 2 - Research Project

Purpose:
The intent is to reinforce your understanding of the constraints of the scientific process by
developing explanations of a supplied scientific observation, designing an experiment to test a
proposed explanation, preparing your supplies, identifying required equipment, doing your
experiments and communicating your results as an article for a scientific journal. The specific
experimental objectives will depend on the observation that your team chooses to explain and the
manner in which your team chooses to test the explanation.
References
Barker, K. 1998. "Chapter 4, How to setup an experiment", At the Bench, a laboratory navigator,
p 69-87 Cold Spring Harbour Laboratory Press, New York, New York.
Barker, K. 1998. "Chapter 5, Laboratory notebooks", At the Bench, a laboratory navigator, p 8999 Cold Spring Harbour Laboratory Press, New York, New York.
Instructions:
1. The project will be done by teams consisting of three or four students.
2. The general requirement is to review previous course projects to identify a research question,
then to devise protocols to address your approach to the observations or problems, prepare the
supplies, carry out the tests and analyse the results. A list of the titles and a list of general
references are included after the materials and methods section in this project outline.
3. The proposed project may not be an exact duplicate of any other posted experiment but you
may repeat an experiment in the Journal of Experimental Microbiology and Immunology
(JEMI) in order to confirm the results before proceeding with your own experiment. You may
develop and use a modification of a published experiment if the modification provides a better
solution or better insight into the problem and your explanation.
4. The number of teams choosing a particular approach or methodology might be restricted.
Each proposal must be different. If two teams suggest the same proposal and same general
approach then the first proposal received will be permitted to proceed and the second team
will need to develop an alternative proposal (unless the second team provides evidence that
the first team pirated their proposal). However, proposals that assess similar ideas by
significantly different approaches or methods are still acceptable.
5. Each team is encouraged to develop the details of their proposed project. However, if you
have a good idea but are unsure how to develop the idea into a testable explanation or develop
a suitable protocol you should discuss the idea with the instructor or the teaching assistant to
see if they have worthwhile advice. Similarly, if you are having trouble choosing an
experiment or you think that you have a suitable experiment but are uncertain whether
13
particular protocols could be attempted with the available equipment or supplies then you
should discuss those concerns with the instructors or teaching assistants to get their advice.
The teaching lab is not equipped to deal with pathogens or eukaryote tissue cultures.
6. Teams proposing projects involving a lot of samples or sampling should do a pretest with a
few samples to ensure that the strain(s) have the correct phenotypes, the results are falling in
the expected assay ranges and that the methods will work and give reproducible results.
7. The results will be presented as a research article suitable for publication in JEMI. Any major
problems or technical errors in the submitted report will need to be corrected before the
manuscript is accepted for publication. The article must be accepted for publication before the
mark for the project is added to your grade.
8. The instructor or the teaching assistant must approve proposals before you start significant
technical preparation. Some basic preparation of media or buffers or glassware can be done in
advance. Ask if you are in doubt about what could be done in advance.
9. The lab will normally be open between 9:00 AM and 5:00 PM to allow you to work on the
project outside the scheduled lab periods if that work is necessary or helpful. However, we
expect some of the work to be scheduled for your registered lab period when assistance will be
available. Your teams lab book may be checked each week by the instructor or the teaching
assistant during your registered lab period. If you work in the lab outside your registered class
there must be at least one other person present if the work involves any potentially hazardous
procedures or equipment.
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10. Week I.
i.
Each student must individually propose a project based on an observation or

suggested follow up experiment described in a JEMI paper that they would like to
investigate and prepare (1) a flow chart and (2) a brief report of approximately 300
500 words that explains their choice. One electronic copy of these individual
reports must be submitted to the MICB 421 Connect assignment drop box by
11:59 PM on the due date (See schedule). When you submit the report use your
initials, surname, day and alphanumeric as the name for the file. For example,
JohnSmithTuesA. Other copies must be distributed to your team mates by the start
of the lab session for your team.
Here is a link to JEMI:

https://www. jemi.microbiology.ubc.ca
You can also review the titles of papers submitted by MICB 447 students last term. These are
posted on MICB 421 Connect. If approved by the authors, the Instructor will send you draft
manuscripts from MICB 447 2015 upon request as these papers are still under revision.
ii.
Begin by preparing a 1 page flow chart on your proposal. A model flow chart as well
as a templated in Powerpoint format is available on MICB 421 Connect. The
flowchart must be uploaded to Connect by 11:59pm on Sunday January 10th 2016.
iii.
Your report should be a maximum size of 1 page in single spaced TNR size 10 font.
The written individual proposal must be uploaded to Connect by 11:59pm on Friday
January 15th 2016. The written proposal must include specific labeled sections for:
-
Identification: your name and your lab day at the top right corner of the page.
Source of the observation: the category of the project and a reference to the
specific observation or problem. These are typically JEMI articles, although
other scientific papers may be permitted with permission from the Instructor.
This reference should be formatted as per ASM 2013 style.
Background: the background necessary to understand / explain the
relationship between the problem and the proposed explanation. (This section
is not expected to be a full review of the topic but it needs enough detail for the
reviewer to understand the logic for your subsequent explanation(s) and
proposed experiment. In most cases this would take approximately 300 or 400
words)
Observation: a brief description of the observation / problem.
Explanation / Hypothesis: a preliminary potential explanation(s) / hypothesis
that might account for the observation that you will work on.
Experimental question(s): state the actual scientific question that will test the
explanation. Include an explanation of how answering the question would
assess the validity of the explanation.
Approach: the general experimental approach for testing your explanation.
(What methods or combinations of feasible methods would be the key way of
getting data to test your explanation? What would the results from that
approach show if the explanation was correct or incorrect?)
Feasibility: the feasibility and outcomes of the proposed approach. (What are
the main technical difficulties? What are the main details that need to be
worked out to know whether it would work? What are the limitations imposed
on the analysis by these methods?)
15
Each student should think about additional project details and requirements and why they wanted
to work on their particular proposal. This additional level of detail is not expected in the
individual proposal but the information might be helpful when the team chooses the topic for the
project they will develop for the rest of the term.
11. Week II.
i.
By Week II you should have selected teammates and been assigned a lab day and bay.
ii.
Please register your team for a 15 minute team meeting on the door to room 138.
Register your team with the TA and be prepared to discuss your flowchart. Only 3
minutes will be allotted to each team members presentation so please be prepared.
The Instructor and TA may provide guidance and ask a few questions for
clarification. Using this feedback, prepare your written proposal.
12. Week III

iii.
To choose a project, compare and discuss the merits of the potential projects proposed
by the different team members. Consider the pros and cons of different projects, the
general interest of the team and alternative explanations that might account for the
observation.
iv.
If your team is sure about the intended project then one choice is sufficient. If the
team is less certain it is better to choose two. For each of these projects the team
should identify the major specific information or general background relevant to
understanding the observations or the preliminary explanations. When the members
draw up this list they should consider what they already know about the topic and
how well they recall details. They should also consider whether their knowledge is
superficial or detailed, whether the knowledge is dated or current et cetera. For
example:
a. What is known about the process that formed the original experiment? Would
knowing more about that process be beneficial? Is it known whether related
processes would work? Is anything known about those processes?
b. Are there known mechanisms involved in the molecular genetics for any of the
components involved in the project. Would a more detailed understanding of
these mechanisms give more insight into potential explanations and processes?
Et cetera.
v.
Each team member should take responsibility for researching / checking some of the
various sets of information that are necessary or helpful for choosing one project and
refining the preliminary hypothesis that is going to be presented at the scheduled
meeting with the TA and the instructor on Week III.
vi.
The team should also consider whether anyone already has a source for some of the
background. The web can sometimes provide some ideas and details but the quality
of information is inconsistent. More reliable sources can be found by doing library
searches for journal articles and compendiums of research facts and methods. The
libraries provide workshops on the use of electronic indexes for finding relevant
journal articles and searching for relevant key words. Some indexes that might be
useful include:
-
Biosis <http://resources.library.ubc.ca/551/ >
16
- Medline <http://resources.library.ubc.ca/139/ >
- PubMed <http://resources.library.ubc.ca/321 >
- Web of Science <http://resources.library.ubc.ca/277/ >
- NCBI Resources <http://www.ncbi.nlm.nih.gov/books/NBK3826/ >
- PubMed <http://www.ncbi.nlm.nih.gov/pubmed/ >
There are more sites at <http://resources.library.ubc.ca/ressubjtitles/S/129/ >
Google Scholar can also be helpful but is less complete than the dedicated research
indexes. In addition, by accessing articles through the UBC library, you go through
the library subscriptions so more prepaid journal access is available for you.
vii.
At this stage you can still consider other projects if the initial choices seem less
suitable when you consider the details.
viii.
After considering the additional information reported by each team member, the team
should decide which problem and which hypothesis or approach will be investigated.
This will be your team project for the rest of the term. When that choice is made
each person on the team should consider the known facts and logic that support the
proposed explanation. Each person should also bear in mind that the eventual
evaluation of the project will consider:
a. Whether the experiment is testing an explanation/model of the observations or
at least setting up the potential to test an explanation/model rather than
dabbling with experimental variables.
b. Whether the proposal is practical. Can it be done within the constraints of
time, expertise and available equipment and supplies?
c. Whether the experiment is worth doing. Some experiments are exciting and
interesting. Some experiments are bland but the results are important to further
understanding. Some experiments are not worth doing because the answers are
either obvious or insignificant. For some experiments the distinctions are most
obvious in retrospect.
ix.
During the scheduled meeting with the TA and / or the instructor during the lab class
the team should be able to:
a. State the observation they intend to investigate
b. Give preliminary explanation(s) of the problem and present testable models
c. Explain what information was sought and discovered by each team member and
how it applies to the potential projects.
d. Discuss the general feasibility of your project with the instructors.
x.
Consider any major additional details that the team will need to look up or work out
in order to finish refining and designing the experiment. Examples of these details
could include relevant strains, chemical requirements, potential hazards, necessary
assays, potential equipment, recipes, scheduling et cetera.
xi.
Assign particular team members specific duties to sort out by the following class.
13. Weeks IV and V

During the scheduled meeting with the TA and / or the instructor during the lab class the
team should be able to:
a. State the problem that has been approved in principle by the instructors
17
b. Provide the potential explanation(s) of the problem
c. Explain the evidence or logic for the explanation
d. State the experimental question that will be specifically addressed and how that
question relates to the original hypothesis or explanation concerning the
original observation.
e. Explain the experimental approach for testing the stated question and the
probable outcomes if the hypothesis is true and the probable outcomes if the
hypothesis is false.
f. Summarize any outstanding issues or changes to the original questions posed in
the preceding week that that need to be addressed.
g. Explain your progress over the preceding week.
18
14. Week VI
i.
By 11:59 PM on the Friday February 12th 2016 the team must submit a detailed
written proposal. In 2500 to 5000 words the report must provide:
-
Identification: the team alpha-numeric and the names of the team members
Overview Chart: a chart that shows how the tests and work fit together as an
overview of the project.
Title: a relevant working title for your experiment (It might change by the
final report).
Introduction and Background: sufficient background to understand the
explanation of the observation, the chosen approach to testing the question and
the outcomes.
Observation and Explanation: the experimental observation and the
proposed explanation that the project is addressing.
Experimental Aims: the experimental question(s) that you are aiming to
answer that will test your explanation / hypothesis of the observation. Include
the potential outcomes if the explanation is correct or incorrect.
Protocol: description of the project that explains what will be done and how
the tests fit together. It would include details of sampling, number of samples,
timing of sampling, types of assays for the different types of proposed
measurements, controls, growth requirements. It is recommended that his be
arranged in a table.
Methods: description of how the assays and tests will be done. It would
include recipes or step by step explanations and details to make
measurements, grow the cultures, and actually assay the samples. Include
quantities and control conditions et cetera. It must be detailed enough to
allow the team to make the supplies in appropriate quantities.
Supplies and Equipment: provides quantities, and condition of the
chemicals, equipment and strains. Any required chemicals or kits or strains
that are not available in the inventories for the class must be clearly identified.
Weekly Time Frame: the detailed schedule of the project work that will be
done by the team each week in order to finish the project on time. Some work
can be overlapped to allow testing of unfamiliar procedures, testing of strains,
taking preliminary measurements to assess response ranges et cetera before
the final experiment is attempted. A Gantt chart may be useful.
Potential Pitfalls: potential problems that might arise to put the work behind
schedule.
Known Hazards: any significant unusual hazards associated with the work
and your means of controlling those hazards so the work is safe.
ii.
This report should be submitted on the MICB 421 Connect assignment drop box.
There should only be one submission for each team.
iii.
The report will be graded for completeness, relevance and readability for each of the
expected sections.
iv.
Submit a separate list of required strains, kits or chemicals that are not in the class
inventories directly to the instructor by e-mail. Include sources, quantities and
costs for each item. Strains that are not available locally can take weeks to arrive so
they need to be requested early in the process. The list should have Required Strains
for MICB 421 or Required Chemicals for MICB 421in the subject heading.
v.
During the scheduled meeting with the TA and / or the instructor during the lab class
for Week V the team should be able to explain the timeframe, the required amounts of
19
supplies and equipment, the strains and the sources of strains that will be needed for
the project.
vi.
The team can start to prepare basic supplies that will be needed for the project.
Weeks VII-through XI.

i.
Over this time the team must finish preparing the required supplies (if they are not
already available). Carry out the experiment. Examine the results. Then use the
observations from your first experiment to re-design or refine the first experiment to
improve or confirm the initial results. Sometimes the second experiment should use
the same general approach with a better sampling strategy, additional controls or
different input. Sometimes the second experiment should use a different approach to
the initial hypothesis. In either version of the second experiment the intent is to refine
your understanding of the initial observation and your proposed explanation of that
observation. In all cases the second experiment must continue to test the idea or an
explanation of the problem or observation made in the first round of testing.
ii.
Starting on Week VIII, prior to the scheduled meeting with the TA and / or the
instructor during the lab class the team should submit a Weekly Summary at
MICB421 Connect by 11:59pm on Sunday.
Weekly Summaries should include:
a. The team name and submission date.
b. Project title (once defined).
c. A summary of the weeks activities as bullet points.
max 1/2 page in single spaced TNR size 10 font.
Each point should concisely explain:
experimental reference numbers from your lab notebook

the rationale for work performed
results, key observations, conclusions
any decisions made and major changes to project
any technical challenges and difficulties
any remaining work with respect to timeline
15. Weeks XI-XIII.
During the scheduled lecture time, each team will present a summary of their project,
including background, results, analysis, conclusions and future directions.
Presentations may be delivered by one or more members of the team. Presentations
should be no longer than 12 minutes maximum (points will be deducted for
presentations exceeding 12 minutes in length). The audience is expected to compose
questions for each presentation. Each team should choose one question to submit to
the presenting team via Connect. Each presenting team can choose to respond to 3
questions submitted by their peers via video. Responses will be posted on Connect.
All team members must attend the lab during their normal scheduled class time to
clean up supplies and work places unless the cleanup was done the week earlier.
20
Completing the project
18.
i.
By 11:59pm on the April 15th 2016 each team must submit a formal journal article
that documents their hypothesis, their experiment, their results and their conclusions.
The article must be written in the style prescribed in Appendix F of the lab manual. If
this article is started in the last week of class it could be a large amount of work.
However, much of the effort can be dispersed if the team coordinator assigns tasks
that initiate the article by early - to- mid-November. For example, even though the
final draft might require revisions to include the final results it is possible to make a
draft version of the introduction as well as the materials and methods section. It is
also possible to prepare preliminary graphs and tables that portray the preliminary
results from the first round of the experiment before doing the second round. It is also
possible to develop a written list of the major thoughts and provisional conclusions
suggested by that data. Even though the final tables and figures need to be embedded
at the end of the word document as pictures pasted into text boxes the preliminary
tables and figures can be prepared in Excel (or an analogous spread sheet program)
and saved so they are easy to update and use when the final experiments are
completed.
ii.
The final report is expected to include at least four journal article references aside
from JEMI articles that are relevant to the explanation of the ideas being tested. To
meet this requirement, it would be useful to continue the library searches and develop
a provisional reference list well before the article is due. An early search might be
helpful for interpreting the observations from the first experiment and designing the
second experiment.
iii.
Streaked plates of all relevant bacterial strains, clones, constructs and plasmids
used in your project must be prepared and submitted to the instructor so the
strains can be preserved and accessible for any future students that wish to
continue your project.
The submitted draft reports will be reviewed in the order that they are received and the
reviewers comments will be returned to the team that submitted the report within a few days.
The team must complete any required revisions and corrections then resubmit the report in
electronic form before the end of April.
21
Materials and Equipment:
-
Lists of inventories of the available equipment, strains and chemicals are available in the
resources section at MICB 421 Connect. There are bound paper copies of this information in
room 104 and 114 Wesbrook.
Major equipment that could be available include:

::-
:-
:-
:::-
:::::::::::-
a variety of centrifuges for microcentrifugation, high-speed centrifugation and ultracentrifugation. Some of this
equipment has restricted access but it can be used as long as you have suitable instruction in the use.
a variety of spectrophotometers. Some machines like the Spectronic 20 are suitable for working with larger volumes
and are well suited for following growth, measuring the results of chemical assays and enzymatic assays but lack the
capacity to measure ultraviolet light. Others work well in the ultraviolet light. These spectrophotometers generally have
a maximum volume of approximately one millilitre but special cuvettes are available to measure 100 microlitre volumes.
Another one has a carousel that can be programmed to rotate in order to automatically read the absorbance of samples in
successive cuvettes. Some of the instruments that read at ultraviolet wavelengths can be set to scan multiple
wavelengths of light and give a reading at the specified wavelengths. Some of the spectrophotometers can be linked to
the SpecX data acquisition program to automatically collect digitized data that can be imported into spreadsheet
programs. The SpecX program can also be adapted to give automatic readings of temperature and ion-probe
measurements
a variety of electrophoresis equipment. There are gel systems for working with protein gels and nucleic acids. The
nucleic acid systems are mostly for submerged agarose gels. The agarose gel systems are available in a range of sizes.
The bigger boxes can hold more samples than the smaller boxes and the samples can be run further to improve the
resolution between bands. However, the smaller boxes run a lot faster and use less agarose so they are much better for
tests that involve fewer samples with well-resolved bands. There is also a Western Blotting apparatus and a special
pulse-field gel apparatus that is used to electrophorese and isolate large molecules such as whole genomes,
chromosomes and viruses.
a variety of incubators, air-shakers, tube rotators and waterbaths to grow cultures. Some incubators are stationary
and are suitable for static incubations of flasks and plates. Some move to allow agitation or aeration of flasks or tubes or
other containers. Some work at ambient temperature. Some have thermostats to work above room temperature. Some
of the waterbaths can be hooked to cooling systems to work below ambient temperature. Some work can also be done in
cold rooms to lower the ambient temperature.
ovens for higher temperature incubations.
digital cameras, an Alpha Imager (geldoc) system and a scanner to create electronic files that store data and images.
The digital cameras can be used in freestanding mode or stabilized in box stands or hooked to some microscopes.
a fluorimeter that measures fluorescence. It is analogous to a spectrophotometer but measures the intensity of light
created by the sample rather than transmitted light. It can be more sensitive than spectrophotometry and has wide
application for a variety of chemical and enzymatic assays.
a spectrofluorimeter that is analogous to the fluorimeter but allows a broader range and combination of excitation and
emission wavelengths.
a luminometer to measure light emitted by luminescent assays.
small capacity bead beaters, large capacity bead mills, grinders, a sonicator and other equipment for breaking cells.
an electroporator to shock and transform cells.
a gas chromatograph (GC) for separating and measuring volatile molecules such as acetic acid, butyric acid and other
metabolic wastes. It can also be used to measure some types of storage products.
a variety of gas tanks and pressure regulators to adjust and control the atmosphere in cultures and reactions.
a variety of ion-probes for measuring pH, oxygen, ammonium and re-dox.
a scintillation counter for quantifying tritium and carbon-14 isotopes. There is also an x-ray developer system for
working with autoradiographs.
two hybridization ovens for working with nucleic acid hybridizations or protein blots.
an ultraviolet light chamber for mutagenesis and binding molecules to specific activated membranes.
polymerase chain reaction machines (PCR).
22
General References that might be Useful to find Project Background

1. Books
Molecular Cloning: A Laboratory Manual, Green and Sambrook 2014.
At the Bench ; a Laboratory Navigator, Barker K.. 1998. A guide to many basic laboratory research skills and
functions.
Difco & BBL Manual , Manual of Microbiological Culture Media , Zimbro MJ, Power DA., (eds.). 2003. A
manual discussing the history, properties and uses of the Difco and BBL media that are commonly used in microbiological work.
Molecular Cloning, 2nd ed., Sambrook J, Fritsch EF, Maniatis T. 1989. A manual molecular biological techniques.
Molecular Biology of Bacteriophage T4, Karam, J.D. and J.W. Drake (eds.) (1994) Reference text concerning T4
properties
Principles and Techniques of Practical Biochemistry, 5th ed., Wilson K. Walker J. 1999. Theory and application of
biochemical techniques.
Short Protocols in Molecular Biology, 4th ed., Ausubel FM et al. 1999. A manual of molecular biological techniques.
2. Catalogues
Bio-Rad Life Science Research Products Information on molecular biology reagents and methods
Fisher Biotech Collection Information on molecular biology reagents and methods
Invitrogen Information on molecular biology reagents and methods
New England Biolabs Information on molecular biology reagents and methods
Promega (Fisher Scientific) Information on molecular biology reagents and methods
Roche Molecular Biochemicals Information on molecular biology reagents and methods
Sigma Catalogue Information on chemicals
3. Websites
UBC Library at http://www.library.ubc.ca
Search for journal articles related to topics by using
Biosis
Medline
PubMed
Web of Science.
These are good places to find journal articles in the topics and to search whether published authors have
published more recent studies.
Google Scholar at http://scholar.google.ca/schhp?sourceid=navclient&ie=UTF-8
Search for research articles and scholarly books
Google at www.google.com
General information search site
EcoCyc and MetaCyc at http://ecocyc.org and http://metacyc.org
Information on metabolic pathways and enzymes in hundreds of different organisms including Escherichia coli
and Bacillus subtilus.
Coli Genetic Stock Center at http://cgsc.biology.yale.edu/
Data base and source for mutant strains of Escherichia coli including the single gene knockout strains in the Keio
Collection.
Hancock Laboratory Methods at http://cmdr.ubc.ca/bobh/methodsall.html
Extensive collection of common assays for molecular and cellular work.
23
Data Presentation and Analysis:
1. The final report for this project must be written as a journal article in a modified form of the
general style used by the American Society for Microbiology for 2013. Examples of this style
include the Journal of Bacteriology, the Journal of Immunology, the Journal of Clinical
Microbiology and the Journal of Virology. Detailed instructions for preparing and submitting
the article are available in the "instructions for authors" for the UBC Journal of Microbiology
and Immunology (JEMI) at the MICB 421 Connect site and Appendix F of this lab manual
2. When the initial draft of the report has been submitted and reviewed by the editors it will be
returned to the authors for any required corrections or revisions. The mark for the project will
not be applied to the grade until the corrected article has been returned to the editors and
accepted for publication in JEMI. All accepted articles will be published and available for
other students in other classes to read and study.
24
Schedules, Performance, Marks
Schedule
The course has two projects (Project 1 or P1 and Project 2 or P2), a lab notebook, an optional
journal, several written reports, an oral presentation, a video communication assignment, several
mandatory scheduled meetings, a WHMIS assignment, an in-class final exam. You will need to
work concurrently on some assignments. Material may be submitted in advance of the due date.
Schedule Overview
Week
Date
Lecture
Lab Phase
Assessment
Due Date
4 8 Jan
1/2
Project 2 Individual Proposal - Flowchart
10-Jan-16
11 15 Jan
3/4
P2 Individual
Proposal
Project 2 Individual Proposal - Written
15-Jan-16
17 22 Jan
WHMIS and Lab Introduction Quiz
14-Jan-16
25 29 Jan
Refworks Assignment
29-Jan-16
1 5 Feb
P2 Team
Proposal / P1
Project
Project 1 Report
5-Feb-16
8 12 Feb
Project 2 Team Proposal
12-Feb-16
15 19 Feb
RW
22- 26 Feb
none
29 Feb 4 Mar
10
7 11 Mar
10
11
14 18 Mar
Exam
In class Final Exam
15-Mar-16
Guest lecture
22-Mar-16
Presentations and Questions (#1/2)
29/31-Mar 16
Presentations and Questions (#3/4)
5/7 Apr-16
12
21 25 Mar
11/12
13
28 Mar 1 Apr
12/13
14
4 8 Apr
14/15
UBC Exam Period (note: there is no exam
during this period for MICB 421)
P2 Research
P2 - Writing
P2 Revisions
Draft Paper
Revised Paper, Lab Notebook
15-Apr-16
30-Apr-16
25
Week
Lecture
Date
Activities for Week

Lecture (8am Tues and Thurs,
*
WESB201)
Team Meetings
Lab
Lab Tours
1/2
4 8 Jan
Tues and Thurs Class
none
3/4
11 15 Jan
Tues and Thurs*** Classes
Team Meeting
17 22 Jan
Only Tues Class
Team Meeting
Project 1 and Lab

Demos
Project 1
25 29 Jan
Only Tues Class
Team Meeting
Project 1
1 5 Feb
Only Tues Class
Team Meeting
Project 1
8 12 Feb
Only Tues Class
Team Meeting
Prep - Project 2
RW
15 19 Feb
No lecture
Team Meeting
Project 2
none
22- 26 Feb
No lecture
Team Meeting**
Project 2
29 Feb 4 Mar
Only Tues Class
Team Meeting**
Project 2
10
10
7 11 Mar
Only Tues Class
Project 2
11
Exam
14 18 Mar
In Class Final Exam on Tues
Team Meeting**
Team Meeting**
Project 2
Project 2
12
13
11/12
21 25 Mar
Tues and Thurs Class
Team Meeting**
12/13
28 Mar 1 Apr
Tues and Thurs Class Pres.
Team Meeting**
Project 2
14
14/15
4 8 Apr
Tues and Thurs Class Pres.
Team Meeting
Project 2
* Team meetings may be optional on some weeks depending on lab actvity. Visit MICB 421
Connect for updates on a week to week basis.
** Weekly Summaries begin on Week 8 and end on Week 13
*** WHMIS and Introductory Lab Quiz In Class
RW = UBC reading week
Detailed Schedule
Week 1 (Jan 4) Select a problem and research background about the problem and the
methods normally used to work with the problem. Submit flowchart via Connect. Attend lab
tour. Recruit team members.
Week 2 (Jan 11) Register your team. Have team photo taken. Sign up for a team meeting
where we will discuss set-up and execution of Project 1. Each person must submit a 1-page
written proposal of the project that they would like to pursue to the drop box at MICB 421
Connect by 11:59 pm. Reference can be added on a separate page.
Week 3 As a team, choose a lead project. Begin to refine your experimental problem, set
objectives, start to develop an experimental plan, check sources of additional information,
consider specific resource requirements (are the types of supplies and equipment
available?), safety constraints.
Weeks 3, 4, 5 Execute Project 1 in the laboratory and compile report for submission on 5
Feb 2016 at MICB 421 Connect by 11:59 pm. As a team, refine and draft Project 2
proposal.
Weeks 5 - 6 - Start to prepare general supplies that will be needed to do the project.
Common supplies may be shared. Request any chemicals or supplies that will likely be
needed but are not available in the class inventory.
26
Week 6 - submit the formal written proposal to the drop box at MICB 421 Connect by
11:59 pm.
Weeks 7 and 8 - begin Project 2 experiments.
Weeks 7 -13 continue experimental work. Each week discuss progress of the results,
observations and problems at the weekly team meeting. Report progress / problems /
changes to proposals. Bring a printed copy of the results and experimental proposals to
each meeting.
Week 11 and 12. Begin writing the introduction, methods and references for the final report.
(Some of these details could be started sooner by writing them out as you encounter them in
the project). Be able to provide an outline of the available results and general outline of the
discussion at the weekly meeting. Clean up lab, stabilize useful cultures and supplies, and
discard other materials.
Week 11 -13 deliver oral presentation of the project. Submit video assignment by 1week post-presentation.
Week 14 meet with Instructor and TA to discuss plans for research article.
Submit draft manuscript for review and grading on 15 Apr 2016 at MICB 421 Connect by
11:59 pm. It will be reviewed, graded and available to be emailed to you by D22 Apr to do
final revisions.
30 Apr 2016 submit the final corrected manuscript (as an electronic copy) and the lab
record book for final grading. If you are away from UBC on a work term make
arrangements before you leave to submit the record book.
27
Checklist for MICB 421
Training, Safety, and Library Assignments
WHMIS and Lab Quiz (15 Jan 2016)

RefWorks Set-up (29 Jan 2016)
Project 1
Notebook (5 Feb 2016)
Project 2
Individual Proposal Flowchart (10 Jan 2016)

Individual Proposal Written (15 Jan 2016)
Team Proposal (12 Feb 2016)
Draft Paper Submission (15 Apr 2015)
Revised Paper and Lab notebookSubmission (30 Apr 2016)
Weekly Summary #1 (28 Feb 2016)

Weekly Summary #2 (6 Mar 2016)
Weekly Summary #6 (3 Apr 2016)
Team Oral Project Presentation

Questions Presenation Set #1
Video Response due 1-week post-presentation
Exams
In Class Final Exam (15 Mar 2016)
Optional
Reflective Journal (submitted Week 3 through Week 13)

Bonus Seminar Summary #1
28
Performance and Marks

Grading Scheme
Weight (%)
Scheme 1 Scheme 2 Scheme 3 Scheme 4
Activity
Due Date
P1 Notebook
5 Feb 2016
10
10
10
10
P2 Individual project
proposal
8 Feb 2016 (flowchart)

and 15 Feb 2016 (written)
P2 Final team project

proposal
12 Feb 2016
15
15
15
15
P2 Oral
Presentations
29, 31 March and 5, 7 or April 2016
15
15
15
15
P2 Draft paper
15 April 2016
20
25
30
35
P2 Revised paper*
30 April 2016
2.5
2.5
2.5
2.5
WHMIS/Lab Quiz**
and RefWorks
Assignment
29 Jan 2016
2.5
2.5
2.5
2.5
In class final exam
15 Mar 2016
20
20
10
10
Weekly summary
Two weekly summaries will be chosen at

random and marked during the term (2.5%
per submission); 0.5% will be deducted for
missed submissions
Optional Reflective
Journal (via Connect)
Submitted each week throughout the term.
NA
NA
Optional Seminar
Bonus
Up to 1.5% of your course grade can be

earned by attending up to 3 seminars at
UBC. See below for details.
Up to 1.5
Up to 1.5
Up to 1.5
Up to 1.5
There will be a 20% penalty for late submissions of the reports unless permission for a late
submission has already been arranged.
Notes:
1. The potential grade for each student will be individually assessed in each of the grading
schemes. The student will be given the higher grade from the different schemes. The
patterns should be read vertically. The total in each vertical column is 100%. Entries
with NA indicate that the category is not applicable for that pattern.
29
2. * There is no grade for the draft report or the lab notebook until an adequate final
electronic report and the lab notebook are submitted.
3. * The lab notebook must be submitted to get the grade.
th
4. If the contribution to the project by some team members is less than 9/10 of the
expected average participation for the team then the project marks for the students
with weak participation will be prorated in proportion to the apparent contribution
to the project.
5. Significant breeches of etiquette or inadequate preparation that affect the safety or
convenience of other students will be penalized by subtracting a penalty mark from the
final grade of the offending student.
6. ** A minimum mark of 80% is required on the WHMIS assignment in order to begin
working in the lab.
Effort and Etiquette

Other research courses such as directed studies expect approximately 20 hours of lab
work per week. The weekly workload for your team should be similar. Some weeks it
might be higher.
Your lab work affects other people in the department that either share the same
equipment, clean equipment, prepare media or instruct. There are seven rules that you
will be expected to follow in order to minimize problems with other people.
1. Clean up balances, centrifuges, and spectrophotometers each time you use them.
2. Clean up your working area including the sinks and balances before leaving the lab.
Put the capped tubes that need to be autoclaved and the uncapped tubes in
separate discard racks in room 112. Remove tape labels and felt pen labels from
flasks and beakers. Use cold water to rinse out all flasks and tubes that contained
dangerous chemicals before you put the flasks on the discard cart for washing.
3. Place working equipment back in the storage areas when you finish.
4. Tell the instructors about malfunctioning equipment so that it can be repaired.
5. Tell the instructor if any particular bottles of chemicals are running low so that the
chemicals can be ordered before there is a shortage.
6. Discard your cultures from the incubator and the refrigerator when the lab work is
completed.
7. Prepare your own project supplies unless you have made formal arrangements to
borrow supplies from another team. Respect other students spaces and supplies.
Participation
30
1. Students are expected to show enthusiasm for their projects and commitment to doing
the best possible job. This commitment includes demonstrating concern for other
team members as well as their individual part of the projects.
2. On average over the term, each member in the team is expected to participate in the
planning, lab work, analysis and writing phases of all the projects.
3. Participation will be assessed by using the weekly summary, the draft report and the
final, peer comments with regard to the participation, and observed effective
participation in the class work throughout the term. Refer to Appendix O for grading
rubric.
4. The average weekly amount of work for each team member over the term is expected
to be 5 or 6 hours per week. Some weeks might be shorter than the average if other
weeks are longer as long as the overall participation balances out every two or three
weeks.
5. Individual students can meet with the instructors if they feel that there is a problem in
the way the workload or effort is distributed. If a member of the team is consistently
not participating in the assignments or blocking participation of others then the other
members of the team should discuss the problem with the instructors.
Laboratory Notebook
The lab notebook must be brought to each lab to keep it up to date and be a record of all
raw experimental details. It is your responsibility to ensure that it is maintained
throughout the term. The TAs and Instructor may check lab notebooks from time to
time throughout the term to provide feedback and to assess progress. Refer to lab
notebook formatting guidelines.
Reflective Weekly Journal (via Connect)

This option expects you to keep track of the various aspects of your learning process as
the course progresses. To keep track of these processes you will be required to write a
short weekly statement of 250 to 500 words that describes the different parts that you
find difficult or time consuming or frustrating or rewarding or appealing in the projects. In
order for the journal to work it must be done each week. Journal submission must be
made each week before the deadline of 11:59 PM Sunday evening on MICB 421
Connect.
Project Proposals and Papers
The requirements for the reports are described in Appendix F of this course manual and
the JEMI link in The UBC Connect eLearning site for MICB 421. Data will not be
specifically graded in the reports. About 40% of the points will be determined by the
quality of presentation details such as the suitability of units, adequate labeling of
graphs, correct arithmetic processing, suitable rounding of values and general
readability. Another 40% will depend on how you analyze, correlate and explain your
actual observations and the potential meaning of your results rather than whether you
31
discover the actual expected answers for the experiments. The last 20% will be
determined by the quality and appropriateness of the concluding statement, the followup experiment and the referencing.
Each report must be submitted by the due date and time. Reports submitted late
will be penalized by 10% per day overtime. Reports will not be accepted once the
material is covered in class or any submitted reports are returned to the class. Missing
reports will be marked as zero when the final grade is calculated. Each marked report
will be returned with written comments and suggestions on how to improve future
reports. If you feel that the mark is less than you expected and you do not understand
the comments in the report, please discuss your concerns about the mark with the
instructor.
Project Presentation, Questions, and Video Responses
The goal of the project presentations is to give each time an opportunity to share their
research with their peers. We recognize that projects will be underway and that data will
represent a work-in-progress. Teams should follow the presentation guidelines described
in lecture. Conclusions should be derived from the presented evidence only (i.e. avoid
speculation or extrapolation). During each presentation, each audience member should
think of / compose 1 or 2 questions. These questions can be asked at the end of the
presentation or can be discussed after class with your team. Each team must submit 1
question for every other team via Connect by 5pm the day after the presentation. Teams
will review the submitted questions and choose 3 to respond to via video. Video response
should be uploaded within 1-week of your presentation. The scores from each rubric will
be tallied to determine your teams grade for the assignment. Each component of this
activity will be marked against a rubric. In addition to contributing to your course mark, this
assignment may have long term value to you as evidence of your ability to communicate, a
highly valuable skill in any field.
Seminar Bonus Option
UBC is a fantastic place for research! As such, in the spirit of a research-driven course
such as MICB 421, bonus marks can be earned for attending seminars. For each seminar
you attend upload a short summary paragraph (3-5 sentences) and you will earn 0.5%
toward your course grade. You can earn a maximum of 1.5% for attending a total of 3
different seminars. Uploaded submissions will be visible to the class via an open blog on
Connect.
Theory Exam
Exam questions could include:
-
any concepts presented or encountered in the lectures.

application of any general type of calculation encountered in lab work such as
growth rate, growth yield, unit conversions, dilutions and data transformations
principles for establishing or designing experiments.
the theory covered in Appendix C, D and E of the lab manual.
the safety concepts and hazards discussed in Appendix B of the lab manual such
as the WHMIS symbols, aerosols and containment.
Some questions might require calculators. Graph paper will be provided with the exam if
it might be useful or necessary to answer particular questions.
32
Grading Rubrics
The following pages have rubrics for the various reports that will be submitted over the
term. The points in the rubrics reflect the relative weighting of each consideration in the
grade for that report rather than the final calculated grade.
Weekly Summaries and Activity Chart

Competent Cell Preparation Project 1
Lab Notebooks Projects 1 and 2
Individual Project Proposal Project 2
Team Project Proposal Project 2
Draft Paper Project 2
Team Oral Presentation Project 2
Questions Project 2
Video Response Project 2
33
Grading Rubric for the Weekly Summaries (Total of 22 Points)
Proposal detail
Clearly meets
expectations.
Few, if any
problems
Acceptable
but definite
weaknesses
Does not meet

expectations.
Significant
weaknesses
Criteria
Identification
Not
applicable
Identifies your submission with

team alphanumeric and
individual names
Working Title
Not
applicable
Current working title for project
Major Progress
Explains the goal and what has

been accomplished; provide
experimental reference numbers
from lab notebook
Major Difficulties
Explains any major difficulties

that have slowed progress or led
to changes in approach
Major Changes
Explains any major changes and

the purpose for the changes.
Remaining Work
Description of the remaining

work and the week by week
staging of the remaining work.
Demonstrates care and critical

understanding throughout
report. Has been checked to
eliminate most spelling,
grammar and syntax problems.
Clearly and concisely presented.
Complete. Readily understood.
Overall
34
Grading Rubric for Project 1 Preparation of Competent Cells (Total of 42 Points) (1 bonus point)
Proposal detail
Clearly
meets
expectations.
Few, if any
problems
Acceptable
but definite
weaknesses
Does not
meet
expectations.
Significant
weaknesses
Identification
Not
applicable
Report formatting
12
Title
Experiment is given a
descriptive title.
States the goal of the project
and the context of the work
with respect to previous and
future work.
Purpose
Materials and
Methods
Results
Figures and Tables
Conclusions
Future Directions
Overall impression
Criteria
Lab notebook is labelled with

team name and names of
individual team members.
Formatting guidelines
described in Appendix F are
followed. Refer to marking
rubric on Lab Notebooking.
All materials and methods are

documented. Relevant
literature is referenced.
All observations are recorded.
Data is presented as standalone tables or figures and is
also clearly described in the
text. Intepretations of data are
stated. In general, results are
written in past tense.
Formatted as per ASM 2013
guidelines for paper
submission. Appendix F.
Bullet point summary of main
findings and points of interest.
Comment on the potential
outcomes and technical
problems that need to be
addressed.
understanding throughout the
report. Correct language,
tenses, and terms. Correct and
appropriate citations.
35
Grading Rubric for Lab Notebooks (total 12 points)
Expected Detail
Clearly meets
expectations.
Does not meet

expectations.
Criteria
Title
Suitable and relevant

to experiment.
Sufficient detail to
be unique and
complete with
regard to the
objectives.
Experiment
Code
Present and accurate.
Page Numbers
Correct
Present and accurate.
All Sections
Complete
Purpose, Methods,
Results, Discussion
and Conclusion
documented.
Table of
Contents
Present and up to
date.
References made
throughout when
appropriate.
Experiment codes or
literature citations.
Present and accurate

on every page.
Signed and dated

with a week of
initial entry.
References
Date
Witness
36
Grading Rubric for the Individual Project Proposal (Total of 31 Points)
Proposal detail
Clearly
meets
expectations.
Few, if any
problems
Acceptable
but definite
weaknesses
Does not
meet
expectations.
Significant
weaknesses
Identification
Not
applicable
Identifies your submission with

your name
Source of observation /
problem
Source of observation/problem is
mentioned and cited in
appropriate ASM style
Criteria
Background
Clearly written background to

understand / explain the
relationship of the explanation to
the problem and the approach.
Observation / problem
Clearly written description of the

selected observation or problem
Explanation / hypothesis
Clearly articulated explanation of

the cause of the observation /
problem that you will work on.
Clearly stated experimental
question(s) that will test the
explanation / hypothesis. Includes
a brief explanation of how
knowing the answer to the
question testing the hypothesis.
Clearly written description of the
general technical approach you
would use to test the experimental
question. Mention the specific
type of methods (PCR,
mutagenesis et cetera).
Experimental Aims
Approach
Feasibility / potential
outcomes
Comment on the potential

outcomes and technical problems
that need to be addressed.
Flow Chart
Flow chart sections are clearly

communicated. Research plan is
logical.

report. Correct language, tenses,
and terms. Correct and
appropriate citations.
Overall impression
37
Grading Rubric for the Detailed Team Project Proposal (Total of 46 Points)
Expected Detail
Clearly
meets
expectations.
Few, if any
problems
Acceptable
but definite
weaknesses
Does not
meet
expectations.
Significant
weaknesses
Proposed Title
Introduction and
Background
Observation and
Explanation
Experimental
Aims
Overview Chart
(Concept Map)
Methods
Supplies and
Equipment
Weekly Time
Frame for
Completing the
Work
Protocol
Criteria
Suitable and relevant to proposal.

Sufficient detail to be unique and
complete with regard to the
objectives.
Clearly written. Addresses
details needed to understand the
explanation, approach and
outcomes for your proposal.
Clearly articulated explanation
for the observation. Suitable
relevance. Builds on an idea
instead of dabbling with a
method.
Clearly written question (or part
of a question) that will test the
explanation. Relates to
explanation and provides
outcome.
Brief overview that shows a
diagrammatic relationship of the
tests within the context of the
experimental question.
Explains what will be done. An
overview of the work and how
the methods fit together to
address the objectives
Explains how the parts / tests
will be done. Choice of suitable
methods for the protocol and
objective. Can be referenced but
should give operational details.
List of supplies and chemicals
needed. Include quantities and
details of preparation of
solutions such as solvation
conditions for your actual
experiment.
Week by week staging of the
work. Allow time to test/work
with unfamiliar procedures or
equipment, testing strains,
determining preliminary values
to assess ranges before
attempting the main experiment.
38
Potential Pitfalls
Known Hazards
References
Participation
Report
Overall
Description of the main obstacles

or difficulty that might be
encountered in the protocol.
Identifies unusual and
significant hazards (if any).
Marginal if reasonable unusual
problems missed. Inadequate if
deadly problems are missed.
Distinguishes routine, minor
risks from real hazards.
At least four background
references supporting the
proposed explanation and
protocol. Proper ASM style.
Includes a meaningful section
describing how each team
member contributed to the work
and the report.
proposal. Has been checked to
eliminate most spelling,
grammar and syntax problems.
39
Grading Rubric for the Draft Paper (Total of 100 Points)
Report detail
Consideration
Title
Clear and Accurate
Abstract
Introduction
Materials
and Methods
Results
Discussion
Conclusion
Clearly meets
expectations.
Few, if any
problems
2
Acceptable
but definite
weaknesses
1
Does not meet

expectations.
Significant
weaknesses
0
Criteria
Relevant
Clear and accurate.

Appropriate format
Relevant details
Clear and accurate
Relevant details.
Appropriate content.
Appropriate depth
Complete and accurate
Appropriate format and

content. Suitable
abbreviations
Consistent and
accurate processing
Appropriate format.
Details are clear and
accurate
12
Appropriate
interpretation of the
results
Appropriate
observations
Has recognized the major

relevant observations
Appropriate comments
Clear and accurate
Relevant
Reasonable depth of
analysis
Accurate deductive
statement
Addresses the
experimental question
Integrated ideas and wrote

about the ideas rather than
simple descriptions
Statements are consistent with
the results, help to understand
the results and relate the results
to the purpose. Statements
show insight. Makes use of
supporting knowledge. Covers
all major observations
Deductive statement proved
by the results rather than
explanations
Conclusion addresses the
experimental question
Concisely conveys intent and

outcome of project
Relates to the study purpose
Easy to read. Consistent with
article. Includes purpose, main
observations and main
conclusion. No abbreviations
or references
Easy to follow. Focused on the
purpose of project. Proper
coverage of background for the
analysis and the discussion.
Does not include parts that
should be in Methods
Mentions relevant details. Uses
paragraph form. Uses citations
where appropriate. Provides
enough detail to follow
approach.
Consistent interpretation
between uses. No processing
errors
Tables and figures processed
with expected details. Suitable
expressed comparable
significance.
Interpretation of trends and
observations is reasonable
40
Future
Directionss
References
Relevant and feasible
Outcomes
Additional, appropriate
and relevant
ASM format, accurate

and appropriate
citation
Participation
Report
Explain how each

author contributed to
the project and the
report.
Overall
Overall impression
Lab
Citizenry
Weekly lab work

assessed by TAs and
Instructor.
+5
-5
Addresses a significant
problem or explanation raised
in the discussion. Relevant to
original purpose.
Four additional references that
provide depth to discussion.
Helpful. Relevant
Cited by number in the
manuscript. Correct ASM
style in listing.
Includes a meaningful section
describing how each team
member contributed to the
work and the report.
report. Correct language,
tenses, and terms. Good
insight into results.
Demostrates safe, courteous,
and organized work habits in
the lab.
Draft papers will be reviewed upon submission for formatting. Papers that do not
need formatting expectations will be returned to the authors for revision prior to
marking. A formatting checklist will be posted to Connect later in the term. Papers
that do not meet minimum formatting requirements will penalized 5% of the final
grade for each round of revision required.
41
Grading Rubric for the Oral Presentation (Total of 34 Points)
Presentation detail
Clearly meets
expectations. Few, if
any problems
Acceptable
but definite
weaknesses
Does not meet

expectations.
Significant
weaknesses
Criteria
Title
Effective title. Title is supported by

data presented.
Introduction
Effectively explains: research

question, significance, observation,
hypothesis, and experimental aims.
Experimental concepts
Effectively explains experimental

question and key concepts.
Data presentation and

interpretation
Effectively explains experimental

set-up (including figures and tables),
observations, and interpretations.
Conclusions and Future

Direction
Conclusions are supported by

presented data. Future directions are
logical, feasible, extensions of
conclusions.
Acknowledgements
Recognize team members,

collaborators, contributors, funding.
Visual aids
Formatting
Appropriate use of presentation

technology (e.g. Powerpoint).
Slides are well designed and easily
interpreted.
All Figures and Tables are formatted
as per ASM style guidelines. All
figures show markers and are
labeled clearly.
Delivery
Speaker(s) sets a reasonable pace,

displays enthusiasm and confidence
(e.g. makes eye contact), and
modulates tone for emphasis.
Timing
Presentation meets time constraint of

maximum ___ minutes. (Time will
depend on number of teams in class)
Presenters effectively address

questions from audience. e.g.
openly considers perspectives,
provide logical, direct responses.
Overall quality of presentation.

Logical flow. Attention to details
such as figure formatting, font size,
and references.
Question and Answer

Period (not included in
___ minute presentation)
Overall
42
Grading Rubric for Questions from Team Presentations (1 point per team)
Team ____________________
Criteria: Full marks will be given for constructive questions that help to improve or promote further thinking
about the project. Questions may be scientific or technical in nature. Question may address specific aspects
of the material presented or inquire about possible future directions or alternate models. Questions must be
respectful and should demonstrate some appreciation for the presentation (e.g. Thank-you for a very clear
presentation. I really like your experimental system where. Have you considered variable X and Y and how
these parameters might impact your interpretation of the results?)
Team
Clearly meets
expectations.
Few, if any
problems
Acceptable
but some
weaknesses
Does not meet

expectations.
Significant
weaknesses
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Comments
43
Grading Rubric for Video Response (Total of 33 Points)

Proposal detail
Clearly meets
expectations.
Few, if any
problems
Acceptable
but some
weaknesses
Does not meet

expectations.
Significant
weaknesses
Criteria
Includes a descriptive
presentation title at the
beginning. Closes with team
names and relevant references.
Video opens with a brief (1-3
minute) summary of the project
outlining key background info,
the research question,
hypothesis, and approach.
Title and
Credits/References
Summary of Project
Originality /
Creativity
Demonstrates some original

thought or presentation style.
Quality of Answers
4.5
Answers address the stated

question. Evidence is presented
clearly. Assumptions and
limitations are acknowledged.
Each questions will be scored
out of 3 points.
Delivery
Video is well paced; vocal tone

is enthusiastic or modulated to
engage viewer / listener.
Appropriate level of
detail
Video explains concepts at a

level that suits the target
audience.
Timing
Not
applicable.
Video presentation meets

maximum 15 minute time
constraint.
Video presentation demonstrates

care and critical understanding.
Overall Impression
44
Appendix A - Basic Methods
I.
Efficient Organization
1.
Arrange your working space so that you can work safely, accurately and
efficiently.
2.
Start the organization by facing the workbench and imagining a line that
divides the surface into a left and right side. Things that are to be used or
held by the left hand should be placed on the left side of the line and vice
versa for the right side.
3.
Things that are used infrequently should be placed further back from the
imaginary centerline than other things that are used more frequently.
4.
You should be able to use the equipment (vortex, pipettes, Bunsen

burner, tubes et cetera) without having to walk around. In a wellorganized bench you should generally be able to reach and use the
equipment while standing in one place.
5.
In aseptic work organize the material and equipment with the Bunsen
burner at the imaginary centerline, about 20-to-30 centimeters from the
front of the bench.
6.
Ensure that hoses and electrical cords do not dangle over the edge of the
bench where they could inadvertently be hooked while working. Be
careful that the flame from the Bunsen burner is not burning either the
Bunsen burner tubing, electrical cords or the over-head counters.
7.
Keep your notebook and flow charts to one side of the workspace, or on
the shelf above the bench or in a drawer out of the way. They should not
be in the center of your work space where they get in the way and
become contaminated with spilled chemicals or bacteria
8.
The organization is dynamic and should change as different stages of the

work proceed. For example, when you are diluting and plating bacteria
for viable plate counts, the saline containing flask is initially up front by the
vortex, but is then moved back out of the way after the dilution blanks are
made. All dilution blanks should be made at once. After the samples are
diluted, the plates should be moved from the back or side areas and
placed between the vortex and the Bunsen burner so they are closer and
easier to use.
45
II.
Aseptic Work
Aseptic technique is used to maintain cultures free of unwanted contaminants and minimize
any chance that the culture will infect you. Since contaminants can not teleport into sealed
culture containers, most aseptic technique will require you to work to rapidly when a culture
tube is open in order to minimize the time that a contaminant has available to fall into the
open culture. Rapid work requires that you know in advance all the manipulations that you
will do once the tube is open in order to avoid wasting time looking up what to do. Aseptic
technique also includes steps to eliminate the contaminants that might fall into the tubes.
These steps include routinely sterilizing the workbench with disinfectant, flaming the open
surfaces of containers to make the surfaces too hot for contaminants to survive, using
sterilized equipment and working in patterns that maintain the equipment in a sterile state.
E. Preparation for Aseptic Work
1.
2.
Personal preparation
a.
Put on your lab coat and arrange your workbench.
b.
Wash your hands at the sink. Use the liquid soap in the 500-ml
polyethylene bottles stored near the taps. This washing step should remove
loose skin cells and other material that can fall off your hands onto your
cultures. Subsequently try to avoid touching your nose or hair since this
contact can contaminate your hands with particles of grease or small flakes
of skin.
c.
At the end of the experiment and any time other time that you want to leave
the lab you should re-wash your hands to prevent anything from your
cultures that might have contaminated your hands from getting transferred
around.
Bench preparation
a.
Pour approximately 10-to-20 ml of phenola (the blue coloured disinfectant in

the squirt bottles at each bench) onto the bench and spread it around with a
paper towel. The damp towel can then be used to wipe and sterilize the
Bunsen burner base and gas line tubing before being discarded into the
waste paper basket.
b.
If you have difficulty controlling manipulations (dripping pipettes, splattering

loops et cetera) or are working with dangerous organisms it is wise to fully
soak a paper towel with disinfectant and leave it on the bench below the
area where the work is being done so that any drips will land in the
disinfectant and be sterilized.
c.
Non-hazardous cultures can be handled by wiping the bench with

disinfectant before and after the transfers. This method has the advantage
of providing a dry surface to work on and minimizes the chance of
contaminating your cultures with disinfectant.
46
F. Flaming
1.
Most transfers of aseptic cultures involve flaming of containers and/or

inoculators. This flaming is a simple procedure but must be done properly to be
effective.
2.
All flaming begins by starting the Bunsen burner and checking that it is adjusted
properly.
3.
Flaming inoculators (loops and stabs)

a.
b.
4.
Hold the inoculator by the handle and briefly pass the metal part of the
holder for the wire loop (or stab) about one centimeter above the colder,
blue-coloured cone of the Bunsen burner flame, then hold the wire end of
the inoculator almost vertically in the hot area of the flame until the wire
becomes red hot.
In most cases the heating is more efficient and unsafe aerosol splattering of
your sample is minimized, if you start the flaming by first holding the region
of the loop where the wire joins the handle in the flame, then drawing the
rest of the wire up into the hot flame after the region of the join becomes hot.
Flaming tubes
Hold the tube in your left hand at about a 45 degree angle from vertical to
prevent contaminants from falling into the culture when the cap is removed from
the tube. Remove and hold the cap with the little finger of your right hand and
then hold the top 1 or 2 cm of the opened tube at a slant in the hot flame of the
Bunsen burner. If the tube is flamed too long, it will subsequently heat up the
metal cap and burn your fingers when you open the tube the second time. If it is
heated too briefly, the surface will not be sterilized. The proper length of time
correlates to the formation and disappearance of a mist of water vapor on the
flamed surface. This time is normally about 2-to-4 seconds if the tube is rotated
with your fingers so that the entire exposed surface at the top is directly heated.
A properly flamed tube should make the metal cap warm but not hot.
47
5.
Flaming flasks
Flasks are similar to tubes except that the opening is usually sealed with a cotton
stopper that does not conduct heat very well. This difference means that the
open surface of the flask can be over flamed without subsequently burning your
hands as long as you subsequently remember to handle flamed flasks by the
base instead of by the hot thinner area at the neck.
Assuming that the flask is held by your left hand, the cotton plugs are generally
removed by curling the little finger of the right hand around the knot at the top of
the plug and pulling up so that the plug will dangle away from your hand. The
plug should not come into direct contact with the palm of your hand and the
remaining fingers of your right hand should be free to manipulate pipettes or
other equipment. Medium size and large flasks are more awkward to hold and
rotate in the Bunsen burner flame than tubes but one way of holding the 125 and
250 ml flasks in the flame is to balance the bottom of the flask against the half
curled up baby finger of your left hand, loosely close your remaining fingers
about the lower part of the flask such that the flask can not fall, then rotate the
loosely held flask by alternately pulling it with your index finger and pushing it
with your thumb.
Start to flame large flasks by tilting the flask so that only one edge is on the
bench. Hold the flask in this position with your left hand, remove the cotton plug
with the little finger of your right hand, then pick up the burning Bunsen burner
with the thumb and index finger of your right hand and move the nozzle of the
Bunsen burner around the stationary flask while directing the hot portion of the
flame against the exposed glass at the top of the flask. As before, the plug
should be held in your finger so that it remains sterile. The plug should not be
held in the palm of your hand or set down on the bench.
6.
Flaming pipette canisters

Pipettes are not usually flamed but some people routinely flame the opened
canisters to keep them sterile. The flaming is done by holding the canister
horizontal and setting the open end in the hot flame of the Bunsen burner for
about 5 seconds. The metal canister readily conducts heat and doesn't need to
be rotated but if the canister is over flamed or repeatedly flamed over a short
time interval the protruding ends of the pipettes can become too warm to handle.
48
During the flaming and handling, the canister should not be tilted upright as this
angle will allow contaminants to drop inside the canister and contaminate the part
of the pipette that gets placed into the sterile solutions and cultures.
G. Aseptic Transfer by an Inoculator
Loops are used for most transfers done to liquid media or the surface of solid
medium. Stabs are used for transfers into solid medium for cultures like motility tests,
citrate utilization tests, triple sugar-iron tests and for transfers of small closely spaced
colonies. The general procedure is the same for either tool. Start by disinfecting the
work area and lighting the Bunsen burner, then assuming that you are right handed,
go through the following steps:
1.
Loosen the caps on any capped tubes.
2.
Hold the loop in the thumb and forefinger of your right hand, sterilize it (see the
preceding pages) and allow it to cool for ten to fifteen seconds to avoid scalding
the cultures and creating aerosols during the transfer.
3.
Tilt the tube or flask containing the original culture in your left hand to about a 45
degree angle, then remove and hold the plug or cap in the curl of the little finger
of your right hand.
4.
Flame the mouth of the open container.
5.
If the culture is in liquid medium, dip the tip of the sterile, cooled inoculator into
the culture fluid. If the culture is on solid agar, touch the tip of the inoculator onto
the agar surface to ensure that it is cooled then touch the growth area.
6.
Lift the tip of the inoculator from the culture tube, flame the mouth of the
container, loosely replace the sterile cap or plug, then set the tube down in the
rack and push down the loose cap with your empty left hand. This way of
capping avoids jarring the inoculator and spraying bacteria around.
7.
Pick up the container of medium to be inoculated in your left hand, remove the
cap or plug and flame the container.
9.
a.
For inoculation of broth, dip the loop below the surface of the broth.
b.
For inoculation onto a solid agar surface draw the loop over the surface of
the agar.
c.
For inoculation of a stab culture stab the wire 1-to-2 cm vertically into the
center of the agar.
Remove the inoculator, flame the mouth of the container and replace the cap.
10. Flame the inoculator.

11. Petri dishes, unlike flasks and tubes are not flamed when they are opened to do
transfers. The bottom with the agar is usually held in the left hand during the
transfer. The top can either be held open by the fingers of the left hand or fully
removed and set upside down on the bench. The most efficient way to remove
49
the lid from plastic plates is to set the intact plate upside down on the bench.
Then pick up the plate at the edges with the tips of your fingers.
H. Aseptic Transfer by Pipette
1.
The general properties for pipetting described are described in the section on
pipetting.
2.
Loosen the caps of any tubes.
3.
Aseptically remove the pipette from the canister or paper wrapping by holding the
top four or five centimeters of the non-calibrated end.
4.
Add the pipette filler to the pipette.
5.
Hold the assembled pipette and filler with your thumb and the first and second
fingers of your right hand.
6.
Take the sample container in your left hand then remove and hold the plug or
cap with the little finger of your right hand. Ensure that the cap or plug dangles
away from your hand and does not get contaminated by contacting your palm or
the pipette filler.
7.
Flame the mouth of the container.
8.
Pipette up a sample.
9.
Flame the mouth of the container.
10. Replace the cap of the container, then set the container down.
11. Pick up in your left hand the container of fresh medium that you wish to inoculate.
Remove and hold the top with the little finger of your right hand, flame the mouth
of the container, deliver the required volume of sample, re-flame the container,
replace the top and discard the pipette.
12. To discard the pipette hold the calibrated end over the large discard jar
containing Wescodyne, remove the pipette filler and then lower the pipette into
the jar. Dropping or throwing the pipettes into the jars can often break off or chip
the ends and can form aerosols of bacteria as the disinfectant enters at one end
and forces air out the other end. Contaminated pipettes should always be
discarded into the appropriate containers and never set down on the bench.
13. The chance of inadvertent pipette drips is reduced by holding the pipette hand
mostly stationary after filling the pipette and moving the containers in the left
hand to or away from the stationary pipette.
14. Spilled culture is cleaned up by first soaking the area with Westosan and then
using paper towels to wipe up the spill and the Westosan.
15. Disinfect the working area of the bench when you finish the work.
50
III.
Mixing Samples
Quantitative work depends on adequate mixing of samples and dilutions in order to
ensure that these solutions are homogenous enough to relate the concentrations in any
measured sample to the concentration in the total. A number of different mixing
methods have been developed.
A.
Mixing with Stirring Rods and Spatulas

This process is the same as using a spoon to mix cream into a cup of coffee except
that the spoon is substituted with a stirring rod or spatula. It is useful if you have to
mash up lumps in the sample but has the disadvantage that the stirring rods are
usually limited in number and must be cleaned between samples and sterilized for
each aseptic transfer. In addition, it is not very efficient compared to some other
methods.
B.
Mixing by Inversion
This mixing is done by sealing the top of the sample container, then alternatively
inverting and righting the container several times. Some people seal tubes or flasks
by pressing their fingertips or palms over the top but this method can contaminate
your hands and the solutions being mixed. A better method is to stretch Parafilm
over the open end to seal the container before covering the top with your hand.
(The Parafilm is a semi-transparent, malleable plastic sheet that behaves like very
heavy duty Saran wrap). This inversion process is a gentle method for complete
mixing. It has the disadvantage of wasting Parafilm, does not break up lumps very
well, and will not work for aseptic containers closed with cotton stoppers or loose
fitting caps.
C.
Mixing by Pipette
To start this method lower the tip of a pipette to the bottom of the solution in the
container, then fill the pipette and draw it up so that the tip is always just below the
surface of the solution as you drain the pipette. This process of filling and draining
is repeated several times to cause the mixing. It works best when the ratio of the
volume of the pipette to the volume being mixed is greater than 1-to-20. Smaller
ratios such as 1-to-50 are not very effective. The method has the disadvantage of
producing extensive aerosols (see the section on Lab Safety). In addition, T.D.
pipettes (see the section on pipettes and pipetting) can not be used to transfer and
then mix since this order will rinse out sample that is supposed to stay in the
pipette. The method does disperse clumps fairly well but large clumps in the
solution will often jam the end of the pipette.
D.
Phage Style Mixing

This is an effective and gentle method developed for dispersing bacteriophage and
other small particles in solution. It will not break up clumps of bacteria very
effectively and isn't normally used for mixing dilutions of bacteria. It is started by
holding the tube between your outstretched thumb and fingers so that the tube is
parallel to your fingers and the cap is nearly in the palm of your hand. Then holding
your hand up so that the tube and your palm are nearly horizontal, then rotate your
wrist around 360 degrees while trying to keep the tube nearly horizontal.
51
E.
Finger Vortexing
This is an effective method for conveniently suspending bacterial cultures in tubes.
It is started by firmly holding the test tube upright between the thumb and second
finger of your left hand one or more centimeters above the liquid in the tube, lightly
resting your forefinger over the top to keep the cap from coming off during the
mixing, then repeatedly pressing the nail of the forefinger of your right hand against
the tube about 1 or 2 centimeter from the bottom, and allowing your finger nail to
slip past the tube and spin the solution. A pencil or pen can be substituted for the
fingernail if the process makes your fingers sore.
F.
Vortexing
This process is like finger vortexing except that a machine is substituted for the
fingernail that strikes the bottom of the tube. The advantage is that the machine
can hit the tube very frequently and cause much more vigorous mixing. To do
vortexing, hold the tube in your left hand as if you were going to finger vortex then
lightly touch the bottom of the nearly vertical tube into the cup at the top of the
machine. The liquid in the tube should begin to swirl and form a whirlpool. If this
swirling does not happen check that your fingers are above the level of the liquid. If
your fingers are high enough, try pressing the tube down more firmly and tilting the
tube to about a thirty or forty degree angle from vertical. You might also try to hold
the tube slightly to one side of the inside of the cup on the machine. The best
mixing is done by causing the whirl-pool to form and collapse a several times by
repeatedly setting the tube into the cup then lifting it up when the swirling is
established for several seconds.
52
IV. Pipettes and Pipetting
E. Classes of Pipettes
All pipettes retain a little bit of liquid on the inside walls after they have been filled and
drained. The two important classes of pipette are distinguished by the calibration
methods used to allow for this retained liquid.
1.
To contain pipettes
a. The letters T.C. printed above the calibrations, in the region where the size and
brand name are listed identifies these pipettes
.
b. These pipettes include all the contained liquid of the pipette in the calibration.
They must be rinsed to get the last drop of sample out of the pipette and do a
quantitative measurement.
2.
To deliver pipettes
a. The letters T.D. printed in the region above the calibrations identifies these
pipettes.
b. They exclude the residual retained liquid from the calibration so that when a
pipette is filled to a mark and freely drained the volume shown at the mark will
drain out of the pipette.
c. Some T.D. pipettes are calibrated so that the pipette must be freely drained and
then blown out with a single puff of air in order to transfer the calibrated volume
between the tip of the pipette and the mark. These blowout pipettes are usually
identified by one or two frosted bands etched into the glass at the top of the
pipette. The T.D. pipettes that do not have etched bands at the top should not be
blown out. They will deliver the volume shown at the mark when they stop
draining, while held vertically with the tip against the side of the receiving vessel.
Some are also designed to just transfer liquid from between two or more
measured marks and not between the end of the pipette and a measured mark.
53
F. Sizes of Pipettes
1.
Most of the standard glass or plastic pipettes used in Microbiology and

Immunology will be blow out T.D. pipettes which deliver 0.1, 0.2, 1, 5 or 10 ml
volumes. Other common sizes include 0.25, 0.5, 2 and 25 ml. The size is
usually shown on the top part of the pipette above the calibrations and is usually
listed to shown the total size and the size between each calibration. For
example:
- 10 ml in 1/10 would mean a 10-ml pipette in which the volume between any two
marks is 0.1 ml.
- 10 ml in 0.1 has the same meaning.
- 2/10 ml in 1/100 would mean a 0.2-ml pipette in which the volume between any
two marks is 0.01 ml.
2.
For normal measurements you should usually choose the size of pipette that is
equal to or bigger than the volume that you are going to measure. For example:
a. The 10-ml pipette is mostly used for volumes greater than 5 ml, but when
making a lot of transfers of 1 or 2 ml you might use a 10-ml pipette if
accuracy isn't too important. Transfers greater than 30 ml are usually done
with a graduated cylinder.
b. The 5-ml pipette is used for volumes that are greater than 1 ml but less than
or equal to 5 ml.
c. The 1-ml pipette is used for volumes between 0.1 ml and 1.5 ml but pipetting
1.2 ml can be done more conveniently and just as accurately with a 5-ml
pipette as with a 1-ml pipette.
d. The 0.2 and 0.1 ml pipettes are best for volumes equal to and less than 0.2
ml, although a 1-ml can be used if accuracy is not too important.
G. Types of Pipettes and Pipetting Aids

There are a large number of types of pipettes and pipetting aids. Several types
are encountered in this department.
1. Common glass and plastic pipettes
a. Graduated pipettes
These are high quality T.D. pipettes calibrated with multiple graduations. Most of
the ones in the department are serological blow out pipettes but some other
types that should not be blown out so you should make a habit of checking the
type of each pipette before it is used. The most common non-blow out type are
the Mohr pipettes that are not calibrated to the tip but some blow out pipettes are
not calibrated at the tip either so it is best to check for the frosted bands.
54
b. Pasteur pipettes
These are non-calibrated transfer pipettes that are used for transferring or
removing liquid. They are made from glass with a relatively low melting point so
that they can be readily heated and drawn out to make very fine tipped pipettes
or sealed off to make fragile stirring rods. They are usually filled and drained by
a simple bulb that attaches at the top, large diameter end.
c. Volumetric pipettes
These T.C. pipettes normally have a single calibration mark and are
characterized by a large bulb in the middle region.
2. Pipetting aids
Pipettes were originally designed so that you could close your mouth around the
top end and create a suction that sucked liquid into the pipette. This mouth
pipetting method is dangerous since it directly exposes your body to any
hazardous material and even if the pipetted solution is harmless there is always
the possibility that the end going into your mouth has been contaminated with
other harmful chemicals. It is also difficult to mouth pipette properly in a sterile
biological hood. These problems have lead to the development of a number of
mechanical devices to fill and drain glass and plastic pipettes. Most of these
pipette filters work by creating a vacuum which draws liquid into the pipette.
They generally work well but are dangerous and inaccurate if they are used
improperly:
Most pipettes are made from hard, brittle glass. This brittleness means that you
must be careful when forcing the ends into the pipette fillers in order to avoid
breaking the pipette and skewering your hand with the remaining shards of glass.
The proper way to insert a pipette into a pipette filler is to hold the pipette within
four or five centimeters of the top end and then carefully twist the pipette into the
holder.
Dripping pipettes are usually due to improper seals between the pipette and the
filler because the cotton filter sticks out of the top of the pipette and interferes
with the formation of a good seal. If this is the problem simply remove the
offending strands. Sometimes the drips are due to chips broken out of the top of
the pipette and the only solution is to replace the pipette. It could also be due to
air leaks at any other junctions in the pipette filler.
Some types of common pipette fillers include:
a. Bel-Art Pi-Pump
This is a polyethylene cylinder with a hole at the bottom and an airtight piston at
the top. The piston is notched so that it can be accurately raised and lowered by
using you thumb to rotate a small knurled wheel located near the top. The hole
at the bottom is designed to hold a pipette in an airtight seal so that when the
piston is raised the vacuum formed in the cylinder will draw air or liquid up
through the pipette. Filled pipettes are then drained by slowly lowering the piston
until the piston is about one centimeter above the bottom, pausing for 1 or 2
seconds to let the liquid still trapped on the walls to flow to the bottom of the
55
pipette, then turning the piston down the rest of the way. To ensure that you can
easily blow out the drained pipette you should start the filling with the piston
already turned up about one centimeter. If you forget to turn up the piston and
residual liquid gets trapped in the pipette you must take the filler off the pipette,
turn up the piston, then reassemble the two parts to blow out the pipette.
The chief drawbacks of these fillers is that different models are required for
different sizes of pipettes and when the piston is lowered too fast, enough liquid
gets left behind on the pipette walls to make the measurements inaccurate. The
yellow filler is for 0.1 and 0.2 ml pipettes. The blue filler is for 1 and 2 ml
pipettes. The green filler is for pipettes 5 and 10 ml pipettes.
b. Valved rubber bulb propipettor

These fillers use three independent valves set in stems attached to a large
rubber bulb to create and maintain controlled vacuums.
To operate them:
56
i.
Press the valve (A) on the top unbranched stem and hold it open until you
squish the air out of the big bulb, then release the valve to keep the air out of
the squished bulb.
ii. Stick the top of the pipette into the large opening at the end of the branched
stem that leads directly out from the bulb.
iii. Immerse the open end of the pipette into the solution and press the valve (S)
between the bulb and the pipette.
iv. When the pipette fills past the desired mark, let go of the valve labeled (S), lift
the pipette out of the solution and adjust the level of the liquid by carefully
pressing the valve (E) on the side branch until the meniscus drains to the
right place. If the bulb fills with air before the pipette is full, press the top
valve and squish the bulb to restore the vacuum.
v. Squeezing the valve (E) on the side branch between your thumb and
forefinger then drains the pipette.
vi. The drained pipette is blown out by continuing to hold the valve (E) open
while concurrently reaching out and sealing the open end of the side branch
stem with the end of your second finger then squeezing the small bulb on this
stem against your fore finger and thumb.
The chief disadvantage of these pipette fillers is that liquid is easily sucked
inside. It is also necessary to move your fingers around to operate them and
they are awkward to blow out if you have short fingers or weak hands.
3. Automatic Pipettes
a. Pipetteman digital automatic pipette
These pipettes are designed to measure a specific volume into a plastic

disposable tip when a spring-loaded plunger at the top of the pipettor is
depressed to a pre-set position and released. To use them, jam a plastic, conical
sampling tip over the shaft of the pipettor, then depress the plunger at the other
57
end of the pipette until it reaches the first stop, a point at which you must press
quite a bit harder to depress the plunger further. Hold the depressed plunger at
this first stop position, insert the end of the plastic tip into the solution without
touching the bottom of the container, then allow the plunger to slowly rise to the
original undepressed position. Once filled, the plastic tip should be touched to
the wall of the container above the liquid in order to drain off any excess
unmeasured liquid clinging to the outside of the tip. Then the cleared tip should
be touched lightly against the inside wall of the receiving vessel about one
centimeter above the level of any liquid in the vessel. The measured sample can
then be drained by slowly depressing the plunger until it reaches the first stop,
pausing for about one second to allow any residual liquid to flow down to the tip,
then blowing out the pipette by depressing the plunger further until it hits the
second stop. In all cases you should eyeball the amount of liquid in the tip before
you dispense it and judge whether the volume looks correct. This judgment is
important because the tips are sometimes poorly sealed to the pipette and might
pick up air instead of sample when the plunger is released. In addition, dense or
viscous solutions are slowly drawn into the tips and will be measured improperly
unless you compensate for the slower flow rate by releasing the plunger quite
slowly.
Some of these types of pipettes are calibrated for only one or two fixed volumes.
Other models are equipped with direct reading volumeters that allow you to set
the pipette for any designated volume by simply changing the displacement of
the plunger. The models used in class are the Pipetteman P-20, P-100 and P1000 series. These pipettes look similar but are readily distinguished by the
model numbers on the coloured stickers at the top of the plungers. The P-20 is
suitable for pipetting volumes between 1 and 20 l at an accuracy range of 0.2
l. The P-100 is suitable for pipetting volumes between 20 and 100 l at an
accuracy range of 0.4 l. The P-1000 is suitable for pipetting volumes between
100 and 1000 l at an accuracy range of 4.0 l. In all three models the desired
volume is set by holding the pipette with the plunger upright and the small
transparent window in the blue pipette handle turned toward you so that you can
see the numbers in the window. While watching the numbers rotate the knurled
black adjustment ring near the top of the handle. The numbers are read from
top-to-bottom in the display and are arranged so the smallest listed digit at the
bottom (in red) for the P-20 is one-tenth of a microlitre. The smallest listed digits
for the P-100 and P-1000 are one microlitre and ten microlitres respectively. The
range for these settings mean that you could set the P-1000 to measure 10 l
and the P-20 to measure 0.1 l, however, the respective displacements of the
plungers would be so small that the volumes could not be accurately measured.
Similarly even though the dial contains more numbers, the pipettes only turn to
about one rotation beyond the designated capacity of the pipette and you will
damage the pipette if you force the adjustment any higher. Within the normal
operating ranges, the accuracy is affected by the way the adjustments are set.
For the best results, when the volumeter is being adjusted for increased volumes
of sample the dial should be turned about one-third of a turn beyond the desired
volume then turned back to the desired volume. When adjusting the volumeter
for decreased volumes, the dial can be turned directly to the desired volume.
58
b.
Eppendorf digital automatic pipettes
59
Different models of Eppendorf automatic pipettes are shown.
To operate them:
ii. Turn the volume-adjusting knob until the desired volume reading
appears in the volume window. Some models equip with lockable
volume-adjusting knob. Push the knob down to lock the set volume. Pull
the knob up to unlock prior to readjust the volume. Attach pipette tip to
the end of the pipette.
iii. Depress the knob to the first stop then immerse the pipette tip slightly
into the sample. Slowly release the knob to return to the original
position. The sample will then be drawn up into the tip.
iv. Place the pipette tip against the inside wall of the collecting container
and depress the knob to the first stop to dispense most of the solution in
the tip. After waiting a couple of seconds, depress the knob to the
second stop to blow out the rest of the solution in the tip.
v. Depress the pipette tip - ejecting knob all the way down to eject the tip
into a suitable container.
H. Pipetting Procedures
60
1. Handling pipettes
The major rule for handling pipettes is to avoid touching the calibrated region. This rule
is to prevent the calibrated part that enters your samples from being contaminated with
bacteria from your hands and chemicals from the bench. It also minimizes the chance
of transferring anything from the pipette to you. The rule seems rather trivial but it is
easy to forget when you are attempting to read a pipette. It is also easy to accidentally
brush the pipette against your lab coat, books, tubes or even the pipette canister while
struggling to remove either the cap or plug of a container in your other hand. For this
reason you should make a habit of holding the filled pipette steady in front of you where
you can readily encompass it in your field of vision and use your other hand to bring the
containers to the pipette.
2. Aseptic removal of pipettes from canisters
There are two acceptable, methods for removing sterile pipettes from canisters.
a. Closed tin method
In this method of the pipette container is sealed with the cap except when a pipette is
being removed. It is done by:
i.
Hold the tin horizontally in your left hand with the cap end pointed towards
your right hand.
ii.
Loosen the cap but do not remove it.
iii.
Holding your right hand palm upward, wrap the last three fingers of your
hand around the cap and pull it free.
iv.
Flame the mouth of the canister.
v.
Rotate the wrist of your right hand about 120 degrees so that the cap is
now mostly pointing away from the canister and your thumb and fore finger
are close to the open end of the canister.
vi.
Shake the canister lightly so that the ends of the pipettes slide out and one
pipette sticks out further than the rest.
vii.
Carefully pick up the end of this pipette with the thumb and forefinger of
your right hand without touching any other pipette and lift the pipette out of
the canister.
viii.
As the pipette is lifted out bend your fingers and twist your wrist so that the
removed pipette ends up parallel to the cap but not touching it and the cap
is positioned to slide back onto the canister.
ix.
Flame the canister and then put the lid back on by holding the right hand
steady and sliding the canister into the cap without touching the sterile
pipette. Set the canister down.
b. Open tin method
61
In this method the canister cap is left off for the duration of the experiment to
make the removal of pipettes faster and more convenient. Some people feel that
it is less aseptic even though contamination does not seem to occur if it is done
correctly. To do it properly:
3.
i.
Hold the tin down then pull off the cap and set it out of the way until the end
of the experiment.
ii.
Shake the tin so that pipettes protrude from the tin about 5-to-8 cm then set
the canister so that the open end slightly overhangs the bench and the
pipettes stick out.
iii.
To remove any selected pipette, lift the pipette up so that it does not touch
any other pipette or the canister then lift it out without touching anything.
iv.
Using this process, the exposed ends of the pipettes probably become
contaminated, therefore the canisters should not be tilted up to any angle
which will allow dust and other junk from the contaminated ends to drift into
the canister and contaminate the other end of the pipettes. Similarly, you
can cause drafts that will carry non-sterile dust into the canisters if you
reach into the tin or return unused pipettes to the tin, or move the open
canister around.
Operation of standard glass and plastic pipettes

a. Fill the pipette to a point above the mark by inserting the pipette tip below the
surface of the liquid and sucking up the liquid with pipette filler.
b. Raise the pipette out of the liquid, tilt the container to steady the pipette
between the walls, then allow the liquid to drain so that the bottom of the
meniscus is exactly at the desired calibration mark on the pipette. During this
process ensure that the pipette is held vertically and that the meniscus is at eye
level when it is finally set since other angles or positions will compromise
accuracy.
62
c. Carefully wipe the bottom end of the filled pipette against a clear, dry area on
the wall of the container to remove any liquid clinging to the outside. All
pipettes, including automatic pipettes, are designed to measure contained
liquid; therefore any liquid clinging to the outside is not part of the measured
volume and must be removed.
d. After clearing the outside of the pipette, check that the meniscus is still set
correctly and that liquid has not dripped out the bottom then hold the pipette
steady and use your free hand to move the fresh container over to the pipette.
If the pipette is almost horizontal when opening the fresh container you will
reduce the chance that fluid will drip out of the pipette if your pipette hand jerks
when a sticky cap is removed.
e. Touch the tip of the pipette against a dry area on the inside wall of the receiving
container and allow the liquid to drain out of the pipette and flow down the wall.
The pipette should not be forced to drain faster than the liquid would freely flow
from the pipette since faster rates will change the accuracy. In addition, the tip
should be touched to the wall in a place slightly higher than the level of any
liquid in the container since lower positions will tend to rinse out the end of the
pipette and alter the accuracy.
f.
If the liquid is being measured between calibration marks hold the pipette
vertical and make sure that the meniscus is at eye level during the final
adjustments. If the pipette is being drained from a calibration mark to the tip,
check whether it is T.C., T.D. or blowout. If it is T.C. it must subsequently be
rinsed by filling and draining diluent from it a few times. If it is T.D. just let it
drain. If it is T.D. and blowout you must allow it to drain then pause for 2 or 3
seconds to let any residual liquid reach the tip and then blow it out one time.
The blow out pipette should be blown out with the tip in contact with a dry
surface of the test tubes, flasks, or Petri dishes in order to transfer the liquid off
the end of the pipette.
g. Either repeat the operation to transfer liquid to another container with the same
pipette or discard the pipette by holding the tip over the discard, pulling off the
pipette filler and lowering the pipette into the discard. Contaminated pipettes
should not be set on the bench but can be stored for subsequent semi-aseptic
or non-aseptic use by standing them tip down in test tubes held in a test tube
rack kept near the back of the bench.
h. If you have a choice of measuring a volume such as 0.3 ml by blowing out the
pipette or by measuring between two calibration lines, the measurement
between the lines is usually more accurate. However, measuring 0.1 ml by
blowing it out of a 0.1-ml pipette is more accurate than using a 1.0-ml pipette
where the 0.1 ml is delivered by measuring between two calibration lines.
i. If measuring approximate, small volumes to several tubes, you can fill a large
pipette to the top mark and dispense liquid to many tubes by measuring
between the calibration lines.
V.
Plating and Enumeration
63
A. Streak Plating for Isolates

1. To streak using liquid cultures as the source.
a. Mark the bottom of the plate at the point where you will deposit a small mount
of organisms. Draw a line that marks off the one-sixth to one-third of the plate
on which the deposit will be placed. Place the plate upside down on your
bench.
b. Aseptically remove a sample of the test culture with the inoculating loop.
c. Pick up the part of the plate containing media and hold it to a 45o angle. Work
in the vicinity of a Bunsen burner.
d. Place a small loopful of organism on the previously marked spot and rub the
sample back and forth over the small portion of the plate that you have marked
off.
e. Turn the plate slightly and use the thin edge of the loop to draw two separate
parallel lines across the streaks in the marked portion of the plate, then wipe
most of the remaining bacteria off the loop by drawing twenty or thirty additional
parallel lines over the first quarter of the empty plate without entering the
marked section of the plate.
f.
Turn the plate slightly and repeat the process. Drag two parallel lines through
the previous set of parallel lines then clean the loop by making twenty or thirty
additional lines without crossing into the first two parallel lines.
g. Repeat the process until the plate is covered with lines

h. When the lines are done, replace the lid, and sterilize the loop.
i.
Incubate the plate in an inverted position at the appropriate temperature.
Pattern of a method of streaking plates
64
2. Streak plating using colonies on solid medium

When you use colonies as the source of the inoculum the loop will pick up more cells
than when you use a liquid culture as the inoculum.
To compensate for these extra cells, the inoculator is usually sterilized after the
inoculum is initially transferred to the plate, and after the first one third of the plate is
streaked.
3. Ideally, streaking should give 30 or more isolated colonies. If you have less isolated
colonies it could mean:
a. The loop was hot enough to kill the inoculum because it was either heated too long
or not cooled long enough. Check that the loop is cool.
b. The inoculum was too thick to isolate in the space available on the plate. Use less
inoculum.
c. The contact angle between the loop and the agar changed during the streaking so
the inoculum was inconsistently transferred from the surface of the loop.
d. The surface of the agar was insufficiently used to wipe off the loop. Use more
lines.
e. The loop was oriented incorrectly so that the streaking was done with the wide
edge of the loop instead of the thin edge.
C.
Enumeration
There are two principle methods for isolating bacteria for enumeration. The inherent error
is about the same but each technique has different advantages and disadvantages.
1. Pour plate method
This method is done by adding bacteria to a plate and then adding molten agar to the
sample and mixing the plate so that the bacteria become scattered throughout the agar
before it solidifies.
a.
Label one empty, sterile Petri dish for each sample and put them right side up on
the bench.
b.
If appropriate, dilute the sample.
c.
Aseptically transfer a measured volume of the dilution being tested in to the bottom
of the dish. This sample is generally between 0.09 and 1.1 ml but the lower limit is
actually set by how accurately you can measure small volumes and length of time
you will take to complete the plate since some bacteria will be killed if the small
samples evaporate. The upper limit is determined by the final concentration of
agar in the plate. If the sample dilutes the agar below about 1.3%, the agar doesnt
solidify firmly enough for a plate.
65
d.
Remove one deep from the 46oC stationary water bath (a deep is the tube
containing 12-20 ml of molten agar for making a pour plate). Wipe the tube with a
paper towel to remove the non-sterile bath water from the outside of the tube.
e.
Raise the lid of the plate and pour the molten agar directly onto the sample without
flaming the tube.
f.
Pick up the plate and tilt the molten agar back-and-forth and side-to-side ten or
twenty times to mix the sample into the agar.
g.
When either the sample seems to be well mixed or the agar is starting to get thick,
set the plate down on the bench, and allow the agar solidify for ten or fifteen
minutes, then close the lid and incubate the plate until the colonies are big enough
to count.
h.
Two or three plates can be done concurrently but be careful that:

i. The work is done fast enough to prevent significant evaporation and killing of any
cells before the addition of agar.
ii. The plates that were stacked up for mixing are separated during the solidification
step so that the plates can cool faster and the cells will not have time to settle out
and make pale ghost-like colonies between the glass and the agar.
i.
Incubated pour plates will usually show three types of colony morphology. Normal
shaped colonies occur on the surface, lenticular colonies show within the agar and
pale, almost invisible ghost-like colonies show at the bottom.
2. Spread plate method

In this method, cells in a sample of bacteria are spread over the surface of a dish
containing solidified agar.
a. Pick out plates that contain a smooth agar surface, one turntable, one glass
spreader, and a small glass beaker containing sufficient 75% ethanol to cover the
flat end of the glass spreader and part of the handle.
b. Label the Petri dish and set it right side up on the top of the turntable. Sterilize the
working end of spreader by placing it in the ethanol to kill any contaminants.
c. Lift the spreader out of the ethanol, tilt it so that the ethanol runs to one corner, and
then drain the resulting drop of ethanol away by touching the spreader to a single
place at the top of the beaker.
d. Slowly pass the spreader through the flame of a Bunsen burner one time so that
any residual ethanol ignites and burns away. The spreader will overheat and kill
cells if it is heated too long or passed through the flame more than once.
e. Set the handle of the spreader in a test tube rack so the spreader is held in mid-air
to cool for at least twenty seconds after the ethanol quits burning.
66
f.
While the spreader cools, open the Petri dish and aseptically transfer a measured
volume of the diluted sample to the central region of the agar plate. This sample
must be less than 0.21 ml because larger samples will not absorb into the agar
surface in an adequate time.
g. Touch the spreader to the inside surface of the lid of the Petri dish and hold it there
for four or five seconds. If the spreader is too hot to use, then a very transient mist
forms on the lid when the spreader is lifted up. If it is too hot, allow it to cool longer
and repeat the test to see if it is cool. When it is adequately cooled, lightly hold the
spreader by the handle so that it rests with only its own weight on the agar, then
use it to smear the sample back-and-forth over the surface of the plate while you
simultaneously rotate the turn table at about ten to twenty revolutions per minute.
Every once in a while use one edge of the spreader to pull the liquid from the
outside edge of the dish back into the center where it can be re-spread over the
surface until it absorbs into the agar.
h. When the sample absorbs into the agar the frictional drag between the spreader
and the surface increases. If you continue to spread the sample beyond this time
the agar will sometimes tear, if you stop too soon the bacteria will grow in the
unabsorbed liquid on the surface and form smeared colonies. It usually takes 30 to
60 seconds to absorb on good plates.
i.
The colonies growing on these plates all grow in the same environment and unlike
the pour plate colonies, when these plates are incubated they should be fairly
similar in appearance.
j.
Freshly poured plates are usually too moist to absorb samples but these plates can
be dried by opening the plates and leaving them inverted over a paper towel in an
incubator for two or three hours.
k. The handle of most of the spreaders is bent so that you can place the working end
of the spreader flat on the agar without holding your hand over the plate.
3. Counting isolated colonies and processing the results
Counting seems like a trivial step but many people get bad results simply because of
poor counting. Your counts should be reproducible within one or two colonies when
any plate is recounted. To do the counts you:
a. Wipe off the bottom of the plate and hold it up to a bright light so that the colonies
are clearly visible.
b. Use a grease pencil or felt pen to check off each counted colony.
c. After counting all the visible colonies turn the plate right side up and carefully check
for unmarked colonies along the perimeter where the agar contacts the glass. This
area is difficult to see from the bottom of the plate and often contains some
colonies.
d. Chains of colonies are usually recorded as a single count unless there seems to be
more than one distinct chain. In that case count each chain as a colony.
e. Counts greater than about 350 colonies can be roughly estimated by counting the
colonies in several square centimeters of plate, averaging the number of colonies
67
per square centimeter and multiplying by either 58 or 65 depending on the type of
plate used in the assay (because there are 58 square centimeters of surface area
in an average plastic Petri dish and 65 square centimeters in an average glass
Petri dish).
f.
In general counts less than 30 are considered inaccurate since the standard
deviation of the count (See the section on evaluating accuracy of data) becomes
progressively more significant as you get progressively smaller numbers.
g. Counts greater than 300 are considered inaccurate because there is a greater
chance that two cells will land close enough together on the plate to look like a
single colony. In addition, higher numbers are more tedious to count accurately
than numbers around 100 so there is greater chance of mistakes.
h. Some people have done empirical plating studies and have proposed changing the
counting range to cover from 25-250, but convention still uses 30-300.
i.
Even if they are not going to be used, counts moderately greater than 300 or less
than 30 should be recorded in your records since they provide a rough estimate of
the number of cells and should roughly correspond to the values determined with
better plates. However, the final calculated results should be done according to
the following rules adapted and abridged from Standard Methods 12th Ed., W.G.
Walter, ed. (1967).
68
Rule 1.
If there is one plate with 30-300 isolated colonies. Count all colonies, including
those of pinpoint size, record the dilution used, and report the total colonies.
Ignore plates at other dilutions that have colony counts outside this range. See
the following Table, Sample 1.
Rule 2.
If more than one plate is prepared for given identical dilutions but only some
plates yield 30-300 colonies, and other plates of the same dilution are greater
than 300 colonies then count all these duplicate plates(s), unless some are
excluded by rule four, and report the arithmetic average. Similarly include values
less than thirty if other plates at that same dilution have greater than thirty
colonies. See the following Table, Sample #11 and 12.
Rule 3.
If plates from two different dilutions both yield between 30-300 colonies calculate
the concentration for each dilution and report the arithmetic average of these
values unless the higher calculated concentration is more than twice the lower
one, in which case report the lower calculated concentration. See the following
Table, Sample#2 and 3.
Rule 4.
If large spreading colonies occur on the plate, count the colonies on

representative portions without any spreader and estimate the total count. This
estimate is only valid if the colonies are well distributed in areas free of spreaders
and the area affected by the spreaders is less than one-half the plate.
Rule 5.
If there is no plate with 30-300 colonies and one or more plates have more than
300 colonies, use the dilution showing counts nearest 300 colonies. See the
following Table, Sample #6 and 10. If the colony number used is much bigger
than 300, suitably qualify your calculated answer.
Rule 6.
If plates from all dilutions yield fewer than 30 colonies each, record the actual
number of colonies on the least diluted sample (unless excluded by rule four) but
report the computed count as less than 30 times the corresponding dilution
factor. For example, if the 1:100 dilution has been used, report Standard Plate
Count per ml as less than 3,000 (3,000). See the Table, Sample #7.
Rule 7.
If none of the plates at any tested dilution show colonies report the results as NC.
If the results are unsatisfactory for other reasons, list the reason.
69
Table 1. Examples for computing standard plate counts per ml
Sample
#
1
2
3
4
6
7
8
10
11
Observed colonies at
Concentration
1:100
dilution
234
293
140
Spreader+
ratio
1:1000
dilution
28
41
32
31
355
Standard
plate count
concentration
23,000
1.4
35,000
2.3
14,000
31,000
360,000
18
<3,000
0
0
NCx
325
25
33,000
175
16
19,000
208
17
12
322
23
30,000
278
29
13
296
40
33,000
378
24
14
138
42
2.4
15,000
162
30
15
274
35
1.4
30,000
230
Spreader+
In cases 1-to-10 only two plates, from two different decimal dilutions, are tested.
In cases 11 -to- 15 two or more plates at each dilution are tested.
Rule
applied
1
3
3
4
5
6
7
5
2
2
2
2,3
2,3
* Concentration ratio is the ratio of the greater to the lesser calculated concentration after
compensating for dilutions. It is applied to plates from consecutive dilution having
between 30 and 300 colonies.
+ If the spreader and the adjoining area of repressed growth cover more than one-half of
the plate, do not use the result.
xNC means not calculated because of no colonies.
C.
Replica Plating for Multiple Tests

This method is used to test the phenotypes of a single set of colonies under two or more
conditions. It usually uses surface colonies that have been isolated by spread or streak
plate but for very accurate work you should re-streak and re-isolate every colony that will
get tested since up to 10% of the original isolated colonies are colonies that arose from
two or more cells and have mixed phenotypes. The method usually involves making grid
plates and then doing the replica plates.
1. Grid plates
a.
Grid plates are prepared by setting a plate over the accompanying grid pattern and
systematically transferring colonies on the original plates to the area over each
number. The purpose is to organize the pattern of growth so that any cells that do
not grow will show up as a gap in the subsequent replica-plating pattern and be
70
much easier to detect than a missing colony in a random distribution of colonies.
The gridding also ensures that the growth for the tests are similar sizes and ages
since colonies on normal spread plates occur in a variety of sizes and
consequently do not transfer with the same efficiency.
b.
Each transfer should be done so that the colony will grow up on the surface as a
short streak about 2 mm long. Be careful not to slice into the agar when making
these transfers since growth below the surface can not be used for replica plating.
c.
The transfers can be done by using sterile toothpicks or stabs. Toothpicks are
discarded after each transfer; stabs are flamed and re-used. Since flamed stabs
take time to cool, some people use two stabs alternately so that one can cool while
the other is used, flamed and left in turn to cool.
d.
You can steady your hand during the transfer by setting a test tube rack about six
cm away from the grid plate and resting the outside edge of your palm on the rack.
You can also shorten the stabs by bending a loop in the wire.
e.
To prevent the plate from wiggling and sliding around on the grid pattern during the
transfers, fold a small piece of tape in half so that both outside surfaces are sticky
then place this tape in the center of your grid pattern and stick the plate over the
71
tape. The folded tape might obscure a few of the numbers in the center of the grid
but you should be able to interpolate these positions.
f.
If the source plate contains more colonies than you intend to grid you should mark
off several small areas on the plate and systematically transfer all the isolated
colonies from one area before going to another area so that you do not
inadvertently pick the easiest or most noticeable colonies and bias the selection.
g.
The completed grid plates should only be incubated until the colonies are about
one-quarter of a millimeter high. Overgrown colonies tend to smear when they are
replica-plated.
2. Replica plates
a. Replica plating involves the transfer of portions of all the colonies on a master plate
or a grid plate, to an inoculation block that is then used to consecutively transfer
portions of each master colony to several successive plates.
b. The inoculation block is prepared by stretching a piece of sterile velvet over the flat
end of a wooden or plastic block that is slightly smaller than the diameter of a Petri
dish. The velvet is held in place with a metal band or an elastic band. The
opened master plate is then inverted over the velvet so that each of the colonies
on the plate comes into contact with the velvet. The master plate is then removed
and the agar in each of the test plates is sequentially pressed into contact against
the velvet. The key to the process is to press hard enough to bring the velvet into
contact with the agar but not hard enough to crush the agar and smear the
colonies. At some light angles the velvet appears moist where it comes into
contact with the agar, try moving the block around to see this effect and use it to
judge how hard to press. In addition, the colonies will often change colour and
small dimples occur where the velvet threads get pushed against the agar. If the
velvet is not in contact with the agar lightly press the bottom of the plate over the
area which is not in contact. The force needed to press the plate onto the velvet
involved is about the same as the minimum force needed to make your finger nail
turn white when you press the fingernail with a pencil.
c. The velvet should be used with the soft, nap side upwards. It is usually wrapped in
aluminum with the nap down so that the package can be opened without
contaminating the working surface of any velvets in the package. Each velvet
should only be handled at the edges.
d. Be careful not to touch the working surface of the velvet when securing it with the
metal band.
e. Make sure that the metal band is pressed low enough on the block that the plate
surface evenly touches the velvet before the edge of the plate touches the metal
band. Some types of bands can be adjusted with a screwdriver if they are too
tight to easily slide over the block and the velvet.
f. Before use each plate should be marked on one edge with a short line which can
be aligned to a second mark on the metal band securing the velvet. This mark will
later allow you to orient the plates relative to each other and the master plate.
72
This orientation will facilitate comparison and is especially important if only a few
colonies grow.
g. Sometimes you can judge whether some of the transfers seem anomalous if you
record the order in which the replica is transferred to each test plate. Usually, a
plate on which everything is expected to grow is done last as a control to judge
whether inoculum was getting transferred.
h. Used velvets should be discarded to the appropriate discard with the nap side
down. The working surface is still heavily contaminated and putting this surface
down helps to contain the contamination until the velvets are sterilized.
i. After the plates have been incubated an easy way to collect the data is to make a
chart which has a column corresponding to each type of plate and a row
corresponding to each colony on the grid plate. Number each row on the chart,
then use a grease pencil to lightly mark the bottom of each plate under the
colonies which have clearly grown, so you can then set the plate over the
numbered grid pattern and check off each numbered position in the chart
corresponding to growth on that plate. The number of checks in each column
represents the number of colonies positive for that feature. The check marks in
each row correspond to the positive phenotypes of different colonies.
j. Sometimes the velvets will transfer sufficient nutrients or cells to show visible
growth even though the cells were not have been able to grow with just the
material in the plate. These colonies which are just due to carryover should not
be recorded as growth. They are usually fainter than colonies due to real growth.
73
Appendix B - Laboratory Safety
A. Laboratory Hazards
Microbiological labs include chemical and biological hazards. Some solutions contain strong
acids, bases, carcinogens, toxic metals and toxic chemicals. You should learn to identify
these materials and handle them appropriately. In some cases they are mixed with other
chemicals and disguised by innocuous names such as Folin-Ciocalteau reagent but they are
still dangerous. To work safely, you should know the components involved in all the
reactions and judge whether any components could have dangerous effects. Some of the
common acids encountered include HCl, H2SO4 and trichloroacetic. Some of the
carcinogens include ethidium and chloroform. Other compounds that react with organic
molecules include orcinol, phenol, copper sulphate and anthrone. Before discarding tubes
containing dangerous chemicals the chemicals must be drained into the sink and flushed
down the drain with lots of cold running water. Paper towels and other paper contaminated
with dried chemicals should be carefully handled since even dry chemicals can still react
with your skin. Long laboratory coats should be worn to minimize the hazards to you and
your clothes.
B. WHMIS
The following section of material has been adapted from the Laboratory, Chemical Safety
Reference Manual produced by the University of British Columbia Department of Health
Safety and Environment (2005).
The Workplace Hazardous Materials Information System (WHMIS) requires
suppliers to provide safety information with their products and requires the University
to educate and train everyone potentially exposed to hazardous materials. The key
elements of WHMIS include:
Labeling - which alerts workers to the identity and dangers of products and to the
basic safety precautions
Material safety data sheets (MSDS) - technical bulletins which provide detailed
hazard and precautionary information; and
Worker education and training.

Products partially exempted from WHMIS include consumer products, cosmetics and
drugs, explosives, pesticides, radioactive substances. These materials are regulated
by separate legislation.
For exempted products, WHMIS regulations requires employers to educate workers
in the safe handling of these products and to use workplace labeling when, for
example, contents are transferred to new containers.
The eight categories of chemicals described in the following six classes are all
covered by WHMIS requirements:
74
Hazard Symbol &
Definition
Class A - Compressed
Gas
Class B Combustible and

Flammable Material
Class C
Oxidizing Material
Class D - Division 1
Poisonous materials;
Causing immediate
and serious toxic
effects
Associated Hazards
an explosion hazard because the gas is being
held in a cylinder under pressure
container can explode if heated in a fire
container may explode if dropped
the material burns; represents a fire hazard
may burn at relatively low temperatures;
flammables will ignite at lower temperatures
than combustibles
may burst into flame spontaneously in air, or
release flammable gas on contact with water
may cause fire when exposed to heat, sparks,
flames or friction
poses fire \ explosion risk in presence of Class
B materials
may cause fire, react violently or cause
explosion in the presence of combustible
materials such as wood and solvents
may react violently with reducing agents
may burn skin and eyes upon contact
potentially fatal substances
may be fatal or cause permanent damage if
inhaled, swallowed or absorbed into body
may burn eyes or skin upon contact
Causing other toxic
effects
not immediately dangerous to health

may cause death or permanent damage as a
result of repeated exposures over time
may be skin or eye irritant or sensitizer
may cause cancer
may cause reproductive or teratogenic effects
Biohazardous and
Infectious Materials
may cause a serious disease resulting in

illness or death
may damage metal
Class E Corrosive material
causes severe eye and skin irritation upon

contact
causes severe tissue damage with prolonged
contact
may be harmful if inhaled
Class F Dangerously
Reactive Material
unstable; may react with water to release toxic

or flammable gas
May explode as a result of shock, friction or
increase in temperature
may undergo vigorous polymerization
Handling Information
do not drop cylinder
keep cylinder away from potential
sources of ignition
store containers in a designated
area
Secure in an upright position
keep away from heat sources and
oxidizing materials
never smoke in vicinity
store in cool, fire-proof area, as
designated by supervisor
keep away from Class B materials

store in designated area
keep away from ignition sources
never smoke in vicinity
wear eye, face, and hand
protection, and protective clothing
handle with extreme caution
avoid contact with skin or eyes;
wear appropriate personal
protective equipment and clothing
avoid inhaling; work in wellventilated area and/or wear
respiratory protection
wash and shower thoroughly after
each use
store in designated areas only
avoid eye, skin contact by using
appropriate personal protective
equipment and clothing
avoid inhaling; work in wellventilated area and/or wear
respiratory protection
store in designated areas
take every precaution to avoid
contamination
handle only when wearing
necessary protective equipment
handle in designated areas only
where appropriate engineering
controls are in place
keep containers tightly closed
avoid skin and eye contact by
wearing eye, face and hand
protection and protective clothing
avoid inhaling; work in wellventilated area
keep away from heat
open containers carefully; do not
drop
store material in designated cool,
flame-proof area
75
Various types of labels or other identifiers are used to alert workers to the hazards and safe
procedures necessary for use of hazardous products in the workplace.
1. Chemical suppliers must prepare a supplier label that typically provides seven pieces of
information:
Product name
Hazard symbols
Risk phrases (e.g. Flammable)
Precautionary statements (e.g. Avoid fumes)
First aid measures (e.g. Wash exposed skin 20 min)
A statement advising that a Material Safety Data Sheet (MSDS) is available
Supplier name
Supplier labels from laboratory supply houses must have:
Product identifier
Risk phrases
Precautionary measures
First aid measures
Reference to availability of MSDS
Supplier labels for laboratory samples with no available MSDS must have the following
information:
Distinctive WHMIS label border

Product identifier
Supplier identifier
Chemical identity of ingredients where known
Emergency telephone number
Workplace labels are required on containers when controlled have been transferred from a
supplier's container. Workplace labels provide three items of information:
Identifier
Safe Handling Information
See Material Safety Data Sheet
Products produced in a laboratory for research, and development work in the same lab, do not
require a workplace label, but every container and reaction vessel must be properly identified.
The information contained on labels and identifiers must not be defaced and must be easy to read
at all times.
An MSDS is a technical bulletin, which must provide detailed hazard, precautionary and
emergency information that includes:
Product information
Hazardous ingredients
Physical data
Fire and explosion hazard
76
Reactivity data
Toxicological properties (health effects)
Preventive measures
First aid measures
Preparation information with date of preparation, name and phone number of
persons or corporate departments to be contacted for additional information
Information must be current since information and risks are subject to change. MSDS more
than three years old should be updated by contacting the supplier, the supplier website or
other sites that link to the supplier information.
The UBC Department of Health Safety and Environment web site at
<http://riskmanagement.ubc.ca/health-safety/chemical-safety/materials-safety-data-sheets>
provides links the Canadian Centre for Occupational Health and Safety MSDS data base and
links to the MSDS listings for many of our major chemical suppliers. This is a subscription
service so you need to go through the UBC Health Safety and Environment rather than
directly to CCOH.
The University of Ottawa has extensive links
<http://www.uottawa.ca/services/ehss/msds.htm >
to
supplier
MSDS
listings
at
<http://www.phac-aspc.gc.ca/msds-ftss/msds63e-eng.php> at Public Health Canada has an

MSDS of microorganisms that describes properties, infection characteristics, disinfection,
handling and other features of pathogenic microbes.
C. National Fire Protection Association Diamond Codes
The NFPA diamond is an alternative safety symbol that shows on American products. The
code consists of a diamond that is made up of four smaller coloured diamonds. Each colour
corresponds to a type of risk. The numbers within three of the coloured diamonds rate the
severity of the risk. The higher numbers have greater risk. The letters in the other diamond
give specific risks.
The red diamond at the top of the bigger diamond represents fire or flammability hazards.
The numbers mean:
0 Will not burn
77
1 Must be preheated for ignition; flashpoint above 93C
2 Must be moderately heated for ignition, flashpoint above 38C
3 Ignition may occur under most ambient conditions, flashpoint below 38C
4 Extremely flammable and will readily disperse through air under standard
conditions, flashpoint below 23C
The yellow diamond at the right of the bigger diamond represents reactivity hazards. The
numbers mean:
0 Stable
1 May become unstable at elevated temperatures and pressures, may be mildly
water reactive
2 Unstable; may undergo violent decomposition, but will not detonate. May form
explosive mixtures with water
3 Detonates with strong ignition source
4 Readily detonates
The white diamond at the bottom represents special hazards. The OX means that it is a
strong oxidizer. The slashed W means that the chemical is reactive with water.
The blue diamond at the left represents health hazards. The numbers mean:
0 Hazard no greater than ordinary material
1 May cause irritation; minimal residual injury
2 Intense or prolonged exposure may cause incapacitation; residual injury may
occur if not treated
3 Exposure could cause serious injury even if treated
4 Exposure may cause death
D. Gloves
Latex, nitrile, polyethylene and vinyl gloves are widely used in many modern laboratories.
They are important to protect you from hazardous materials and to protect materials from
you. For example, the surfaces of chemical containers can be contaminated with residues
of the chemical. These chemicals can be harmful if they transfer to your hands.
Conversely, your skin is the source of many bacterial contaminants. Skin is also the source
of sweat that contains nucleases and nucleic acids that affect polymerase chain reaction
assays, restriction endonuclease assays, plasmid isolation, and so forth. In each of these
cases the gloves work as a barrier to minimize the chance of moving the material or microbe
from one site to another site where it can cause problems. However, the gloves will only
work if you are aware of the role and continue to minimize inadvertent transfer and
contamination. If you put on the gloves then handle the contaminated chemical bottle then
pick up a water bottle with the same gloves the chemical can transfer to the water bottle and
cause problems for other people that touch the bottle without using gloves. Similarly, if you
use gloves to protect the PCR reaction but touch your pen or your desktop with the gloves
you have probably contaminated the outside of the gloves with nucleic acid and nucleases
that could affect the PCR. This means that the gloves should only be used when they are
needed and you should be aware of all the things that the gloves touch and whether those
things compromise the purpose for using the gloves.
78
Even though the different types of gloves seem to be interchangeable they are made with
different chemicals. This difference is important because some chemicals will cross through
some types of gloves but not others. Latex gloves are suitable for excluding particulates
such as bacteria and viruses and minimize contamination of PCR reactions by cells from
your skin chemicals but they are permeable to many chemicals. Nitrile gloves are generally
suitable for alcohols, ammonium fluoride, ethidium bromide, hexane, hydrochloric acid,
perchloric acid, phosphoric acid, trichloroacetic acid, potassium hydroxide, sodium
hydroxide, water soluble materials, dilute acids and bases and for particulates but dissolve
in chemicals such as dimethyl sulfoxide. Vinyl gloves tend to be suitable for organic
solvents such as benzene, carbon tetrachloride, chloroform, and xylene, for water soluble
materials, dilute acids and bases and for particulates. Polyethylene gloves exclude
particulates and tend to be inert to many chemicals but fit poorly.
The glove section of the chemical hygiene site at the University of Florida at
<http://www.ehs.ufl.edu/Lab/CHP/ > states that no glove may be used as protection from all
chemicals. A glove may protect against a specific chemical, but it may not protect the
wearer from another. If a glove protects the wearer, it will not protect the wearer forever, as
the glove material will deteriorate then provides several rules for choosing gloves and using
gloves as well as providing a number of links that lead to charts relating the types of gloves
to chemical compatibility.
Some of those rules for glove use in the labs state:
Wear the correct gloves when needed.

Wear gloves no longer than 2 hours.
Wash hands once gloves have been removed.
Discard disposable gloves once removed. Do not save for future use.
Dispose of gloves into the proper container (biologically contaminated gloves will need to
go into a autoclave bag); while other chemically contaminated gloves will not.
Remove gloves before touching personal items, such as phones, computers, pens and
ones skin. Remember the designated area rule where science does not mix with
personal space (ones desk or lunch space). Gloves used in research are considered
science.
Do not wear gloves out of the lab. If gloves are needed to transport anything, wear one
glove to handle the transported item. The free hand is then used to touch door knobs,
elevator buttons, etc. If you are wearing gloves to protect your sample from you and
are in the hall, no one else understands this and will be concerned about the items you
have contaminated with those gloves.
You should also be aware that gloves can cause chemical contact dermatitis, allergic
contact dermatitis and allergic sensitivity. The first is a dry, itchy, irritated skin condition.
The second is a delayed hypersensitivity similar to the effects of poison ivy. The
hypersensitivity is an immediate allergic response. The individual risk for these problems is
unknown. There is some association between food allergies and allergic contact dermatitis.
There is also an association to the dust in the gloves as if the allergies arise from latex
particles that are breathed in when the gloves are snapped when they are removed or
discarded. The safety policy at Oxford specifically states: All latex gloves present a risk of
causing irritation, sensitization or allergic reaction in susceptible individuals, although this
risk is reduced in gloves with lower levels of latex protein and process chemicals. Powdered
latex gloves carry an additional risk to sensitized individuals, because the latex protein
leaches into the powder and becomes airborne when gloves are removed, or may be carried
79
around on the wearer's clothing. This may affect others in the vicinity, not just the person
wearing the gloves.
The important point is that the gloves are a benefit and a risk. They should not be used
when it is not necessary to use them. If you do use them and your hands become irritated it
is important to consider using alternative types of gloves or using glove liners in order to
eliminate the irritation.
E. Laboratory Techniques
The following information has been abridged from the Laboratory Safety Monograph - A
Supplement to the NIH Guidelines for Recombinant DNA Research that was compiled by
the U.S. Department of Health, Education and Welfare in 1979. The recommendations were
developed to reduce the risks involved in working with potentially hazardous infectious
disease causing organisms. Some of the rules will not be followed in this lab course since
you will be working with benign strains of pathogenic bacteria. You should still be aware of
all the hazards and safety recommendations involved in even simple laboratory operations.
1. Aerosols
Fewer than 20 percent of known laboratory infections can be attributed to a documented
accidental exposure. Risk assessment studies, however, have demonstrated that aerosols
consisting of tiny particles of liquid and/or solid dispersed in air are created by most
laboratory manipulations that involve microorganisms. These results suggest that
inhalation of undetected aerosols may contribute to occupational illness among laboratory
workers who have handled biohazardous materials.
Operations that have the potential to create hazardous aerosols include:
2.
blowing the last drop of a liquid culture or chemical from a pipette.

removing the cap from a bottle of a liquid culture or suspension immediately after
vigorous shaking.
removing cotton plugs from flasks and centrifuge tubes.
decanting the supernatant fluid after centrifugation.
resuspending packed cells by shaking or mixing.
inserting a hot wire loop into a culture.
opening wet Petri dishes.
opening a freeze-dried culture.
shaking and blending cultures or infected tissues in high speed mixers.
streaking an inoculum on a rough agar surface.
sonic disruption of cells.
Safe Pipetting
The fluids that are handled in pipettes are frequently infectious, toxic, corrosive or
radioactive agents. Safe pipetting techniques are required to reduce the risk for exposure
to these materials. One hazard associated with pipetting is the application of mouth
suction. More than 13 % of known laboratory accidents that resulted in infection was due
to oral aspiration during a pipetting. Additional hazards are created by liquid dripping from a
pipette to a work surface, by mixing cultures with alternate suction and blowing, by forceful
ejection of an inoculum onto a culture dish, or by blowing out the last drop. High-speed
photography has shown that an aerosol of approximately 15,000 droplets is produced when
80
the last drop of fluid in the tip of the pipette is blown out with moderate force. The aerosol
hazard associated with pipetting procedures can be reduced by use of safe techniques,
Biological Safety Cabinets, and mechanical pipetting aids.
a. Safety pipetting aids
There are many commercial safety pipetting aids. Some systems use disposable tips
and some a small vacuum pump. The latter usually incorporate a filter system to
prevent the escape of potential hazardous aerosols and a valve to prevent the sample
from being accidentally drawn into the pump. In selecting a pipetting aid, consider
whether the unit can be sterilized with steam as well as to the ease of reading the
meniscus, controlling the flow of fluid, filling, discharging, and changing pipettes. In
addition, it should not contribute to fatigue of the finger and wrist and it should leave the
third and forth fingers free to manipulate plugs and bottle caps.
b. Pipetting practices:
Use some type of pipetting aid rather than mouth pipetting.
Work with biohazardous or toxic fluids in a safety cabinet or hood.
Plug pipettes used for the pipetting of hazardous materials with cotton (even
when safety pipetting aids are used).
Do not bubble air through biohazardous liquids with a pipette.
Do not mix biohazardous material by alternate suction and expulsion through a

pipette.
Do not forcibly expel biohazardous material out of a pipette.
Place a disinfectant-soaked towel on the working surface to catch any drips.

Autoclave the towel after use.
Use mark-to-mark measurements in order to avoid aerosols caused by blowing

out the last drop.
Minimize aerosols by discharging pipettes as close as possible to the fluid or

agar level and running the contents down the wall of the tube or bottle.
3. Centrifuging
a.
Activities such as filling centrifuge tubes, removing cotton plugs and rubber caps from
tubes after centrifugation, removing the supernatant and resuspending the cells, are
capable of releasing aerosols into the environment. The greatest hazard associated
with centrifuging biohazardous materials is created when a centrifuge tube breaks
and releases the fluid into the centrifuge chamber.
b.
Safe Centrifuging Practices:

i.
Do not use tubes with cracks or chipped rims.
81
4.
ii.
Avoid pouring the supernatant material from centrifuge tubes. If you must do so,
wipe off the outer rim with a disinfectant afterwards; otherwise, in a subsequent
step, biohazardous fluid may be spun off as droplets that form an aerosol. A
suction system with appropriate in-line safety reservoirs and filters is preferable
to pouring from centrifuge tubes or bottles.
iii.
If the pellet is infectious microorganisms or other hazardous material resuspend it

by using a swirling, rotary motion rather than shaking. If vigorous shaking is
essential to suspend the material or achieve homogeneity, allow a few minutes
for the aerosol to settle before opening the container. Shaking always
contaminates the closure; thus, there is the added hazard of liquids dropping
from the closure or running down the outside of the container. A Biological
Safety Cabinet with gloves in place may be required to assure safety.
iv.
Avoid filling the centrifuge tube to the point that the rim, cap, or cotton plug
becomes wet with culture. This level is well below the top of the tube when tubes
are placed in slanted (angle) rotors.
v.
Screw caps or caps that fit over the rim outside the centrifuge tube are safer than
plug-in closures. Some fluid usually collects between a plug-in closure and the
rim of the tube. If the rim or screw-capped tubes are soiled and sealed
imperfectly, some fluid will escape down the outside of the tube.
vi.
When tubes are balanced, the buckets, trunnions and inserts, including any
disinfectant solution or water added for balancing, should be included in the
procedure. The basic concern is that the centers of gravity of the tubes are
equidistant from the axis of rotation. To illustrate the importance of this, two
identical tubes containing 20 g of mercury and 20 g of water, respectively, will
balance perfectly on the scales; however, their performance in motion is totally
different, leading to violent vibration with all its attendant hazards.
Blending and Mixing

Aerosols are created by most laboratory operations concerned with blending, mixing,
stirring, grinding or disrupting materials such as cells, tissues, blood samples, freezedried sera, and environmental samples. In one investigation the air was sampled
during the operation of a 500-ml blender. The results still showed an aerosol of
Serratia indica 5 minutes after the blender was turned off.
Magnetic mixers, although generally operated at slower speeds than blenders and not
designed to create a turbulent, macerating action, are capable of creating aerosols if
the material mixed produces bubbles or foam. Magnetic mixers provide a
comparatively gentle, swirling action, but the mixing of infectious, oncogenic,
allergenic, or toxic materials should be considered a potentially hazardous operation.
F. Personal Practices
Appropriate personal hygienic practices in the laboratory reduce physical injury or disease
and raise the quality of the laboratory work by reducing the possibilities for contamination of
experimental materials. The reasons for many of the following recommended precautions
and practices are obvious, but every rule is not applicable to every specific laboratory.
Consequently, what might be forbidden in one laboratory might be only discouraged in
another and be permissible in a third. Nevertheless, adherence to habitual safe practices
82
even when seemingly not essential, provides a margin of safety against unrecognized
hazards. The following guidelines minimize the risks of working in laboratories:
1. Do not store or consume food, candy, gum, and beverages in the laboratory.
2. Keep hands away from mouth, nose, eyes, face, and hair to minimize self-inoculation.
3. Tie long hair back or use a head cover to protect the hair from swinging into Bunsen
flames, machinery, chemicals or cultures and reduce the facial contamination
inadvertently caused by brushing the hair out of your face.
4. Store personal items, such as coats, hats, storm rubbers or overshoes, umbrellas,
purses, etc., outside the laboratory.
5. Only use books and journals returnable to the institutional library in the clean areas.
6. Promptly wash hands after removing protective gloves and clothing. Tests show that
despite use of gloves microbial or chemical contamination is frequently present, due to
unrecognized small holes, abrasions, tears, entry at the wrist, or solvent penetration
through the gloves.
7. Do not wear jewelry in the laboratory, as it will interfere with the hand washing
procedure. If worn, it could become contaminated and cause the contamination to be
brought to the home.
8. Do not use disinfectant wash or soap to the point of causing roughening, desiccation or
sensitization of the skin.
9. Do not work with biohazardous material when you have a fresh or healing cut, abrasion,
lesion of the skin or any open wound.
10.
Beards retain particulate contamination more persistently than clean-shaven skin. A

clean-shaven face is also essential to the adequate fit of a facemask or respirator
when the work requires respiratory protection.
G. Chemical Disinfectants
1.
Characteristics of Common Chemical Disinfectants

Inactivation of microorganisms by chemical disinfectants may occur by either
coagulation or denaturation of protein; lysis; or inactivation of an essential enzyme by
the oxidation, binding, or destruction of enzyme substrate. The relative resistance of
the action of chemical disinfectants can be substantially altered by such factors as:
concentration of active ingredient, duration of contact, pH, temperature, humidity, and
presence of organic matter. Depending upon how these factors are manipulated, the
degree of success achieved with chemical disinfectants may range from minimal
inactivation of target microorganisms to sterility within the limits of sensitivity of the
assay systems employed.
Ineffectiveness of a disinfectant may also be due to the failure of the disinfectant to
contact the microorganism rather than failure of the disinfectant to act. If you place an
item in a liquid disinfectant, you can see that the item is covered with tiny bubbles.
The area under the bubbles is dry, and microorganisms in these dry areas will not be
83
affected by the disinfectant. In addition any microorganisms under spots of grease,
rust or dirt on the object will not be contacted by the disinfectant. Scrubbing items
immersed in disinfectant and incorporating surface-active agents enhance contact.
2.
Properties of Some Common Disinfectants

a. Alcohol
Ethanol and isopropanol at a concentration of 70-85% (v/v) are common
disinfectants. Higher concentrations tend to dehydrate the bacteria and are
less bactericidal. They work by denaturing proteins and are somewhat slow
in their germicidal action. They are effective against lipid-containing viruses.
b. Formaldehyde
Formaldehyde for use as a disinfectant is usually marketed at a 37%
concentration of the gas in water solution called formalin or as a solid
polymerized compound called paraformaldehyde. Formaldehyde at a
concentration of 5% active ingredient is an effective liquid disinfectant.
Formaldehyde at 0.2 to 0.4% can be used to inactivate viruses in the
preparation of vaccines. Formaldehyde loses considerable disinfectant
activity at refrigeration temperatures. Formaldehyde vapor made by heating
paraformaldehyde is an effective space disinfectant for sterilizing rooms or
buildings.
c. Phenol
Phenol is not often used as a disinfectant because the odor is unpleasant and
a gummy residue remains on treated surfaces. However, phenol homologs
and phenolic compounds are the basis for a number of popular disinfectants.
The phenolic compounds such as Phenola are effective disinfectants against
some viruses, rickettsiae, fungi and vegetative bacteria. The phenolics are
not effective against bacterial spores.
d. Quaternary Ammonium Compounds
An example is the Westosan disinfectant used for disinfecting the bench
surface. They are cationic detergents. They are bacteriostatic,
tuberculostatic, sporostatic, fungistatic and algistatic at low concentrations.
They are bactericidal, fungicidal, algicidal and virucidal against lipophilic
viruses at higher concentrations, but are not tuberculocidal, sporicidal or
virucidal against hydrophilic viruses. They are neutralized by anionic
detergents, such as soap and will lose effectiveness in the presence of
proteins. They have the advantages of being odorless, nonstaining,
noncorrosive, stable, inexpensive and relatively nontoxic.
e. Chlorine
This strong oxidizing agent is a fast acting disinfectant effective against all
microorganisms, including bacterial spores. It combines with protein and
rapidly looses effectiveness. It is also corrosive to metals and will gradually
lose strength so that fresh solutions must be prepared frequently. Most
chlorine disinfectants use sodium hypochlorite as a base and an excellent
disinfectant can be prepared by diluting household bleach from 5.25% to 525
84
ppm hypochlorite and adding a nonionic detergent to a concentration of about
0.7 percent. Recent research shows that chlorine can combine with some
types of organic molecules to form mutagenic halo-organic products.
f. Iodophors
Wescodyne is an iodophor disinfectant based on a complex of iodine and
long chain alcohol. It is used in the discards for the glass pipettes. It is used
at 9.1%. For some purposes it is used at 25 ppm to 75 ppm but this small
amount of iodine can be rapidly inactivated by extraneous protein present in
the discarded samples.
H. Disposal Procedures for Sharps, Glass and Biomedical Waste UBC Site
1. Sharps and needles
a. All sharps and needles must be collected in plastic sharps containers
b. When containers are full and the lid securely closed and snapped in place, take
to the buildings designated area for disposal. DO NOT place containers in the
glass waste only cans.
2. Glass Waste
a. Dispose of glass waste in 5-gallon metal cans labelled GLASS WASTE ONLY
and lined with a clear plastic bag.
b. When the cans are full, choke off and securely tie the bags. Glass waste
MUST NOT extend past the top of the can.
c. Lab personnel take the cans to the buildings designated area for disposal
d. Low level radioactive glass waste is disposed of as detailed in the
RadionuclideSafety and Methodology reference manual.
e. Discarded PLASTIC chemical/reagent bottles that are not contaminated should
be packaged in plastic bags then contained in cardboard boxes. Mark boxes
PLASTIC WASTE FOR INCINERATION and take to the buildings designated
area for disposal.
3. Biomedical
All biomedical waste packaged in red plastic or autoclave bags are rendered
non-infectious, tagged and taken to the buildings designated area for pick up
and incineration. DO NOT dispose of these bags in the regular garbage.
4. Disposable Syringes
a. All disposable syringes MUST be disposed of as special waste.
b. Infectious or biohazardous syringes must autoclaved or chemically
decontaminated prior to disposal.
85
c. After disposal of the needle in a red sharps container, package plastic syringes in
clear autoclave bags. Label as PLASTIC WASTE FOR INCINERATION
indicating name, phone number and contents of the bag.
I.
Radiation Safety and Working Rules

Most of the rules about using radioactivity are common sense. The chief problem is that
the long-term effects of even low doses of direct radioactivity are unknown. Consequently
you should assume that any amount of radiation is not good for you and take precautions
to minimize your exposure. Some of the precautions for handling low levels of
radioisotopes are included in the following rules that have been abridged and adapted
from the U.B.C. Reference Manual for Radionuclide Safety and Methodology (Department
of Health, Safety and Environment, 1994).
1.
Contain the radioactivity. The radioactivity should only go where you consciously
want it to go, even during spills, and you should know where it is at all times.
2.
Plan your experiment carefully. Minimize the number of steps where you handle the
radioactivity.
3.
Do all the manipulations on trays lined with plastic-backed absorbent paper that will
contain any spilled samples. The purpose of the tray is to contain any accidental
spills; consequently you should not carelessly contaminate the surface. If you have
any contaminated equipment such as stirring rods, forceps and pipettor tips that you
wish to save and re-use, set them down so that the contaminated part rests on a small
piece of aluminum foil on the tray. This foil is the only area on the tray that should
become contaminated. The parts of the equipment that are probably uncontaminated
should rest on the paper, not the alumina. In some cases it is convenient to fold
ridges into the alumina before you use it to prevent the tools from sliding together, this
is particularly important if you are saving pipette tips that were used for different
samples.
4.
Avoid direct physical contact with any radioactive material. Wear lab coats at all times.
Use gloves when you handle anything that has been in direct contact with radioactive
material. This contaminated equipment could include things like the culture tubes, the
pipettor tips and the forceps.
5.
If you handle non-radioactive items such as notebooks, pens, door knobs, water taps,
waterbaths, water bottles, centrifuge lids et cetera with your gloved hands you could
contaminate them with any radioactivity that might be on the gloves. This
contamination could transfer back to your hands when you handle the same
equipment in later experiments when you are not wearing gloves. Consequently you
should be conscious of what you are handling at all times. If it is something that is not
radioactive and should have no probability of becoming radioactive do not touch it with
a glove that has touched a tube with a radioactive sample. If you remove your gloves
after touching a radioactive sample, replace the gloves because the palm and the
corresponding finger area might be contaminated.
86
6.
For some experiments it is convenient to use one hand for handling possible
radioactive material and the other for handling non-radioactive material.
7.
If you use a pin board to hold filters for samples, use forceps to handle the filters while
impaling them on the pins.
8.
Take extra care when doing any transfers. If a spill occurs use a red grease pencil to
draw a circle around the contaminated area and then immediately notify the instructor
so that it can be properly cleaned up.
9.
When the work is completed, the radioactive waste and contaminated supplies or
equipment should be isolated and discarded to the appropriate areas. It needs to be
sorted out since different things are disposed differently. Some is cleaned, some is
burned and some is buried.
10. Contaminated material left at the bench should be marked with a piece of tape
bearing a radiation warning signal. A piece of tape with one symbol is as good as a
piece bearing several symbols and is a lot easier to clean up in the end.
Wash your hands thoroughly before leaving the lab for any purpose. Even if you were
careful, your hands might have become contaminated if the gloves had small holes or if
someone else didnt work carefully.
87
Appendix C Units, Graphs and Calculations
A. Units and Prefixes
1. Units of concentration measurement
Concentration represents the amount of a material or solute in a volume of solvent.
Measurements of concentrations are usually simple, but some people find
concentration units confusing because they occur in several different forms and are
sometimes mistakenly thought of as amounts. Since all concentration
measurements represent the same general concept, the units should be
interchangeable, but some units are more convenient for representing specific
measurements than other units. The common units of concentration are grams per
liter (g/L), moles per liter (moles/liter), molar (M), molar equivalents per liter (eq/liter),
normal (N), percent weight per volume (% w/v), percent volume per volume (% v/v),
parts per million (ppm), pH, absorbance (A), optical density (O.D.) and relative
concentration (X). When one mole of material is dissolved in enough solvent to
make one liter of solution the concentration can be expressed as moles per liter or as
molar. A molal represents one mole of material dissolved in one kilogram of solvent.
The units of equivalents per liter and normal both represent the moles of protons per
liter when strong acids are present in solution and allow for the fact that a one molar
solution of an acid which produces one H+ per molecule will be less acidic than a
one molar solution of an acid which produces two or more H+ per molecule. Percent
usually refers to the number of grams of solute per hundred ml of solution (% w/v)
but can mean milliliters of solute per hundred ml of solution (% v/v). Parts per million
represents milligrams of solute per liter of solution. pH units represent the minus log
of the moles of hydrogen ions per liter of solution. Absorbance values are inversely
proportional to the relative transmission of light intensity through a solution and
correlate to the concentration of molecules per volume of that solution. Optical
density is also inversely proportional to the relative transmission of light intensity
through a solution, only it correlates to the concentration of light scattering particles
per volume of solution. Relative concentration compares the concentration of a
given solution to another solution containing the same chemicals and expresses the
concentration as a multiple of the solution to which it is compared. For example the
concentration of each chemical in a 0.5X solution is half as concentrated as the
concentrations in the standard solution. A 10X solution would be ten times more
concentrated than the 1X solution to which it is compared. A 1X solution usually
represents the standard working concentration but exceptions do occur where the
working concentration is some other proportion of the stock solution.
2. Units in Tables and Graphs
Good tables and figures are generally uncluttered and easy to read. For example,
the table or figure of rough results might show units of mg/ml but if most of the
measurements yield concentrations in the g/ml range then you should express the
data in units of g/ml. Try to avoid a lot of zeros or exponents that will clutter up the
data and interfere with easy comparisons. To choose the most appropriate units,
round off the numbers (described in the section on the Reliability of Results in the
middle section of this appendix) and express the numbers in scientific notation. After
the rounding, look for potential common repeated exponents within each set of data
then simplify the presentation by choosing the appropriate prefixed units which
88
include the most common repeated exponent. For example, the volumes two
thousand, twelve thousand, and seventeen thousand milliliters could be respectively
expressed as 2, 12 and 17 times 103 milliliters but they are better expressed as two,
twelve, and seventeen litres because the bigger unit has less zeros to clutter up the
table. In general it is better to express SI units by adjusting the prefix to avoid the
need for expressed exponent multipliers. Some of the standard prefixes for
modifying the root symbol of units such as gram (g) or litres (l) are shown in the
following table.
Multiplying
Factor
Prefix
Symbol
10
12
10
peta
tera
P
T
109
106
103
giga
mega
kilo
G
M
k
10-3
10-6
10-9
milli
micro
nano
m
or u
n
10-12
10-15
10-18
pico
femto
atto
p
f
a
15
Two non-SI units that frequently show in some literature are centi (c) such as
centimeter (cm) and deci (d) such as decilitre (dL). A centi prefix indicates 10-2 of
the named unit. A deci prefix indicates 10-1 of the named unit.
Sometimes numbers in a table have a common exponent that can be left in the
column heading to simplify the comparison of the critical part of the numbers.
When you use multipliers in the column headings be careful to consider the sign
of the exponent relative to the position of the units because the sign depends on
the position relative to the units. For example, the following values in each row of
the following table are all the same value:
g/ml
g/ml
x10-4 g/ml
(g/ml) x 10+4
mg/ml
200
1400
2x102
14x102
2.0
14.0
2.0
14.0
360
36x 101
3.6
3.6
0.20
1.40
0.36
The last column shows the preferred style because it is clear, simple and unambiguous.
Multipliers expressed as 100 and 101 are needlessly complex and should not be used.
The first column is acceptable but has more digits than necessary to express the values.
The center columns are cluttered and awkward listings.
89
B. Graphs
There are three types of graphs that you will normally encounter in microbiological
research. The simple X-Y line graph and the semi-logarithmic X-Y line graph are
usually used if you expect a progressive, continuous trend between the sets of
information in the X-axis and the Y-axis and you can validly expect to interpolate
between measurements. The histogram is used where you have discrete points
relating the information in the X-axis and the Y-axis but there is no reason to believe
that an interpolation between the two points is valid or meaningful. In all three types
of graphs, the fixed variable that you are controlling is usually plotted on the X-axis
and the variable that is measured but not directly controlled is plotted on the Y-axis.
1. Simple X-Y line graphs
a.
These graphs have two linear axes that are scaled to cover the range of
measurements for each axis.
b.
Scales are usually set up so that the control datum line shows an approximate
visual impression that is either horizontal, vertical or forty-five degrees depending
on the expected relationship between the fixed control values and the
uncontrolled variable.
c.
The sample values should be represented by large discrete symbols

approximately two millimeters across. Different shaped or textured symbols
should be used for different sets of values so that the information belonging to
each set can be readily identified.
d.
Normally each graph is limited to less than five different sets of data so that the
trends are easy to see.
e.
Unless the data represents discrete and independent trends, each line in the
graph should be an interpretation of the probable data pattern that represents the
real trend between the points if the measurements were done continuously and
consistently throughout the experiment. This way of using the data means that
some points might be considered incorrect and off the line but most of the points
should be on or near the line.
f.
Choosing the lines through points in graphs is frequently complicated because

some measured values are inaccurate and the real trends are masked by the
error. However, there are several considerations that help to simplify making
probable interpretations.
i.
Try to avoid drawing any lines in which the trends are completely dependent
on the position of one point. In some cases, one-point trends are real but
most reliable trends are usually including three or four datum points and one point trends are usually due to a single bad datum point.
ii.
Some scales magnify minor differences in measurement and seem to show a

very complicated relationship between the graphed variables when the
apparent differences are not real and the real trend is a simple line. This type
of problem can be reduced by attempting to estimate the error limits
associated with your measurements and then including those estimates as
90
error bars in the graphed points. In some assay results the error limits can be
estimated as 95 and 99% confidence limits, in other cases you will get an
adequate estimate by averaging duplicate measurements and calculating an
error range.
iii.
Usually the origin of the graph has some meaning or implication that can be
assessed by looking at the two graphed variables before deciding whether
the graphed line should go through the origin or intercept either axis. For
example, if you were plotting the number of progeny phage produced at
different multiplicity's of infection you would normally expect to see no
progeny phage produced when no infecting phage were added to the culture.
Therefore you would expect the line from that relationship to graph through
the origin and if the line apparently intercepts the Y-axis then you either have
bad data or you have phage contamination in the environment.
iv.
Most biological results show simple, progressive trends when plotted in

graphs. If your points can only be connected with a squiggly line you
should consider the possibility that one or two points in the graph could be
erroneous and try to successively fit three-fifths of your points to:
:::::-
A straight line through the origin (y = mx)

A straight line with an axis intercept (y = mx + b)
A concave hyperbolic curve (x . y = k).
An exponential increase (log y = x or y = log x)
An exponential decrease (1 = x.log y)
In many cases, limitations of the data make the hyperbolic curves difficult to
distinguish from the exponential curves but the distinction is not essential
unless you are trying to mathematically describe the results. The more
important concept in making interpretations is to decide on the simplest
apparent trend of the results and avoid lines with several randomly changing
one-point trends.
v.
Standard curves are line graphs that are used in a variety of assays to
convert results expressed in one type of unit to the equivalent value in
another type of unit. They are usually prepared by taking several samples
with known values in one type of unit and then measuring the same samples
in a way that gives their measurements in the second type of unit. The two
types of values for each sample can then be plotted against each other to
form a graph which can be used to convert values in one type of unit to the
corresponding unit values when only one type of measurement has been
done. These standard curves are easiest to use when the graphed
correlation is linear and can easily be described with one of the line formulas.
However, even non-linear standard curves can be used to do
interconversions by simply looking up the corresponding Y co-ordinate (value
on the y-axis) for the point on the graphed line corresponding to the known X
co-ordinate (value on the x-axis) or vice versa.
vi.
When data is portrayed in several different graphs or mathematically

transformed to compensate for non-linear features of the measurement it is
still the same information and should be interpreted consistently between the
different types of graphs. Interpretations between graphs might look different
91
but observations seen in one graph must be consistent with observations in
other graphs using the same data.
vii.
The figures should include keys that correlate different graphed symbols to
the appropriate reaction conditions. In these keys identify the active
component of the test condition rather than the arbitrary number given to that
test during the experiment. For example, if you added lactose to a culture of
cells growing on glycerol in flask number one it is better to correlate the
symbol in the key to lactose treatment rather than flask number one.
Generally the keys should be boxed off or positioned so that the symbols in
the key cannot be confused with actual data points. It is also important to
identify when something is added, if the addition is done after the
observations begin. For example, if you plot the growth of a culture which
grew on glycerol for fifteen minutes and then was treated with lactose you
should use an arrow to show when the lactose was added. This arrow will
give you a direct visual reference to show how long any effects due to the
addition take to occur and make any effects showing up prior to treatment
more obvious.
viii.
Freeform interpretive lines can be drawn in Excel graphs by duplicating the

values you intend to graph before plotting one set of values as points without
lines and the other set as lines without points. The values in the latter set can
be adjusted to make the line fit the perceived pattern of the points shown by
the former set of values.
2. Semi-logarithmic line graphs

a.
These are the same as the simple X-Y line graphs except that the distance
intervals on the Y-axis are at logarithmic intervals and are shown progressively
closer together for progressively bigger numbers. This spacing means that these
graphs will show any constant exponential relationships as straight lines but
changing exponential relationships should still form a curved line.
b.
A logarithmic scale has no zero because the logarithm of zero is at minus infinity
and can only be approached but not reached.
c.
Semi-log plots can be made by converting the values to the log value before
plotting them on a regular linear X-Y graph. They can also be plotted on graphs
that distort the space along the Y axis so that the points occupy the same
relative position that they would have if they were converted to a log value and
plotted on a linear scale. However, since the distorted scale on this type of graph
does not actually convert the observed values to logarithms and any values
manipulated for calculations revert back into the normal non-logarithmic
relationship unless you actually convert them to logarithms.
3. Histograms (bar graphs)

a.
Each value is plotted as a distinct point that is joined to the X-axis with a vertical
line or column.
b.
Usually the two axes are set out so that the graph appears square or rectangular.
92
c.
Choose a range and a scale of each axis that amplifies any patterns.
d.
Samples which are being compared, for example the test and control values at a
number of time intervals, can be plotted with the adjacent columns for the test
and control for each interval or as two totally separate graphs. If the different
samples are plotted side-by-side, the columns are usually shaded differently so
that they can each be readily and distinctly seen.
e.
In a histogram the ordinate (X-axis) is a continuous variable and the adjacent

columns of the graph touch each other. In a bar graph the ordinate variables are
not continuous and the adjacent columns do not touch each other.
4. Graphs (figures) in reports

a.
Reduce the clutter in the figures and tables and enhance the readability of the
data by putting the common multipliers in the column headings and in the axis
labels just before the units.
b.
Each axis on the graphs should name the concept plotted on that axis as well as
the units. The title should indicate the intent or purpose of the graph rather than
just repeat the labels from each axis. Check for scale distortions by thinking
through the value of each number. For example, the interval between exponents
such as 106 and 1 x 107 is sometimes mistaken as equal to the interval between
1 x 107 and 2 x 107 instead of being one-tenth the interval.
c.
Divide the labeled intervals on each graph axis by using simple, recognizable unit
multiples such as 2 or 5 or 10 rather than oddball intervals like 2.32 or 4.5.
d.
The figures should include keys that correlate different graphed symbols to the
appropriate reaction conditions. In these keys identify the active component of
the test condition rather than the arbitrary number given to that test during the
experiment. For example, if you added lactose to a culture of cells growing on
glycerol in flask number one it is better to correlate the symbol in the key to
lactose treatment rather than flask number one. Generally the keys should be
boxed off or positioned so that the symbols in the key can not be confused with
actual data points. It is also important to identify when something is added, if the
addition is done after the observations begin. For example, if you plot the growth
of a culture which grew on glycerol for fifteen minutes and then was treated with
lactose you should use an arrow to show when the lactose was added. This
arrow will give you a direct visual reference to show how long any effects due to
the addition take to occur and make any effects showing up prior to treatment
more obvious.
e.
Be careful to include all of the data. Sometimes related information collected in

different parts of the protocol is relevant to the idea being tested elsewhere and
should also be included with that data. For example in a chemical assay such as
the Bradford protein assay the blank is used to adjust the spectrophotometer but
it is also the absorbance reading of an infinitely diluted protein sample and
should be included in the as a datum point at (0,0).
f.
Since lines in graphs are your interpretations of the true pattern shown by the
data you need to be careful that you are drawing the line shown by the data
rather than the line that you expect the data to show. You can disagree with the
93
interpretation indicated by the data in the write-up but it is important to recognize
the trends indicate by the data in the graphs and tables.
i.
Since the interpretation depends on readily seeing the data, each point
must be large enough to be readily visible (approximately 2-3 mm
across).
ii. The line that is the interpretation must be clearly visible.

iii. The interpretation should normally include 3/5th of the datum points
without relying on one-point trends or creating an unreasonable bias in
the pattern of error around the interpretation. For example each time that
a point is not on the line you are saying that you considered the point to
be in error relative to where it should have been if the measurement was
done accurately and reproducibly. The points that you consider
erroneous should normally scatter throughout the graph rather than being
limited to just the low end of the graph. Similarly, the error is usually
neither all above the interpreted line nor all below the interpreted line.
iv. Once a point is considered in error in one graph it should show the same
relative error in other graphs where it shows up as a datum point. When
data is reused in several graphs the interpretations might look different
but the interpretations are not independent and must be consistent in the
treatment of the patterns of interpreted error. For example if the line goes
between two points in one graph it should still go between those points if
the same data is transformed and reused in another graph. In addition,
the percent difference between each observed point and the
corresponding imaginary point on the interpreted line should remain the
same in each interpretation of each graph using the same data.
g.
During the analysis any descriptions of the interpretations the data should
correspond to the interpretation shown by the lines in the graphs. You can
disagree with the interpretation and explain why you believe it is wrong but the
interpretations in words and figures must correspond.
C. Reliability of Results
1. Theory of Measurements
The trends and patterns which make up the natural laws and rules of science are based
on the ability to do measurements and resolve the patterns in those measurements by
making graphs and deriving predictive equations which reproducibly describe the
correlations between the measured values. Therefore, good measurements are
important for doing good science. However, even good measurements have the
limitations that are pointed out in the following essay that has been adapted and
abridged from the Experimental Biology by R.W. Van Norman (1963):
If we repeat our best measurements several times, experience teaches us
that we should not expect exact duplication of results. Slight human
variation, small changes in the instruments, and other fluctuations, some of
which are too small to be noticed, will combine to affect the final
94
measurement. Our series of several numbers will be close to each other but
not identical. If only random variations affect the results, a graph of a large
series of measurements tends to follow the normal curve shown in the
following figure. The higher a point on the curve, the more frequently that
value is found. A skinnier curve indicates a more precise measurement.
Precision of measurement refers to the closeness of agreement among the
various values.
If the measurement is accurate, the numbers obtained cluster around the real
and true value obtained by averaging the results of a large number of
measurements. Some individual numbers will be larger, some smaller, but
taken together the set of numbers gives a useful approximation of the true
value. If you failed to notice that someone had broken off the end of your
meter stick, you might make precise but inaccurate measurements.
Some kinds of measurement require all the precision available but other kinds do not
need such precision, so the extra effort is wasted. It is quite possible to spend too much
time in careful measurement if, for example, the living material changes during the
measurement. Sometimes when work is done too carefully and slowly factors such as
evaporation and growth significantly changes the concentration. A reaction rate might
be found by measuring the concentration of one of the reactants every five minutes but
the estimate of that rate will not be very good if it takes three minutes to do the
measurement. The largest common error in biological measurement is the 5-to-10%
variability caused by natural variation in the living material.
95
2.
Significant Figures
Since the results are limited by the measurements the range of expressed digits in the
number conventionally indicates the estimated precision of any collection of
measurements. These expressed numbers are called the significant digits. For
example, if you weigh a cell pellet on a balance that weighs to one-tenth of a gram and
get a result of 1.6 gram then that number indicates that the result is good to two
significant digits and the actual mass is between 1.55 gram and 1.649 gram. When the
same sample is weighed on a top loading balance that measures to one-hundredth of a
gram, the result might read 1.63 gram and that means that the actual mass is between
1.625 gram and 1.6349 gram and has three significant figures. Similarly, if the same
sample is measured on a balance good to one-tenth of a milligram the result might read
1.6327 gram and has five significant digits so that the actual answer is between
1.632649 and 1.63274 grams. The important point is that the result from the more
precise machine gave a more detailed measurement. The result from the cruder
machine should not be expressed as 1.6000 gram because that degree of precision was
not shown by the crude measurement and is actually wrong since the more precise
measurement shows the actual result as 1.6327 gram.
Generally, the precision of the measurement is taken as the lowest measured increment
in the scale of the measuring instrument. For example, a pipette with a scale in 0.01 ml
increments would be able to measure volumes to two significant digits beyond the
decimal point and the inherent variation of the measurement is at the third significant
digit. However, this general rule for estimating precision becomes complicated when the
measurements are done on machines with non-decimal increments or machines such as
spectrophotometers that have non-linear scales. The estimation is further complicated
when measurements of several different instruments are used to calculate the final
result. Therefore, a more general rule for determining significant figures is to estimate
the combined probable error of the measured result and then round off the answer so
that the lowest expressed digit in the number is the digit affected by the probable degree
of error. If the estimated error is around 10% then round the values to 2 significant
digits. If the estimated error is around 1% then round the values to 3 significant figures.
However, since the purpose of doing measurements is to compare results there is no
point expressing comparable numbers to different degrees of significance and therefore
you should round off all of the numbers to reflect the least accurate result in the entire
set. For example it is not useful to compare 1.6 g to 0.0578g since the first number
could be anywhere between 1.5500g and 1.6499g and consequently the observed
difference at the second, third and fourth digits can not be adequately assessed. The
proper comparison is to round off the 0.0578g to 0.1 and then compare 0.1g to 1.6g.
The three rules for rounding off numbers are:
a.
When the digit to be dropped is less than 5, leave the last significant digit
unchanged.
b.
If it is more than 5, increase the last significant digit by one.
c.
If the digit to be dropped is exactly 5, the last significant digit is left as an even digit
by rounding up or down accordingly; e.g., 0.635 = 0.64; 0.545 = 0.54.
96
3.
Estimation of Error
a.
Mean Absolute Deviation

This estimation is the average of the absolute differences between the mean
answer and each individual measurement. (Mean is another name for arithmetic
average). If the results have a large variation then the mean absolute deviation will
be larger than the mean absolute deviation in a better set of results where each
answer is very close to the average value. It measures how well the measured
results cluster around the average result.
An example of the mean absolute deviation calculation can be done using pH
measurements of a solution that gave successive readings of: 6.71, 6.75, 6.74,
6.77, 6.73 and 6.74. The average value derived by adding all the values together
and dividing by six is 6.74. The individual deviation between each measured value
and the mean value is: 0.03, 0.01, 0.00, 0.03, 0.01 and 0.00 respectively and the
average deviation obtained by adding the individual deviations together and
dividing by six is 0.013. Therefore the result of the measurements is expressed as
6.74 + 0.013 pH units. An extra digit is retained in the error estimate to avoid
distorting the apparent range of the error.
b.
Standard Deviation
This estimation of the reliability of an answer is based on the observation that a
series of measurements tends to form a bell shaped distribution about the mean
value when the number of occurrences at each observed value are plotted against
the corresponding observed value. These plots are commonly referred to as
normal curves and they are important because they show that a series of
measurements with a lot of error have lower and wider bell curves than another
series with less error. Therefore, the relative error of the measurement can be
estimated by comparing the height of the normal curve at the mean value to a
chosen width of the curve that includes a consistent portion of the total curve. In
practice the comparison is done by determining the area of the curve necessary to
contain a consistent fraction of the total area since the area is proportional to the
height and width of the curve. If the data is bad the width is higher than the height
and contributes more to the total area and vice versa if the data is good. The
consistent portion of the bell curve that is used amounts to the 68% of the area of
the curve closest to the mean value and the comparison is called a standard
deviation. A more detailed definition of standard deviation is:
s=
where:
Y2 (n -1)
s
Y2
n
= standard deviation
= sum of the square of each of the individual
deviations from the mean value
= total number of samples
This formula is only suitable for results using several samples and should not be
used for experimental tests using only one or two samples. When the
measurement involves populations such as the colonies of a plate count or the
97
observed disintegrations in a radioactive sample then the standard deviation is
closely approximated by simply taking the square root of the total number of counts
or colonies in the observed sample. For example the standard deviation of a plate
count showing 100 colonies is 10 colonies. The standard deviation of the average
value for two or more plates is approximated by adding up the colonies on all the
plates, taking the square root of that total and then dividing the square root by the
total to give a decimal fraction which can be converted to a percent.
c.
Confidence Limits
Confidence limits modify the calculated standard deviation to estimate the range of
values that should be considered to be reasonably sure of including the true
answer. Obviously, one standard deviation estimates the uncertainty, but one
standard deviation is a range that only includes 68% of the expected answers and
will not include the real value almost one-third of the time. However, for large
samples two standard deviations include 95% of the possible observed values.
Therefore, you can estimate the necessary range of answers to be sure of
including possible answers 95% of the time by simply multiplying the standard
deviation value by two. Similarly, if you want to be 99% sure of including possible
answers then multiply the standard deviation by 2.6.
For plate counts and radioactive counts where the standard deviation is roughly
approximated by the square root of the observed counts, the approximate
confidence limit is calculated by multiplying this approximate standard deviation by
two to get the 95% confidence limit and by 2.6 to get the 99% confidence limit. For
example, a plate count showing 144 colonies in the plate has a standard deviation
of 12 colonies and 95% of the time any repeated assay of that sample is going to
be within 24 colonies of the answer. Therefore, 95% of the time the possible
answers range between 120 to 168 colonies and the 95% confidence limit is 144 +
24 colonies. To estimate the standard deviation of an answer based on the
average of more than one plate, assume that the colonies all come from one plate
and find the square root of the total colonies of all the plates. This calculation
becomes complicated if the plates were done at different dilutions but you can still
complete the calculation by expressing the standard deviation as a percent of the
total number of colonies because the percent standard deviation will apply to the
final answer. For example, one plate at 10-6 final plated dilution has 36 colonies
and another plate at 10-5 final plated dilution has 289 colonies. The average
answer is 3.24 x 108 cells per ml and the standard deviation is:
289 + 36
(289 + 36) X 100% = 5.5% of the average answer.
d.
Combining of error in processed results

When two quantities such as plate count values or radioactive counts are added
or subtracted, the uncertainty of the result is less than the sum of the individual
error because some error may partially cancel. To allow for this cancellation
effect the uncertainty of the combined result is usually calculated as the square
root of the sum of the squared values of the individual standard deviation for
each value in the calculation. For instance, given that a control plate count at 10-
98
final plated dilution showed 144 colonies and a test plate count at 10-3 final
plated dilution showed 225 colonies then the standard deviation of each plate
count is the square root of each colony count divided by the corresponding
dilution. The standard deviation of the difference between the two results is the
square root of the sum of the square of each individual standard deviation so that
after appropriate rounding the combined result equals:
2
225
225 144
144
3
2 2
3
10 10
10
10
2
225
144
225 144
3 2 3 2
10
10
10
10
210,000
225.0 10 1.4 10
6
= 210,600 15,000 colonies/ml
When quantities are multiplied or divided, the error combines differently because
each error is equally weighted in the answer. This equal weighting is included in
the error calculation by converting each error to percent before it is squared and
added to the other values. For example, if the test result in the plate count
example was divided by the control plate count value, then the standard
deviation of the ratio after appropriate rounding equals:
225
225 144
144
10 3 10 2 10 2
10
225
144
3 6.7% 2 8.3%
10
10
225 144
3 2 (6.7%) 2 (8.3%) 2
10
10
15.6 44.4% 69.4%
156
. 10.7% of 156
.
156
. 167
.
99
4.
Chauvenet's Criterion
Sometimes sets of data contain an outlier (a value that does not seem to fit the general
pattern)) that deviates so much from the mean value of the set that they distort the
observed mean and the standard deviation. When this distortion arises, it would be
beneficial to discard the outlier. However, indiscriminate discarding of all the values that
do not fit the expectations of the analyst can also distort the final results. The criterion
established by Chauvenet implicitly states that an outlier should be discarded if it has a
deviation from the mean that exceeds "n" times the standard deviation where "n" is a
multiplier determined by the number of readings in the set of data.
For example, in a set made up of 10 readings, if the difference between any readings
exceeds the average reading multiplied by the "n" value 1.96, then that reading should
be discarded and the mean should be recalculated using the remaining 9 values.
The criterion can also be described as a ratio where a value is discarded if the ratio of
the value to the mean value exceeds "n".
X1
Xm = n
where:
X1 = potential outlier
Xm = mean value for all the samples
n = maximum value accepted by Chauvenet's Criterion
Table of the "n" values used by Chauvenet's criterion for rejecting a value from a data
set
Number of values
10
15
25
Corresponding "n"
value
1.15
1.38
1.54
1.65
1.73
1.80
1.96
2.13
2.33
D. Growth Calculations
1. Most exponential bacterial growth is mathematically defined by the formula:
ln 2 ln Xi ln Xo
g
T
where
= growth rate
= doubling time or generation time
Xi = final concentration of bacteria
100
Xo = initial concentration of bacteria

T
= the time elapsed between Xi and Xo
ln
= natural logarithm
It is important to note:
a. Growth rate is usually expressed per hour.
b. Doubling time is usually expressed in minutes
c. The concentration of bacteria can be in cells per ml, optical density, mg/ml or any
other concentration unit, as long as it is the same for both measurements. If the
growth is balanced all of these different growth parameters double at the same time.
d. The formula can be rearranged to the following form after taking the exponent of both
sides of the formula :
Xi
Xo
e. Both growth formulas are useful for predicting cell concentrations from known growth
rates and determining how long it will take a culture to re-grow upon inoculation.
f.
The doubling time can be read directly off semi-logarithmic growth plots by simply
checking the time that it takes for the measured component to double. For example,
the time it takes to grow from concentration 2 to 4 or 3 to 6, etc. The semi-log graph
is also useful for checking whether the growth was constant since constant
logarithmic growth forms a straight line on a semi-log plot. However, a curved line
on the graph could be due to a changing rate of logarithmic growth.
g. The growth rate formula can be rearranged to give T = ln Xi/Xo by taking the natural
logarithm of both sides of the equation. This rearrangement shows that there are no
concentration units left in the linearized growth rate because the values are ratios
and therefore the concentration units on the right side of the equation will cancel out
in the calculation. This effect means that the correct units of growth rate () are
relative increase per hour or per hour if the elapsed time is in hours.
2. The yield for bacterial growth is mathematically defined by the formula:
X1 X0
S0 S1
where Y
= growth yield
X1 = final concentration of bacteria

Xo = initial concentration of bacteria
101
S1 = the final nutrient or substrate concentration
So = the initial nutrient or substrate concentration
It is important to note:
a. The formula vaguely resembles the growth rate description but it is not logarithmic
and does not include time. It reflects the efficiency of the use of the measured
nutrient or substrate (S) for producing growth (X). It is not than a rate.
b. The nutrient or substrate (S) can be generalized to represent any supplied nutrient
such as phosphorous, sulfur, carbon, nitrogen and so forth.
c. The growth can be measured in a variety of units
E. Dilutions and Dilution Schemes

1. Dilutions
a.
Dilutions are fractions in which the final relative concentration of the sample
appears in the numerator and the original undiluted concentration appears in the
denominator. They can be expressed in a variety of forms:
1
200 ,
1
1
2 x 100 ,
1
-2
2 x 10 ,
5 x 10-3 , 0.005 ,
1 1
1
2 10 10
one-in-200 and one-to-199

1-in-200, 1-to-199
all express the same dilution
b.
Dilutions represent decreases of concentration and only occur if you decrease

the amount of stuff in the volume of solution, if you keep the amount constant but
increase the volume, or if you concurrently decrease the amount and increase
the volume.
c.
The key to most dilution calculations is to recognize that:

A
C=V = AV
where
C = concentration of stuff in your solution

A = amount of stuff in your solution
V = volume of your solution
This point is important since any dilution is simply the ratio of the final
concentration (CF) to the initial concentration (CI) so that the preceding formula
can be substituted to give the formula in which:
102
CF
AF AI
final relative dilution = C = CF CI = V V
I
F
I
d.
(AF VI)
= (A V )
I F
In most cases where a portion of a culture (or anything else) is being diluted to
make a new culture the amount of bacteria in the pipette is equal to the amount
of bacteria that you will end up with in the flask. In addition, the concentration of
bacteria in the pipette is the same as the original concentration in the stock
culture since pipetting does not affect concentration. This means that the
preceding formula can be rewritten to the form:
CF
[(AF VP)]
[AF VP]
=
=
CI
[AP VF] [AP (VS + VP)]
where:
CF =
final concentration of the inoculum
CI =
concentration of the inoculum
VF =
volume of the new culture after adding the inoculum
VP =
volume of solution pipetted from the inoculum culture
VS =
volume of new medium before adding the inoculum
AF =
final amount of bacteria
AP =
initial amount of bacteria in the pipette but since AF = AP then:

CF
VP
VP
=
=
CI
VF
VS + VP
This new formula will allow you to do most dilution calculations. To use the
formula you need to distinguish whether the values are amounts, volumes or
concentrations. You also need to keep in mind that the final volume in the flask
is equal to the volume that is already there plus the volume that you added so
that you must decide whether supplied volumes are the total final volumes or the
pre-addition volumes.
e.
Since dilutions are fractions, once you know the relative dilution and the
corresponding concentration of any diluted sample, the original concentration can
be calculated if you divide the observed concentration by the dilution.
103
CF
CI
f.
= dilution; CI = CF dilution
The calculations for concentrating solutions work the same as the calculations for
diluting solutions except that the observed concentration goes up instead of
down. Relative concentrations are usually expressed as the fold increase. For
example, a 5-fold or 5X concentration would be five times more concentrated as
the starting solution.
2. Dilution schemes
These are simply the outlines showing the specific volumes of diluent and
sample that will be mixed to achieve the final dilution. Some schemes are more
practical and useful than others.
a.
A one in a million dilution could be done by adding 1 ml of the sample to 999,999

ml of diluent. However, this method is not very practical since you can
conveniently do three sequential transfers of 1 ml to 99 ml in which you first take
1 ml of original to 99 ml of diluent and then take 1 ml of the first dilution to 99 ml
of diluent and then take 1 ml of the second dilution to 99 ml of diluent and arrive
at the same final dilution. The sequential method uses far less diluent and is far
easier to measure. This process of sequential diluting is called a serial dilution
and it is usually written out as if it was a multiplication. For example a one in a
million dilution done as one in one hundred serial dilutions would be:
(1/100) x (1/100) x (1/100)
b.
Since dilutions are fractions, the final dilution resulting from a serial dilution is the
product of all the numerators involved in each dilution divided by the product of
all the denominators of all of the dilutions. For example:
i. Three serial 1-in-10 dilutions which are the same as
(1/10) x (1/10) x (1/10) have a final dilution of 1/1000 or 10-3
ii. Three serial 5-in-7 dilutions which are the same as
(5/7) x (5/7) x (5/7) have a final dilution of 125/343
c.
It is important to distinguish between minor differences in wording such as 1-in10 dilution and 1-to-10 dilution. The latter is a 1-in-11 dilution. A 1-to-9 dilution is
the same as a 1-in-10 dilution.
d.
It is easier to keep track of serial dilution steps if most of the steps are done as 1in-100 or 1-in-10 dilutions and only one of the dilutions, if any, is done at the odd
ratio necessary to make the products of the numerators and the products of the
denominators work out to the values in the desired final dilution.
104
e.
Routine serial dilutions for plate counts can be done in any volumes that can be
accurately measured but in most cases using standard glass pipettes, it is faster
and more efficient to routinely do 1-in-10 as either 1 ml -to-9 ml or 0.5 ml-to-4.5
ml and to do 1-in-100 as either 0.1 ml-to-9.9 ml or 0.05-to-5 ml. In the last case
the difference in accuracy between using 5 ml and 4.95 ml of diluent is negligible.
f.
It is best to arrange the steps in a serial dilution scheme to do a relatively high

dilution first so that you do not waste a lot of sample. For example the serial
dilution (1/100) x (1/10) is better than (1/10) x (1/100) even though both give the
same final dilution.
g.
The final step in a serial dilution must have enough volume to perform the
intended tests. For example, you should not do a 1-in-5 dilution in a volume of 5
ml if you need 6 ml of diluted sample.
h.
If you list down the final dilution, it is very easy to develop the serial dilution
scheme by first figuring out how many 1/100 dilutions to do and then figuring out
how many 1/10 dilutions and figuring out the odd dilution, if any is required.
Once all that is done, look at the numbers, check whether you could easily
combine the 1/10 step and the odd dilution into a single step, then transpose the
minimum necessary steps to give the best order. For example:
a dilution of 1-in-6000 = 1/6000 = (1/100) x (1/10) x (1/6)
The 1/10 and 1/6 steps can be combined as a single 1/60 step that can be
readily done by adding 0.1 ml to 5.9 ml. The final order could be re-arranged to
1/60 x 1/100 if you needed more than 6 ml of solution in the final dilution.
i.
If you do a dilution and put 0.1 ml on the plate you should see the same number
of colonies as if you did an additional one-tenth dilution and put 1 ml of this new
dilution on the plate. This volume effect is called a plating dilution even though it
actually represents a plating volume. The effect is important since it can save
time and equipment and simplify some odd dilutions, for example, 57-in-100
could be done as 5.7 ml-to-4.3 ml and plate one ml. It could be done more easily
in a single step by plating 0.57 ml instead of 1 ml (if you are using pour plate
method of plating).
j.
Because of plating dilutions you must distinguish between the statements, a final
plated dilution and final dilution which is then plated. The former combines the
plating volume used in the plating step, the latter does not include the volume
plated although the volume plated will need to be used in calculating the original
values. The final plated dilution equals the plating dilution times the final dilution
and has units of ml/plate while the final dilution is just a ratio of concentrations
and has no units. The final plated dilution should actually be called a final plated
volume, but the former name is more widely used.
k.
Results from plate count assays are usually expressed per ml of original culture.
In some special cases such as samples from milk and seawater very low results
are expressed as colonies per liter or as colonies per cubic meter of sample.
105
3. Stock solutions
A stock solution is a solution that is more concentrated than the actual working
concentration. Stock solutions are prepared when:
-:
a range of concentrations of the same reagent is desired and it is easier to

prepare and dilute one initial solution instead of weighing out ingredients for
several solutions
-:
very large volumes of reagents are required and it is not practical to store the
large volumes of diluted solution
-:
the necessary mass for a dilute solution is too low to accurately weigh on the
balance but can be accurately prepared in a more concentrated form and diluted
to the correct concentration.
-:
the same solution could be combined with a variety of other chemical solutions to
easily prepare a range of unique chemical solutions. For example, a stock of a 1
M Tris solution could be diluted to give 0.1 M Tris. It could also be diluted and
combined with EDTA to make a different type of solution.
F. Sample Problems with Dilution and Growth Calculations

A short set of assorted problem samples involving dilutions, unit conversions and growth
rate calculations are included in the next few pages. Some of the problems are simple,
some are repetitive and some seem complex. Most can be answered using the
information in the section on units and dilutions and the section on growth calculations,
but you might also need to be familiar some general information on pipetting, plate
counts and chemical assays. All the questions can be answered with simple
mathematics so if you end up with a complex solution or do not get any solution, ask the
instructor or teaching assistants for help, because you should be able to do all of these
types of calculations. Some answers and clues for solutions are provided on the pages
following the questions.
Questions:
1.
Given a culture of 6x108 cells per ml:a.
What is the final plated dilution necessary to get:

i. 15 colonies per plate?
ii. 35 colonies per plate?
iii. 105 colonies per plate?
iv. 500 colonies per plate?
b.
If you must plate 0.1 ml volumes, what is the final dilution in the tube that was used to
make each of the preceding plated dilutions?
106
2.
c.
If you must plate 0.12 ml volumes, what is the final dilution in the tube that was used to
make each of the preceding plated dilutions?
d.
If a concentration of 0.6 O.D.460 corresponds to about 5 x 108 cells/ml, what is the

concentration of the supplied culture in O.D. units?
e.
If you dilute the supplied culture to 10-7 what volumes would need to be plated to get the
colony numbers listed in question a? Which of these volumes would be suitable for a
normal pour plate procedure? Which of these volumes would be suitable for a normal
spread plate procedure?
Given that a supplied culture shows a concentration of 0.25 O.D. when diluted 1-to-5 and that a
concentration of 0.6 O.D. corresponds to about 5 x 10 8 cells/ml then:a.
Calculate how many cells are present per ml of supplied culture.
b.
If you plate 1 ml, to what extent must the supplied culture be diluted to give:-
c.
i.
90 colonies per plate?
ii.
iii.
If you want to start a new culture at a concentration with an O.D. of 0.07

then:i.
What volume of the supplied culture must be added to 10 ml of medium?
ii.
What volume of the supplied culture must be added to 26 ml of fresh medium?
iii.
If you want a final volume of 25 ml then:- How much medium do you use?
- How much of the supplied culture do you use?
3.
Start with a 27 ml culture. Take 1 ml and add it to 8 ml of saline, then make a reading and
observe a concentration of 0.17 O.D.460 for the diluted sample.
a.
How much of the supplied culture should be used to make a new culture with an O.D. of 0.1
in a final volume of 22 ml?
b.
How much of the supplied culture must be added to 22 ml of medium to make a new
culture with a starting concentration of 0.1 O.D.?
c.
What is the range of additions that could be used to prepare a culture with a final volume of
30 ml and a starting concentration between 0.15 and 0.18 O.D.?
d.
Repeat question c assuming that the flask already contains 30 ml of medium and you
want a final concentration of between 0.15 and 0.18 O.D.?
e.
What is the final concentration in O.D. units when 1 ml of the culture is added to 17 ml of
medium?
107
4.
5.
Determine the corresponding number of bacteria per ml of the original culture if the following
numbers of colonies were observed when samples were diluted and plated.
a.
Get 35 colonies after serial diluting 0.1 to 9.9 three times and then plating 0.3 ml.
b.
As in a but plate 1 ml.
c.
As in a but plate 0.1 ml.
d.
As in a but plate 0.7 ml.
e.
Get 47 bacteria after diluting 0.1 to 9.8, then 0.1 to 9.8, then 1 to 8 and plating 0.5 ml.
f.
As in e but plate 1 ml.
g.
As in e but plate 0.1 ml.
h.
As in e but plate 0.3 ml.
Determine the volume of a 20% stock solution of glucose that is needed to make the following
diluted glucose solutions.
a.
12 ml at 300 g per ml.
b.
1.5 ml at 0.1 mg/ml.
c.
0.5 ml at 10 pg/ml.
d.
5 ml at 15%.
e.
10 ml at 6 mM. (The free molecular weight of glucose is 180.2 daltons).
f.
A solution that is 10 mM.
g.
13 ml at 10 fg per ml.
h.
22 ml containing 40 ng of glucose per ml.
i.
11 ml containing 4 x 10-6 gram per ml.
j.
6 ml containing 15 micromoles.
6.
Repeat question 5 but work as if the given volumes are the volume of diluent that the glucose will
be added to rather than the final volume of the solution.
7.
Some of the calculated volumes in questions 5 and 6 are very small and consequently it would be
necessary to use intermediate stock solutions to do the dilutions with normal glass pipettes.
8.
a.
Which volumes seem too small to use directly?
b.
Decide what would be a suitable concentration for each intermediate stock solution and
then determine the dilutions that are necessary to get those intermediate stock solutions.
Determine the final plated dilution if you:a.
Make 4 serial 2-in-7 dilutions and plate:-
108
b.
c.
9.
10.
i.
0.2 ml.
ii.
1 ml.
iii.
0.5 ml.
Make 5 serial 2-to-7 dilutions and plate:i.
0.15 ml.
ii.
1.2 ml.
iii.
0.6 ml.
Make two sequential one-to-nine dilutions and plate:i.
0.05 ml.
ii.
0.08 ml.
iii.
0.16 ml.
Determine the original starting concentration of bacteria for each dilution in

question 8c if the incubated plates showed:a.
50 colonies each
b.
120 colonies each
c.
280 colonies each
If each plate in question 8a was made by pouring the entire contents of a

deep containing 20 ml of 1.4% agar, determine:a.
The final concentration of agar in each plate.
b.
The final amount of agar in each plate.
11.
Determine the minimum number of dilutions that are needed to ensure that an unknown glucose
sample that has a glucose concentration between 10 ug per ml and 50 mg per ml will give at least
one useable result in an anthrone assay if the anthrone assay has a useable range of 27.7
molar to 138.7 molar, then list the unknown range covered by each dilution of the unknown
sample.
12.
Determine the minimum number of dilutions necessary to get at least one plate with useable
counts if the sample that is to be plated contains between 900 and 4 x 10 7 cells per ml, then list
the range of possible concentrations of the sample that are covered by each dilution.
13.
A culture is inoculated with 0.2 ml of an overnight culture that had a concentration reading of 0.5
O.D.460 when diluted 1-in-4. The inoculated culture reads 0.05 O.D.
a.
What is the volume of media that was present in the flask before the inoculum was added?
109
14.
b.
If a concentration of 0.24 O.D. had actually been desired, how much inoculum should have
been added to the start and how much more inoculum should be added to the culture that
is reading too low?
c.
If a turbidity of 0.035 O.D. had been desired, how much inoculum should have been added
at the start and how much fresh medium should be added to the inoculated flask to reduce
the concentration to the desired value?
When an inoculum culture reaches 0.65 OD460 units, it will be used to start a second culture at a
final density of 0.14 OD460 units.
(a)
What volume of sterile broth should be added to the second flask so the final culture
becomes exactly 10 ml after the inoculation?
(b)
If the initial volume of medium in the new flask is already 12 ml before adding the inoculum,
how much inoculum should be added?
(c)
If the inoculum turbidity reading is off and you get a reading of 0.11 OD 460 when you add 1
ml of inoculum to the 12 ml of medium in the flask, how much more inoculum should be
added to get the intended start turbidity of 0.14 OD460?
15.
Given that two ml of inoculum culture with a turbidity of 250 Klett units is inoculated 1-in-12 into
fresh medium then grown for 25 minutes to give a turbidity of 45 Klett units, what volume of fresh
medium must be added to this freshly grown culture to give a culture with a turbidity of 30 Kletts?
16.
A two ml culture containing 5 x 109 bacteria per ml will be diluted 1/200 with fresh medium then
grown for 6 hours to get a turbidity of 0.6 OD 460 (which corresponds to a concentration of 4 x
108 bacteria per ml) before being infected at an MOI of 0.2. If the phage stock used for the
infection produced 60 PFU when 0.2 ml of a final plated phage dilution of 10-9 per plate was
incubated for eight hours, what volume of undiluted phage stock must be added to the culture at 6
hours to initiate the proper infection?
17.
During an electrophoresis experiment, a sample of culture supernatant taken from a test culture
at 15 minutes and measured in a Bradford protein assay containing one ml of 1/20 diluted sample
and two ml of Coomassie blue dye gave a reading corresponding to 40 microgram of protein in
the Bradford assay tube.
18.
(a)
What is the concentration of protein in the 15-minute undiluted sample?
(b)
What volume of water and the undiluted 15 minute sample must be combined to get 2
microgram of protein in the well of the electrophoresis gel if you mix 30 microliter of
diluted sample with 10 microliter of 4x loading buffer before boiling the mixture and
putting 20 microliter of the boiled mixture into the electrophoresis well?
(c)
Given that the supernatant sample contained only a single secreted protein with a free
molecular weight of 79 kilodaltons, how many moles of this P79 enzyme was added to
the electrophoresis well in the preceding question?
If a culture has a doubling time of 20 minutes, then:a.
What is the specific growth rate? (in hours).
b.
How long would the culture take to go from 0.02 O.D. to:-
110
i.
ii.
iii.
iv.
0.6 O.D.?
0.8 O.D.?
0.9 O.D.?
1.2 O.D.?
c.
If you want the culture to take 5 hours to reach 0.6 O.D., what should be
the starting concentration?
d.
If the inoculum in the preceding question was at 1.0 O.D., what volume
should have been added to 10 ml of fresh medium in order to make the
correct starting turbidity in the new culture?
19.
Repeat question 18 but use a generation time of 150 minutes.
20.
If a culture is at 0.15 O.D. and will increase to 0.25 O.D. in 10 minutes then:a.
What is the doubling time?
b.
How long will the culture take to reach 0.5 O.D.? (assume constant growth and
proportionate turbidity).
c.
How long will the culture take to reach 0.85 O.D.? (make the same assumption as in b).
d.
What is the growth rate? (in hours).
e.
Assuming that there are 10-9 O.D. -ml per cell, how long would the initial culture take to
reach 2 x 108 cells per ml.
f.
How many minutes earlier than the 0.15 O.D. reading was the culture at 5 x 10 7 cells per
ml?
g.
If a cell weighs 2 pg on average, how long will it take from the 0.15 O.D. reading before the
culture contains 1.2 mg of cells per ml? (assume that there are 10-9 O.D.-ml per cell and
there was constant exponential growth).
h. What is the time that the culture will reach growth saturation (stationary phase) if the culture
quits growing at 5 x 109 cells per ml?
111
Answers:
1.
a.
i.
ii.
iii.
iv.
b.
i.
2.5 x 10-8 ml/plate.

5.8 x 10-8 ml/plate
1.8 x 10-7 ml/plate
8.3 x 10-7 ml/plate
ii.
iii.
iv.
2.5 x 10-7
5.8 x 10-7
1.8 x 10-6
8.3 x 10-6
c.
i.
ii.
iii.
iv.
2.1 x 10-7
4.9 x 10-7
1.5 x 10-6
6.9 x 10-6
d.
about 0.7 O.D.
e.
i.
ii.
iii.
iv.
0.25 ml
0.58 ml
1.75 ml
8.33 ml
The last two volumes are too large for normal pour plating. None of the volumes are suitable for normal
spread plating.
2.
a.
The undiluted culture is 1.5 O.D., this is about 1.25 x 10 9 cells per ml.
b.
i.
ii.
iii.
7.2 x 10-8
2.16 x 10-7
4.0 x 10-7
c.
i.
ii.
iii.
0.49 ml
1.27 ml
a. 23.83 ml
b. 1.17 ml
3.
a.
b.
c.
d.
e.
1.44 ml
1.54 ml
2.94 ml minimum to 3.53 ml maximum
3.26 ml minimum to 4.00 ml maximum
0.085 O.D.
4.
a.
b.
c.
d.
e.
f.
g.
h.
1.2 x 108
3.5 x 107
3.5 x 108
5.0 x 107
8.3 x 106
4.1 x 106
4.1 x 107
1.4 x 107
5.
a.
1.8 x 10-2 ml
112
b. 7.5 x 10-4 ml
c. 2.5 x 10-11 ml
d. 3.8 ml
e. 5.4 x 10-2 ml
f. volume of stock varies with the volume of new solution prepared but the answer requires 9 x 10 -3 ml of
stock per ml of new solution.
g. 6.5 x 10-13 ml
h. 4.4 x 10-6 ml
i. 2.2 x 10-4 ml
j. 1.4 x 10-2 ml
6.
a.
b.
c.
d.
e.
f.
g.
h.
i.
j.
0.018 ml
7.5 x 10-4 ml
2.5 x 10-11 ml
15 ml
0.05 ml
volume is not supplied in the original question
6.5 x 10-13 ml
4.4 x 10-6 ml
2.2 x 10-4 ml
1.4 x 10-2 ml
Because of the small volumes involved, many of these answers are the same as for question 5. Differences
develop when volumes are bigger.
7.
The volumes for b, c, g, h, i and j are too small for normal glass pipettes to measure directly. There are a
number of different intermediate dilutions that could be conveniently made to get new stock solutions that
could be used to make the final desired solutions.
8.
a.
i.
ii.
iii.
1.33 x 10-3 ml/plate

b.
i.
ii.
iii.

6.5 x 10-4 ml/plate
c.
i.
ii.
iii.

a.
i.
ii.
iii.
1.0 x 105 CFU/ml

6.3 x 104 CFU/ml
3.1 x 104 CFU/ml if 50 colonies observed.
b.
i.
ii.
iii.
2.4 x 105 CFU/ml

1.5 x 105 CFU/ml
c.
i.
ii.
iii.
5.6 x 105 CFU/ml

3.5 x 105 CFU/ml
a.
i.
13.9 mg per ml after plating 0.2 ml
9.
10.
113
ii.
iii.
iv.
b.
11.

In each case there is 280 mg of agar.
The standard has a range of 5-to-25 g per ml, therefore it will take at least 6 tubes. The exact dilutions
used will depend on your choice of starting values but one possibility is to make:a.
1-in-2 to cover 10-to-50 g per ml
b.
c.
1-in-50 to cover 250-50-1250 g per ml
d.
e.
1-in-1250 to cover 6.25-to-31.25 mg per ml
f.
1-in-6250 to cover 31.25-to-156.25 mg per ml
The important concept in this problem is to reduce the sample into overlapping concentration values that are
covered within the range of the standard when diluted.
12.
The normal assay range is 30-300 colonies per plate. This could be achieved with a number of dilution
schemes but one possibility is to make:a.
b.
c.
d.
e.
1-in-30 to cover 9 x 102-to-9 x 103 cells per ml

1-in-3 x 104 to cover 9 x 105-to-9 x 106 cells per ml
1-in-3 x 105 to cover 9 x 106-to-9 x 107 per ml
a.
7.8 ml
b.
Should have added l.06 ml. Have already added 0.2 ml. Therefore add another 0.86 ml.
c.
Should have added 0.139 ml of inoculum and now need to add 3.43 ml of medium. By calculating the
volume final that is needed to convert the added inoculum to 0.035 ml you can then calculate the
extra volume of medium to add by subtracting the net culture volume that is already present in the
flask from the volume that should be present. This difference is the volume of medium that should be
added.
14.
a.
b.
c.
7.85 ml
3.29 ml
need 1.3 ml total, add 0.3 ml more
15.
12 ml
16.
0.53 ml
17.
a.
b.
c.
800 g/ml
25. l of water, 5 l of undiluted sample
2.53 x 10-11 mole
18.
a.
2.08 per hour
b.
i.
ii.
iii.
iv.
13.
1.64 hour
1.76 hour
1.81 hour
1.95 hour
c.
1.83 x 10-5 O.D.
d.
1.83 x 10-4 ml
114
19.
20.
a.
0.28 per hour
b.
i. 12.2 hour
ii. 13.2 hour
iii. 13.6 hour
iv. 14.6 hour
c.
0.15 O.D.
d.
1.76 ml
a.
b.
c.
d.
e.
f.
g.
13.6 minutes
0.39 hour or 23.6 minutes
0.58 hour
3 per hour
0.096 hour
22 minutes
Convert the mass per ml units to cell per ml units, then convert the cell per ml units to O.D. (or viceversa). Time works out to 0.46 hour.
1.17 hour
h.
115
Appendix D - Technical Theory

I.
Centrifugation
General Information
1.
Centrifugation works by placing the samples into a cylinder called a rotor and
then spinning the rotor on its axis. The particles in the sample suspension are
subjected to an outward angular acceleration that will cause the particles to
accelerate out of solution towards the outer most point of the centrifuge tube.
In some rotors the tube is held horizontal and the outer most point is the
bottom of the tube. In other rotors the tube is vertical or partially vertical so
that the outer most point is the wall. In the partially slanted tubes the sample
moves out to the outer wall then slides down the wall towards the bottom
outside edge of the tube.
2.
The angular acceleration is commonly called the centrifugation force or force

of centrifugation even though it is an acceleration rather than a force. The
actual acceleration depends on the speed and size of the rotor such that:
2R
F r w2 r
60 sec
min
centrifugation
where:
F = angular acceleration per second squared due to

w
r
R
= angular velocity of the rotor in radians

= radius of the rotor
= rotor speed in revolutions per minute
= normal mathematical constant pi
3.
The angular acceleration of centrifugation is often expressed as "relative

centrifugation force" (RCF) by dividing the angular acceleration per second
due to the centrifugation by the normal acceleration per second due to gravity
and listing the result with the units "times g" or "x g".
4.
The angular acceleration of centrifugation is important because larger

centrifugation forces will sediment smaller molecules faster than smaller
centrifugation forces. Since the velocity is proportional to the angular
acceleration. However the actual velocity that a particle achieves is dependent
on the other factor such that:
v = s * F = (Pv *(Pd - Md) * F ) (6 * Mv * Pr)
where:
= velocity of the suspended particles
= sedimentation coefficient. For many biological materials such as

ribosomes, RNA, and viruses this coefficient has a characteristic
constant value around 10-to-the-minus-13 seconds and are
expressed in S units or Svedberg units (which are 10-to-the-
116
F
Pv
Pd
Md
Mv
Pr
B.
minus-13 seconds) in order to avoid complicating results with big

exponents.
= angular acceleration per second squared due to centrifugation
= volume of the sedimenting particles
= density of the sedimenting particle
= density of the medium that the particles are suspended in.
= mathematical constant pi
= viscosity of the medium
= radius of the particle being centrifuged
5.
Since distance is velocity multiplied by the elapsed time, the length of time to
sediment a material out of solution is derived by dividing the distance that the
particles will have to move by the velocity. The result is called the clearing
time of the solution.
6.
The value of the centrifugation formula is that it shows that the results are
dependent on a few specific variables. This means that results can be
improved by simply changing the time of the centrifugation, the speed of the
centrifugation, the viscosity of the solvent or the density of the solvent.
7.
The sedimentation coefficient of bacteria in water is approximately 60,000

Svedberg units (S). This high value means that bacteria usually sediment at
fairly low rpm of the centrifuge.
8.
At very high speeds the friction between the rotor and the air around the
centrifuge cause heating problems. Consequently the high-speed
ultracentrifuges are normally operated with the rotors in a vacuum chamber.
Types of Centrifuges
1.
Centrifuges fall into three categories according to rotor design and three
categories according to speed.
a.
The rotor categories include:

i.
Swinging bucket rotors that are made so that the sample holder is
hinged and swings up during the centrifugation so that the sample
holder is horizontal and all of the sample pellets to the bottom of
the container.
ii.
Fixed angle rotors that are made so that the sample holder stays
in a constant position and the sample pellets against the outward
side of the container.
iii.
Continuous flow rotors that are designed so that sample can be
pumped in and out of the rotor while the rotor is spinning. This
arrangement allows large volumes to be centrifuged since the
particles in the samples will pellet against the outward wall of the
spinning rotor and the solvent will simply pass right through.
b.
The speed categories include:

i.
ii.
iii.
Clinical centrifuges with a maximum speed of 3,000 or 4,000 rpm.

High speed centrifuges that go up to 20,000 or 25,000 rpm.
Ultracentrifuges that go above 25,000 rpm.
117
2.
Medium speed centrifuges that hold one or two milliliter samples are called
microfuges.
II. Electrophoresis
1.
Electrophoresis is a method for separating molecules that depends on the

observation that a charged molecule in an electric field between two
electrodes will migrate to the electrode with a charge opposite to the charge of
the molecule.
2.
The velocity (v) of the molecule arises because the charged molecule will be
accelerated by an electrical field (F). This acceleration is proportional to the
charge on the molecule and proceeds until an opposite force created by the
friction between the molecule and the environment balances the electrical
field. At that point the velocity is constant such that:
v = ((F * Pq) f )
where:
3.
v
F
Pq
f
= velocity of the molecule

= electrical field
= charge on the molecule
= frictional coefficient
The electrical field is proportional to the voltage between the electrodes and
the distance between the electrodes. The friction depends on the size of the
molecules and the viscosity of the environment. This means that the formula
can be expressed in more detail such that:
v = ((F * Pq) f )
= (((E d) * Pq) f )
= (((E d) * Pq) 6 * Pr* Mv )
where:
4.
v
F
Pq
f
E
d
Pr
Mv
= velocity of the molecule

= electrical field
= charge on the molecule
= frictional coefficient
= voltage between electrodes
= distance between electrodes
= mathematical constant pi
= effective radius of the molecule
= viscosity of the medium or the environment
There are several important observations in the preceding equations. These

observations include:
a.
The velocity of each separating molecule is directly proportional to

voltage and charge.
118
b.
c.
The velocity of each separating molecule is inversely proportional to

size, viscosity and distance between electrodes.
The velocity for each molecule is constant for given conditions but
molecules that differ in size and/or charge will separate during
electrophoresis because they move at different velocities and will be at
different distances from the original position after any given time.
5.
If a sample is placed in a simple buffer solution and electrophoresed, the

frictional force is usually so low that all the molecules will migrate very rapidly
and will not separate well. This problem of resolution is further aggravated
since any separated material will easily be re-mixed together by convection
currents and even mild shaking of the buffer. Consequently samples are
electrophoresed in buffers containing some sort of inert stabilizing material to
increase frictional force and eliminate the possibility of re-mixing. The
common stabilizing materials include -: filter paper, starch gels, Sephadex
beads, cellulase acetate powder, acrylamide gels, agarose gels, and
combinations of acrylamide and agarose in gels.
6.
Each of the stabilizers has different properties that affect the separation and
each type has particular advantages and limitations. The most popular types
of stabilizers are the agarose and acrylamide gels.
a.
Acrylamide gel is a jelly-like plastic produced by reacting acrylamide

molecules with other acrylamide molecules and methylenebis(acrylamide) to form a three dimensional, convalently cross-linked
network. The extent of the cross-linking depends on the concentrations
of both components, but the methylene-bis(acrylamide) is usually kept
constant at 5% and only the acrylamide concentration is varied to alter
the cross-linking. The degree of cross-linking is important because it
determines the size of the holes or pores available in the gel for the
sample molecules to migrate through. Such pore size is important
because:
i.
ii.
The frictional coefficient described in the electrophoresis equation

is partly dependent on the relative sizes of the pores and sample
molecules. Consequently the gels that are used must be set up to
have pore sizes that are appropriate for the size of molecules
being separated.
The molecules will not move into the gels if the pore sizes are too
small.
In some gels the acrylamide solution is mixed and poured into the
support in a way that results in a higher degree of cross-linking at one
end than at the other end. These types of gels are called gradient gels.
They have the advantage of concurrently resolving a wider range of
molecular sizes than gels with uniform acrylamide concentrations.
b.
The agarose is a type of purified agar. It is a natural linear polymer of

galactose and 3,6-anhydrogalactose. It is not covalently cross-linked
like acrylamide but the agarose polymer strand is extensively hydrogen
bonded to adjacent strands to form a rigid, porous gel. The size of the
pores in these gels is inversely proportional to the agarose concentration
119
and they behave like the pores in acrylamide. However, the pores are
usually bigger than the largest useful pores in the acrylamide gel and
consequently agarose is usually used for separating bigger molecules
and molecular complexes than acrylamide can separate.
7.
None of the stabilizing materials are very mechanically strong and

consequently they need to be supported so that they hold together during the
electrophoresis.
a.
The agarose and acrylamide gels are either:

i.
ii.
iii.
Cast inside open ended glass tubes to form tube gels.

Cast as a sandwich between two glass plates to form slab gels
that can be used in a vertical position.
Cast on a flat tray to form a slab gel that is used in a horizontal
position.
b.
The paper stabilizers are usually just wetted with buffer and flattened
onto horizontal glass or plastic sheets.
c.
The powdered cellulose acetate is commercially bonded onto plastic

sheets.
d.
The Sephadex beads are simply slurried and poured into a tray.
8.
In addition to all of the other properties of gel electrophoresis, the gels

sometimes contain formamide or the ionic detergent sodium dodecyl sulfate
(SDS) in order to denature the sample molecules and remove secondary
structures which affect the effective size. The negatively charged SDS will
also coat the protein molecules in the sample and mask the natural charge of
the proteins. Consequently the proteins in any gel containing SDS will have a
uniform negative charge density generated by the SDS coating and will
separate according to size. In the absence of SDS the charge on a protein
varies with the type of amino acids present in the protein and the separation
will depend on the net charge as well as size. In contrast, nucleic acid
molecules normally have the same charge densities and normally separate by
effective size even without SDS. In general, the movements of proteins in
SDS gels and nucleic acids in normal gels are inversely proportional to the
logarithm of the size. However, this limited proportionality breaks down when
the ratio of molecular size to pore size becomes either too big or too small.
9.
When setting up a gel to do electrophoresis the samples are usually mixed

with sucrose or glycerol and one or more charged dyes. The mixture is then
placed in small holes or wells in the gel that keep the sample in place in the
gel until the electrophoresis is started. The small, charged dyes such as
bromophenol blue, bromocresol green and xylene cyanol are added because
they migrate faster than most of the sample molecules during the
electrophoresis and provide a simple visual indication of how fast and how well
the separation in the gel is occurring. The sucrose or glycerol is included to
increase the density and viscosity of the samples so that the initial diffusion of
the samples is reduced and the material stays in the wells until the
electrophoresis is started.
120
10.
The separated molecules in a gel are identified and detected by a variety of

methods. These methods include:
a.
b.
c.
Using radioactive samples so that separated molecules can be detected

by doing autoradiography or by cutting the gel into narrow strips and
then doing scintillation counting on each strip to determine which ones
contain radioactive material.
Using stains such as Coomassie Brilliant Blue, colloidal gold, silver stain,
and ethidium bromide to selectively colour the separated material but
not the gel.
Putting an overlay containing enzyme substrates or antibodies against
specific enzymes over the gel and then marking the places where
something in the gel reacts with the appropriate reagent in the overlay.
III. Enzyme Assays

An enzyme is a protein catalyst that converts substrate(s) to product(s). The activity
is measured as the gain of product or the loss of substrate. In order to do the
measurements there must either be a change in a measurable feature during the
activity or the product(s) must be able to be separated from the substrate(s) so they
can be independently measured.
During some assays there is a gain or loss of colour that can be measured as
changes of absorbance. There could be a conversion of solids to gases such as
oxygen or hydrogen that will separate from the solution to change gas pressure or
gas volume. There could also be formation of charged molecules that alter
conductivity or formation of molecules such as reducing sugars that can be
measured because they give distinct chemical reactions. The important point is that
some enzyme reactions cause changes that are easily measured.
When the products can not be measured independently from the substrates, the
molecules need to be separated before they can be measured. Some of the tools
that have been used to do these molecular separations include: thin layer
chromatography (TLC), high pressure liquid chromatography (HPLC), gas
chromatography (GC), electrophoresis, trichloroacetic acid solubility, ammonium
sulfate solubility, dialysis, DEAE binding, hydrophobicity column binding, affinity
columns for lectin binding and antibody binding.
The study of enzymes allows direct understanding of the function with regard to
inhibitions, rates, substrate specificity, impact of pH and impact of temperature.
They also allow enzymes such as nucleases proteases, ligninases, lactamases and
amylases to be used as tools to clean and isolate molecules. Products from other
enzyme activities such as restriction endonucleases form products that are valuable
for analytical patterns. Others are used as sensors, activators, release agents and
lytic agents.
In order to assess the properties or make use of enzymes all the enzyme
measurements depend on the conversion of one or more substrates into one or more
products. However since most enzyme activities are reversible some assays
measure the conversion of the natural product into the natural substrate.
121
(Si + (SiiSn)) (Pi + (PiiPn))

The results of the conversions from substrate to product or product to substrate are
typically expressed as enzyme activity per ml or as specific enzyme activity per mg
of enzyme material where enzyme activity is the amount of product produced per
minute.
Many common enzymes show relatively simple kinetic patterns with regard to
substrate availability. The most common trend is a hyperbolic increase in enzyme
activity as the concentration of substrate increases. These types of hyperbolic
curves for enzyme activity are called Michaelis-Menton kinetics. They can be
linearized by plotting the inverse of the substrate against the inverse of the
corresponding enzyme activity. For enzyme studies these inverse plots are called
Lineweaver - Burk plots. The Lineweaver - Burk transformations of the hyperbolic
curves are important because they allow researchers to predict two constants that
can characterize different enzyme properties. The intercept on the Y-axis is the point
where the maximum amount of substrate is theoretically available. Since enzyme
activity increased as substrate increased, the point where the maximum theoretical
amount of substrate is present must also be the point where the maximum amount of
activity is present. This intercept is (1/ Vmax). The X-axis intercept on the fourth
quadrant is the (-1/ Km). The Km is a substrate concentration. It characterizes how
well the enzyme binds the substrate. A high value means that the enzyme does not
recognize a substrate as well as when the Km is low. In contrast, the Vmax is a rate
of product formation. It characterizes how well the enzyme converts bound substrate
to product.
If substrate is not limiting and the amount of enzyme is doubled then the observed
enzyme activity and the observed Vmax will double but the observed Km will remain
the same. This effect is important for three reasons.
1. It means that the enzyme assay measures the concentration of functional
enzyme in a solution whenever the substrate is unlimited. This means that as
long as the reaction conditions are the same an assay that shows twice as much
activity must contain twice as many functional enzymes.
2. If the amount of substrate is limiting and the enzyme is in excess then the
enzyme activity directly correlates to the amount of substrate. A reaction that is
twice as fast as another reaction must have twice as much substrate present.
3. The specific activity of an enzyme is constant and independent of the
concentration of enzyme as long as reaction conditions are constant and the
substrate is in excess.
Enzyme activity is affected by the environment and different environments affect
different enzymes in different ways. In addition to the enzyme and the substrate
many assays include accessory molecules such as:
1. Metal cofactors such as Zn+2, Mn+2, Mg+2, Co+2 et cetera.
2. Ionic stabilizers such as NaCl or (NH4)2 SO4
122
3. Chelators such as EDTA and citrate
4. Organic cofactors such as ATP, NADH, FAD et cetera
5. Bovine serum albumin (BSA) to stabilize the protein.
6. Reducing agents such as mercaptoethanol and dithiothreitol to minimize
oxidation and stabilize the protein.
7. Buffers such as acetate, carbonate, phosphate, borate, glycine, Tris, Mops,
HEPES, PIPES and MES to stabilize the pH.
The accessory molecules have effects on the reaction aside from the intended
purpose. Some behave like products or substrates and inhibit some reactions.
Some can activate or inactivate enzymes. For these types of reasons conditions that
are suitable or ideal for one enzyme will not work for another.
IV. Growth Media
1.
All growth media must supply the mineral and organic material necessary to
support the biosynthetic properties of the cultured cells. The detailed
requirements vary with the different organisms but invariably include sources
for nitrogen, sulfur, phosphate, magnesium, sodium, potassium and carbon.
Depending on the actual ingredients involved the media will be classified as
defined or undefined.
2.
The defined media are usually simple solutions of specific pure salts that
contain one or more specific pure carbon containing molecules as a
carbon source.
3.
The undefined media are usually rich media that include crude, organic
mixtures that might vary from batch to batch and therefore are not as
functionally reproducible as the chemically defined media are. Some of the
organic mixtures used in undefined media include.
a.
Acid-hydrolyzed casein (Casamino acids). This material is prepared

from the milk protein casein and is a rich source for most amino acids
except tryptophan, cystine, asparagine and glutamine. The first two
amino acids are destroyed during the acid hydrolysis. The last two are
hydrolyzed to yield aspartic acid and glutamic acid. The specific
composition varies with the hydrolysis conditions and purity of the
original casein. It usually contains significant levels of vitamins and
sodium chloride.
b.
Peptones are prepared by enzymatically hydrolyzing proteins. Peptones

are not as rich as Casamino acids but include a variety of polypeptides
and seem to be essential for good growth of some microbes. Common
peptones include:
i.
Tryptone and casitone are casein hydrolyzed by a mixture of

pancreatic proteases that is dominated by trypsin and
chymotrypsin.
123
ii.
iii.
c.
Proteose peptone is a proteolytic digest of beef muscle tissue.

Soya peptone is a proteolytic digest of soybean meal and is rich in
peptones, carbohydrates, calcium and B vitamins.
Extracts are the water-soluble material obtained by soaking ground up

beef and yeast cells. The extracts of yeast are rich in vitamins, purines,
pyrimidines amino acids, carbohydrates and micronutrients.
4.
Liquid media is usually prepared in flasks or tubes. Solid media is usually the
same as liquid media except that it is gelled by adding l.5 - 2% agar and is
usually dispensed in tubes or Petri dishes.
5.
Some components of the media serve chemical purposes. The phosphate is

used as a growth factor but also stabilizes the pH in minimal media. Amino
acids or peptides in rich media work as carbon sources and buffers. Citrate
and EDTA frequently occur in minimal media to stabilize the divalent ions like
magnesium so they do not precipitate out of solution. Other chemicals are
added as colour indicators of re-dox states or relative acidity.
6.
Rich media are made from empirically defined mixtures of organic components
that provide C, N, P, S, and O, vitamins and trace elements for the bacteria as
well as buffer the growth media against extensive pH changes.
7.
Minimal media consist of pure chemically defined components that provide the
necessary materials for the cells to grow. Specific media are not equally
applicable or sufficient for all types of cells or even for all strains of the same
species.
8.
The Difco & BBL Manual , Manual of Microbiological Culture Media , Zimbro,
M.J. and D.A.Power, (eds.) (2003)and Manual of Methods for General
Bacteriology, Gerhardt, P. et al., (eds.) (1994) contain recipes for several
defined and undefined media that are suitable for the growth of prototrophic
Escherichia coli, Salmonella typhimurium and Bacillus subtilis. Auxotrophic
strains would require appropriate additional supplements.
a. When rich media are being prepared it is normal to add each sequentially
listed component to a stirring solution and ensure that it dissolves before
adding the next component. At this stage the solution contains
approximately 2/3 to 3/4 of the final volume of water. Once all the
ingredients are added and the acidity is adjusted with NaOH or HCl
additional water is added to bring the solution to the final volume.
b. Minimal media are also prepared by sequentially adding the components
listed in the recipe and ensuring that each component is fully dissolved
before adding the next component. However the preparation is a bit more
complex because divalent ions will frequently precipitate the phosphate
ions if they are in the same solution when they are autoclaved. In addition,
components such as Mg++, other divalent ions and tryptophan will react
with carbohydrates such as glucose and agarose to form dark hydrolysis
products that are inhibitory for some bacteria. These problems can be
reduced by including chelators such as citrate to bind the divalent ions.
However, the precipitation and reactivity problems can be avoided by
124
simply preparing separate stock solutions of the incompatible components
then combining the separate stock solutions after they are autoclaved.
This is the reason that a minimal medium such as M9-glycerol is prepared
by combining appropriate volumes of three independently autoclaved stock
solutions of the simple salts, the magnesium sulfate and the glycerol to
give a single solution with the specified concentration of each ingredient.
V. Buffers
1.
Buffers are zwitterions that stabilize the pH of the solution. They are used
extensively in in vitro systems to ensure that the pH stays within acceptable ranges
for growth, enzyme activity and chemical stability.
2.
The supplied zwitterion is usually effective within plus or minus 0.6 pH unit of the
pKa. This means that a range of different ions are needed to cover the natural range
of biological activity. If the pH of the solution is likely to drop during the incubation
then a zwitterion is chosen that has a pKa that is a bit below the starting pH of the
solution. If the pH is likely to rise during the incubation then a zwitterion is chosen
that has a pKa a bit above the starting pH of the solution.
3.
Traditional buffers include molecules such as:

Buffer Molecule
Citric acid
Relevant pKa
3.1 and 4.8 and
5.4
Acetic acid
4.8
Cacodylic acid
6.3
Bicarbonate
6.1 and 10.2
Phosphate
2.0 and 6.7 and

12.3
Tris (hydroxymethyl)aminomethane
8.1
Glycine
2.4 and 9.6
Boric acid
9.2
Potential Problems
Biological nutrient, chelator of
Ca++, Mg++
Biological nutrient, weak
chelator of Ca++, Mg++
Toxic, reacts with SH groups
Pecipitates divalent ions,
equilibrates with gas phase,
limited solubility
Nutrient, chelates Ca++ and
Mg++, inhibits some enzyme
reactions, toxic to animal
cells
Temperature dependent pKa,
interferes in some enzyme
reactions, reacts in some
chemical assays for proteins,
penetrates membranes
Nutrient, pKa at extremes
Toxic, complexes with vicinol
diols
125
4.
The ideal buffer should be

a.
b.
c.
d.
5.
6.
inexpensive, readily available and readily soluble in water

biologically inert, non-toxic and impermeable to cells
chemically and biologically stable so it will not break down or form precipitates
have a pKa that is independent of temperature or concentration
The chemist N.E. Good developed a series of organic biological buffers in the range
from 6.0 - 9.0. These buffers are more expensive than the traditional buffers but they
have minimal membrane penetration, minimal absorbance in the visible and UV
spectrum, minimal changes in pKa due to concentration, temperature or other
chemical interactions. Some of the common Good buffers include:
Buffer Molecule
Relevant pKa
Potential Problems
MES
6.2
PIPES
6.8
ACES
6.9
MOPS
7.2
HEPES
7.6
Can form radicals
Tricine
8.2
Binds metal ions
Can form radicals, limited

solubility
Binds metal ions
When preparing buffers it is important to consider the influence of the preparation on

the buffer and the needed characteristics of the buffer. For example:
a. The adjusted pH of bicarbonate buffers is changed by autoclaving.
b. The pH of concentrated or crystalline buffers can change when the solutions are
diluted.
c. The pH of most buffers can be adjusted by adding NaOH or HCl. Buffers used
for electrophoresis should only be adjusted with one of the two buffer ions.
d. Buffers without mineral ions such K+ or Na+ can be prepared by adjusting the pH
with tetramethyl ammonium hydroxide instead of KOH or NaOH.
e. Buffers such as acetic acid and bicarbonate can be driven off in the gas phase.
Other buffers such as tris and phosphate accumulate as a solution dries out.
Residual salts from NaOH and HCl will also accumulate.
7.
The names of buffers need to be used carefully. Tris, Tris base, Tris HCl, Trizma,
THAM, are all synonyms of variants of Tris hydroxymethyl amino methane. The
important consideration is whether it is the base or the base neutralized with HCl.
The neutralized base is called Tris HCl. Tricine is a completely different molecule.
8.
A major natural intracellular buffer is the amino acid glutamate.
126
VI. Ion Probe Measurement
A.
pH Probe
1.
pH is the unit for the minus log of the molar hydrogen ion concentration, but is
generally thought of as the degree of acidity. It is important since the hydrogen ion
concentration affects the ionization of other molecules and consequently influences
biological properties such as enzyme activity, ion availability and membrane stability.
[Acidity]pH = - log [H+-]M
where:
[Acidity]pH
[H+-]M
= acidity concentration in pH units

= molar hydrogen ion activity
2.
The three principle ways to determine pH include using the Henderson-Hasselbalch

equation, measuring specific colour changes and using pH meters.
3.
The Henderson-Hasselbalch equation shows that:

- log [H+-] = pH
pH = pKa + log [A-] [HA]
where:
pH
pKa
[A ]
[HA]
= minus log of the hydrogen activity

= minus log of dissociation constant of the acid
= concentration of the conjugate base
= concentration of undissociated acid
The equation can be useful but the calculations are tedious for routine use and are
difficult to apply to many biological solutions that are complex mixtures with poorly
defined pKa.
4.
Some weak acids such as nitrophenol, phenolphthalein and methyl red produce
coloured tautomeric structures when they dissociate. Since the degree of
dissociation depends on the pH these molecules will have different characteristic
colours at different pH and therefore the approximate pH of a solution can be
estimated by looking at the colour of the solution when these molecules are present.
Sometimes the molecules are dried onto paper strips that can be wetted by the test
solutions. In other cases, such as MacConkey media, the colour indicator is added
directly to the solution.
5.
The most convenient way to measure pH is a pH meter. This machine depends on

the observation that when two different solutions of ions are connected together by a
salt bridge there is an electrical potential between the chambers that can be
measured with a voltmeter. The strength of the electrical potential depends on the
ratio of the ion concentrations in the two solutions and is described by the Nernst
equation.
127
where:
E
R
T
F
[A]
[B]
RT
A
In
F
B
= electrical potential difference between the

electrodes
= molar gas constant
= temperature
= Faraday constant
= concentration of ions in one chamber
= concentration of ions in the second chamber
This equation shows that when you know the temperature and the ion concentration
of one solution you can measure the ion concentration in a second solution by simply
measuring the potential voltage and correlating that measured voltage to the voltage
produced by known concentrations. However, many solutions contain a complex
mixture of ions and the measured potential voltage is likely to reflect an overall
average of all of the ions instead of the hydrogen ions. This problem is gotten
around by the observation that some types of glass selectively bind different ions and
some types of glass will only bind hydrogen ions. If you make a closed tube of this
special glass and then fill up the tube with solution and immerse the end of that tube
in a more basic solution then all of the hydrogen ions bound to the outside of the
special tube will go into the basic solution. The vacated binding sites on the outside
of the tube will be filled by binding hydrogen ions from the inside wall of the tube.
This initiates a cascade that sequentially draws hydrogen ions from further inside the
wall until eventually the inside surface of the wall is depleted and draws hydrogen
ions from the solution inside the tube. Conversely, when the tube is put into an
acidic outside solution the bound hydrogen ions on the inside will leave the glass and
enter the inside solution. This effect means that the only ion changing concentration
inside the tube is the hydrogen ion and the concentration of that ion is dependent on
the hydrogen ion concentration of any solution that is placed outside the tube.
Therefore the hydrogen concentration of the outside solution can be selectively
measured by actually comparing the potential voltage due to hydrogen ions inside of
the tube and a standard solution of hydrogen ions.
Unfortunately, the hydrogen ions inside the special tube are difficult to measure
directly since the hydrogen half-cell has no potential voltage. Therefore the internal
hydrogen ion concentration is measured by coupling the concentration of hydrogen
ions to a second chemical reaction. More specifically, the internal solution contains
AgCl and a silver electrode in 0.l M HCl. The HCl is fully dissociated while the AgCl
is only weakly soluble. When the tube is in an alkaline solution the hydrogen ions
bind to the glass, and the Cl ions interact with silver ions generated by the electrode
to form insoluble silver chloride. Consequently, the decrease in internal hydrogen
concentration will result in a measurable decrease in internal chloride ion
concentration. Conversely, when the outside hydrogen ion concentration is high, the
inside hydrogen ions on the glass go into solution and are available to preferentially
interact with the chloride ions so that the chloride ions enter into solution and the
silver ions reassociate with the silver electrode. Therefore the hydrogen ion
concentration in the external solution directly affects the internal chloride ion
concentration and can be specifically measured by comparing the internal chloride
ion concentration to a standard solution containing saturating concentrations of
highly soluble KCl and AgCl.
128
The tube that contains the special glass is called a sample electrode. The other tube
containing the reference solution of KCl is called a reference electrode. However,
many samples are too small to immerse two large glass electrodes inside. Therefore
the two electrodes are frequently combined into a single double chambered glass
tube which holds the sample solution at the bottom in one chamber and the
reference solution in the separate upper chamber. This double chambered electrode
is called a combination electrode.
Since the pH meter is intended to measure hydrogen ion activity there is no need to
record the results in millivolts before converting to the pH units. Therefore most
instruments automatically convert the results and give direct readouts in pH units
instead of millivolt units.
B. Other Probes
The ammonium probe is like a pH probe surrounded with an extra, external solution-filled
chamber that is covered by a membrane which is selectively permeable to ammonia gas
but not to ammonium ion. When samples are made alkaline by adding NaOH, the
alkaline conditions force the natural equilibrium between ammonia and ammonium to
strongly favour ammonia. The ammonia can selectively diffuse across the membrane into
the chamber where the low pH of the fill solution favours the re-formation of the
ammonium ion. However, when the ammonia forms the ammonium there is a
proportional consumption of free protons in the fill solution and the net pH of the fill
solution rises. The increase in pH is measured by the pH probe inside the chamber and
observed as a proportional net change in the electrical potential (E). The important point
is that the ammonium probe is a pH probe that becomes selective for ammonium ions by
setting up conditions so that only the ammonia gas can alter the pH of the solution
surrounding the probe. Other ion selective probes are based on similar principles.
Other common probes include oxidation-reduction probes and oxygen probes.
VII. Nomenclature
A.
Strain Names
Common strains of bacteria such as Esherichia coli have been used in different
laboratories around the world for almost a century. During this time they have
undergone spontaneous and induced mutations and genetic manipulations. These
changes are important because they mean that the original isolates designated as
Esherichia coli strain B, Escherichia strain K-12 and Escherichia coli strain C are
potentially quite different than when they were first isolated and strains of K-12
manipulated by different researchers are related but potentially different populations.
To keep track of the properties of the stocks in different labs each lineage of
genetically manipulated bacteria are called strains and each strain is given an
identification code to keep track of the different properties. For example:
1. Some examples of the thousands of strains of Escherichia coli K-12
include: HB101, DH5, INV , VC10, HfrH, HfrC, O11', 1776 , LD5,
LD5456, Bug 6, MG1665, W3110 and EMG2. The widely used B23 strain
is a hybrid B strain with the phage receptors from the K-12 lineage.
129
2. There is no overall logic to the codes. Some reflect strain features, some
are the researcher's initials, some are functional properties, some are
named after places, some are named for historical events and some are
spoofs. Each one reflects a unique strain and related strains within a
laboratory will probably have related designations. However, similar codes
can refer to quite disparate organisms. For example, there is a Bacillus
subtilus L15, a Salmonella typhimurium LT2 and an Escherichia coli LD5.
The important point is that the strain designation is indicative of particular
lineages of bacteria and different strains might have different properties
that can affect experimental results.
3. Wild type isolates from the environment are usually designated with similar
identification codes in order to track them during the study. However,
sometimes the codes reflect a property used in the identification system.
For instance, some species are identified by using virus susceptibility or by
using antibodies. An example of the latter code in Escherichia coli
O157H7. This code means that the isolate type 157 "O" antigen and type
7 "H" antigen but two isolates with the same antigenic features could be
very different in the other genes.
B.
Genetic Nomenclature
The genotypic features of bacteria are identified by a convention of three - letter
mnemonics followed by an upper case letter followed by a number. The mnemonic
usually reflects a property of the function or pathway for the gene. The upper case
letter designates different genes with the same mnemonic and the number refers to
different mutations within the designated gene. For example: rpoA1 and rpoA2 are
different mutations in the -subunit protein of the RNA polymerase of Escherichia coli.
The rpoB1 is a mutation in the -subunit of the RNA polymerase while rpsL1 is a
mutation in the protein L of the large ribosomal subunit and pro is a gene in the
pathway for proline biosynthesis
By convention the three-letter mnemonic for a genotype should be written in lower
case letters and either italicized or underlined. The upper case letter and the Arabic
numeral are written in normal forms rather than italics. Since each genotypic
mnemonic usually refers to a genetic mutation the only qualifier is to indicate a wild
type gene. For example, dna refers to a mutated gene for DNA polymerase while
dna+ refers to a wildtype gene. There is no dna- designation because the qualifier is
not needed.
Similar conventions apply to phenotypic features except that the mnemonic is written
in normal form and the first letter is capitalized. Various modifiers that describe the
phenotypic features will typically qualify this phenotype. For example there could be
Rpo+, Rpo-, Rpots, Rpoamber, et cetera to describe wild type, mutated, temperature
sensitive and t-RNA suppressible phenotype for the RNA polymerase of an
Escherichia coli. The symbol means a deletion of the following gene, means that
the genes in the following brackets are fused, and are symbols for insertions of
genetic elements.
More complete descriptions of the nomenclature are described in the instructions to
authors for the UBC electronic Journal for Microbiology and Immunology (JeMI) and
130
the January issue of all recent journals published by the American Society for
Microbiology (journals such as the Journal of Bacteriology).
The physiological function of various mnemonics in the descriptions of different
Escherichia coli strains can be found in websites such as the Coli Genetic Stock
Center in Yale University at http://cgsc.biology.yale.edu/ . The functions can also be
located in published reviews such as the Escherichia coli genetic map in the journal
Microbiology and Molecular Biology Reviews (such as Berlyn, M. "Linkage map of
Escherichia coli K12" Microbiol. Molec. Biol. Rev. 62, 814-984 (1998)) or the book
Escherichia coli and Salmonella typhimurium by F. Neidhardt (1987). Additional
information is available at EcoCyc in the BioCyc website at http://ecocyc.org/
The strain libraries of mutated Pseudomonas aeruginosa available from the Robert
Hancocks lab are described in different references.
The University of Washington PAO1 mutant library is described in Proc. Natl. Acad.
Sci. U. S. A. 100:14339-14344.
The UBC (Hancock) PAO1 mutant library is described in Genome Res. 15:583-589.
The Harvard University PA14 mutant library is described in Proc. Natl. Acad. Sci.
U. S. A. 103:2833-2838.
VIII.
Protein Isolation
Many cellular functions are due to the activity of proteins. However, most proteins differ in
function from one another and in order to understand how particular individual types of
protein work they must be isolated from all the other types of proteins in a cell. Generally
such isolations sound simple but in practice they are complicated because even though
proteins differ in function there are thousands of cellular proteins and most of these
thousands of proteins are chemically similar. In addition, most proteins must first be
released from the cell in order to isolate them and many become inactive when released.
Consequently most purification schemes are trial and error processes which must
successively consider a means of releasing the proteins from the cell, a means of
stabilizing the activity and a means for separating one released protein from all the rest of
the released proteins.
A.
Protein Release from the Cell

1.
A few of the proteins produced by bacteria are secreted through the cell
membrane and released into the culture media. These proteins can easily be
separated from cell bound proteins by simply centrifuging the culture so that the
cells are pelleted and only the free, exocellular enzymes are left in the
supernatant. Examples include some cellulases, some proteases, some
lactamases (penicillinases) and some amylases.
2.
Some proteins are loosely contained in the periplasmic space and can be
differentially released by either:
131
3.
a.
Spheroplasting the cells so that the outer membrane is leaky. The

periplasmic proteins can then diffuse into the media and be separated from
the cell bound protein by centrifugation.
b.
Osmotically shocking the intact cells by washing them repeatedly in pure

distilled water, EDTA or a high concentration salt solution such as one molar
lithium chloride. All three methods decrease the stability of the outer
membrane and make it relatively leaky.
Internal cytoplasmic proteins are released by smashing the cell. There are a
large number of ways to cause them to break but they generally fall into either
enzymatic, chemical or physical breakage categories.
a.
The most common enzymatic methods involve lysozyme and penicillin

treatments. In the former method the outer membrane of the cell is usually
permeabilized by repeated cycles of freezing and treatment with EDTA then
the enzyme lysozyme is used to hydrolyse the bonds in the peptidoglycan
sacculus. Once the sacculus is weakened the internal osmotic pressure of
the cell will cause the cell membrane to swell and burst. However, if the
cells are treated in normal media the swelling is so gradual that the cells only
burst in a few places and then the resulting large membrane fragments
partially re-seal into vesicles and entrap a large portion of the cytoplasmic
material. Consequently the best breakage occurs when cells are osmotically
protected with sucrose during the lysozyme treatment so that they form
osmotically fragile spheroplasts. The spheroplasts are then burst by rapidly
adding extra buffer and diluting away the sucrose.
The method using penicillin treatment is analogous to the lysozyme method
except that it requires growing cells and relies on the endogenous cellular
autolytic enzymes to weaken the protective sacculus when the penicillin
inhibits the formation of new peptidoglycan bonds in the growing cells.
b.
The chemical methods for breaking cells include treating them with bases or
acids such as TCA. These methods cause effective breakage but frequently
inactivate and destroy the proteins.
c.
The physical methods rely on physical force to shear or crush the cell. Most
of these methods release a lot of heat and can denature the proteins if the
samples are inadequately cooled during the breakage. Examples of these
breakage methods include sonication, French press, Gaulin homogenizer,
Hughes press, bead mills and grinding. The sonication works by using an
ultrasonic generator to transmit high frequency sound waves into the sample
of cells. The sound waves cause rapid, but variable streaming currents in
the solution and any cells caught at the interface between opposing currents
can be yanked in both directions and torn apart. In addition, the pressure
changes caused by the oscillating sound waves result in microcavitation at
the cell surface and the decreased air pressure within these
microcavitations puckers the membrane surface and pulls it into small
pieces.
The French press and the Gaulin homogenizer both work by exposing the
cells to pressures of 10,000-to-20, 000 lbs per square inch and then
132
transferring the cells back to normal atmospheric air pressure. This switch
in pressure breaks the cells because the compressed gases in the solution
under high pressure equilibrate across the cell membrane and then these
gases expand faster than they equilibrate out across the membrane when
the pressure drops back to normal. These internal expanding gases create
enough force to explode the cell.
The Hughes press works by freezing a sample of cells and using high
pressure to squeeze the frozen sample through a narrow crack in a steel
plate. This squeezing crushes the ice and the resulting ice fragments shear
the cells as the mixture of the ice and cells moves through the crack.
Grinding is usually done in a simple mortar and pestle. It works by crushing
and shearing the cells between the moving surfaces of the mortar and the
pestle. However, most bacteria are so small that they fit into slight
depressions on the grinding surfaces and escape crushing. Consequently,
the grinding is usually done in the presence of small beads or ground
alumina particles that will be compressed around the cells, even when they
are in the depressions on the grinding surfaces.
Ball mills, bead mills, bead beaters, bead bashers and FastPrep machines
work mix the cells in a small container with a high proportion of violently
agitated steel balls or glass beads. The beads are then violently agitated by
shaking the container or swirling them with rapidly rotating blades so that the
beads bounce around. The bacteria are smashed open whenever they are
crushed between two beads or balls colliding together.
4.
B.
Membrane associated proteins can be solubilized by breaking the cells to release

cytoplasmic proteins and then treating the remaining membranes with
detergents, organic solvents such as butanol, or chaotropic salts such as
guanidinium chloride. All of these treatments reduce hydrophobic bonding and
increase the solubility of hydrophobic material in water. However, all the
treatments are not equally effective under all conditions and all detergents are
not equally effective since they are actually a broad class of chemicals which
differ in detergency, ionic charge and chemical composition. The major
detergents used in biology include the anionic detergents deoxycholate and
sodium dodecyl sulfate (SDS); the ionically neutral detergents such as Triton Xl00, Nonidet, Brij-35 and Tween; the cationic detergent
hexadecyltrimethylammonium bromide; and the zwitterionic detergent CHAPS.
Some detergents such as SDS denature the solubilized proteins and are
generally not useful for isolating active enzymes.
Stabilization of Released Enzyme Activity

Proteins released from a cell are usually in an unnatural environment and can lose
activity if the new environment has not been empirically modified to minimize damage to
the protein. Some of the important environmental factors include the presence of other
proteins, pH, oxidation, solvent polarity, solvent ionic strength, and temperature.
133
1.
Proteins in the solution affect the stability of the released enzyme in several different
ways:
a.
Proteins in solution interact ionically and hydrophobically with other proteins to

form aggregates that stabilize each other. The effectiveness of these aggregates
will change as different participating proteins get removed during the purification.
b.
At low concentrations, the soluble protein absorbs at the surface of the solution
and denatures. This is important because purification reduces the total protein in
the solution and increases the risk of damage for the protein being isolated.
Sometimes additional unrelated purified proteins such as bovine serum albumin
(BSA) are added to the partially isolated protein to increase the total protein
concentration in the solution and reduce the loss of the desired protein.
c.
Some of the released cellular proteins might be proteases that will hydrolyse the
desired proteins in the solution. Adding chelators such as EDTA, phenanthroline
or hydroxyquinoline to the buffers can minimize proteolysis due to
metalloproteases. Serine dependent proteases such as trypsin are similarly
inhibited by diisopropylphosphofluoridate (DIPF) or phenylmethylsulfonyl fluoride
(PMSF). However some other types of enzymes also require metal ions as
cofactors or have critical serine residues in the active site. Specific chemicals
that are added to the buffer to inhibit the proteases might inhibit these types of
enzymes. Therefore, many samples are protected from proteolysis by simply
keeping them chilled in an ice bath so that any protease activity is minimized.
2.
The pH of the reaction buffers affects the enzymatic activity of many enzymes.
Consequently, enzymes are usually suspended in buffers that maintain the samples at
the pH optima of the reaction. However, this pH optima for activity might not
correspond to the pH where the enzyme activity is most stable. In addition, the
buffers used to maintain the pH are chemicals and can have direct chemical effects
on the enzyme reactions. For example, Tris is a strong nucleophile. It can drive
reactions forward by substituting for the water in the nucleophilic attack that separates
the covalent enzyme-substrate intermediates that are present in many enzymatic
reactions. Similarly, phosphate is a product in many enzymatic reactions and
phosphate buffers frequently competitively inhibit those activities by increasing the
phosphate concentration and driving the reactions backward.
3.
Many enzymes become inactivated when reduced sulfhydryl groups in the protein
become oxidized and form disulfide bonds with other sulfhydryl groups in either the
same molecule or other molecules. Including reducing agents in the buffer solutions
minimizes this problem. Common reducing agents include mercaptoethanol, cysteine,
glutathione, thioglycolate, dithiethreitol (Cleland's reagent), and polyvinylpyrrolidone.
4.
Some enzymes such as alkaline phosphatase are most stable in high ionic solutions
and are kept in buffers containing high concentrations of salts such as KCl and
(NH4)2SO4.
5.
Many membrane associated enzymes and some cytoplasmic enzymes such as RNA
polymerase are more stable in buffers with less polarity than water. The reduced
polarity is achieved by including materials like glycerol or sucrose in the enzyme
solutions.
134
C. Isolation of the Released Enzyme Activity
Proteins all consist of amino acid polymers and consequently have many similar features
but they also differ from one another with regard to relative solubility, charge, size, function
and sequence. These differences are the basis for the techniques used to separate one
type of protein from another. However, a single technique usually separates different
proteins on the basis of only one property such as size or charge and each technique will
only separate the proteins that differ for that property. Consequently, a single purification
technique usually produces a mixture of proteins which are similar for the property selected
by that technique and several techniques which select for different protein properties must
be consecutively applied to the initial mixture of released proteins in order to purify the
desired protein.
1.
Separation based on solubility

a.
Salt effects
i.
At low concentrations, salts such as NaCl and KCl increase the solubility of
proteins by interacting ionically with the protein. This effect is called "saltingin."
ii.
Other salts interact ionically with the dipole moment of the water molecules of
the solution and effectively reduce the amount of water available to dissolve
other molecules such as proteins. Consequently, as the salt concentration
increases the proteins precipitate out of solution. Some salts do this
precipitation very effectively; some do it very poorly since the effect depends
on the way each salt interacts with the water molecules. However, in terms
of protein purification the salts ammonium sulphate and zinc chloride are
more useful than many other salts since at any given concentration they
selectively precipitate some proteins but not others. This selective
precipitation allows you to partially purify some proteins by simply adding
enough ammonium sulphate to precipitate all the less soluble proteins, then
increasing the salt sufficiently to precipitate the desired protein but not other
proteins that are even more soluble. However, the technique involves a lot of
empirical variables. Some proteins are permanently denatured if the salt is
added too fast. Others are denatured if the salt is added too slowly. Some
proteins will precipitate over broad ranges of salt concentration and even the
good precipitates can entrap soluble protein. In addition, the available water
depends on the quantity and nature of other competing proteins in the
solution. Consequently, the salt precipitation method is good for removing
protein from large volumes of sample and it works relatively quickly but it
usually needs to be coupled to other purification methods for a complete
purification.
b.
Solvent effects
i.
Organic solvents such as ethanol, acetone and methanol dissolve in water

and reduce the amount of water available for dissolving proteins in the
solution. In addition, the solvents reduce the dielectric constant of the
water and consequently increase the internal charge interactions of the
proteins. Both effects cause many proteins to precipitate.
135
c.
2.
ii.
The extent of the precipitation depends on the ionic strength of the solution,
the solvent and the temperature. It works best with acetone in low ionic
strength solutions at cold temperatures such as minus twenty degrees
Celsius.
iii.
Some hydrophobic proteins dissolve better in organic solvents than in water

and will not precipitate.
iv.
Many proteins are irreversibly denatured by organic solvent precipitation,

especially if the solvent concentration becomes too high.
v.
Polyethylene glycol is used to selectively precipitate some proteins and

protein complexes such as bacteriophage.
Other effects on solubility

i.
Polycations such as protamine sulfate and streptomycin sulfate bind to

negatively charged proteins and causes them to precipitate.
ii.
Some proteins are relatively resistant to denaturation by alterations in pH or

temperature and can be separated from proteins that denature and
precipitate under equivalent pH or temperature conditions.
Separation based on charge

a.
Ion exchange chromatography

This technique is based on the observation that proteins are ionically charged
(most have a net negative charge at pH 7) and will interact ionically with any
material in the solution which has an opposite charge. The strength of this
charge interaction depends on the net charge of the protein and consequently
varies from one protein to another. In addition:
i.
The proteins with stronger charge affinity will bind longer than ones with weak
affinity and compete for the available binding sites.
ii.
The ionic interaction can be competed with the salt NaCl. The proteins with
stronger affinities for binding will take more NaCl to be competed.
iii.
Protein interaction with the binding sites can be altered by changing the pH
since increasing the pH makes the proteins more anionic (negatively
charged) and decreasing the pH makes the proteins more cationic (positively
charged).
These observations mean that if you take a charged molecule and chemically
fasten that molecule to small, chemically inert, insoluble particles, then proteins
that can bind to that charged molecule will stick to the insoluble particles and
become insoluble. These bound molecules remain bound to the particle until
they are released by competition from the appropriate concentration of NaCl or
by changing the pH. This binding is important because the strength of the
surface charge on different proteins can be different. If you bind a complex
mixture of proteins to charged insoluble particles, the proteins with the weakest
136
charges will be released at a lower concentration of NaCl than the strongly bound
proteins and successive washes by buffers with successively increased
concentration of NaCl will contain different types of protein.
In practice, the insoluble particles are usually stuck into an open-ended column
designed to retain the charged particles but let buffer and soluble molecules
wash through. The protein solution is then poured into the column and the
proteins that bind will get trapped in the column while the rest will wash through.
The bound proteins are then released and eluted by pouring successive washes
of increasing NaCl concentration through the column and collecting each wash or
fraction of a wash into a separate tube such that differently charged proteins
become separated and end up in different tubes. When the NaCl washes consist
of separate and discrete concentrations (such as 0.l M and then 0.2 M NaCl) the
elution is called a step elution. If the NaCl concentration is arranged to increase
as a continuous gradient the elution is called a gradient elution.
The inert insoluble particles that hold the charged molecules in the column are
usually made from cellulose, agarose, polystyrene, acrylamide, dextran, or glass.
These materials are usually ground up or formed into small beads such that there
are spaces between the packed beads that allow the wash buffers to readily flow
through the column. The common charged molecules fastened to the particles in
the column include:
Cationic groups (+)
:- Aminoethyl (AE)
:- Diethylaminoethyl (DEAE)
Anionic groups (-)
:- Carboxymethyl (CM)
:- Phospho
:- Sulphopropyl (SP)
Ideally the separation of different proteins by the ion exchange column only
depends on interaction with the insoluble charged groups. In practice some
proteins will also interact directly with the insoluble particles. Consequently it is
important to know the type of particle as well as the charged group. Therefore
ion exchange systems are usually described by naming both parts (ie: CMcellulose, DEAE-cellulose, DEAE-Sephadex, CM-Sephacryl). In addition, the
flow rates of the buffer through the column are frequently inconsistent or slow in
systems using open columns and some high performance ion exchange systems
have been designed to seal the columns and precisely pump the buffer past the
particles. One of these high performance systems uses low pumping pressure.
It is called a Fast Protein Liquid Chromatography (FPLC) system. Other systems
use high pumping pressures and are called High Performance Liquid
Chromatography (HPLC) systems. Both the HPLC and FPLC use the basic
principles of ion exchange chromatography, they just have better control of the
movement of the buffer washes so that the ion exchange is more rapid, more
efficient and more precise than in an open column.
b.
Electrophoresis
The general technique for separating charged molecules by electrophoresis is
described in the earlier part of this appendix. The described technique indicates
that molecules are separated according to relative size and charge density. In
137
addition, another type of electrophoresis is done by using special buffers called
ampholines to create stable, immobilized pH gradients from one end of the gel to
the other. When mixtures of protein are electrophoresed in these gels the
different proteins will only migrate until they reach the places in the gel where the
pH is equivalent to the respective isoelectric constants (pI) of the individual
proteins. At that place, each protein has no net charge and will quit moving.
Consequently proteins with different isoelectric points will form bands at different
places in the gel. This technique is called isoelectric focusing. It is quite
expensive but it does give good resolution.
In some instances the resolution of the standard electrophoresis and the
isoelectric focusing are sequentially combined. This is combined technique gives
a two-dimensional gel or 2-D gel. The process is started by electrophoresing the
sample in a narrow isoelectric focusing gel. The narrow isoelectric focussing gel
is then placed along the top of a standard acrylamide slab gel and
electrophoresed (so the proteins migrate at a right angle to the direction they
moved in the original gel. The proteins that had similar pI and banded together in
the first gel will then migrate into the second gel and separate according to size
and charge density.
3.
Separation based on size

a.
Acrylamide gel electrophoresis separates protein molecules with respect to size if

the proteins have first been denatured with sodium dodecyl sulfate so that they
are coated with the detergent and have a constant charge density. However, this
is a poor technique for isolating active enzymes since the denaturation is
frequently irreversible.
b.
Most proteins are so small that centrifugation is not a practical technique for
separation but it is used for isolating large protein complexes such as ribosomes.
c.
Proteins can also be separated according to size by forcing them through

membrane filters that have holes that limit or cut-off the size of the molecules that
pass through the filter. For example, a Millipore Micro-YM 50 filter selects for
molecules with a molecular weight of less than 50,000 daltons, a Millipore MicroYM 10 filter selects for molecular weights under 10,000 daltons and a Millipore
Micro-YM 3 filter selects for molecular weights under 3,000 daltons. Since the
pore sizes are quite small, most solutions do not pass readily through these
filters. This problem is gotten around by putting the filters in special holders and
using either high pressure or centrifugation to force the solutions through the
membrane pores. A bigger problem is that the filters are only sold in a limited
number of pore sizes. Consequently the separation of different sizes of protein is
limited and the filter systems are more useful for concentrating solutions of
proteins rather than for separating different proteins. The filters are also useful
for removing salt from protein solutions since salt is so much smaller than most
proteins. De-salting can also be done by dialysis.
d.
The best general system for separating enzymes according to size is gel
permeation chromatography. This process is based on the observation that
when a mixture of different molecules is poured through a column filled with
appropriately sized porous beads, the smaller molecules will take longer to elute
from the column than the larger molecules. Since the different sizes elute at
138
different times, each size can be collected into separate test tubes by periodically
changing the collection tubes during the elution. This difference in elution time
occurs because the buffer flowing through the column passes around the beads
much easier than it flows through the beads and there are many more small
holes than large holes in the porous beads. This difference means that the large
molecules tend to be excluded from the beads and stay in the main current
flowing through the column, while the smaller molecules frequently diffuse into
the abundant small holes in the beads and are out of the current until they diffuse
back out.
The system works over a wide range of sizes but if the holes in the beads are too
big, the smaller molecules tend to diffuse into the beads too frequently and this
slowly eluting material is poorly resolved because it becomes spread over very
large volumes. Consequently the beads are normally chosen to have holes
which are just big enough to include the size of the desired molecules. In this
way the bigger molecules are excluded from the beads and flow out ahead of the
desired proteins. The desired proteins and similar sized proteins in the
remaining mixture diffuse into the beads enough to get resolved by slight
differences in size. The much smaller molecules still diffuse too much for good
resolution but this problem is not important for that separation since these
molecules will elute long after the desired molecules elute from the column.
The beads are usually made from dextran, agarose, or acrylamide and are
respectively sold as Sephadex, Sepharose or Bio-GelA, and BioGelP.
4.
Other types of column chromatography

a.
Hydrophobic chromatography is based on the observation that proteins in high

salt concentration buffers bind to beads coated with hydrophobic material such
as phenol and can be eluted by decreasing the salt concentration in the buffer.
Different proteins need different minimum salt concentrations to bind and will
elute at different salt concentrations. The process can be done with fractions
from ammonium sulphate fractionation and consequently eliminates the dialysis
step normally required before putting these fractions onto other columns like
DEAE-cellulose.
b.
Reverse phase chromatography is also based on interaction with beads coated

with hydrophobic material but relies on the observation that tyrosine and
phenylalanine groups interact hydrophobically and that the strength of the
binding is proportional to the number of these particular amino acids exposed on
the surface of the protein. The bound material is then released by successive
washes with different concentrations of propanol at pH 2.0. This elution is fairly
severe and does denature many proteins.
c.
Lectin chromatography relies on the observation that special proteins called

lectins will interact reversibly with specific sugar residues. If these lectins are
immobilized on beads in a column they will bind and retain any glycoproteins
which contain the appropriate sugar residue and let all other proteins wash
through. The bound glycoprotein can be liberated by increasing the salt
concentration in the buffer or competing for the binding site by including high
concentrations of the sugar recognized by the lectin. Lectins can be isolated by
using columns that contain the appropriate immobilized sugar residues. Some
139
common lectins include conconavalin A, lentil lectin, soybean lectin, Helix poma
lectin and wheatgerm lectin. The first two recognize glucose and mannose
sugars, the soybean lectin recognizes galactosamine, the Helix poma lectin
recognizes N-acetygalactosamine and the wheat germ lectin recognizes Nacetylglucosamine.
d.
Immunoglobulins against specific protein sequences can be immobilized on

beads and used to specifically bind and isolate the proteins with that sequence.
The bound proteins can then be eluted from the beads by increasing the salt
concentration or changing the pH of the buffer wash. Specific antibodies can
also be used to directly precipitate desired proteins so that they form large
complexes that can be isolated by centrifugation.
D. Protein Measurement
There are eight common assays and many variants for measuring protein. Each
assay has significant advantages and disadvantages that make it more suitable or
less suitable for any given protocol. The choice of any particular assay should
consider the purpose of the measurement, the influence of potential interfering
chemicals in the sample, the sensitivity, and the specificity. It should also consider
technical factors such as the skill needed to do the assay, the number of samples, the
cost per sample, the availability of equipment and the speed.
The major common assays include:
1. Bradford Assay (Anal. Biochem. 72,248(1976)). It is also called the Coomassie
blue dye-binding assay or the Coomassie blue protein assay. It depends on
measuring a change in absorbance that occurs when Coomassie brilliant blue
G250 interacts with protein. It measures down to 10 ug/ml protein and takes
about 10 to 15 minutes to perform. However it is affected by pH of the samples,
detergents such as Triton or SDS. The response depends on the composition of
the protein so the same concentration of different proteins with different
compositions could give different values. Some proteins do not bind Coomassie
blue and will not react in the assay.
2. Lowry Assay (J. Biol. Chem. 193, 265 (1951)). It depends on the reduction of
Folin's reagent by copper ions chelated to peptide bonds and tyrosine and
tryptophan. It measures down to 10 ug/ml and takes about an hour to perform. It
also reacts with phenol, ammonium, mercaptoethanol, amino acids, Tris and
guanine. The response varies with the aromatic amino acid composition of the
measured protein.
3. Biuret Assay (J. Biol. Chem. 177, 751 (1953)). It depends on the colour formed
when copper ions bind to peptide bonds. It is relatively insensitive and only
measures down to 1mg/ml protein but the reaction occurs within a few minutes so
it is very fast. It also reacts with Tris and ammonium so these types of molecules
interfere. However, it gives about the same response with all proteins and is
insensitive to variations in the amino acid composition of the protein.
4. Bicinchoninic Acid Assay (Anal. Biochem. 150, 75 (1985)). It is a chromogenbased assay analogous to the Lowry assay but substitutes bicinchoninic acid for
Folin's reagent. It is sensitive to 0.2 ug/ml and takes about an hour to perform.
140
The incubation temperature can control the extent of colour development so the
assay can be made more sensitive or less sensitive by changing temperatures. It
also has interference from ammonium and EDTA. Detergents do not affect it so it
is good for working with membrane proteins.
5. Fluorescamine Assay (Arch. Biochem. Biophys 155, 213 (1973)). It depends on a
fluorescent molecule binding to amines in the protein. It is very sensitive and
measures down to 5 ng/ml in a short time. It has interference by amino acids,
peptides and other primary amines. The reactivity will vary with the amount of
basic amino acids in the protein since each basic amino acid will increase the
number of free amines. It also requires a fluorimeter to measure the fluorescence.
6. 280 or 280/230 Absorbance Assays. Depends on the absorbance of tyrosine and
tryptophan at the wavelengths 280 and 230 nm of light. It is sensitive to 10 ug/ml
and is very rapid since it no reactions are involved. It requires relatively pure
samples since many other molecules such as nucleic acids absorb light in this
range of wavelengths.
7. 210 or 190 Absorbance Assays. Depends on the absorbance of peptide bonds at
the wavelengths 210 and 190 nm of light. It is sensitive to 1 ug/ml and is very
rapid since it no reactions are involved. It requires relatively pure samples since
many other molecules that absorb light in this range of wavelengths. It also
requires spectrophotometers and cuvettes that are not available in many
laboratories.
8. Kjeldahl Nitrogen Assay. Depends on trapping and titrating the ammonium
released when the sample is hydrolyzed. It is sensitive down to 10 ug/ml but it
takes about a day and has interference by ammonium, bases, and other
molecules with primary amines. It is frequently used for measuring protein in
complex solid samples.
IX. Radioactivity
A. General Information
1.
There are two common systems for describing amounts of radioactivity.

Current Canadian publications and regulations use the International Systems
of Units (SI) based on the bequerel unit but all the old literature, some of the
new literature and many of the American chemical supply centers for
radioisotopes use a non-SI system based on the curie. You should be familiar
with both systems.
1 becquerel (Bq) = 1 nuclear disintegration per second = 60 DPM.
10
12
1 curie (Ci) = 3.7 x 10 becquerels = 2.2 x 10 DPM.
2.
Both types of units can be qualified with prefixes to eliminate large exponents
in the designations. For example a radioactive sample of 106 disintegrations
per second (DPS) = 1 megabecquerel (MBq) = 27 microcuries (Ci). For a
more complete listing of the correct prefixes, refer to the Table in the units and
prefixes section of Appendix C - Calculations and Graphs.
141
3.
The units generally show the degree of radioactivity present and not the
amount of the molecule. This means that equivalent amounts of radioactivity
of two isotopes with different half-lifes will not represent the same total amount
of molecules. The molecules containing the isotope with the shorter half-life
will be less common because more short - lived isotopes will disintegrate in the
time taken to disintegrate each long - lived isotopes.
4.
Specific activity of an isotope represents the average ratio of the radioactivity

to the total amount of the type of molecule that was labeled. For example, the
specific activity of radioactive glucose would represent the average
radioactivity per total amount of glucose present. This proportion is usually
expressed as millicuries per millimole of the labelled type of molecule.
Sometimes it is expressed per milligram of labelled molecule. The important
thing to notice is that the measurement reflects the average radioactivity for
that molecule and can readily be changed by adding unlabeled molecules.
These unlabelled molecules are called carriers.
Specific activity can also be chemically changed by altering the ratio of
radioactive to non-radioactive isotopes within a single molecule. For example,
a glucose molecule labelled in one of the carbon positions would be less
radioactive than a glucose molecule labelled in all six of the carbon positions.
B. Nomenclature of Radioactive Molecules

The most common nomenclature is that adopted by the American Chemical Society.
The symbol for the isotope is placed in square brackets directly attached to the front
of the chemical name or the part of a compounded name that represents the actual
labeled portion. Standardized Arabic numeral or Greek letters preceding the symbol
of the isotope are used to indicate the position of the isotope within the molecule. If
the symbol is preceded by a capital "U", it indicates that the molecule is statistically
uniformly labeled at all the possible places. A capital "G" indicates that on average
all sites are labeled but they are not uniformly labeled. Some examples are:
[U-14C] Adenosine 5'-triphosphate where all the carbon positions are labelled.
[adenine-U-14C] Adenosine 5'-triphosphate where all the carbon positions in
the adenine but not the ribose are labelled.
[2-3H] Adenosine 5'-triphosphate where the "2" position on the adenine is
tritiated.
Adenosine 5'-[-32P] triphosphate where the phosphate closest to the base is
labelled.
Adenosine 5'-[-32P] triphosphate where the phosphate furthest from the base
is labelled.
C. Properties of Some Isotopes
There are a large number of radioactive isotopes but only a few such as 3H, 14C, 32P,
35S, and 131I are normally used in biological experiments. These isotopes all have a
142
different average instability and differences in the characteristic observed half-lives
and the maximum specific activities. The beta emitting isotopes such as 3H, 14C, 32P
and can also be distinguished from one another since each type produces beta
particles () with different and characteristic average kinetic energies during a nuclear
disintegration. For example, 3H produces particles with a low average energy while
32P produces particles with a high average energy. This distinction however is
complicated by the fact that the energy from any single disintegration is conserved by
being inconsistently split between a -particle and an anti-neutrino. This means that
the actual amount of energy in any one -particle can range from zero to the
maximum available for that type of isotope and even though the average energy of
the -particles from 32P is much higher than those from 3H or 14C, the actual energy
of a given particle from any of the different isotopes could be the same.
D. Measuring Radioactivity
There are a number of different methods for detecting radioactivity. Most of the
practical methods use the energy released in the radioactive nuclear disintegrations
to cause either: the ionization of gases which can be measured as electric impulses;
the oxidation of photographic film which can be detected as blackening of the
emulsion; or the production of measurable pulses of light from specific light-sensitive
solvents. The first method is used in Geiger counters; the second is used in
autoradiography. The last method is called scintillation counting.
1.
Scintillation counting
In scintillation counting the radioisotope is submerged in a vial containing a
scintillation fluid composed of a solvent such as toluene and one or more light
reactive chemicals called fluors. When a -particle collides with a solvent
molecule, the solvent molecule absorbs some of the energy and is temporarily
activated. The activated molecules then return to normal by emitting the
acquired energy as faint, short wavelength light. This light is difficult to detect in
the scintillation counters. But it can temporarily activate the primary fluors that in
turn return to normal by emitting light at a longer more usable wavelength. In
some cases, secondary fluors are included in the scintillation fluid to further
amplify and shift the wavelength of light from the primary fluors. This means that
each disintegration can be detected as a flash of light, however, the signals aren't
all the same since high - energy -particles can stimulate more solvent molecules
and produce more photons of light than low - energy -particles. This difference
can be detected since the scintillation counter measures the amount and duration
of light produced and converts it to a proportionally amplified electronic signal.
This means that the high-energy particles that produce more light produce a
higher voltage electronic signal. These electronic signals are then analyzed with
voltage discriminators that have preset upper and lower limits. Any signal
outside the range is ignored and any signal that falls within the chosen range is
recorded as a count. These counts can be recorded for a variety of times but are
usually expressed as counts per minute (CPM). The observed number of counts
will obviously be dependent on the chosen range of the voltage discriminators
and a range is usually chosen that covers the range of the energy from the
isotope being examined.
143
Unfortunately, the scintillation counter is not 100% efficient. The electronics are
not sufficiently sensitive to measure all the disintegrations and the entire process
is susceptible to quenching. Quenching is the general name for anything that
interferes with the efficiency of the detection of disintegration. It commonly
includes:
a.
Chemical quenching which is caused when chemicals in the sample that

do not fluoresce consume the -particle energy. It also includes chemicals
in the sample that interact directly with the solvent and prevent it from
scintillating.
b.
Colour quenching occurs when coloured molecules in the sample absorb

the light produced by the solvent before the machine can detect the light.
c.
Water quenching occurs because many samples are hydrophilic and are
not readily miscible with the hydrophobic solvent in the scintillation fluid.
This means that the hydrophilic samples will preferentially tend to dissolve
into any residual water that is present and only the high energy -particles
will have enough energy to pass through the water layer and interact with
the solvent. This type of quenching can be reduced by thoroughly
dehydrating the samples or by adding detergents to the scintillation fluid to
promote mixing of the hydrophobic solvents and the samples.
2. Quenching compensation
The problem with quenching is that it reduces counting efficiency and makes it
harder to relate the observed counts per minute detected by the scintillation counter
to the actual number of radioactive disintegrations per minute (DPM) that have
occurred. There are four normal ways to get around this problem. The internal
standard ratio method works by counting the sample and then adding an additional
known amount of radioactivity and recounting the sample. The effect of quenching is
then calculated by recognizing that the difference between the two counts should
correspond to the added amount of radioactivity and that the ratio of the actual
observed increase in counts to the actual added radioactivity represents the counting
efficiency of the sample for both sets of counts. The external standard ratio method
is similar except that a known amount of an isotope that produces high-energy
electrons called -particles is placed beside the sample vial after the first count.
Then when the sample is recounted the external high energy -particles travel
across the vial and increase the counts in proportion to the counting efficiency in that
vial. The third common method for quench compensation is called the channel-ratiomethod.
The channel-ratio-method relies on the observation that some isotopes such as 14C
emit -particles when the neutron decays into a proton and that different -particles
can have different energy levels (ie. When 14C decays, a neutron in the isotope
looses an anti-neutrino and an electron to become a proton. These changes convert
unstable 14C to stable 14N but each conversion is not equal because the energy
conserved by the electron in one decay can be different than in another decay). The
pattern observed when you plot the observed energy for many different -particles
against the number of times the -particles with that energy are observed is called
the -spectrum. Quenching lowers the energy pattern for the observed -spectrum.
For example, the solid line in the following graph shows the -spectrum of
144
unquenched 14C, the dashed line shown the -spectrum of a moderately quenched
14
C and the dotted line is a highly quenched sample.
The important thing to notice in the preceding figure are that the quenched samples
show relatively fewer particles with higher energy. If you set the detectors of the
scintillation counter so that one detector detects particles with energy between
something like 0-to-0.035 MeV and another detects particles with energy between 0
and 0.155 MeV the ratio of the counts for the two detectors will be different for the
quenched and unquenched samples. The ratio changes because there is a unique
proportion of counts in each channel for each quenched sample. Since the ratios are
unique, any known ratio can be converted to counting efficiency by using empirical
standard calibration curves.
The fourth method is the H-number analysis. It combines properties of both the
channel ratio method and the external standard method. It works by placing a
radioactive cesium ball adjacent to the outside of the scintillation vial containing the
sample. The cesium produces gamma rays that cross into the vial and collide with
the toluene molecules in the scintillation fluid. These collisions cause the toluene
molecules to eject Compton electrons. Populations of these Compton electrons
behave like -particles and have a characteristic -energy spectrum which shrinks in
quenched samples and produces reproducible -spectrum patterns for any given
degree of quenching. By using a large number of scintillation detectors which are set
to detect different energy levels it is possible to determine the inflection point of the
curve in the Compton electron -spectrum in any given sample. The difference
between the observed inflection point in the -spectrum and the same inflection point
in the spectrum of an unquenched sample is proportional to the degree of quenching.
By comparing this H-number to built-in standards in the machine memory, it is
possible to directly express the value as counting efficiency and some machines
directly convert the observed CPM on the printout to DPM. This conversion is easy
to calculate since:
calculated DPM = CPM counting efficiency (expressed in
decimal form)
145
However, the scintillation counters also register a low level of spurious counts that
were not due to your sample and these background counts must be subtracted from
the calculated DPM to get the real DPM. Since the background counts are usually
low and random the actual background could vary from between zero-to-one
hundred and fifty for repeat counts of the same sample. The background is also
different for different machines but most machines in use over a normal counting
range have an average background of about 80 DPM. Some assays include blank
samples; these will include the background values and so will automatically
compensate for the background when you subtract the blank values from the test
values.
E. Incorporation Properties
1.
Assays based on radioactivity measure the presence of the radioisotope

rather than the molecule that contains the isotope. This distinction is
important because supplied radioactivity at lower specific activity will give a
slower rate of uptake and incorporation of the isotope compared to a test done
with the same amount of radioactivity provided with higher specific activity.
2.
The specific activity is the ratio of total radioactivity compared to the total
amount of that type of molecule (labeled and unlabeled). It is usually specified
in becquerels or millicuries per mmole of molecule.
3.
If the specific activity is too high it might not be taken up or used by the cells if
the chemical presence of the molecule is well below the Km for the transport
of the molecule or the Km for the enzymes that will use the molecule. To
ensure sufficient chemical quantities of a molecule, radiolabeled molecules
are frequently diluted with non-radiolabeled molecules of the same type.
These non-radiolabeled molecules are called carriers. If too much carrier is
added the specific activity will be too low and the radiolabeled molecules will
have little chance of being used compared to the non-radiolabeled molecules
so the incorporation will be very low.
4.
Radiolabeled molecules are usually provided as either

a. Continuous label in which the radioactive molecules are provided at the
beginning of the test. Samples are then removed at assorted times after
the start of labeling to follow the radioactivity as it incorporates throughout
the test. This approach allows a large amount of radioactivity to
accumulate and is easy to compare because the internal specific activity of
the isotope is stable. However, it has the disadvantage that the
accumulated material might represent different classes of molecule than
the initial labeled molecules. For example the majority of the incorporation
is in mRNA when RNA is measured for a short period of time because
most of the transcription at any one time is that class of RNA. However,
since mRNA is unstable, the majority of the radioactivity will accumulate in
the rRNA if the labeling is prolonged for more than fifteen or twenty
minutes.
b. Pulse labeling where each test sample is removed and then individually
treated with the label. This label usually is provided for a short period of
time compared to the metabolic changes being monitored. It allows good
146
observations of unstable processes and initial patterns but it is seldom a
steady state condition and minor differences in the timing of the
measurements or the amount of supplied label can significantly alter the
apparent rates.
c. Pulse chase labeling where the label is provided at the beginning of the
test, allowed to accumulate for a short period of time then diluted with a
large amount of unlabeled carrier molecule to lower the specific activity.
This type of labeling combines the properties of the preceding methods. It
is good for monitoring the transformation of molecules from one form to
another.
5.
Alternative elemental methods of labeling molecules include the use of stable,

heavy labels such as 13C or 15N. These types of isotope are cheaper than
radioactive isotopes but they are less convenient to measure and monitor
since they are usually measured by using NMR or mass spectrometry.
6.
Molecular methods of labeling molecules include antibody affinity, lectin

affinity, avidin binding, polyhistidine affinity and two hybrid systems. These
methods are useful for following the production of specific proteins and
carbohydrates rather than intermediary metabolism. The molecular changes
done to allow avidin binding, polyhistidine affinity and two hybrid systems
might alter the normal molecular functions of the proteins involved.
IX. Spectrophotometry
A.
General Information
1.
Spectrophotometers measure sample concentrations by comparing the

intensity of light that passes through a solvent in the presence and absence of
dissolved or suspended sample. If the sample contains molecules which
absorb the incident light or particles which scatter the incident light then less
light passes straight through the sample than the empty solvent and this
difference shows up as a decrease in the ratio of directly transmitted light.
More particularly, as the concentration of interacting material in the solution
increases the ratio of transmitted light intensity decreases such that:
where:
2.
c
T
I
Io
1
when I Io = T
T
= concentration of sample
= transmittance, in decimal fractions
= intensity of light after passing through the sample
= intensity of light in the absence of sample
The preceding formula shows that the concentration of material in a solution

can be measured in transmittance units simply by shining a light through the
sample. However, transmittance units are not very convenient to use since
they are not linear. Fortunately, transmittance results follow an exponential
decrease with increasing sample concentration and can be transformed into a
positive linear relationship by taking the minus logarithm of the measured
147
results. These transformed units are called absorbance units and since they
are more convenient than transmittance for comparing concentration
measurements most contemporary spectrophotometers are designed to
mechanically or electronically perform the transformation and automatically
express the results in an absorbance scale.
3.
In actual practice the transformed results are only linear at relatively low
concentrations (which range from 0 to a maximum of between 0.6 and 4
absorbance units depending on the quality of the spectrophotometer).
However, as long as the samples are diluted sufficiently to fall in the linear
range of the transformation when taking readings then:
A logT
c
k
c k A k logT k 2 log%T
where:
c
k
= concentration of the sample in standard units.

= an empirical constant that depends on how the
solutions of material interact with the light being used

x
= multiplication symbol, times
A
= concentration of the sample in absorbance units
%T = transmittance expressed as a percent instead of a
decimal
This formula showing a direct relationship between normal concentration
measurements and absorbance is commonly called Beer's Law. It is adequate
to describe most absorbance measurements. However, other researchers
have shown that the actual loss of light transmittance depends on the distance
through the sample as well as the sample concentration. Most
spectrophotometers are consequently designed to hold standardized cuvettes
so that the distance that the light passes through the sample remains constant
and does not interfere with the concentration measurements. However, some
measurements can only be done on samples with odd dimensions and faint
samples can sometimes be measured by using cuvettes with longer light paths.
These special light path modifications are allowed by the more complete
description of absorbance measurements provided by the combined BeerLambert Law -:
logT a l c A
where:
T = transmittance as a decimal fraction

a = absorptivity coefficient, a constant characteristic of the
molecule being measured and the measuring condition.
x
l
c
= multiplication symbol
= distance the light passes through the sample
= sample concentration in common concentration units
148
A = sample concentration in absorbance
4.
The absorptivity coefficient in the Beer-Lambert Law is frequently called an

extinction coefficient. The actual units will vary with the nature of the material
measured and the type of units that the results will be expressed in. However,
the most common coefficient is the molar extinction coefficient. This
coefficient represents the absorbance of a one molar solution of a given
chemical measured in a one-centimetre long path length of light at the
specified wavelength of the measurement. It is used to convert absorbance
units to molar units.
5.
In many instances such as measurements of results in chemical assay the

specific absorptivity coefficient is unknown and might vary between repetitions
of the assay. In those cases, conversion between concentration
measurements in mg/ml or molar and absorbance is determined by including
samples of known concentration in the assay so that an empirical absorptivity
coefficient can be determined. Alternatively, the unknown concentrations can
be calculated as long as the absorbance measurements are done in the range
of the spectrophotometer where Beer's Law is obeyed by the measured
solution. In this range the quotient of the absorbance of a known sample
divided by the known concentration of that sample must equal the quotient of
the measured absorbance of an unknown sample divided by the concentration
of that unknown sample.
6.
Loss of transmittance occurs when molecules in solution absorb the incident

light or when particles in solution scatter the incident light. In both cases the
measured results are inversely and exponentially related to the concentration
of material in the samples. This means that both types of results can be
transformed by the same minus log formula to give a linear correlation
between the transformed measurements and concentration. Therefore both
kinds of material can be directly measured on the absorbance scale of the
spectrophotometer despite the fundamental differences in the way the light is
lost. However, the amount of light absorbed by molecules is much more
dependent on the choice of wavelength of light used for the measurement
than the amount of light scattered by particles. For example, DNA absorbs
light of a wavelength of 260 nm but has no effect on light with a wavelength of
400 nm or higher. In contrast, when light is scattered by particles, all visible
wavelengths will cause some scatter and even though shorter wavelengths do
scatter better than longer wavelengths there are no abrupt changes in
effectiveness such as those that characterize light absorbance by molecules.
These differences mean that it is important to recognize the that light interacts
differently with different samples and even though both types of interaction are
measured on an absorbance scale of the spectrophotometer only the solutions
of molecules are actually absorbing and referred to as absorbance. The
measured turbidity caused by particles in solution should be referred to as
optical density or O.D. Obviously, the units of absorbance and O.D. are
similar and some people do use them interchangeably but the concepts
involved in the measurements are clearer if you recognize the difference in the
loss of the transmitted light and keep that difference in mind by using the
different units appropriately.
149
7.
Strong absorbance of light in the visible wavelengths (~380-780 nm) is

generally associated with coloured molecules containing metal ions,
conjugated bonds and double bonds. For example, nucleotides, aromatic
amino acids, metalloenzymes, cytochromes, carotenes and photosynthetic
pigments all show selective absorbance. Many of these compounds absorb
the blue wavelengths and are yellow or red coloured solutions. In contrast
suspensions of small, non-pigmented particles such as ribosomes are viruses
are a smoky blue colour. Suspensions of larger particles such as nonpigmented bacteria are a dirty white colour.
8.
Since absorbance is dependent on the wavelength of light used in the

measurement, spectrophotometers must be designed to isolate specific
wavelengths in sufficient quantity to be used for measurements. This
requirement has lead to several important features in the design.
a.
The light source must produce a bright light that has fairly uniform
intensity at all wavelengths so that all of the selected wavelengths are
readable and the machine does not need major changes in zeroing
when the selected wavelengths are slightly changed.
b.
The white light from the source is usually split into light with specific
wavelengths by passing through a prism or reflecting it from a diffraction
grating to form a spectrum. The specific wavelengths are then selected
by positioning the prism or diffraction grating so that the emerging
spectrum hits an opaque plate which has a narrow vertical slit which is
just wide enough to pass a small segment of total spectrum. The light
that hits the plate is blocked and only the light selected to pass through
the narrow slit gets used for subsequent measurements. Different
wavelengths are selected by turning the prism or diffraction grating so
that different parts of the spectrum line up with the slit.
c.
Diffraction gratings produce multiple partly overlapping spectrums and

the blue light from the secondary spectrum overlaps the red light of the
primary spectrum. Consequently spectrophotometers which use
diffraction gratings usually pass the separated light through a red filter to
absorb out the interfering secondary blue light when readings are done
at red wavelengths (red filters only transmit red light).
d.
A few types of spectrophotometers use fixed prisms that direct the

separated light spectrum onto an array of about four hundred separate
sensors that are arranged so that each sensor detects a different portion
of the spectrum. These spectrophotometers can do simultaneous
measurements at all of the various wavelengths. The more usual
spectrophotometers that use slits to separate out light with distinct
wavelengths usually only have one detector and can only measure
absorbance at one wavelength at a time.
e.
The detector is usually a photomultiplier that is sensitive enough to react

to the small amount of light at the selected wavelength. Consequently
slight amounts of external light significantly affect the results and usually
the readings must be done with the samples inside dark sample
compartments to avoid problems from extraneous light sources.
150
B.
9.
A colorimeter is similar to a spectrophotometer except that the light is

separated by filters that transmit broad ranges of wavelengths instead of the
narrow ranges produced by prisms. The difference means that the detector
does not need to be as sensitive since the selected light contains more
wavelengths and is more intense than the selected light in a
spectrophotometer. These differences mean that slight amounts of external
light are usually not picked up by detector in the colorimeter so the readings
can be done with samples out in the open.
10.
Nephalometers are similar to spectrophotometers, but the phototube is

positioned at a right angle to the incident light path. Consequently the
phototube measures the scattered light instead of the transmitted light.
Therefore nephalometers are useful for measuring turbidity but not
absorbance.
Problems and Compromises in Spectrophotometer Design

1.
Stray light
a.
Spectrophotometers use filters, slits, prisms and diffraction gratings to

try to produce pure light of specific wavelengths. However, slight
mechanical imperfections in the selection system can result in
contamination of the selected wavelengths of light with undesired
wavelengths of light. These unwanted and unselected wavelengths of
light are called stray light.
b.
Stray light is important because it can include wavelengths of light which

do not interact with the sample and consequently limit the correlation
described by Beer's Law. Theoretically, Beer's Law predicts an infinite,
linear correlation between absorbance and concentration. In practice
however, many spectrophotometers only give good agreement up to 0.6
A units and even good spectrophotometers only give correlation up to
about 4.0 A units. This disagreement with Beer's Law usually occurs
because the amount of light being measured at high absorbance
readings is quite small and even a slight amount of stray light will result
in significant error in the reading. For example -: If you examined two
different samples in a spectrophotometer that isolated absolutely pure
wavelengths of light and the readings produced were 79% T and 1% T,
the same samples would have readings of 80% T and 2% T if they were
measured in a spectrophotometer with an additional 1% stray light. The
difference does not seem like very much but when the results are
transformed to absorbance units, the corresponding values read 0.1 and
1.7 A units instead of the expected 0.1 and 2.0 A units. The absorbance
transformation magnifies the slight error in readings at higher
concentrations and at increasing concentrations of the samples there is
a progressively greater tendency to significantly underestimate the real
concentration measurement predicted by Beer's Law. This
transformation also means that even though some spectrophotometers
can linearly measure readings up to 4. 0 A units, the results might not be
reproducible or accurate above 1 A unit because very little light is
actually being transmitted through the sample and even minor changes
151
in that transmittance can have massive effects. The software in modern
spectrophotometers automatically compensates for these stray light
effects but the measured amount of transmission is still very small so
that very large readings are still subject to uncertainty because of minor
changes in the reading of the light.
d. Other causes for deviation from Beer's Law besides stray light
include -:
2.
i.
Limitations in the formation of chromogen (light absorbing molecule)

from the components in the sample being examined. (If the reactive
component is much more plentiful than the reagent or the reagent is
unstable in the assay conditions).
ii.
Interaction of the chromogen with itself at higher concentrations and the

consequent formation of particles which scatter the light and compound
the loss of transmittance.
iii.
Changing interaction of the sample with the solvent.
iv.
Change of refractive index of the solution as the sample concentration

increases.
v.
Changes of pH or ionic strength that alter the chromogen interaction with

the light.
Slit width
a.
The width of the slit used for selecting the wavelengths affects the
amount of stray light in the reading since wider slits allow more
wavelengths at the selected reading.
b.
Many compounds have characteristic absorbance patterns (action

spectra) which visually show up when absorbance readings of single
samples are taken at different wavelengths of light and plotted against
the wavelengths of light used for the measurement. Typically such
graphs will show characteristic maxima and minima which can be useful
for identifying and characterizing compounds. In addition, knowledge of
the patterns sometimes allow two compounds in a single solution to be
measured independently as long as one of the compounds has an
absorbance peak that differs from the peaks of the other compound.
However, wide slit widths allow a wider range of wavelengths to pass
through the sample and consequently broaden the apparent absorbance
maxima and reduce the resolution between peaks.
152
Appendix E Teams and Teamwork
Teams
Most of the work in the MICB 421 lab course is done by teams. One of the reasons to work
in teams is to split the labour and conserve resources. Another reason to practice work in
teams is because most careers including professions such as science, business, medicine,
dentistry, optometry, law, and teaching depend on teams and teamwork.
Whenever you work in teams you should think a bit about how your team functions and
whether there are ways to make your team more effective because an effective team is
more productive than an individual. Sometimes novices just think of teams as a group of
people working together but an effective team can be distinguished from a group because
effective teams have:
1. Goals that focus the team effort
2. Rules for enforcing expectations of the team members
3. Roles for team members
4. Strategies and agreements to achieve the goals.
Sometimes rules and roles will switch as the projects develop but they are still the best way
to ensure that the team can function efficiently. The scientists that study complex team
behavior report that teams tend to follow a predictable pattern of behavior and development.
They suggest that all teams tend to work well initially then struggle as work loads,
expectations and problems arise but then start to become more effective again as the
members develop strategies to share the effort, resolve team problems, share solutions and
anticipate future problems. They also suggest that teams can become more effective more
rapidly if they establish ground rules at the beginning for the participating members. These
ground rules:
1. Establish schedules and meeting times and expected levels of participation at the
initial team meetings before conflicts and problems arise. By establishing the rules in
advance the individuals on the team have less chance of feeling that the other team
members are specifically picking on them when a rule is made to address a problem.
2. Establish consequences or penalties for neglecting meetings and skipping
commitments.
3. Expect all members to be accountable and reliable.
4. Establish and promote respect for other opinions, solutions and opinions within the
team.
5. Establish a decision making process within the team so that different opinions will be
proposed and effective solutions or choices can be made.
6. Establish and define roles such as leader, facilitator, scribe and devils advocate.
These roles can change and different team members can take on different roles.
However, all teams require a leader to receive information, summarize information
153
and distribute information, test agreement of members and develop consensus for
the plans to achieve the goal. They require a facilitator that encourages all team
members to participate in the decisions and planning by monitoring and soliciting
each members ideas. They need a scribe to record agreements and discussions so
that there are documents that state this information when the need for the
information arises. They also need a devils advocate to question proposals and
propose alternatives to ensure that the team is not too complacent.
When projects are simple and solutions are simple it is generally easy to make choices and
draw up team strategies. When projects are more complex there are more choices and
more solutions. There is also more chance of hearing different opinions and approaches
from the team members so there is a need to have a mechanism to develop team decisions.
There are several established mechanisms for making team decisions that have different
advantages and disadvantages.
1. The autocratic approach where the leader makes a choice and tells the other team
members what to do is fast but sometimes the other team members disagree with
the decision and do not work wholeheartedly towards the goal.
2. The consultative approach where the person needing to make a decision asks for
advice from some of the other experts on the team but still makes the decision is
also fast but might not take all the possibilities into account, might not make the best
decision and might not have support from all team members.
3. The democratic approach where everyone on the team voices an opinion or solution
before the team members vote on a preference is fast but the losers might be
unsatisfied with the vote.
4. The consensus approach where the problem is defined and all members have the
opportunity to keep modifying the proposals until everyone can accept the solutions
is slow but it does reach an outcome that everyone on the team can accept even if it
is not their first choice or approach.
To reach a consensus the team needs to define the problem then describe all the criteria
that the solution would ideally need to address. For MICB 421 these criteria could include
things such as time, effort, skill development, present skills, the general kind of project area
et cetera. The team members should then brainstorm, gather facts and suggest options or
opinions. The pros and cons of different opinions are then summarized in terms of the
criteria that the solution needed to address. At that stage the solutions or the criteria can be
revised, clarified, combined and consolidated. If it seems as if a consensus has been
reached you can vote then ask for reservations. If there are reservations but the team
members with reservations can accept the decision then the team solution can proceed. If
there are reservations that some team members cannot accept then the proposal should be
reevaluated by gathering more facts and compromises until a new solution arises.
Choosing Teams
Team work in MICB 421 becomes more important and explicit as the term proceeds.
You will be allowed to choose your own teams as long as the team has three or four
members, there are fewer than seven teams for each scheduled lab section and all the
class members for that day are on a team. Students lacking a team might be put
together or added to teams that only have three members.
When you agree to participate on a team you should consider some important details
154
1. Teams of three are easier to coordinate but teams of four have more people to
shoulder the work.
2. The single biggest stress on the previous teams working in the course was due
to differences in study habit and effort within the team. For example, students
that tended to procrastinate have difficulty working on teams with students that
like to plan and schedule work well in advance of the due dates. Similarly,
students that work to get by have trouble working with students that are trying to
maximize their effort and grades. In order to minimize these types of problems
we have prepared the following set of questions. We will not expect you to turn
in your answers to us but within your potential team you should each consider
the answers and honestly decide whether you could work comfortably for a whole
term with a person that could give that sort of answer. Some differences might
be more acceptable than others. Sometimes friends are not the best choices for
teammates if their goals and study habits are significantly different than your
goals and habits.
3. Many teams expect all members to contribute to the work and the ideas. Some
teams become stressed when an experienced student tries to take over the
whole project and does not allow the rest to contribute. It is better to have
everyone involved. This involvement might take some training but it spreads out
the work load so more can be done over shorter periods of time. This allows the
project to progress more efficiently.
4. Teams will be able to choose to work on any of the supplied categories described
in the project folder. However, these categories cover a wide range of different
areas. Teams that work on a project that interests everyone on the team tend to
be more effective than teams where the project was chosen by some members
but it was not interesting to others.
Some questions that you should ask and answer with your potential team members
when you consider joining a project team include:
Study times
i.
ii.
iii.
iv.
Do you prefer to work in the morning (8-12), or afternoon (noon-8) or the

night (midnight - 6)?
Do you prefer to work long continuous hours or do you prefer shorter spurts
with breaks (when necessary occasional long hours can be done but only
when absolutely necessary)?
Do you prefer to plan and schedule work to complete it well within the allotted
time or do you tend to work better under pressure and allow work to
accumulate until the deadlines?
Do you have lots of time to work on the projects or do you have limited time
available to work on the projects?
Study habits
i.
ii.
iii.
Do you work to get by, do you work to optimize overall performance in all
courses or do you work to do well in everything?
Do you prefer to brainstorm and discuss details in the team to pool
knowledge or do you prefer to think through details independently?
Do you prefer to work independently or do you prefer to work with other
students?
155
iv.
v.
Do you like detailed directions or do you like to figure out operational details?
Will you be available and willing to do your share of the work on the project in
February and March when mid-terms are scheduled? Will you do as much
work as the rest of the team each week? How many hours will you contribute
each week?
Work habits
i.
ii.
iii.
Do you tend to do careful, fastidious, detailed work or do you tend to work to

get the job done?
Do you like to be in charge or do you prefer taking directions from others?
Do you prefer teams where ideas are discussed and everyone works together
to reach a consensus `or teams where the work is authoritatively divided up
by the coordinator and each person works independently on their portion of
the work?
Interests
i.
ii.
iii.
iv.
What sort of research interests you?

What project areas were you considering?
How does the category of your proposed project fit with the interests of the
other students on the team?
What do you hope to get out of the project?
Rules
i.
ii.
iii.
What rules and penalties would you accept and expect for the team?
What will you contribute to the intellectual development and practical
development of the team project?
What do you expect for leadership?
156
Appendix F Records and Reports
Project Reports
The final project report will be published as an article in the UBC Journal of Experimental
Microbiology and Immunology (JEMI) so these reports must use the style required by
JEMI. This style is adapted from the style for the American Society for Microbiology
(ASM) journals for 2013 and subsequent years. Requirements are described in the
instructions for authors for any ASM journal such as J. Bacteriol. at:
http://jb.asm.org/site/misc/journal-ita_org.xhtml#01.
The draft manuscript for the project will be done in the same style. In each report you
are expected to concisely interpret the major observations in your data and then analyze
the interpretations by relating them to your basic background knowledge and the specific
objectives of the experiment.
The grade for the submitted reports will consider:
- grammar
- logic and accuracy
- formatting and style
- effectiveness of the presentation details (appropriate tables, graphs, graph
choices).
- analysis and explanation of appropriate details within the data
- consistency and completeness of the analysis
- conciseness
Each report must include the title of the experiment, the names of the authors, the
affiliation of the authors, an abstract, an introduction and labeled sections for material
and methods, results, discussion, future experiments, acknowledgements and
references. Examples of these features can be seen in the most recent volume of JEMI.
1. Title
The intent of the title is to capture the readers attention and allow potential readers
to know whether the article might be relevant to their interests. The title should be a
single sentence that should convey the essence of the study. Use the fewest
possible words to accurately describe the paper and provide enough information for
an indexing service such as Medline. There should be no jargon or abbreviations.
2. Abstract
The abstract is a short form of the paper. It contains the objective, main method,
summarized results and the main conclusion. It is intended to give potential readers
a better idea of the objective of the study, the approach to the study, the main
observations and the main conclusions. It is written in the past tense as a single,
self-contained, short paragraph without references or abbreviations.
3. Introduction
The intent of the introduction is to provide enough background to understand and
evaluate the purpose and the results of the reported study and justify the purpose
157
without further literature review. It is not intended to be a review but there should be
sufficient literature information to orient the project by providing the essential details
that allow the reader to understand the hypothesis being reported, the reason that
anyone would study those details, the direction of the eventual discussion and a
specific statement of the scientific purpose of the study. It must include a formal
written statement that gives the scientific purpose that will be addressed and
answered by the experimental results. It should define any specialized terms or
abbreviations.
4. Material and Methods
The details in this section must be written in paragraphs rather than point form but
Tables and Figures can be included to concisely present details that are too wordy to
write out. It should be written in the past tense and should not include any results,
just the details needed to know how the reported work was done.
Provide enough detail to understand how the assays were done. Where appropriate
the operational details should be referenced to existing published articles or
published books but there should be enough information that the reader can
understand the specific approach without looking up a lot of references. If the
materials and methods were identical to the descriptions in the references then you
should make a short statement to that effect and cite the reference. If there were
any significant changes then you can indicate that the materials and methods were a
modified version of the protocol in the reference then cite the reference and briefly
explain the modifications. Use the past tense. Provide catalogue #s and supplier
source for any significant chemicals, kits and supplies. Provide recipes or references
to the recipes for the solutions used for growth or reactions. Give names and
properties of constructs by taking the first letter of each surname on the team and
linking it to the number of the winter session and a unique number for that strain
(initials/session year/clone #, ie: AABF081 or KPST082). Use titles and labels for any
Tables and Figures.
5. Results
a. The introduction and the methods explain how you got results. The results
section is used to present the data and comment on the patterns. The tables and
figures for the report will depend on what was done and seen in your project. In
addition, there should also be a few paragraphs that point out and comment on
the main observations and problems in the presented results. These paragraphs
should be succinct summaries that point out the meaning and patterns of the
observations rather than simply re-describing the graphs and tables in words.
You are expected to consider the experimental purpose and pick out what seems
to be the most relevant observations, problems, paradoxes and quirks from the
data. Then briefly compare those observed effects to your expected results.
You should also comment on the quality of the result and the main observations
and the main relationships of the trends but reserve the explanations of the
trends for the discussion section. Similar effects should be considered together
and treated as a single observation so that space is not wasted simply repeating
the same thing. For example, imagine that you are examining the effect of drugs
on polymerase activity and observe that one drug speeds up the activity and two
drugs slow it down. In the report it is better to observe that different drugs have
different effects since some decrease activity and some increase activity, instead
158
of breaking the information into three totally separate and isolated observations.
Since the results comment on measurements and observations done in the past
they must be written in the past tense.
b. Wherever possible, the observations should be related to each other and
compared to the controls. All comparisons should be quantitative. For example,
if something becomes two times bigger than the control values you should say
that it is 2x bigger or 200% bigger or twice as big instead of just saying that the
value got bigger.
c. The tables and figures are intended to organize most of the data into a form that
is easier for you to analyze, compare and examine for patterns. You should not
include extra tables that simply repeat information already presented in other
figures and tables.
d. Rules for making the figures are included in the units, graphs and
calculations section in Appendix C. We will expect you to understand and
apply those rules when you prepare and submit the reports.
e. Figures should be numbered in Arabic numerals and identified with relevant,
informative titles at the bottom of the figure. A useful title is a sentence that
indicates the purpose or intent of the information or trend in the graph rather than
simply repeating the axis labels. For example: the title Effect of carbon dioxide
levels on the growth rate of E. coli in aerated minimal media is more useful and
informative than the title Growth versus time or OD versus time. Figures
should be identified by the capitalized abbreviation such as FIG. 2 rather than
Fig. 2 or Figure 2.
f.
The tables should be identified with upper case letters, Arabic numerals and
informative titles at the top of the table.. For example, TABLE 2. Strains and
plasmids used in this study. rather than Table II. Strains and plasmids used in
this study.
g. When data from a particular experiment is referred to in the discussion, the

relevant data should be clearly identified by referring to the appropriate figure or
table number. This referencing can be done in a several ways, for example:
Figure 12 shows that the growth rate doubled after the addition of the amino acids.
or
The growth rate doubled after the addition of the amino acids (Fig. 12).
or
The affinity of Listeria monocyogenes to attach at different temperatures is
shown in Fig.1
or
The amino acid uptake increased 3 fold after glucose was added (Table 2).
h. You should note that even though the identification of the actual TABLE or
FIG. is capitalized, the reference statements in the discussion and results
use Table and Figure or Fig.
159
i.
Good tables and figures are generally uncluttered and easy to read. Choose
units and prefixes that try to avoid a lot of zeros or exponents that will clutter up
the data and interfere with easy comparisons. If most of the measurements yield
concentrations in the ug/ml range then you should express the data in units of
ug/ml. Try to avoid a lot of zeros or exponents that will clutter up the data
and interfere with easy comparisons.
j.
The figures should include keys that correlate different graphed symbols to the
appropriate reaction conditions. In these keys concisely identify the active
component of the test condition rather than the arbitrary number given to that test
during the experiment. Since the only intent of the key is to identify the datum
points to correlate the result to the conditions identified in the title or the axis
labels, the key should not repeat any unnecessary information that is already
presented in the title or the labels. The keys should be boxed off or positioned so
that the symbols in the key cannot be confused with actual data points.
Frequently it is more convenient and effective to place the information about the
symbols in the title rather than in a separate key.
6. Discussion
a. The discussion is an analysis of the observations and interpretations in the
results. You are not expected to review the experimental technique or theory in
this section or repeat the results. You are expected to discuss what could cause
the effect, explain what the effect indicates is probably happening, suggest
alternate explanations, explain why the effect is important and judge whether the
effect and interpretations seem reasonable. Comment on the principles
demonstrated by the results. Point out items that do not make sense and provide
an explanation. Show how your results and interpretations agree or disagree
with results in previous publications. Discuss the implications/significance of
your work. The observations and problems should be ranked so that the more
important ones are discussed first and the less important ones last. Trivial
observations and obvious minor problems should not be discussed.
b. The final paragraph in the discussion should be a conclusion. Conclusions are
different then summaries. For a conclusion, summarize the evidence then make
a brief statement about the general idea that your data has proved,
assuming that the data is correct. This statement must be based on the
observed facts and should only be two or three sentences long. It must not
include untested explanations of your results and must not be a discussion
because facts do not need a discussion to explain them. If your tentative
conclusion seems to be inconsistent with known facts you should bring that up
somewhere in the earlier part of the discussion.
c. The entire project report should normally be done in less than 4000 words. If
your report is significantly longer then you are approaching the report wrong and
doing unnecessary work.
d. All abbreviations except for standard chemical abbreviations and the
standardized symbols for units should be identified the first time that the
abbreviation is used. Usually an abbreviation is identified the first time by writing
out the word(s) in full then putting the abbreviation in brackets after the word(s).
You can then use just the abbreviation for the rest of the report. If you do not use
160
the abbreviation after the first time when it was defined then there was no need
to include the abbreviation.
7. Future Directions
What is the next step that should be done? What are problems that should be
avoided by future researchers? When you discussed your results you should have
provided explanations of important observations and problems. Briefly outline an
experiment that will test one of the most important explanations or paradoxes that
you have actually proposed in your discussion section to account for some of your
observed results or problems. Do not include operational details such as volumes,
temperatures, times et cetera but give a brief statement of how the explanation will
be tested and the expected results if the experiment supports or refutes your
explanation. For example, if the test involved electrophoresis, explain how the result
would test your explanation and what it would show if your explanation was correct
or incorrect. It is not acceptable to merely repeat the experiment (even though this
might be necessary).
8. Acknowledgments
Indicate the financial support of the Department of Microbiology and Immunology,
University of British Columbia.
9. References
The references in the list of project categories are intended to give you some
background on the potential projects. Additional background can be located by
using the library search programs. Web of Science, PubMed, Medline or Google
Scholar are good starting places. At least two newer, relevant journal citations
are expected for your project report. Somewhere between ten to - thirty
references should normally be present. The intent of a reference is to provide
necessary background to understand your arguments or support one of your
statements with facts. Any reference material that is used to expand or support
arguments in the discussion section of the reports should be properly and completely
cited in the style of the American Society for Microbiology journals such as Journal of
Bacteriology and Journal of Immunology. If a reference is not cited or used in the
introduction, methods or discussion it should not be included in the report because
the information in it was not applied or needed.
When citing references you must use the standard journal abbreviations
available at: http://www.efm.leeds.ac.uk/~mark/ISIabbr/ or
http://home.ncifcrf.gov/research/bja/
The formal abbreviation for the on-line Journal of Experimental Microbiology and
Immunology (UBC) used for publishing MICB 421 and MICB 421 manuscripts is J.
Exp. Microbiol. Immunol. The informal abbreviation JEMI should not be used in
the reports.
The program RefWorks or analogous reference tracking programs can be beneficial
for developing reference lists but you still need to ensure that the listed references
are in the appropriate style and format.
161
If you used a mutant strain of Pseudomonas aeruginosa from one of the libraries
available from the Robert Hancocks lab in Project 2 then you must include the
appropriate original reference to those strains in your report.
The University of Washington PAO1 mutant library is described in:
Jacobs M A, Alwood A, Thaipisuttikul I, Spencer D, Haugen E, Ernst S, Will O,
Kaul R, Raymond C, Levy R, Liu CR, Guenthner D, Bovee D, Olson MV, Manoil
C. 2003. Comprehensive transposon mutant library of Pseudomonas aeruginosa.
Proc. Natl. Acad. Sci. U. S. A. 100:14339-14344.
The UBC (Hancock) PAO1 mutant library

Lewenza S, Falsafi RK, Winsor G, Gooderham WJ, McPhee JB, Brinkman FSL,
Hancock REW. 2005. Construction of a mini-Tn5-luxCDABE mutant library in
Pseudomonas aeruginosa PAO1: A tool for identifying differentially regulated genes.
Genome Res. 15:583-589.
The Harvard University PA14 mutant library

Liberati NT, Urbach JM, Miyata S, Lee DG, Drenkard E, Wu G, Villanueva J, Wei
T, Ausubel FM. 2006. An ordered, nonredundant library of Pseudomonas
aeruginosa strain PA14 transposon insertion mutants. Proc. Natl. Acad. Sci. U. S. A.
103:2833-2838.
10. The reviewed draft manuscript must be corrected before it can be accepted for
publication in JEMI. The grade will not be applied until the revised report has been
resubmitted and the corrections are accepted.
11. The final submitted copy of the manuscript will be converted to two column format
and the figures and tables will be moved to appropriate places in the text after you
submit the electronic copy. You should not convert it to two column format.
C.
Format Requirements of the Project Reports

The draft and the final copy of the formal journal article reports for project #2 must be
formatted as JEMI articles. To facilitate that process we need you to present the report
for project 2 formatted as described below:
Title:
No heading
Capitalize first letter of each major word (unless it is a gene name that should be lower
case). Do not capitalize connecting words such as are, of, from, the, a, and by unless they
are the first word in the title
Use 16 pt, bold, Times New Roman font
Centered, single spaced
Put one line space before authors
Authors names:
No heading
Alphabetical listing, each separated by a comma, there is no and before last author
162
12 pt, bold, Times New Roman font

No space before institutions
Institutions of authors:
No heading
Use 10 pt, italics, Times New Roman
Department, University
Put one line space before abstract
Abstract:
No heading
Use 10 pt, bold, Times New Roman for writing
Use 1 margins on both sides
Use left alignment,
Make first line indent 0.13 (first notch on tab ruler)
Put two line spaces before main body
Introduction:
No heading
Use 10 pt, Times New Roman for writing
1 margins on both sides
0.13 indents for beginning paragraphs
Use justify alignment
Put one line space before Material and Methods section
MATERIALS AND METHODS:
Put in the heading name All CAPS, 8 pt, bold Times New Roman, one line space before
text.
Subheadings are put in line with the text of the paragraphs to form the first sentence in
the paragraph Bold font, followed by a period. Make 0.13 indent
Main text - 10 pt Times New Roman
Use 1 margins on both sides
Use 0.13 indents for beginning paragraphs
Use justify alignment
Put one line space before Material and Methods section
RESULTS:
Same as Materials and Methods section except use 10 pt font throughout.
DISCUSSION:
Put in a heading All CAPS, 10 pt, bold Times New Roman, one line space before text.
Main text is the same as in the Results section (10pt Times New Roman, 1 margins,
0.13indent, justified)
One line space before Future Directions section
FUTURE DIRECTIONS:
Same formatting as Discussion section

Include the heading
ACKNOWLEDGEMENTS:
Put in a heading all CAPS, bold 8 point font
163
Use 8 pt font
REFERENCES:
Put in the heading All CAPS, bold, 8 point font

Number references in order of first appearance in the report. (Prior to 2013 the style placed
the authors in alphabetical order but that style changed for 2013).
Cite the number in text with parentheses eg. (1).
Follow ASM formatting. See examples in Journal of Bacteriology
Use justified 8 pt font. Numbering should not be bold or italics.
Indent first line with a margin at 1 and the left tab (bottom tab) set at 0.25
Figures and Tables:
Place figures and tables at the end of the document. They will be moved into the text
when the files are converted to two column format. To simplify that move these items should
be sized to either fill a half width or full width of the page. If it is legible at half a page width
and the details are readable/visible at a half page then make it at that size.
Tables:
Type out title separate from the table. Center it above the corresponding Table.
Title should be 8 pt font. With non-bold CAPS for the TABLE and number, and non-bold
for sentence structure for the title, eg. TABLE 1. Effect of sodium salicylate on cell
growth of E. coli B23.
Tables should not contain vertical lines or borders
If the Table does not contain enough information to justify a table then write out the
material in the appropriate section instead of using a table.
Figures:
For figures make sure the axes and legends are legible.
Figures should be in black and white or grayscale (not coloured unless the colours are
different when the document is printed in grayscale).
Use Times New Roman font throughout the figure if possible.
Type out the title separate from the figure and place it below the figure.
Title should be 8 pt font with all CAPS for FIG, non-bold for the FIG and number, bold
for the descriptive first sentence of the title, non-bold for any additional info included
with the title. eg. FIG. 1. Effect of a kanamycin on E. coli B23 capsule
concentration. Black bars refer to the samples collected at 80 minutes of incubation.
Justify alignment of the title.
If there is text such as lane numbers or band identification that have been added to an
image, aside from the title, include this text with the image so that the complete figure
can be moved for formatting.
Modifying or annotating the images in PowerPoint then copying and pasting the
annotated images into a text box as a pic files to a word document helps to make figures
more legible.
Participation Reports
When the team project proposal, the draft manuscript and the final manuscript are submitted there
must be a separate page attached to the end of the submission that explains how each author
contributed to the work and how much time each author contributed to the work and the
preparation of the report. This section should be about half of a page long.
164
Lab Notebook Guidelines: Form and Function

If it isnt documented, it didnt happen.
The laboratory notebook is a critical tool in science. Systematically documenting data and ideas
can serve as (1) a memory aid for the primary researcher, (2) a forum for the researcher(s) to
organize, refine and record thoughts, concepts, and methods, (3) a communication tool between
researchers over time (sometimes decades) and within teams, and (4) and a legal document,
especially when audits occur and intellectual property rights are concerned. Clearly, it is of
paramount importance to maintain and pay careful attention to the quality of your lab notebook.
Maintaining a lab notebook may seem simple, however, in order for this tool to yield long term
value a standard process for each organization must be established. In an academic setting the
Principal Investigator may apply lab standards. In an industrial setting the Quality Assurance
department will set a standard as part of Good Documentation Practice (GDP), provide GDP
training to employees, ensure compliance, and manage the overall documentation and filing
process.
There isnt a perfect model for a standardized lab notebook system. Each lab notebook system
will be customized depending on factors such as lab/company size, the type of data typically
being collected, regulatory requirements, and the budget available (e.g. some sophisticated
laboratory information software options can be costly). In general, a lab notebook system must
establish and maintain a standard for documentation and filing.
The following standards have been developed for use in the MICB 421 laboratory setting. These
practices have been adapted from an effective industrial model open to audit by regulatory
bodies such as the Food and Drug Administration (FDA) and the International Standards
Organization (ISO). Lab notebook records were maintained both electronically (for quick
access, searches, sharing, and the option to copy/paste information) and in paper notebooks (as a
back-up to guard against potential corruption and obsolescence of digital files as well as to
comply with regulatory requirements). The MICB 421 approach will be a hybrid digital/paperbased lab notebook system. Formatting and content details are first described followed by a
suggested approach for maintaining your teams lab notebook. Two pages from a mock lab
notebook are included at the end of this Appendix.
Keep in mind that each laboratory you work in will set its own lab notebook standards, partly
based on historical methods, regulatory requirements, and compatibility with the type of research
being done.
The Book
It is recommended that a notebook with a stitched binding be used. Ringed binders are generally
not acceptable as its too easy to tear out pages. A hard cover may protect your work from spills
and bending. The size of the notebook isnt critical although larger pages may be more
convenient if printed pages are going to be taped in. Buying a book with pre-numbered pages is
strongly recommended; as youll see below, page numbers are critical to notebook organization.
Carbon copy options arent recommended as inserted pages and photos wont be transferred.
Electronic lab notebook options exist and it is likely that over time they will be adopted as
standard practice. However, working with electronic devices (e.g. tablets) in a lab setting brings
a new set of considerations (e.g. spills and contamination) which need to be addressed. Ensuring
that electronic data is secure and backed-up is also a consideration; cloud based data storage will
likely address this issue.
165
Pens and Printers
Use pens and printers that can stand up to the dangers of the lab such as water, ethanol, methanol
and acetone spills. Ball point pens are often acceptable but should be tested on a case by case
basis. Felt tip pens and permanent Sharpies should be avoided as they may resist water but not
solvents. Laser printers are recommended; ink jet print is water soluble. Once youve found
good options for pens and print-outs, stick to them.
Coding Your Notebook
Your lab notebook should be coded on the cover and spine with the initials of your teammates
followed by the number 715. This code will be recorded in a spreadsheet to facilitate lab
notebook searches in the future.
For example: Dan, Cathy, Kang, and Dave would label their notebook: DCKD715.
Note: the 1 represents MICB 421 and the 15 represents the year 2015.
You should also include your name, e-mail, phone number, Department, and course number on
the inside cover.
Table of Contents
The first 2 pages of your notebook should be dedicated to the Table of Contents section. This
section should include columns for experiment title, experiment code, and respective page
numbers. The Table of Contents should be updated as each experiment is concluded.
Project Codes
Every major project should be given a code and be listed on the last page of the Table of
Contents. Each page should have the project code noted in the right hand side of the header
section.
Project should take the form: P### where # is a whole number starting at 1.
Note: For MICB 421 you only have 1 major project so the code is simply P001.
Experiment Code
Each experiment should be given a unique code. This code should begin with the notebook code
(e.g. DCKD115) dash ###, where # is the page number upon which the experiment started.
Examples:
An experiment starting on page 1 of the notebook should be coded DCKD715-001.
An experiment starting on page 113 of the notebook should be coded DCKD715-113.
The experiment code should be in the top left hand side of the header space on every page that
the experiment appears.
The experiment code can be very* useful when labeling reagents, electronic data files, and
preparing summaries of work.
Dates
The date of data entry should be recorded on every page in the footer. The date should take the
form Day-Month-Year (e.g. 02 Sep 14).
166
Page Numbering System
Page numbering is useful for organizing multiple experiments, even when they are being carried
out simultaneously. Every page should have a page address recorded at the top left of the page
and the bottom right. Every experiment begins with the address Start in the top left corner of
the first page. At the bottom right corner of the page, the page number address to which the
reader should turn to next is recorded. This top to bottom / page to page reference process
continues sequentially through the notebook (e.g. pages 1 4). If a second experiment starts
before the first one finishes it occupies the next available pages in the notebook (e.g. pages 5
8). When the first experiment resumes, it is documented in the next pages of the notebook (e.g. 9
13) and the page numbering system is used to link it to the previous work. When the
experiment finishes and all documentation has been recorded the address End is tagged at
lower left hand side of the last page. This system is illustrated on page 153i and 153j. The Table
of Contents at the front of the notebook is then updated.
Title
Every experiment should be given a descriptive title. The title needs to only appear on the first
page of the experiment and should be preceded by the experiment code.
For example:
DCKD714-001
Western Blot Analysis of Putative Gene Y Deletion Mutant in E. coli strain B23
Purpose
This section should briefly (usually 1 -2 paragraphs) explain the background and rationale for the
experiment and point to relevant references. Linking experiments through careful referencing is
very important. The Purpose section should almost always contain references to other
experiment codes or primary papers from the literature. Experimental purposes may be as
simple as preparing a buffer for a future experiment.
Methods and Materials
Similar to a research paper, this section must contain descriptions and/or references to all
protocols and formulations. For sensitive procedures it may be useful to record lot numbers of
reagents (e.g. in the case of antibodies) and descriptions of equipment (for example, pipetteman
or balance numbers). In Industry this information may be critical for compliance with regulatory
standards. This information is also necessary for compiling your Materials and Methods section
of your paper. Be sure to expand all calculations. Future experiments may reference the
experiment code where the primary calculation was made. It can sometimes be more efficient to
handwrite calculations in a space reserved in a printed sheet.
Results and Observations

All observations and data should be captured in this section. It is recommended that fillable
tables be created in excel and printed prior to beginning experiments. Often the Purpose,
Methods and Materials, and Results and Observations tables are drafted and printed before
entering the laboratory. As the experiment unfolds the researcher records data and observations
real time. Label all figures and tables, including standards, ladders, and controls. Style
guidelines for figures and tables required for final publications should be followed to ensure
efficiency when preparing presentations and papers. Often tables, figures, and photos are
167
printed from excel, cut to size, and taped into the notebook. The interface between any taped
item and the notebook should be initialed to flag the location if the inserted items were to be
removed or fall out. Staples are not recommended.
Very large data files, such as diffraction data and high resolution images, can be named with the
experiment code and saved on a computer in a folder labeled with the Project Code and date.
Similar filing approaches can be used for samples and specimens.
Discussion
This section should recap the major observations and results. The Discussion may include
thoughts and speculative ideas if clearly identified as such.
Conclusions
The Conclusions should state exactly what the experiment shows based on the available data.
Avoid speculation here. This section is often written in bullets.
Future Directions
State what the next experiment(s) (if any) should be.
Signing and Witnessing Notebook Entries
At the bottom of every page a space near date should be reserved for the primary author of the
experiment to sign to confirm authenticity. Within a week of the notebook entry another
member of your team should carefully proof read the experiment and flag any errors or
omissions. Once the author makes the necessary corrections, the proof reader signs and enters
the date at the bottom of the page as a witness. Time sensitive entries such as potential patent
ideas should be signed and witnessed the day the information is entered into the notebook as
timely completion may be used as evidence of first discovery.
Minor Corrections and Typos
Small entry errors such as typos can be corrected by drawing a single line through the error and
correcting it in ink. The correction should be initialed and dated by the author.
Experiment Extensions and/or Changes in Conclusions
Larger adjustments to experiments (e.g. conclusions) should either be explained in writing by
extending the experiment via amendment of the End page or by starting a new experiment with
clear reference to the previous experiment.
Skipped pages and blank spaces
Pages should not be left blank. If a page accidently gets left blank the author should draw a link
through the blank page and initial and date it. A similar approach is taken with large blank
spaces.
Concepts, Discussions, E-mail, and Letters
In addition to experiments, other items such as concepts, discussions, e-mail and letter
correspondence should be entered into your notebook. For example, if a strain or plasmid is
received from another lab it is convention to include a copy of the actual e-mail or letter in your
notebook. Further, models or late night ideas should be included in your notebook; these may
prove valuable later.
168
Suggested Approach
The following is a suggested approach to organizing and maintaining your MICB 421 lab
notebook:
Each experiment is initiated by creating an electronic document named with the experiment code
and brief description which is saved in an experiment folder within a weekly project folder.
The document is formatted with the experiment code, title, and date. The purpose and methods
sections are completed electronically BEFORE the experiment begins. It is strongly
recommended that tables and areas for entering observations are included. An experiment
should not begin unless this portion of the notebook is complete. Writing up this material prior
to the experiment allows ideas to be refined, procedures to be reviewed and scrutinized, and time
management planning.
The experiment is then carried out and data and observations collected. Data is then analyzed,
formatted, or labeled prior to entry into notebook. All paper is cut to size with scissors and taped
neatly into notebook. Do not use staples.
Digital files, such as excel spreadsheets and photos, are named with the experiment code and title
and stored in experiment folder on your computer.
The Results, Discussion, and Conclusion are completed electronically. All sections are printed
and taped into notebook.
Once complete the author should apply all formatting and enter the experiment into the Table of
Contents. The witness should carefully scrutinize the entry for omissions, clarity, and errors
prior to signing off.
Previous students have found that cloud based options such as Google docs are an effective place
to share and store files.
At the end of the course both the paper lab notebook and the digital files will be submitted and
filed in the course library for use by other students.
MICB 421 Lab Notebook Expectations and Assessment

In MICB 421, notebooks will be assessed from time to time by the TAs and Instructor. Required
formatting points for MICB 421 notebooks are described in Appendix G. A mock notebook is
depicted on page 152 and 153. As long as these standards are met, other styles/methods (e.g.
electronic notes) will be considered acceptable if deemed (by the Instructor) to be equivalent or
superior.
References
Lab notebooks are an important, interesting and evolving scientific tool. For other thoughts and
suggestions in this area the following reference are suggested:
http://colinpurrington.com/tips/academic/labnotebooks
http://www.nature.com/scitable/blog/bioscience-elearning/to_lab_book_or_not
http://postdocexperience.scienceblog.com/2012/11/19/electronic-lab-notebooks/
http://www.gradhacker.org/2013/08/23/experiments-with-an-electronic-lab-notebook/
http://www.nature.com/news/going-paperless-the-digital-lab-1.9881
169
1
Start Expt
Code
Project Code
Title
Purpose
Material and Methods
DO
DO
DO
Results
e.g.
tables
DO
Results
e.g. figures
Results
Signature
Date
Witness
Date
Go to 2
170
2
From 1 Expt Code
Discussion
DO
Conclusions
DO
Future Directions
DO
Initials / Date
Signature
Date
Witness
Date
End
171
Oral Presentations
Information will be distributed during lecture.
172

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1

Project 1 Preparation of Competent E. coli

Materials and Equipment

Schedules, Performance and Marks

Appendix A - Basic Methods

Preparation for Aseptic Work

III. Mixing Samples

V. Plating and Enumeration

Streak Plating for Isolates

Appendix B - Laboratory Safety

Appendix C Units, Graphs, and Calculations

Units and Prefixes

Sample Problems with Dilution and Growth Calculations

Appendix D - Technical Theory

Protein Release from the Cell

Appendix E Teams and Teamwork

Appendix F Records and Reports

Department of Microbiology and Immunology

William Ramey / David Oliver

Course Operation and Rules

9. To further your ability to work in scientific teams.

Do not eat or drink in the laboratory.

Do not take cultures out of the laboratory without permission.

Project 1 Preparation of Competent Esherichia coli

Project 2 - Research Project

Each student must individually propose a project based on an observation or

Here is a link to JEMI:

12. Week III

Biosis <http://resources.library.ubc.ca/551/ >

13. Weeks IV and V

Weeks VII-through XI.

experimental reference numbers from your lab notebook

15. Weeks XI-XIII.

Major equipment that could be available include:

General References that might be Useful to find Project Background

Project 2 Individual Proposal - Flowchart

Project 2 Individual Proposal - Written

WHMIS and Lab Introduction Quiz

Project 2 Team Proposal

In class Final Exam

Presentations and Questions (#1/2)

Presentations and Questions (#3/4)

Activities for Week

Tues and Thurs Class

Tues and Thurs*** Classes

Only Tues Class

Project 1 and Lab

Only Tues Class

Only Tues Class

Only Tues Class

Only Tues Class

Only Tues Class

In Class Final Exam on Tues

Tues and Thurs Class

Tues and Thurs Class Pres.

Tues and Thurs Class Pres.

Checklist for MICB 421

Training, Safety, and Library Assignments

WHMIS and Lab Quiz (15 Jan 2016)

Notebook (5 Feb 2016)

Individual Proposal Flowchart (10 Jan 2016)

Weekly Summary #1 (28 Feb 2016)

Team Oral Project Presentation

In Class Final Exam (15 Mar 2016)