Combined Practical Exam & Final Exam Review

Test for fermentation of carbohydrates & aerobic/anaerobic respiration

● Labs 7 and 8: Differential (Biochemical) Tests: used for identification of bacteria by
analysis of different biochemical properties; know the basis for the tests, what they test &
how to interpret the tests, including any addition of reagents:
○ Tests for fermentation of carbohydrates & aerobic/anaerobic respiration:
■ O-F (oxidation-fermentation) Test: differentiates bacteria on the basis of
fermentative or oxidative metabolism of carbohydrates
● O-F medium contains glucose & peptone and a pH indicator
bromothymol blue – yellow @pH 6; green @pH 7.1; blue @ 7.6
● Two tubes of O-F media are stab-inoculated per test organism;
O-F is semi,solid, wire loop must be inserted into agar (¾ depth)
● Following inoculation, one tube is overlaid with oil (anaerobic); the
other tube does not have an oil overlay (aerobic tube).

■ Phenol Red glucose/sucrose/lactose: determines the ability of a

bacterium to ferment sugars.
● Fermentation → production of acidic end products; media turns
yellow due to the phenol red pH indicator.
● Non-fermentative bacteria → no color change (remains red) or
turns alkaline (pink color) due to degradation of peptone.
● Durham in the medium tests for gas production
● Will use phenol red broth to test for ability of bacterium to ferment
glucose, sucrose, & lactose.
 ■ MR VP broth: contains peptone, glucose, & phosphate buffer
● Methyl Red test: checks for mixed acid fermentation
that overpowers the buffer and lowers pH → verified by
addition of methyl red pH indicator. Immediate red
color formation is positive; orange or yellow is
negative.
● Voges-Proskauer: checks for fermentation of
glucose to neutral end products acetoin and
2,3-butanediol. Addition of VP reagents (A & B)
detect acetoin to form a red color. *Formation of
cherry red color is positive; wait 30 minutes.

■ Nitrate Reduction: detects for anaerobic respiration using nitrate as

terminal electron acceptor (reduced to nitrate & beyond); understand
interpretation of test → add reagents; no color or red color & addition of
zinc

Test for utilization of a nutrient: Citrate Test

● Citrate @ sole C source & ammonium phosphate as sole N source; Simmons
Citrate agar is a defined medium
● Tests for ability of organism to use citrate as a sole carbon source
● If the bacteria grows on citrate agar, the nitrogen source ammonium phosphate
is converted to ammonium ion which turns the bromothymol blue pH indicator
blue (basic).
● Citrate test one of the IMViC tests for differentiating the Enterobacteriaceae
mentioned earlier

● Urea Hydrolysis Test: detects presence of urease

enzyme. pH change from yellowish to purplish (basic)
due to ammonia production (hydrolysis of urea)
● Motility Test Medium (Red Indicator TTC): is a semisolid medium designed to detect
bacterial motility.
○ The agar concentration of 0.4% (lower than the typical
1.5%) provides for a solid media but allows for movement of
bacteria.
○ TTC (tetrazolium salt) is added to the medium to facilitate
interpretation of results.TTC is an artificial electron acceptor
that the bacteria can use; when oxidized it is colorless and
soluble – when reduced, it turns red.

● Combination Differential Media: allow for multiple biochemical

determinations in one test
○ SIM: determines 3 bacterial activities – sulfur reduction, indole
production and motility:
■ Sulfur reduction to H2S: via cysteine desulfurization or by
thiosulfate reduction. H2S forms black ppt (FeS) in
presence of FeSO4
■ Indole: from catabolism of tryptophan substrate. Add
Kovac’s reagent→ Red color = positive.
■ Motility: motility determined by growth emanating from the
stab-inoculation site (or not, if non-motile)

● KIA:
○ Glucose-only fermenter (red slant/yellow butt): fermentation turns entire
medium yellow (acidic); after 12 hrs glucose runs out & in the aerobic portion (on
the slant) bacteria use peptones turning slant basic (red).
○ Glucose & Lactose fermenter (yellow slant/yellow butt): lactose is 10x higher
concentration; utilization of this amount of lactose makes it much more acidic.
○ Non-fermentation of either glucose or lactose: utilizes peptones and
alkalinizes the medium (turns red). An obligate aerobe only turns the slant red; a
facultative anaerobe turns slant & butt red.
○ Gas production from fermentation visualized as cracks in the medium
Lab 9:
● Chemical germicides are substances designed to reduce the numbers of microbes,
specifically pathogens, in two ways:
○ Disinfectants: for use on inanimate objects (non-living substances)
○ Antiseptics: for use on living tissues
○ Efficacy of chemical germicides in reducing pathogen numbers is commonly
tested via the use-dilution test
● The use-dilution test is a standard method to test the effectiveness of a disinfectant or
antiseptic against pathogens.
● The protocol uses glass beads coats with living bacteria that are exposed to varying
concentrations (dilutions) of test disinfectants, then transferred to growth media.

Lab 10
● Quantitative techniques estimate the quantity of cells in a sample.
● This can be done by directly counting cells or by counting colonies formed by cells
growing on solid media; the latter is a viable count method.
● Measure # cells/mL of sample; CFU = colony forming unit is the standard quantitation
unit for plate counts.
● A CFU can arise from a single cell, or group of cells, depending on the species.
● Bacterial cultures at high cell density require dilution of the sample to a range that yields
a countable number of colonies on a plate.
● Successive dilutions of the original sample (i.e., serial dilutions) are carried out to obtain
an optimum quantity of cells that can be counted once plated out and grown.
● Antibiotics are natural antimicrobial agents produced by microorganisms (bacteria and
fungi primarily). The Kirby-Bauer method, or disk diffusion method, is a tool for
measuring the effectiveness of antibiotics and other antimicrobial agents against
pathogenic microbes.
● The test utilizes paper disks impregnated with the antibiotic which are then placed on a
plate inoculated with a lawn of particular bacterial species.
● As the plate is incubated, the antibiotic diffuses from the disk into the surrounding
medium and forms a concentration gradient.
● Bacterial resistance***
● If the bacterium is susceptible to the antibiotic, a clear zone of inhibition will appear
around the disk where growth has been inhibited.
○ The size (diameter) of this zone depends upon the sensitivity of the bacteria to
the antibiotic; can assess as resistant, intermediate, or sensitive.
● Technique: want a thick lawn of bacterial growth on the plate - use cotton swab.
○ Dip swab into culture, swab entire plate
○ Repeat: turn plate 90 degrees, dip
same swab into culture and swab again
● Reliable, consistent results require
standardized procedure: (a) use of
Muellar-Hinton agar poured to a depth of 4mm
(allow for optimal diffusion of antibiotic); (b)
broth cultures of bacteria of specific turbidites
(use McFarland turbidity standards).
● Purpose: understand the basis of the Kirby-Bauer method for measuring the
effectiveness of antibiotics against bacterial strains and how it is performed and how
results are interpreted
Just in case
● Lab 2
○ Exercise 3-1 - Microscopy; be familiar with Microscope
parts/functions/transport
○ Parts of microscope
■ Magnification: an increase in the apparent size of the image, is required
to view an individual cell in a colony
■ Total magnification: objective lens magnification x ocular lens
magnification
■ Size scale: micrometers (um; also microns) & nanometers (nm) are
metric units typically used in reference to microbe size and to the size of
their structural components.
■ Refraction: light by lenses results in magnification
● Condenser
● Objective lens
● Ocular lens
■ Resolution: refers to clarity/sharpness of the image
■ Limit of resolution: the minimum distance two points can be to still be
viewed as separate points; also called resolving power
■ Numerical aperture: refers to the light gathering of the objective lens and
the condenser
■ Ocular lens: the eyepiece through which you view the specimen
■ Objective lens: different magnifications (scanning 4x , low 10x , high-dry
40x, oil immersion 100x)
■ Condenser: focuses light onto specimen
■ Parfocal: the ability of the microscope to stay in focus when changing
magnifications
○ proper microscope procedure - for both handling a microscope and to view
a specimen on a slide
■ Transport: carry with two hands - one on the arm of the microscope, one
under the base; gently place on table
■ Cleaning: use only lens paper to cleans lenses, including condenser; do
not use paper towels
■ Operation:
● Raise condenser to highest position; open iris diaphragm
● Turn microscope lamp on; adjust to maximum intensity
● Move the scanning (4x) or low power (10x) objective lens into
place
● Place slide on stage; center specimen over opening
● Adjust iris diaphragm to yield
optimum illumination
● To focus image, first coarse focus,
then fine focus
 ● *bacterial specimen: process to oil immersion lens; place drop of
immersion oil on specimen. Submerge oil objective
● Objective lenses are parfocal
■ When finished:
● Lower stage & remove slide; prepared slides - return to box;
others - dispose of in proper receptacle
● Move scanning objective into place
● Lower stage and center; clean lenses with lens paper only

● Lab 3
○ Exercise 1-3 - Growth requirements for microbes and media types
■ Growth requirements for microbes
● Proper nutrients must be available
● Oxygen or other gasses must be available, as required
● Moisture is necessary
● The medium must have appropriate pH
● Proper temperature relations must prevail
● The medium must be free of interfering bioburden
● Contamination must be prevented
● Must contain carbon, nitrogen, inorganic
phosphate and sulfur, trace minerals, water,
vitamins
■ Complex media - Contains one or more complex
nutrients of undefined composition → yeast extract;
or enzymatic digests of beef or plant
■ Defined media - The exact chemical composition is
known

○ Exercise 1-3 & 1-4 - Aseptic technique and culture transfers and streak
plate methodology
■ Pure culture- a single type of organism growing without contaminations
■ Want to obtain a pure culture of desired microbe on solid media
■ Employ aseptic techniques to minimize contamination. Flaming the wire
loop is an example of aseptic technique.
■ Aseptic technique: disinfecting lab surfaces, limiting exposure to plates
by closing lid, sterilizing equipment, avoiding
breathing on cultures, flaming the neck of
bottles to prevent the climbing of bacteria
 ■ Dilution streak method - plate is “seeded with cells; mechanical dilution
of cells as they are spread on the plate surface. Goal → to obtain isolated
colonies. Reflame loop before changing direction of streaking.

■ Streak plate method- 4 quadrants using wire loop. The inoculation

source can be from liquid, plate, or slant.
1. Sterilize loop by flaming
2. Gently scraping off a colony
from the plate to collect it on
the loop
3. Transferring the bacteria to
the agar plate
4. Streaking the bacteria back
and forth across the plate,
gradually moving away from
the initial area
5. Repeating the process in
subsequent areas of the plate, rotating the plate 90
degrees each time. Reflame loop before changing direction
of streaking
6. * if your plate is contaminated, there may be random
colonies from the edges, or growth in an area where you
didn’t streak

● Lab 4
○ Exercise 3-4 - Simple staining and Exercise 3-5 - Negative staining
■ Staining enhances contrast and resolution, reveals details of cellular
morphology and arrangement. Stains bind to different cell structures and
molecules
■ Basic dyes: positively charged dyes (methylene blue, crystal violet) bind
to negatively charged bacterial cell walls, making them visible under the
microscope and the inside of the cell will be dyed
■ Nigrosin is acidic which means they are negative so they will not enter
the cell. This means the outside of the cell will be colored
■ Heat-fixed smear: kills the bacteria and fixes them to the slide but can
distort the shape if overheated
■ Bacterial morphology: know the terms such as cocci (spherical), bacilli
(rod-shaped), and spirilla (spiral-shaped), and arrangement like chains
and clusters
Staining specimens for microscopy
1. To highlight cells to observe morphology and structures: do a SIMPLE STAIN
- Prepare smear of specimen and heat fix
- BASIC DYE: positively charged→ attracted to negatively charged cells
 - Types: crystal violet, safranin, methylene blue
2. For cell morphology, size measurement, presence of capsule: do negative stain
- Smear and fixing NOT required; mix live cells with acidic dyes
- Acidic dye: negatively charged→ remain outside cells; colorless cell against dark
background
- Types: nigrosin, eosin, congo red
- Purpose:
- Determine morphology & cellular arrangement
- Typically for cells sensitive to heat-fixation during smear preparation
- Minimal distortion of cells (no heat-fixation involved)
- Uses acidic dye: nigrosin
Preparing heat-fixed smear:
1. Flame wire loop
2. Place a small drop of water (not too much) on a clean slide using an inoculating loop. If
you are staining from a broth culture, begin with step 2.
3. Aseptically add bacteria to the water. Mix in the bacteria and spread the drop out. Avoid
splattering the emulation as you mix. Flame your loop wire when done.
4. Allow the smear to air dry. If prepared correctly, the smear should be slightly cloudy.
5. Using a slide holder, pass the smear through the upper part of a flame two or three
times. This heat-fixes the preparation. Avoid overheating the slide as aerosis may be
produced.
6. Allow the slide to cool, then continue with the staining protocol.

A differential stain is where multiple dyes are used to differentiate between different types of
microorganisms or cellular structures. Gram staining is an example of differential staining.

Preparing a simple stain:

1. Begin with a heat-fixed emulsion. More than one organism can be put on a slide.
2. Cover the smear with stain for the designated time (1 minute). Use a staining tray to
catch excess stain. Be sure to wear gloves.
a. Available stains: crystal violet, methylene blue, and safranin (all basic dyes)
3. Grasp the slide with a slide holder. Rinse the slide with water. Dispose of the excess
stain according to your lab’s practices.
4. Gently blot dry in a tablet of bibulous paper or paper towels. (alternatively, a page from
the tablet can be removed and used for blotting). Do not rub. When dry, observe using oil
immersion.
Preparing a negative stain
1. Begin with a drop of acidic stain at one end of a clean slide. Be sure to wear gloves.
2. Aseptically add organisms and emulsify with a loop. Do not over-inoculate and avoid
splattering the mixture. Sterilize the loop after emulsifying.
3. Take a second clean slide, place it on the surface of the first slide, and draw it back into
the drop.
4. When the drop flows across the width of the spreader slide…
5. Push the spreader slide to the other end. Dispose of the spreader slide in a jar of
disinfectant or sharps container.
6. Air dry and observe under the microscope. Do NOT heat fix.

● Lab 5
○ Exercise 3-6 - Gram stain and Exercise 3-9 - Spore Stain
■ Differential stains: allows for detection of differences between bacteria
and serves as an aid in identifying bacteria
● Also provides information on cellular morphology and arrangement
on their structural features
● Types of differential stains are the gram stain, acid-fast stain,
spore stain, and capsule stain
● Typically consists of a series of steps utilizing a primary stain,
counter stain, and often (though not always) a decolorizing agent
■ Gram stain: most common differential stain; differentiates many bacterial
types on basis of cell wall differences
● Gram positive: higher content of peptidoglycan and
cross-linkage, allows for retention of primary stain (crystal violet)
and resists decolorization
● Gram negative: less peptidoglycan, higher lipid content;
decolorization w/alcohol extracts lipids, increasing porousness of
cell; results in loss of primary stain.
● Crystal violet is the primary stain, iodine is the mordant,
decolorization removes crystal violet from gram- cells, safranin is
used to counterstain gram-cells
● Common mistakes: if you wait too long, cells age and lose their
ability to retain color. If you wait too long with the decolorizer and
use too much, the gram positive cell can start to lose its dye too.
Stains are on for 1 minute, decolorizer is for 15 seconds. Blot dry
after rinsing with water when you use the decolorizer and then
proceed with counterstain step

■ Endospore: is a dormant form of bacteria that is formed under conditions

of nutrient starvation and/or other physical, metabolic stress. Spore
formation is a complex process involving many genes. Spores are highly
resistant to heat, desiccation, and radiation.
 ■ Two major groups of spore formers are Bacillus and Clostridium, both are
primarily soil microbes with Clostridium being anaerobic and Bacillus are
mainly aerobic or facultative anaerobes.
● Clostridium: has known pathogens that forms toxins → botulism,
tetanus
■ Three different forms depending on state of stress: free spores, cells
containing spores, and vegetative.
■ The spore stain uses heat to allow the dye (malachite green) to penetrate
the spore; safranin is used to counterstain. Microscopy reveals
green-colored spores and red vegetative cells
■ 1. Begin with a heat-fixed emulsion. As many as four small emulsion can
be put on one slide
■ 2. Cover the smear with a strip of bibulous paper. Apply malachite green
stain. Steam for 5 minutes or more, if necessary.
■ 3. Grasp the slide with a slide holder. Remove the paper and dispose of it
properly. Gently rinse the slide with water.
■ 4. Counterstain with safranin stain for 1 minute. Rinse with water.

○ Exercise 3-7 Acid fast stain and Exercise 3-8 - Capsule stain
■ Acid fast stain: used for the Mycobacterial species. The basis for acid
fast stain: the presence of unusual lipids in the cell wall of Mycobacterial
species called mycolic acids. Mycobacterial types are the cause of
tuberculosis and leprosy.
■ The Kinyoun method utilizes a highly concentrated carbol fuchsin solution
(the primary stain) whose chemical nature allows it to penetrate the waxy
acid-fast cell wall mycolic acids; acid-fast bacteria resist decolorization by
acid alcohol
■ Non-acid-fast bacterial must be counterstained with methylene blue in
order to be seen
■ 1. Begin with heat-fixed emulsion
■ 2. Apply Kinyoun carbolfuchsin stain for 5 mins. Perform this step with
adequate ventilation and wear gloves.
■ 3. Grasp the slide with a slide holder. Gently rinse the side with distilled
water.
 ■ 4. Continue holding the slide with a slide holder. Decolorize with acid
alcohol until the run-off is clear. Gently rinse the slide with distilled water.
■ 5. Counterstain with methylene blue for 1 min.
■ 6. Gently blot dry with bibulous paper. Do not rub. Observe under oil
immersion.

■ Capsule Stain: Capsules are generally polysaccharide in nature and are

often found in bacteria that are pathogenic. Streptococcus pneumonia,
anthrax. Capsules allows them to avoid host defenses.
Oral bacteria capsule allows it to adhere to teeth and
form a biofilm.
■ 1. Begin with a drop of congo red stain at one end of a
clean slide. Add a drop of omit serum.
■ 2. Aseptically add organisms and emulsify with a loop.
Do not over inoculate and avoid splattering the mixture.
Sterilize the loop after emulsifying.
■ 3. Take a second clean slide. Place it on the surface on
the first slide and draw it back into the drop.
■ 4. When the drop flows across the width of the spreader
slide, push the spreader slide into the other end
■ 5. Air dry and do NOT heat fix
■ 6. Flood slide with Maneval’s stain for 1 minute. Rinse with water
■ 7. Gently blot dry with paper. Do not rub
■ 8. You should see a red background with a white circle around a cell. That
represents a capsule.

● Lab 6: Selective and Differential Media

○ Selective media: contains chemical components that inhibit certain bacteria,
favors the growth of others.
○ Differential media: can visualize metabolic differences between bacterial types
● Selective media for Gram + bacteria:
○ Phenylethyl Alcohol Agar (PEA) (selective) phenylethyl alcohol inhibits gram
negative organisms by interfering with DNA synthesis
○ Columbia CNA + 5% sheep blood (differential) colistin and nalidixic acid
suppresses growth of gram - . Addition of blood permits differentiation of
hemolytic activity. Streptococcus genus are differentiated by hemolytic reactions
on blood agar
S.Pyogenes: scarlet fever, strep throat
S. Mutans: dental caries endocarditis
S. Pneumonia: pneumonia, meningitis

○ Mannitol Salt Agar (MSA) (Differential & selective); differentiates mannitol

fermenters (yellow) & non-fermenters. High salt favors staphylococcus sp.
● Selective media for Gram - bacteria
○ MacConkey’s Agar (MAC) lactose fermenter (Differential and selective). Inhibits
gram + via bile salts and distinguishes between lactose (purple) and non lactose
(colorless) fermenters

○ Eosin-Methylene Blue Agar (EMB) (Differential and selective). Dyes inhibit

growth of gram +. Lactose fermenters produce purple, Strong acid production
results in metallic green, weak lactose is pink/purple.

○ Hektoen Enteric Agar (HEA) (Differential and selective). Indicators of lactose

fermenters (yellow coliforms) as well as H2S production
● Nutrient agar is used as a control because it is a general-purpose medium that supports
the growth of a wide range of non-fastidious microorganisms