SUMMER

MIDTERM MICROBIOLOGY (LABORATORY)

Terminology
MICROBIOLOGY
 The study of microscopic organisms (MICROORGANISM), such as bacteria, viruses, archaea, fungi and protozoa.
 This discipline includes fundamental research on the biochemistry, physiology, cell biology, ecology, evolution and clinical aspects of
microorganisms, including the host response to these agents.

Parasitology
 The scientific discipline concerned with the study of the biology of parasites and parasitic diseases, including the distribution,
biochemistry, physiology, molecular biology, ecology, evolution and clinical aspects of parasites, including the host response to these
agents.
Applied microbiology
 a scientific discipline that deals with the application of microorganisms and the knowledge about them. Applications include
biotechnology, agriculture, medicine, food microbiology and bioremediation.

Virology
 The scientific discipline concerned with the study of the biology of viruses and viral diseases, including the distribution, biochemistry,
physiology, molecular biology, ecology, evolution and clinical aspects of viruses.

Microorganism or microbe
 A microscopic organism, which may be single-celled or multicellular.

Microscopy
 The technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye (objects that are
not within the resolution range of the normal eye.
 Microscopy is the science of investigating small objects and structures using such an instrument.
USING METRIC SYSTEM TO EXPRESS THE SIZES OF MICROORGANISM

METRIC SYSTEM
 Used to express the sizes of microorganism
 Primarily micrometer SI symbol: μm (micron) and nanometer

Meter= is the basic unit of meter system

1 meter= 39.4 inches
10 (101) = decimeters
100 (102) = centimeter
1000 (103) = milliliters
1 million (106)= micrometers
1 billion (109) = nanometers

Example Between 1 μm and 10 μm:

 1–10 μm – length of a typical bacterium

 10 μm- Size of fungal hyphae
 5 μm – length of a typical human spermatozoon's head
 3–8 μm – width of strand of spider web silk
 about 10 μm – size of a fog, mist or cloud water droplet

 10 to 55 μm – width of wool fibre

 17 to 181 μm – diameter of human hair
 70 to 180 μm – thickness of paper

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  The sizes of bacteria and protozoa are usually expressed in terms of MICROMETERS.
For example; spherical bacterium (coccus; pl. cocci) =1 um in diameter.
Example: rod-shaped bacterium (bacillus;pl.bacilli) = about 1 um wide x 3 um long
 The size of viruses are expressed interms of nanometers.
 The most of the viruses that cause human disease range in size from about 10 to 300 nm
Example; Ebola Virus = cause hemorrhagic fever, 1000nm (1um)
 Some very large protozoa reach a length of 2000 um (2mm)

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OCULAR MICROMETER
 Used to measure microorganism in Microbilogy laboratory,
 A tiny ruler within the eyepiece (ocular) of the compound light microscope
 a glass disk that fits in a microscope eyepiece that has a ruled scale, which is used to measure the size of magnified objects. The physical
length of the marks on the scale depends on the degree of magnification.
 It must be calibrated before using it.
 It can be used to measure lengths and widths of microbes and other objects in specimen slide

Relative Sizes of microorganism

ORGANISM DIMENSION APPROXIMATE SIZE (um)

VIRUSES (MOST) DIAMETER 0.01-0.3
Bacteria
 Cocci (spherical bacteria) Diameter Average =1
 Bacilli (rod -shaped bacteria) e.g , Escherichia coli (width x length) Average =1x 3
Filaments (width) 1
Fungi
 Yeasts e.g Candida albicans (diameter) 3-5
 Septate hyphae (hyphae with cross-walls) width 2-15
 Septate hyphae 9hyphae without cross- width 10-30
walls)
Pond water protozoa
 Chlamydomonas Length 5-12
 Euglena Length 35-55
 Vorticella Length 50-145
 Paramecium Length 180-300

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  Volvox Diameter 350-500
 Stentor Length (extended) 1000-2000

MICROSCOPES
 An instrument used to see objects that are too small to be seen by the naked eye.
 The human eye, a telescope, a pair of binoculars, a magnifying glass, and a microscope can all be thought of as various type of optical
instruments.
 Used to observe tiny objects that cannot be seen at all with the unaided human eye.
Microscopic
 Means invisible to the eye unless aided by a microscope.
 The scale of objects and events smaller than those that can easily be seen by the naked eye, requiring a lens or microscope to see them
clearly.

RESOLVING POWER OR RESOLUTION

 A limit to what can be seen using optical instrument

TYPES OF MICROSCOPES

1. SIMPLE MICROSCOPES
 Containing only one magnifying lens.
 Also called magnifying glass.
 It is actually a convex lens of small focal length, which is used for seeing the magnified images of small objects.
 When using magnifying glass usually appear about 3-20 times larger than the object’s actual size.

Principle of Simple Microscope

A simple microscope works on the principle that when a tiny object is placed within its focus, a virtual, erect and magnified image of the object
is formed at the least distance of distinct vision from the eye held close to the lens.

Uses of Simple Microscope

Following are the important uses of simple microscope:

1. The simple microscope is commonly used by watch makers to see the magnified view of small parts of a watch.
2. It is also used by the jewelers to see the magnified view of the fine parts of jewelry.
3. Simple microscope is used to see the enlarged image of letters of a book, textures of fibers or threads of a cloth.
4. Simple microscope is used to see the magnified view of different particles of different types of soils.
5. It is used by palmists to see enlarged view of the lines of our hand.
6. Simple microscope is used by skin specialists to find out various diseases of skin.
7. It is also used to see the details of stamp and engravings.

Example, the microscope of Anton van Leeuwenhoek, believe that his simple microscope had a maximum magnifying power of about 300 x (300
times)

2.Compound Microscope
 A microscope that contains more than one magnifying lens.
 Usually magnifying objects about 1000 times.
PHOTOMICROGRPHS
o PHOTOGRAPH taken through the lens system of compound microscopes

Compound Light Microscope

o The compound microscope use from built –in light bulb (visible light )
o It is the wavelength of visible light 9apprximately 0.45 um) that limits the size of objects that can be seen using the compound light
microscope.
o Contains two magnifying lens system

OCULAR LENS = eyepiece or ocular use to magnify power of x10.

EARLY COMPOUND MICROSCOPES

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HANS JANSEN (1590-1595)
 Optician in Middleburg, Holland ,often credit for developing the first compound microscope.

ZACHARIAS
 Son of Hans Jansen,later took over production of the jansen microscope
 Jansen microscope contain two lenses and achieved magnification of only 3x to 9x.

MARCELLO MALPHIGI in Italy and ROBERT HOOKE in England

 Use three-lenses system compound microscope
 Both publisehed paoers between 1660- 1665 describing their microscopic finfings.
 1665 :The book called Micrographia ,Hooke described a fossilized shell of a foraminiferan – type of protozoan and two microscopic
fungi

PARTS OF COMPOUND MICROSCOPE AND FUNCTIONS

1. Eyepiece: The lens the viewer looks through to see the specimen. The eyepiece usually contains a 10X or 15X power lens.
2. Diopter Adjustment: Useful as a means to change focus on one eyepiece so as to correct for any difference in vision between your two
eyes.
3. Body tube (Head): The body tube connects the eyepiece to the objective lenses.
4. Arm: The arm connects the body tube to the base of the microscope.
5. Coarse adjustment: Brings the specimen into general focus.
6. Fine adjustment: Fine tunes the focus and increases the detail of the specimen.
7. Nosepiece: A rotating turret that houses the objective lenses. The viewer spins the nosepiece to select different objective lenses.
8. Objective lenses: One of the most important parts of a compound microscope, as they are the lenses closest to the specimen.
9. A standard microscope has three, four, or five objective lenses that range in power from 4X to 100X. When focusing the microscope, be
careful that the objective lens doesn’t touch the slide, as it could break the slide and destroy the specimen.
10. Specimen or slide: The specimen is the object being examined. Most specimens are mounted on slides, flat rectangles of thin glass.
11. The specimen is placed on the glass and a cover slip is placed over the specimen. This allows the slide to be easily inserted or removed
from the microscope. It also allows the specimen to be labeled, transported, and stored without damage.
12. Stage: The flat platform where the slide is placed.
13. Stage clips: Metal clips that hold the slide in place.
14. Stage height adjustment (Stage Control): These knobs move the stage left and right or up and down.
15. Aperture: The hole in the middle of the stage that allows light from the illuminator to reach the specimen.

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 16. On/off switch: This switch on the base of the microscope turns the illuminator off and on.
17. Illumination: The light source for a microscope. Older microscopes used mirrors to reflect light from an external source up through the
bottom of the stage; however, most microscopes now use a low-voltage bulb.
18. Iris diaphragm: Adjusts the amount of light that reaches the specimen.
19. Condenser: Gathers and focuses light from the illuminator onto the specimen being viewed.
20. Base: The base supports the microscope and it’s where illuminator is located.

CHARACTERISTICS OF VARIOUS TYPES OF MIICROSCOPES

TYPE RESOLVING USEFUL CHARACTERISTICS

POWER MAGNIFICATION
BRIGHTFIELD 0.2000 um 1000 x  used to observe the morphology of organism such as
bacteria ,protozoa, fungi, and algae in living (unstained) and
nonliving (stained ) state.
 Cannot observe organism less than 0.2 um in diameter or
thickness such as spirochetes and viruses.
DARKFIELD 0.2000 um 1000x  Unstained organism are observed against a dark
background
 Useful for examining thin spirochetes
 Slightly more difficult to operate than brightfield
PHASE-CONRAST 0.2000 um 1000x  Can be used to observe unstained living microorganism
FLUORESCENCE 0.2000 um 1000x  Fluorescent dye attached to organism
 Primarily an immunodiagnostic technique 9
immunofluorescence
 Used to detect microorganism in cells ,tissues and clinical
specimens
TRANSMISSION ELECTRON 0.0002 mm (0.2 200,000x  Specimen is viewed on a screen
(MICROSCOPE (TEM) nm)  Excellent resolution
 Allows examination of cellular and viral ultrastructure
 Specimen is nonliving
 Reveals internal features of thin specimens
SCANNING ELECTRON 0.0200 mm (20 10,000x  Specimen is viewed in the screen
MICROSCOPE(SEM) nm)  Gives the illusion of depth (three-dimension)
 Useful for examining surface features of cells and viruses
 Specimen is nonliving
 Resolution is less than that TEM

3. ELECTRON MICROSCOPES
 USE AN ELECTROMN BEAM AS ASOURCE OF ILLUMINATION AND MAGNETS TO FOCUS THE BEAM
 a type of microscope that uses a beam of electrons to create an image of the specimen.
 It is capable of much higher magnifications and has a greater resolving power than a light microscope, allowing it to see much
smaller objects in finer detail.
 They are large, expensive pieces of equipment, generally standing alone in a small, specially designed room and requiring
trained personnel to operate them.

Types of Electron Microscopes

1. Transmission Electron Microscope (TEM)

 involves a high voltage electron beam emitted by a cathode and formed by magnetic lenses.
 The electron beam that has been partially transmitted through the very thin (and so semitransparent for electrons) specimen carries
information about the structure of the specimen.
 The spatial variation in this information (the "image") is then magnified by a series of magnetic lenses until it is recorded by hitting a
fluorescent screen, photographic plate, or light sensitive sensor such as a CCD (charge-coupled device) camera.
 The image detected by the CCD may be displayed in real time on a monitor or computer.
 produce two-dimensional, black and white images.

2. Scanning Electron Microscope (SEM)

 produces images by detecting secondary electrons which are emitted from the surface due to excitation by the primary electron beam.
In the SEM, the electron beam is scanned across the surface of the sample in a raster pattern, with detectors building up an image by
mapping the detected signals with beam position.
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 MAGNIFICATIONS ACHIEVED USING THE COMPOUND LIGHT MICROSCOPE

OBJECTIVE TOTAL MAGNIFICATION ACHIEVED

WHEN THE OBJECTIVE IS USED IN
CONJUCTION WITH A 310 OCULAR
LENS
X4 (Scanning objective 40x
X10 (low-power objective) 100x
X40 ( high-power objective) 400
X100 ( oil-immersion objective 1000x

Sample Preparation
Cryofixation - freezing a specimen rapidly, typically to liquid nitrogen temperatures or below, that the water forms ice. This preserves the specimen
in a snapshot of its solution state with the minimal of artefacts. An entire field called cryo-electron microscopy has branched from this technique.
With the development of cryo-electron microscopy, it is now possible to observe virtually any biological specimen close to its native state.

Fixation - a general term used to describe the process of preserving a sample at a moment in time and to prevent further deterioration so that it
appears as close as possible to what it would be like in the living state, although it is now dead. In chemical fixation for electron microscopy,
glutaraldehyde is often used to crosslink protein molecules and osmium tetroxide to preserve lipids.

Dehydration - removing water from the samples. The water is generally replaced with organic solvents such as ethanol or acetone as a stepping
stone towards total drying for SEM specimens or infiltration with resin and subsequent embedding for TEM specimens.

Embedding - infiltration of the tissue with wax (for light microscopy) or a resin (for electron microscopy) such as araldite or LR White, which can
then be polymerised into a hardened block for subsequent sectioning.

Sectioning - the production of thin slices of the specimen. For light microscopy, the sections can be a few micrometres thick but for electron
microscopy they must be very thin so that they are semitransparent to electrons, typically around 90nm. These ultra-thin sections for electron
microscopy are cut on an ultramicrotome with a glass or diamond knife. Glass knives can easily be made in the laboratory and are much cheaper
than diamond, but they blunt very quickly and therefore need replacing frequently.

Staining - uses heavy metals such as lead and uranium to scatter imaging electrons and thus give contrast between different structures, since many
(especially biological) materials are nearly "transparent" to the electron beam. By staining the samples with heavy metals, we add electron density
to it which results in there being more interactions between the electrons in the primary beam and those of the sample, which in turn provides us
with contrast in the resultant image
Freeze-fracture and freeze-etch - a preparation method particularly useful for examining lipid membranes and their incorporated proteins in "face
on" view. The fresh tissue or cell suspension is frozen rapidly (cryofixed), then fractured by simply breaking or by using a microtome while
maintained at liquid nitrogen temperature. The cold, fractured surface is generally "etched" by increasing the temperature to about -95°C for a few
minutes to let some surface ice sublime to reveal microscopic details.

Sputter Coating - an ultra-thin coating of electrically-conducting material, deposited by low vacuum coating of the sample. This is done to prevent
charging of the specimen which would occur because of the accumulation of static electric fields due to the electron irradiation required during
imaging. It also increases the amount of secondary electrons that can be detected from the surface of the sample in the SEM and therefore
increases the signal to noise ratio. Such coatings include gold, gold/palladium, platinum, chromium etc.

CONTROLLING MICROBIAL GROWTH IN VITRO

FACTORS THAT AFFECT MICROBIAL GROWTH

MICROBIAL GROWTH
 Affected by many different environmental factors, including the availability of nutrients and moisture, temperature, pH, Osmotic
pressure, barometric pressure and composition of atmosphere.

1. AVAILABILITY OF NUTRIENTS
 ANY NUTRIENTS ARE ENERGY SOURCES; ORGANISM will obtain energy from these chemical; ls by breaking chemical bonds.

NUTRIENTS

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  Serve as sources of carbon, oxygen, hydrogen, nitrogen, phosphorous and sulfur as well as other elements (e.g. sodium, potassium,
chlorine, magnesium, calcium and trace elements such as IRON, Iodine and zinc.) that are usually required in lesser amounts.
 About 25 of the 92 naturally occurring elements are essential to life.

2. MOISTURE
 Water is essential for life
 Cells consist where between 70 and 95% water.
 All living organism require water to carry out their normal metabolic processes and most will die in environments containing too little
moisture.
 There are certain microbial stages (e.g. bacterial endospore, and protozoan cysts) however, that can survive the complete drying process
(DESICCATION).

3. TEMPERATURE
 Every microorganism also has a minimum growth temperature, below which it ceases to grow and a maximum growth temperature,
above which it dies.

a. THERMOPHILES
 Microorganism that grow best at high temperature
 Organism that loves heat
 Found in hot springs, compost pit, and silage as well as in and near hydrothermal vents at the bottom of the ocean.
 Ex: thermophilic cyanobacteria = certain other types of bacteria and algae cause many of the colors observed in the near-boiling hot
springs
HYPERTHERMOPHILES = organisms that favor 100 degrees Celsius. ex. Pyrolobus fumarii.

PHILES = Meaning to love something (acidophiles ,alakaliphiles)

B. MESOPHILES
 Microbes that grow best at moderate temperature.
 Includes most of the species that grow on plants and animals and in warm soil and water.
C. PSYCHROPILES
 Prefer cold temperature. They thrive in cold ocean water.
 T high altitudes, algae (often pink). Can be seen living on snow.

PSYCHROTROPHS
 Refrigerator temperature (4 degrees Celsius): bread molds, for example)

PSYCHRODURIC ORGANISMS
 Microorganism that prefer warmer temperature, but can tolerate or endure very cold temperature and can be preserved in the frozen
state.

4. POWER OF HYDROGEN
 Refers to the acidity or alkalinity of a solution
 Microorganism prefer a neutral or slightly alkaline growth medium (Ph 7.0-7.4)
ACIDOPHILES = can live in the stomach and in pickled foods, prefer a Ph OF 2-5.
 Fungi prefer acidic environments.
ALKALIPHILES = prefer alkaline environment (ph greater than 8.5). found in the intestine (ph approximately 9.0) . Ex vibrio cholera = ph 8.

5. OSMOTIC PRESSURE AND SALINITY

Osmotic pressure
 The pressure that is exerted on a cell membrane by solutions both inside and outside the cell.
 The ideal situation is that the pressure inside the cell is equal to the pressure is that the pressure inside the cell is equal to the pressure of
the solution outside the cell.
Hypertonic
 When the concentration of solutes in the environment outside of the cell is greater than the concentration of solutes inside the cell.

OSMOSIS
 The movement of solvent through a permeable membrane, from a solution having a lower concentration of solute to a solution having a
higher concentration of solute.
CRENATION
 The loss of water causes the cell to shrink.

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PLASMOLYSIS
 The cell membrane and cytoplasm shrink away from the cell wall.
 Inhibits bacterial cell growth and multiplication.

HYPOTONIC
 When the concentration of solutes outside a cell is less than the concentration of solutes inside the cell, the solution in which the cell is
suspended

HEMOLYSIS
 If sufficient water enters, the cell will burst (lyes)

PLASMOPTYSIS
 If the pressure becomes so great that the cell ruptures

ISOTONIC
 When the concentration of solutes outside a cell equals the concentration of solutes inside the cell.

HALOPHILIC
 Microbes that actually prefer salty environment
 Halo= referring salt and philic= to love Ex. Vibrio cholera.

6. BAROMETRIC pressure
 Some thrive at normal atmospheric pressure (about 14.7 pounds per square inch or psi)

BASOPHILES baro= referring to pressure. Thrive deep in the ocean and in oil wells where the atmospheric pressure is very high. Ex: Archeans

7. GASEOUS ATMOSPHERE

a. OBLIGATE ANAEROBES
 killed by the presence of oxygen.
Capnophiles= require increased concentrations of carbon dioxide usually 5-10 % CO2.

b. OBLIGATE AEROBES
 prefer the same as atmospheric as human (about 20 -21 % O2 and 78 – 79% N2 with all other atmospheric gases combined representing
less than 1 %.)
 microaerophilic are also require oxygen, they require reduced concentration of o2. Around 5%.
________________________________________________________________________________________________

CULTURING BACTERIA IN LABORATORY

Instrument used in Culturing Bacteria

A. Compound Light microscope
B. Petri dishes contain solid culture media
C. Tubes containing liquid culture media
D. Bunsen burners
E. Wire inoculating loop
F. Bottles of staining reagents
G. incubators

A. BACTERIAL GROWTH
 REFERS INTO THE INCREASE in the number of organism rather than an increase in size.
 When organism reaches its size, it divides by binary fission.
Binary fission = (Bi meaning into two daughter cell. Each cells will split into half and become identical cells)

On Solid medium, in binary fission continuous through main generation until colony is produced.

BACTERIAL COLONY = a mound or pile of bacteria containing millions of cells.

GENERATIO Time = the time it takes for one cell to become two cells by binary fission. It varies from one bacterial species to another

E. coli, V. Cholerae, Staphylococcus and streptococcus

 Generation time is 20 minutes
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Pseudomonas and Clostridium = may divide every 10 minutes
M. tuberculosis = may divide only every 18 to 24 hours

Rapid growers = bacteria with short generation time.

SLOW GROWER = bacteria with long generation times

FACTORS THAT INFLUENCE THE GROWTH OF MICROORGANISM IN THE BODY, IN THE NATURE, OR IN LABORATORY
1. TEMPERATURE
2. pH
3. moisture content
4. available nutrients
5. characteristic of other organism present

 therefore, the number of bacteria varies with season, rainfall, temperature, and time of day.

FASTIDIOUS = HAVE COMPLEX NUTRITIONAL REQUIREMENTS. SPECIAL mixture of vitamins and amino acids or some organism may not grow in
artificial culture this include:
a. OBLIGATE INTRACELLULAR PATHOGENS= such as viruses, ricketssias and chlamydias
b. PROPAGATE OBLIGATE INTRACELLULAR PATHOGENS IN LABORATORY = inoculated in live animals, embryonated chicken eggs or cell
culture
c. Other organism that will not grow on artificial medium = Treponeman pallidum (syphilis) and Mycobacterium leprae (Leprosy)

B. CULTURE MEDIA
 USE IN LABORATORY TO CULTURE BACTERIA
ARTIFICIAL CULTURE OR SYNTHETIC
 USE in laboratory, occurs naturally prepared in laboratory
CLASSIFICATION OF CULTURE MEDIA
1. Chemically defined medium
 One in which all the ingredients are known: this is because the medium prepared in the laboratory by adding a certain number of grams
of each of components
 Ex: carbohydrates, amino acids and salts

2. COMPLEX MEDIUM
 One in which the exact contents are not known.
 Contain ground up or digested extracts from animal organs (Ex; heart, livers, brains.), fish yeast and plants, which provide the
necessary nutrients, vitamins, and minerals.

3. ENRICHED MEDIUM
 A broth or solid medium contain a rich supply of special nutrients that promotes the growth of fastidious organism
 Usually prepared by adding additional extra nutrients to a medium called nutrient agar.
NUTRIENT AGAR
a. BLOOD AGAR = nutrient agar plus sheep red blood cells
 Bright red, with hemoglobin
b. CHOCOLATE AGAR = nutrients agar plus powdered hemoglobin
 Brown, considered to be more enriched than blood agar because the hemoglobin is readily accessible
 Usually use to culture important, fastidious bacterial pathogens like Neisseria gonorrhea and H. influenza that will not grow in
Blood agar.

4. SELECTIVE MEDIUM
 Added inhibitors that discourage the growth of certain organism without inhibiting the growth of the organism being sought.
 Example;
a. mc Conkey agar = inhibits the gram-positive bacteria and selective for gram-negative bacteria,
b. Phenyethyl alcohol (pea) agar and colistin – nalidixix acid agar (CAN) = inhibit growth of gram-negative bacteria and selective positive
bacteria
c. Thayer –martin agar and Martin-Lewis agar =chocolate agar containing extra nutrients plus several antimicrobial agents. Selective for
Neisseria gonorrhea
d. MAnnitol agar (MSA) = only salt tolerated (haloduric) bacteria can grow.

5. DIFFERENTIAL MEDIUM
 Permits the differentiation of organism that grow in the medium
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  Example: MC CONKEY AGAR = frequently used to differentiate among various Gram-negative bacilli that are isolated from fecal
specimens. Gram- negative bacteria able to ferment lactose
 Produced pink colonies, whereas those unable to ferment lactose produce colorless colonies
 MANNITOL AGAR = USE TO SCREEN STAPHYLOCOCCUS aureus. I turns the original pink medium to yellow because of its ability
to ferment mannitol
 Blood agar is also a Differential medium because it used to determine the type of hemolysis (alteration or destruction of red
blood cells) that the bacterial isolate produces.

CATEGORIES OF CULTURE MEDIA

1. LIQUID MEDIA
 Also known as broth
 Contained in tubes and are often referred to as tubed media.
2. SOLID MEDIA
 Prepared by adding agar to liquid media and then pouring the media into tubes or petri dishes where the media solidifies.
 Bacteria ARE THEN GROWN ON THE SURFACE OF THE AGAR- CONTAINING SOLID MEDIA
 A COMPLEX POLYSACCHARIDES that is obtained from a red marine alga: it is used as a solidifying agent ,much like gelatin is
used as a solidifying agent much like gelatin is used as a solidifying agent in the kitchen.

BLOOD AGAR= Enriched and differential

Mc Conkey and MSA = selective and differential
PEA and CAN = enriched and selective
THAYER MARTIN AND MARTIN-LEWIS AGAR= highly enriched and highly selective

Thioglycollate Broth (THIO)

 A very popular liquid medium for use in the bacteriology laboratory
 Supports the growth of all categories of bacteria from obligate aerobes to obligate anaerobes

C. INOCULATION OF CULTURE MEDIA

 Culture media are routinely inoculated with clinical specimens

INOCULATION
 A liquid medium involves adding a portion of the specimen to the medium
 Inoculation of solid or plated medium involves the use of sterile inoculating loop to apply a portion of the specimen to the surface of the
medium:a process commonly referred as STREAKING
STREAKING= proper method of inoculating plated media to obtain well-isolated colonies

STERILE TECHNIQUES
 Practice when it is necessary to exclude all microorganism from a particular area, so that the area will be sterile.
 Contaminants = unwanted organism

INCUBATION
 After media is inoculated they must be incubated
INCUBATOR= a chamber that contains appropriate atmosphere and moisture level and set to maintain the appropriate temperature

1. Carbon Dioxide Incubator = an incubator top which a cylinder of CO2 is attached

2. Non-CO2 Incubator= an incubator containing room air thus, it contains about 20-21% O2
3. Anaerobic Incubator= an incubator containing an atmospheric devoid of oxygen.

D.BACTERIAL POPULATION COUNTS

 To determine the degree of bacterial contamination in drinking water, milk, and other foods
 The microbiologist determines the total number of bacterial cells in the liquid (the total number would include both viable and dead
cells.)
 Determine the number of viable (living) cells.
Instrument use:
1. spectrophotometer= a beam light is passed through the liquid.

E. BACTERIAL POPULATION GROWTH CURVE

 Any particular species of bacterium may be determined by growing a pure culture of the organism in a liquid medium at constant
temperature.

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FOUR PHASES
1. LAG PHASE
 THE FIRST PHASE OF THE GROWTH, DURING WHICH the bacteria absorb nutrients, synthesize enzymes and prepare for cell division.
 The bacteria do not increase in number during the lag phase.
2. LOG PHASE
 (logarithmic growth phase) or exponential growth phase
 The bacteria multiple rapidly that the number of organism doubles with each generation time.

3. STATIONARY PHASE
 The culture is at its greatest population density

4. DEATH PHASE (Decline phase

 The concentration of toxic waste products continues to increase and the nutrient supply decrease.
 The culture may die completely and the other organism continue to survive.

INHIBITING THE GROWTH OF MICROORGANISM IN VITRO

Terminology
STERILIZATION
 The complete destruction of all microorganism including cells, spores and viruses.

DISINFECTION
 The destruction or removal of pathogens from nonliving objects by physical or chemical methods
 Chemical agents also used to eliminate pathogens

Disinfectants = chemical used to disinfect inanimate objects.

Antiseptics = solutions used to disinfect skin and other living tissues

Sanitization= the reduction of microbial population to levels considered safe by public health standards such as those applied to restaurants.

1. MICROBICIDAL AGENTS
 CIDE- OR CIDAL = REFERS TO KILLING
GERMICIDAL agents= germicides
BIOCIDAL AGENTS = biocides
MICROBICIDAL AGENTS = microbicides

BACTERICIDAL AGENTS = BACTERICIDES, DISENFECTANTS THAT SPEcIFICALLY KILL BACTERIA BUT NOT NECESARRILY BACTERIAL ENDOSPORES.

SPORICIDAL AGENTS = kill bacterial endospore

FUNGICIDAL AGENTS= kill fungi
ALGICIDAL AGENTS = used to kill algae in swimming pools and hot tubs.
VIRICIDAL AGENTS= destroy viruses
PSEUDOMONICIDAL AGENTS = kills pseudomonas species
TUBERCULOCIDAL AGENTS= kills M. tuberculosis

2. MICROBISTATIC AGENTS
 A drug or chemical that inhibits growth and reproduction of microorganism

BACTERIOSTATIC AGENT
 Specifically inhibits the metabolism and reproduction of bacteria.

LYOPHILIZATION
 Process that combines dehydration and freezing
Lyophilized materials = frozen in vacuum

SEPSIS= Refers to the presence of pathogens in blood or tissues

ASEPSIS = the absence of pathogens

ANTISEPSIS = the prevention of infection
ANTISEPTIC TECHNIQUE = developed by Joseph LISTER, refers to the use of antiseptic

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3. STERILE TECHNIQUE
 Practice when it is necessary to exclude all microorganism from a particular area, so that the area will be sterile

USING PHYSICAL METHODS TO INHIBIT MICROBIAL GROWTH

1. HEAT
 The most practical, efficient and inexpensive method of sterilization of those inanimate objects and materials that can withstand high
temperature

TWO FACTORS
A. temperature
B. time

1. THERMAL DEATH POINT (TDP)

 Any particular species of microorganism is the lowest temperature that will kill all the organism in standardized pure culture within
specified period.
2. THERMAL Death Time (TDT)
 The length of time necessary to sterilize a pure culture at a specified temperature
Types of Heat
A. DRY HEAT
 Baking in thermostatically controlled oven provides effective sterilization of metals, glassware some powders, oils, and waxes.
Incineration = Burning, an effective means of destroying contaminated disposable materials.

B. MOIST HEAT
 Heat applied in the presence of moisture as in boiling or steaming, is faster and more effective than dry heat and can be accomplish at
lower temperature.

AUTOCLAVE = large metal pressure cooker that uses steam under pressure to completely destroy all microbial life.

2. COLD
 Most of the microorganism are not killed by cold temperature and freezing but their metabolic activities are slowed.
 Refrigeration merely slowed the growth of microorganism it does not completely inhibit

3. DESSICATION
 Foods been preserved by drying

4. RADIATION
 The sun is not a particularly reliable disinfecting agent because it kills only those microorganisms that are exposed to direct sunlight.
 The rays of the sun include the long infrared rays, the visible light rays, and shorter UV rays

5. ULTRASONIC WAVES
 Frequently use means of cleaning and sterilization of delicate equipment
 It consists of tanks filled with liquid solvent.

6. FILTRATION
 Filters of various pore sizes, used filter or separate cells, larger viruses, bacteria, and certain other microorganism from the liquid or gases
in which they are suspended.
7. Gaseous Atmosphere
 It is possible to inhibit growth of microorganism by altering the atmosphere in which they are located

USING CHEMICAL AGENT TO INHIBIT MICROBIAL GROWTH

1. DISINFECTANT
 The use of chemical agent to inhibit the growth of pathogens either temporarily or permanently.

2. ANTISEPTIC
 Reduce the number of organism on a surface, it does not penetrates pores and hair follicles to destroy microorganism residing there.
 Applied to the site of surgical incision to destroy local microorganism.

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