LABORATORY 2

Basic Bacteriologic Techniques

I. OBJECTIVES

1. To master viewing bacterial specimens with the microscope. Practice using oil
immersion to focus on specimens.

2. To become familiar with routine bacteriologic procedures: sterile transfer,

inoculation of media, and slide preparation.

3. To utilize the streak-plate method for isolation of individual bacterial colonies

from pure and mixed broth cultures.

4. To gain familiarity with the Gram stain procedure.

4. To perform antibiotic sensitivity testing using different microorganisms.

6. To prepare an acid-fast stain.

7. To observe demonstrations of bacterial stains routinely used in clinical

microbiology laboratories for organism identification.

II. BACKGROUND

All of the bacteriology laboratories in this course will rely heavily on the successful use of the
light microscope and oil immersion lens to examine the morphology and staining
characteristics of bacteria. The skills developed in this laboratory section should be carried
with you during your clinical years and will be useful in the care of your future patients. The
ability to stain and identify bacteria in clinical specimens such as sputum, CSF, and wound
cultures will benefit your patients and enhance your performance as a medical student and as
a resident on wards. Therefore, focus your attention on learning the basic bacteriologic
methods presented during laboratory exercises.

Bacteria are everywhere! On the bench tops, in water, soil and food, on your skin, in your
ears, nose, throat, and intestinal tract (normal flora). The diversity of bacteria present in our
environment and on and in our bodies is incredible. When trying to study bacteria from the
environment, one quickly discovers that bacteria usually exist in mixed populations. It is only
in very rare situations that bacteria occur as a single species. However, to be able to study
the biochemical, morphological, and physiological characteristics of an individual species, it is
essential that the organism be separated from the other species that are normally found in its
habitat. In other words, we must establish what is called a pure culture of the microorganism.

A pure culture is defined as a population containing only a single species or strain of bacteria.
Contamination means that more than one species is present in a culture that is supposed to
be pure. Contamination does not imply that the contaminating organism is harmful, it simply
means that the contaminating organism is unwanted in the culture that you are trying to
isolate and study. Special techniques, commonly referred to as aseptic pure culture
techniques, must be used to obtain a single isolated strain for study.
Historically, bacteria have been the cause of some of the most deadly diseases and
widespread epidemics of human civilization. A tremendous amount of time and resources
were used to develop methods of treatment for infectious diseases. Antibiotics have
dramatically changed the prognosis of patients exhibiting symptoms of bacterial infection.
Vaccines were developed that are able to prevent the spread of infectious disease in
populations. Advances in technology and molecular medicine (automation, polymerase chain
reaction) have added greatly to the clinical microbiology diagnostic armamentarium.
However, clinical microbiology remains an interpretive science in which an individual’s
experience and judgment are pivotal. It pays to understand the biology of bacterial
pathogens and how they are able to produce disease.

Some infectious diseases are distinctive enough to be identified clinically. However, most
infectious agents can cause a wide spectrum of clinical symptoms in humans. Conversely, a
patient may present with symptoms that may be attributed to infection with any one of many
pathogens. For example, Influenza virus infection causes a wide variety of respiratory
syndromes that cannot be distinguished clinically from those caused by streptococci,
mycoplasmas, or more than 100 other viruses.

Therefore, identification of a specific infectious pathogen typically requires the clinical

microbiology laboratory. The clinical microbiology laboratory will test patient specimens for
microorganisms that are, or may be, a cause of the symptoms experienced and will provide
information (when appropriate) about the in vitro activity of antimicrobial drugs against the
microorganisms identified.
Importance of the clinical laboratory. Specimens are collected from a symptomatic patient and submitted to
the laboratory for characterization and identification. The diagnosis is determined by the use of many types of
evaluation: microscopic, culture, serology, and genetic. Additionally, it is helpful for the clinician to know which
antibiotics the pathogen is susceptible to (http://gsbs.utmb.edu/microbook/ch010.htm).
A. OPERATION OF THE MICROSCOPE

Note that there are three objectives: a low power (10X), medium or high dry power
(40X), and oil immersion objective (100X). The oil immersion objective is always
used for the morphological study of bacteria. The three objectives may be
distinguished by the difference in focal length or numerical aperture, as indicated in the
following table:

Common Focal Length Numerical Lens Eye Piece Final

Designation Aperture Magnification Magnification Magnification

Low Power 16.0 mm 0.25 10X 10X 100X

High Dry 4.0 mm 0.65 43X 10X 430X
Oil Immersion 1.0 mm 1.25 (in oil) 97X 10X 970X

http://www.hendrix.edu/homes/fac/sutherlandM/CellWeb/Techniques/microparts.html

The following directions should be followed routinely when operating the microscope:

1. Rotate the low power objective (yellow stripe) into place and put the slide on the stage.

2. Move the low power objective down as close as possible from the slide using the
coarse control and adjust upwards until the specimen is in focus as seen through the
ocular. Find the specimen first with the 10X objective.

3. Light adjustments may be necessary. It is possible to place the light source in focus
by raising and lowering the condenser. Center the light source.

4. Adjust the condenser and iris diaphragm to a position giving the strongest even light.

5. Focus on the specimen with the coarse adjustment finishing with the fine adjustment.
6. Once in focus, you are now ready to use the oil immersion lens. Move from the yellow
stripe, to the red stripe, and finally to the white stripe objective. To focus the oil
immersion lens:
a. Select an appropriate field.
b. Open the iris wide.
c. Turn the 40x objective away enough to place a drop of oil on the specimen/
slide. Place a small drop of immersion oil on the specimen.
d. Swing the oil immersion lens into the oil.
e. Use only the fine focus to bring the object into focus. Never use the coarse
focus when the oil objective is in place. The slide could break and the objective
could be damaged.
f. At the end of the class period, clean off the objective with lens paper.
7. Again, note that the oil immersion lens must be used for examining bacteria. Do not
attempt to identify bacteria under lower power; you will be seeing only artifacts.

Wipe the upper lens of the eyepieces with clean lens paper both before and after use.
The oil should always be wiped from the oil immersion lens with lens paper prior
to putting away the microscope. Microscopes should be clean, with the low power
objective in place when put away.

B. ROUTINE BACTERIOLOGIC PROCEDURES: STERILE TRANSFER,

INOCULATION OF MEDIA, AND SLIDE PREPARATION

Sterile transfer of bacteria and inoculation of media

Since diagnostic bacteriology is concerned essentially with the isolation and

identification of single species of bacteria, techniques must be acquired and employed
assiduously in order to ensure that contamination does not occur. Sterile transfer
refers to the techniques used to move bacteria from single colonies or from a pure
culture source to another culture media or test tube.

With any type of microbiology technique (i.e. working with and culturing bacteria), it is
important not to introduce contaminating bacteria into the experiment. Because
contaminating bacteria are ubiquitous and are found on fingertips, bench tops, etc., it
is important to avoid these contaminating surfaces. When working with a
microorganism, the round circle at the end of the inoculating loop and the surface of
the agar plate should not be touched or placed onto contaminating surfaces. Proper
sterile technique will also help ensure safe microbiological practice. Use the following
guidelines for all transfers of bacteria during this laboratory course.
1. The inoculating loop and needle generally are used for making transfers. The
same methods apply to both.

Source: http://www.antonides.nl/en/entogen.htm

2. To transfer a culture, flame the loop until it glows. Allow the loop to cool
sufficiently so that the organisms picked up by the loop will not be killed (10-15
seconds).

3. After the transfer is completed, sterilize the loop carefully. To avoid splattering
infectious material, first dry and gradually burn the material in the inner (cooler)
zone of the flame.

4. Flame briefly the mouth of test tubes, flasks, etc. both before and after each
transfer. Learn how to remove and replace screw caps or metal caps with the
same hand that holds the loop. (Your TA will demonstrate this elegant
technique.)

5. Work while seated, avoid unnecessary movement and confine manipulations to

the vicinity of the flame.

6. Practice and attention to detail will allow you to work both rapidly and
accurately. (If you get an opportunity, observe Jan West’s masterful technique.)
C. TYPES OF CULTURE MEDIA

A satisfactory culture medium must contain sources of carbohydrate, nitrogen,

inorganic salts and, in certain cases, vitamins or other growth factors. These may be
supplied by meat infusions, beef extract, and peptone. Certain bacteria require the
addition of other substances, such as serum and blood. Carbohydrates may be
desirable at times. Dyes may be added as indicators of metabolic activity or because
of their selective inhibitory powers.

1. Broths (liquid) – maintain growth of most organisms

2. Agar (solid) – culture plates, slant or stab tubes

3. Kinds of media:

• General purpose – agar or broth. Supports growth of most organisms, e.g.,

nutrient agar and trypticase soy agar
• Enriched media - Certain organisms require growth- promoting ingredients
such as blood, glucose, serum, egg, etc. The media containing ingredients
which enhance their growth-promoting qualities are enriched media, e.g.
blood agar, chocolate agar and Loeffler medium.
• Enrichment media - Enrichment media are liquid media containing chemical
constituents which inhibit some normal flora and allow pathogens, which may
be present in very small number in the specimen, to grow unhampered and
thus enriching them. Isolated colonies of these organisms may be obtained by
subculturing onto solid media. An example of enrichment media is selenite
broth used for primary isolation of enteric bacteria.
• Differential - usually agar. Differential media have chemical constituents
which characterize different bacteria by their special colonial appearances in
the culture e.g. MacConkey agar contains lactose as a substrate and neutral
red as an indicator. Bacteria fermenting lactose produce acid and this will
change the color of the indicator and thus the colonies will turn red. The red
lactose fermenting colonies can be differentiated from the pale non-lactose
fermenting colonies.

Examples of agar plates used for culturing bacteria.

Source: http://www.netpath.net/~billbsr/Page26.htm
 Frau Hesse and the Use of Agar in Microbiology

Fanny Angelina Eilshemius was born in 1850 in New York to a wealthy Dutch immigrant family. As
a young woman, she toured Europe. While in Europe, she met and married a German doctor,
Walther Hesse (1874). Dr. Hesse joined Robert Koch's laboratory in 1881 to study the new
science of microbiology. Frau Hesse, nicknamed Lina, was a talented artist and drew illustrations
for her husband's publications.

At that time, gelatin was used as a solidifying agent for microbiological media. Gelatin plates
frequently melted on hot days and many of the bacterial isolates digested (liquified) the gelatin.
According to Wolfgang Hesse (ASM News Vol 58#8 p. 425-428, 1992), a descendant of Dr. and
Frau Hesse, Dr. Hesse asked his wife why her jellies and puddings stayed solid in warm weather
when his microbiological media did not. She told him that she used agar-agar as a solidifying
agent when she made jellies in hot weather. She had learned of agar-agar as a youngster in New
York from a Dutch neighbor who had immigrated from Java. Dr. Hesse subsequently tried agar as
a solidifying agent in bacteriological media and found that it worked wonderfully. Since then, agar
has been the standard solidifying agent for microbiology.
D. SLIDE PREPARATION
In order to examine the bacteria in mixed or pure culture under the light microscope, it
is necessary to fix them to a glass slide and stain them.
1. Cleanliness of Slides. Microscope slides must be clean and free from grease
so that satisfactory films may be obtained. A clear slide will permit a drop of
water to spread into a thin film.
2. Division of Slides. For convenience, several films may be prepared and stained
on one side. Use a glass-marking pencil to divide the slide into sections on the
side opposite to that on which the films are to be made.
3. Cultures in Fluid Media. Transfer one or more loopfuls of the material to a slide
to form a film about a centimeter in diameter. Deposit and spread the material
gently to minimize disruption of cellular arrangements.
4. Cultures on Solid Media. Transfer one or more loopfuls of tap water to the
slide. Using the loop or the inoculating needle (the latter is preferred), transfer
a small portion of the bacterial colony or culture fluid next to the drop of water.
You should not see lumps of material on the slide. Mix the two and spread
gently to form a thin film.
5. Fixation. Place slid on dryer until it is completely dry. This fixes the organisms
to the slide and kills them.
Practice is required to prepare films that are suitable for staining and microscopic
examination (see below). A satisfactorily stained preparation will allow examination of
individual cells and of cellular arrangements. The usual error involves films which are
too thick, so that only clumps and masses of material are seen and cellular
morphology is obscured. Keep in mind that a single loopful of a heavy broth culture
will contain 106 -107 organisms.
E. ISOLATION OF MICROBIAL SPECIES IN PURE CULTURE

Bacteria are rarely found as pure cultures in nature. Many clinical samples submitted for
analysis to the microbiology laboratory are mixtures of organisms. Within these mixtures may
reside both pathogenic (disease-causing) and non-pathogenic bacteria. An organism must
first be isolated and grown in pure culture before it can be studied accurately. Plate cultures
offer a means of isolating pure species of organisms in a simple manner. The following two
methods are generally used: 1) the streak-plate method, and 2) the selective inhibition
method.

Streak-plate Method -- This method is used most commonly.

a. The inoculum is transferred to an area near the periphery of the plate.

b. The inoculum is spread back and forth over the surface of about one-fourth of
the plate.

c. The plate is now turned 90°.

d. Sterilize the loop in the flame, allow it to cool, and touch it to the last streak on
the original inoculum.
e. With this diluted inoculum, streak the second quadrant of the plate and, without
flaming the loop, continue streaking the third and then the fourth quadrant of the
plate (this procedure will be demonstrated).

f. Invert the plate and incubate.

Isolated colonies of bacteria will appear on the surface of the agar after incubation.
Each colony usually represents the growth from a single organism. A pure culture
may be obtained from a well-isolated colony by transferring a portion with a wire
needle to an appropriate culture medium.
F. SEPARATION OF ORGANISMS FROM MIXED CULTURES

Separation of different bacteria in a mixed population is accomplished by spreading an

appropriate sample of the mixed culture on the surface of a solid agar medium in such
a way that the individual bacteria are well separated from one another. If the medium
is nutritionally adequate, the isolated bacterial cells will multiply in a fixed position to
form a clone composed of the progeny from a single organism. The clone or colony
produced is usually of sufficient size to be visible.

When a particular organism is sought, it is often possible to use a medium or

conditions which will support the growth of the organism in question and at the same
time prevent the growth of undesired bacteria by exclusion of required nutrients or by
addition of selective inhibitors or both. This principle will be illustrated in this
laboratory exercise through the use of the following media:

1. Blood agar plates (BAP), a general medium.

2. Columbia CAN Agar (CNA), contains colistin and nalidixic acid, which inhibit
DNA replication of Gram-negative organisms.

3. MacConkey agar (Mac), a selective and differential medium. MacConkey agar

is selective in that it inhibits the growth of Gram-positive organisms due to the
presence of bile salts and crystal violet. It is differential in that Gram-negative
bacilli that ferment lactose (coliform bacilli) produce red colonies and non-
lactose fermenters produce colorless colonies. The basis for this differentiation
is that when coliforms ferment lactose (the only carbohydrate in the medium),
acid is produced in the area of the colony. The acid precipitates the bile salts
and allows absorption of neutral red (pH indicator in medium), which turns red
to purple at acid pH.

4. Mycosel medium contains cycloheximide and chloramphenicol, which inhibit

protein synthesis in prokaryotic cells. Growth of non-pathogenic fungi and
bacteria are inhibited. These inhibitors do not penetrate pathogenic fungi and
yeast cell walls.

G. GRAM STAIN

Of the staining techniques routinely employed in diagnostic microbiology, the Gram

stain is most widely used. The principle of this stain has remained unchanged since
the procedure was devised in 1884 by Dr. Christian Gram. The technique, however,
has been modified over the years in keeping with improvements in the qualities of
dyes.

Bacteria either retain the primary stain crystal violet (which has a bluish-purple color)
in this procedure, and are designated Gram-positive, or they retain only the second
dye, usually safranin (reddish pink) and are designated as Gram-negative.
Briefly, Gram-positive organisms have cell walls whose permeability properties are
changed by exposure to alcohol or acetone, so that the crystal violet-iodine complex is
not removed by subsequent washings with this solvent for short periods of time.

In the case of Gram-negative bacteria, the crystal violet, in combination with iodine,
stains the cell, but because of the nature of the cell wall, the alcohol or acetone
treatment removes the stain. The colorless cells are then stained with a dye of a
different color.

Because of changes that occur in the walls of bacteria as a result of aging and death,
old or dead Gram-positive cells may stain Gram-negative. This is especially true of
certain species of Gram-positive bacteria and may occur in clinical specimens from
patients who have been given antibiotic therapy.

Gram + Cell Wall Gram - Cell Wall

 Gram Stain Procedure

Reagents:

Primary Stain: Crystal violet solution

Mordant: Iodine and potassium iodine solution
Decolorizer: 95% ethyl alcohol and acetone, 1:1
Counter Stain: Safranin O solution

Method: Paine and Thomas (1963). Gram staining without the clock.
New England Journal of Medicine 269: 941.

a. Do not look at the clock.

b. Apply crystal violet to the heat-fixed smear. Allow stain to remain on

the slide only as long as it takes to replace the bottle of stain.
Immediately wash the slide with tap water.

c. Flood the slide with Gram's iodine solution, leaving the iodine on the
slide only as long as it takes to replace the bottle. Immediately wash the
slide with tap water.

d. Apply 4 or 5 drops of decolorizer (alcohol or acetone) and immediately

wash the slide with tap water. Apply the decolorizer carefully and do not
try to decolorize the thick parts of the smear. The thin areas of the
smear, which are the only areas that can be examined satisfactorily, will
then be over-decolorized.

e. Apply safranin, leaving the stain on the slide only as long as it takes to
replace the bottle, and immediately wash the slide with tap water. Blot
the slide dry (do not wipe!) and examine microscopically.

Although the Gram stain is easy to perform, several factors influence the results, e.g.
thickness of the film, amount of decolorization, and the age of the culture employed.
Constant practice is required in order to obtain consistent results. This is the most
important skill you will master in this course. Take the time to learn it well.

Gram + Gram –
 Steps in Gram Staining:

PURPLE PINK
(http://www.spjc.edu/hec/vettech/vtde/ATE2639LGS/Gramstain.htm)
 Gram Stain and General Microscopy
Troubleshooting Guide
Problem
Slide is almost focused, but is Inadequate or no
hazy or unclear
Slide is clear under 10X but
cannot be focused under oil
Organisms appear to have a
halo around them or cannot
be focused
Slide is in focus, then a
shadow appears
Floating material is seen
Nothing is visible on the slide
Excessive deposits of stain
crystals on slide
Gram positive organisms
have odd angular shapes
Gram negative organisms
look more like needles than
rods
Specimen appears mixed with
Yeast growing in the
large oval cocci when it
staining reagents
should be one organism
Gram positive cells appear
Gram negative
Gram negative organism
appears Gram positive
Mixture of Gram positive and
Gram negative organisms
from a pure culture
Probable Cause Corrective Action
There should be enough oil to make a seal
immersion oil between the slide and the 100X objective
Slide is upside down Check slide and flip over if necessary
Oil was added before Make a new slide. Allow slide to dry longer
slide was dry or blot more carefully before adding oil
Air bubble trapped by
Rotate objective back and forth
lens
Objective scraped the Clean objective; gently remove oil from
smear slide and refocus more carefully
Smear too thin Remake with a thicker smear
Slide wiped to dry it Blot slides; never wipe
Smear was not heat
Repeat; heat fix the smear
fixed
Inadequate rinsing Repeat; wash off one stain completely
between steps before adding the next
Repeat: Do not allow dye to dry on the
Viewing crystal violet
slide. Do not allow crystal violet to remain
crystals
on slide longer than suggested times
Viewing safranin
crystals
Repeat after the instructor has filtered the
reagents
Overdecolorization Decrease decolorizartion time
Non-viable organisms Use a fresher culture
Underdecolorization Increase decolorization time
Smear is too thick Remake smear
H. ANTIBIOTIC SENSITIVITY TESTING

The simplest and most widely used method for antibiotic susceptibility testing is the
antibiotic disc agar diffusion procedure. Sterile filter paper discs impregnated with
specific concentrations of various anti-microbial agents are placed on an agar plate
which has been inoculated with the culture to be tested. After overnight incubation,
zones of growth inhibition appear around the disc. The size of the inhibition zones
depends upon:

1. Diffusibility of the antibiotic

2. Test conditions
3. Inherent susceptibility of the test strain

Under very specific standard conditions the size of the zone can be used as a
measurement of the susceptibility of the strain. The Kirby-Bauer standard method is
based on the comparison of zone sizes with quantitative dilution tests as well as a
correlation with achievable drug levels for each antibiotic (Bauer, Kirby, Sherris, and
Turk [1996], Am. J. Clin. Path. 45: 493).

I. ACID FAST STAIN

Acid fast cells contain mycolic acid (a saturated fatty acid) in the cell walls.
Mycobacteria and certain species of Actinomycetes resist staining with ordinary dyes.
Moreover, these organisms, once stained, retain the dye with great tenacity, so that
treatment with alcoholic mineral acid fails to remove the dye. Non-acid-fast bacteria
are readily decolorized by the acid-alcohol. Kinyoun's method eliminates the use of
heat, which is required by the Ziehl-Neelsen technique. You will use Kinyoun method
as given below:

Reagents
ƒ Basic fuchsin 4.0 gm
ƒ Phenol crystals 8.0 gm
ƒ Alcohol, 95% 20.0 ml
ƒ Distilled water 100.0 ml

Method
a. Prepare the smear in the usual manner.
b. Flood with basic fuchsin and allow to sit undisturbed for 5 minutes;
rinse with water.
c. Flood with acid alcohol and allow to sit undisturbed for 2 minutes;
rinse with water.
d. Apply the methylene blue counterstain to the slide and allow to sit
for 1 minute; rinse with water.
e. Air dry (DO NOT BLOT) and examine under microscope.
 Acid-fast bacteria will appear as bright red organisms against a blue background.

This is an acid fast stain of Mycobacterium tuberculosis (MTB).

Note the red rods— hence the terminology for MTB in histologic
sections or smears: acid fast bacilli.

Microscopically, Mycobacterium avium intracellulare

infection is marked by numerous acid fast organisms
growing within macrophages. Lots of bright red rods are
seen, particularly in macrophages, in this acid fast
stain of lymph node.

J. DEMONSTRATIONS OF SPECIFIC STAINS AND MICROSCOPES

There are a number of specific stains that are useful in a clinical microbiology
laboratory in order to identify specific structures of a microorganism. Various bacterial
structures may be visualized in the light microscope with the use of specific staining
techniques. Certain of these structures are of importance in the identification of
pathogens.
1. CAPSULE STAIN
Many microorganisms secrete a highly viscous gum-like material which adheres
to the cell wall. In some instances, as in the Pneumococci, the presence of a
capsule is associated with virulence. The chemical structure of the capsule is
often used as a basis for classification within a bacterial species.

2. SPORE STAIN
Spores are formed primarily by species of Bacillus or Clostridium and survive
harsh environments, remaining dormant for many years, until conditions for
growth become more favorable. Spores are able to withstand periods of
desiccation, high temperature, UV light, toxic chemicals, and a lack of suitable
nutrients. Because a spore’s outer coat is an effective barrier to chemicals,
spores generally stain poorly. However, the use of very hot dyes apparently
allows the coat to expand and allow the dye to permeate.
3. FLAGELLA STAIN
Flagella are long filamentous appendages on organisms classified as non-
fermenters. They are too thin to be seen by ordinary microscopy unless heavily
coated with special stains containing a precipitating agent such as tannic acid,
which make the flagella thicker and more visible.

4. CELL WALL STAIN

The bacterial cell wall is a fairly rigid structure that preserves the integrity of the
protoplast. It plays an important role in metabolism and cell multiplication. In
this stain, the normally negative bacterial cell surface is made positive by
treating it with a cationic surface agent. The positive cell surface will now bind a
negatively charged chromophore of an acidic dye. The cell wall stain indicates
the presence of cell walls, which is important in the identification of bacterial
species that lack a cell wall (mycoplasmas).
5. METHYLENE BLUE STAIN
Useful in the identification of bacteria such as Corynebacteria that contain
inclusion bodies, also called metachomatic granules. Bacteria will stain a light
blue, and metachomatic granules, if present within the bacterial cell, will stain a
deeper blue.

6. DARKFIELD
Darkfield microscopy is used to examine living microorganisms that are mostly
invisible with a brightfield microscope. It uses a special condenser and
reflected light so the specimen appears bright against a black background. This
type of microscopy is particularly useful for primary syphilis identification in
exudate.
III. PROCEDURE
Day 1
Step 1 Teaching assistants will demonstrate and answer your questions regarding:
a. Standard operation of the microscope.
b. Sterilization of an inoculating loop with an open flame.
c. Transfer of cultures from tube to plate and how to streak plates for
isolation of colonies.
Step 2 Isolate colonies from pure cultures of microorganisms (Work independently)
Pure cultures of representative microorganisms in broth and agar will be
provided: Escherichia coli (labeled E. coli), Staphylococcus aureus (labeled S.
aur), Candida albicans (labeled C. alb).

a. Streak the E. coli, S. aureus, and C. albicans for isolation on a

individual blood agar plates (BAP). Use a different plate for each
organism! Invert labeled plates and place in containers at the end of
the tables for overnight incubation at 37|C.

b. Prepare a Gram stain from each of the three organisms from the pure
broth culture. Follow the Gram Stain protocol on page 30 and record
your results on page 37.

Candida albicans Staphylococcus aureus

Budding structures (arrows) Gram (+) cocci in clusters

Escherichia coli
Gram (-) bacilli
 Colony Morphology Gram Stain Microscopic
on BAP/Size, Color Gm + = purple Morphology
Gm - = pink
1. E. coli

2. S. aureus

3. C. albicans

Step 3 Gram stain each of the three organisms growing on the trypticase agar plates
(Work independently)

Step 4 Isolate individual colonies from a mixed culture of microorganisms

(Work independently)
A mixed culture consisting of Escherichia coli, Staphylococcus aureus, and
Candida albicans will be provided. Four types of media will be supplied: Blood
agar, Coumbia CNA agar (CNA), MacConkey agar, and Mycosel agar.

http://www.indstate.edu/thcme/micro/basic.html

a. Streak the mixed culture on the different media plates supplied using the
streak dilution method and incubate at 37°C for 18 hours.

b. Make a smear of the mixture and stain according to the Gram Stain protocol.
Identify the different morphologies and their staining characteristics.

Step 5 Antibiotic Sensitivity Testing (work in pairs; 1 large plate per two students)
Dilutions (1:100) of broth culture of various organisms will be provided for
completion of the antibiotic sensitivity testing.
a. Using a sterile cotton swab, inoculate a large agar plate with the diluted
culture. Streak the surface evenly and completely in three directions to
 provide a solid lawn of growth. You should make sure the surface of the
agar is completely covered with the organism.

b. Place antibiotic discs on the agar with an automatic disc dispenser. Note:
Be sure to use the appropriate set of discs for Gram-positive and Gram-
negative organisms. Seat the discs firmly into the agar with forceps that
have been flamed and cooled. Be careful not to scar the agar. It will not
require force to seat the discs into the agar. Label plates. Leave at the
table to be picked up and incubated overnight.

Day 2
NOTE: It is essential that you master the Gram stain and streak-plate isolation methods.
They are necessary in order to identify unknowns that will be coming up in future laboratory
sessions. If you are still having trouble staining the microorganisms, ask your TA for help and
keep trying.

Step 1 Verify that isolated colonies were obtained on plates streaked from cultures.
• Record notes about the colonies. Are they large, small, pinpoint?
• Do they have a particular color? Do they produce an odor?
• Did they grow on some plates, but failed to grow on others?
• Are they dull or shiny in appearance?
• Are they raised on the plate or are they flat?

Step 2 Use an isolated colony to perform a Gram stain for each organism.
• Record whether the organism is Gram positive or Gram negative.
• What shape are the microorganisms?
• Do they form clusters, or are they spread out across the field?

Step 3 Evaluate organisms growing on the different media plates by performing a

Gram stain for each.
• Record observations about the colonies growing on the different types of
media on page 40. Also, complete the chart on p. 40 regarding selective
and differential media.

Step 4 Interpret the antibiotic sensitivity plates

• Measure the diameter (in mm) of the clear zone surrounding the antibiotic
disc.
• The chart on page 41 will provide the information necessary to determine
whether the organism is resistant, intermediate, or sensitive to the
antibiotic.
• Record your results on pages 42 and 43.
 S. aureus exhibits different sensitivities to antibiotics.
http://www2.austin.cc.tx.us/microbugz/48antibio.html

Step 5 Perform an Acid Fast Stain on the sample provided

a. A slide from a clinical specimen will be provided. Perform acid-fast staining

by the Kinyoun method (as described on page 33).
b. Examine your slide for the presence of acid-fast organisms, and compare
your results with others at your table.

Step 6 Demonstration of specific stains.

Demonstration slides will be set up on microscopes in the center of the

laboratory. Observe each staining technique and record your findings in the
table on page 44.
 Selective Media
Media: Contains: Inhibits: Selects for: Differentiates Mechanisms of
between: differentiation:

1. PEA

2. MacConkey

3. Mycosel
 INTERPRETATION OF DISK SUSCEPTIBILITY TESTING
Measure the diameter of the clear zone surrounding the antibiotic disc to the nearest mm. The
following chart will provide necessary information as to whether it is sensitive or resistant.

Antibiotic Disc Potency Resistant Intermediate Sensitive

Amikacin (AN) 30 μg 14 15-16 17

Amoxacillin/Clavulanic
Acid (AMCL) 30 μg 19 ------- 20 or more

Ampicillin (AM)
Gram-negative and
and Enterococci 10 μg 11 or less 12-13 14 or more
Staphylococci 20 or less 21-28 29 or more
Others 11 or less 12-21 22 or more

Cefotaxime (CTX) 30 μg 14 15-22 23

Cefotetan (CTT) 30 μg 12 13-15 16 or more

Ceftazidime (CAZ) 30 μg 14 15-17 18

Cephalothin (CF) 30 μg 14 or less 15-17 18 or more

Clindamycin (CC) 2 μg 14 15-20 21

Erythromycin (E) 15 μg 13 23

Gentamicin (GM) 10 μg 12 or less 13-14 15 or more

Imipenem (IPM) 13 14-15 16

Levofloxacin (LVX) 5 μg 13 14-16 17

Minocycline (MI) 30 μg 14 15-18 19

Oxacillin (OX) 10 11-12 13

Penicillin-G (P)
Staphylococci 10 μg 20 or less 21-28 29 or more
Other organisms 10 μg 11 or less 12-21 22 or more

Piperacillin/Tazobactam (PIP) 17 μg 17 18-20 21

Sulfa/Trimeth (SXT) 30 μg 12 or less 13-16 17 or more

Tobramycin (NN) 10 μg 12 15

Vancomycin (VA) 30 μg 8 or less 9-11 12 or more

 ANTIBIOTIC SENSITIVITY RESULTS

Gram-Positive Organisms

Sensitive = S Intermediate = I Resistant = R

ORGANISM
Antibiotic S. aureus S. epidermidis Enterococcus

AMCL

CF

CC

LVX

OX

MI

SXT

VA
 ANTIBIOTIC SENSITIVITY RESULTS

Gram-Negative Organisms

Sensitive = S Intermediate = I Resistant = R

ORGANISM
Antibiotic Pseudomonas E. coli Klebsiella

AN

AM

CTX

CTT

CAZ

GM

IPM

LVX

SXT

PIP

NN
Record your observations of the demonstrations here (use drawings)
Staining Technique Microscopic Appearance Organism Demonstrated

1. Capsule Stain

2. Spore Stain

3. Flagella Stain

4. Cell Wall Stain

5. Methylene Blue Stain

6. Darkfield

7. Phase contrast
QUESTIONS YOU SHOULD BE ABLE TO ANSWER:
1. Why is the Gram stain said to be a differential stain?

2. What are the differences between a Gram-positive and a Gram-negative cell wall?

3. Can a Gram-positive cell ever stain Gram-negative?

4. What are some different stains that can be used to help identify an organism?

5. How might a physician use antibiotic sensitivity when treating a patient?

6. Why should one not lay an inoculating loop on the table?

7. Why is it important to use aseptic transfer techniques in a clinical microbiology

laboratory?

“Typhoid Mary’s Gallstones To Blame”

American Society for Microbiology General Meeting, Florida, May 2001.

Nearly 100 years ago at least 10 outbreaks of typhoid fever in New York City were traced to the apparently health cook,
Mary Mallon. Typhoid Mary, as she later became known, may have been an efficient reservoir for the disease because her
gallstones were coated with typhoid bacteria, researchers told this week’s annual meeting of the American Society for
Microbiology in Orlando, Florida.

Thanks to improved sanitation and antibiotics, typhoid fever, which is caused by the bacterium Salmonella typhi, is no longer
common in the developed world. Where it does occur, it causes fever and sometimes death if untreated.

Between 3 and 5% of those infected show no signs of illness and become carriers of the disease. The reason for their
amazing ability to harbor and pass on the bacteria, sometimes for years, had until now been a mystery.

One thing carriers often have in common is gallstones. So microbiologist Angela Prouty took sterile gallstones from patients
at the University of Texas Health Science Center in San Antonio, where she works, and infected them with S. typhi. The
bacteria rapidly formed tough biofilms over the surface of the gallstones.

Bacteria in a biofilm are tightly bound to one another and to the surface they coat. Tenacious biofilms of S. typhi on
gallstones could explain why some people harbor the disease so well, says Prouty. “It’s a very good way for the bacteria to
keep put and not get washed away,” she says.

Bile and gallstones were essential to induce S. typhi to form biofilms. Say Prouty: “We tried it with pebbles and it didn’t
work.” The bacteria’s predilection for the gallbladder makes good evolutionary sense. Bile aids digestion by draining from
the gallbladder into the lower intestine. ”The bacterium can shed itself back into the environment,” say Prouty.

Now Prouty needs to demonstrate the presence of S. typhi in biofilms of the gallstones of infected patients, says Salmonella
microbiologist Brad Cookson of the University of Washington in Seattle. “But it’s a step towards understanding the
interaction and its significance,” he says.

Even if there had been antibiotics back in Typhoid Mary’s day they would have been of little use. Most antibiotics do not get
to the gallbladder, and those that do can rarely break of biofilms. “The only way to get rid of the bacteria is to get rid of the
gallbladder,” Prouty says.

http://www.nature.com/nsu/010524/010524-12.html