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Microbiology Lab Packet

Students are responsible for all the material in this lab packet.
Last reviewed: January 2016

MCB 3020 Laboratory Rules

1. Backpacks, purses, and other personal effects will be placed on the rack near
lab entrance when you first come in to lab. Paper and writing utensils are
2.
3.
4.
5.
6.
7.

provided at the bench.


No food (including gum) or drink allowed in the lab.
Close toed shoes must be worn at all times
Long pants (to ankles) and sleeves (mid-arm or longer) must be worn to lab.
Goggles will be provided in your bins, and must be worn at all times.
Gloves are provided and should be worn at all times.
Lab coats are provided and should be worn at all times. Lab coats are NOT to

be removed from lab.


8. Cell phone use is not allowed in lab at any time. They should be on silent and
put away. In the event an emergency should arise and you need to use your
cell phone, let your TA know, remove your gloves and lab coat, wash your
hands, and leave the lab.
9. There will be no talking during the lecture portion of lab sessions. Paying
close attention during the lectures will improve your total grade for the
course.
10.Long hair should be tied back during lab.
11.Test tubes should never be carried without a test tube rack. Do not pick up
test tubes by the cap.
12.In the event of a spill do not move. Notify the TA and people around you
immediately.
13.Cough drops, eye drops, and other forms of oral medication may not be
consumed during lab.
14.Wash hands before and after each lab.
15.Decontaminate benches before and after each lab.
16.Never remove organisms or materials from this lab unless directed by
instructor.
Students wearing inappropriate attire, those not wearing proper PPE, or those found eating or drinking may
be asked to leave the lab.
Signature: _______________________________________
Printed name: ___________________________________________

Date: _____________________
Lab section: ________________

MICROSCOPE BASICS

Simple Microscope: one lens


Compound Microscope: two lenses
1. Ocular-eyepiece lens (usually 10x)
2. Objective-nosepiece lenses (commonly 4x 10x 45x 100x (oil immersion))
o Total Magnification = Objective Magnification x Ocular Magnification
Important Parts of the Microscope:
Framework includes the arm and base
o Structural parts of the microscope which support the basic frame.
Stage holds the slide
o The mechanical stage clamps the slide and moves the slide around the stage.
Lens system includes
o Oculars eyepiece lenses (usually 10x magnification)
o Objectives lenses attached to rotatable nosepiece common magnifications of 4x, 10x, 45x (low
power, high-dry objectives) and 100x (oil immersion lens).
Parfocal microscope focusing adjustments are not to be made when changing objective
lenses.
Oil immersion lens uses oil with approximately the same refractive index as glass to
prevent light loss due to diffraction (bending of light rays) which would occur if light
traveled from one refractive index to another (e.g. glass to air).
As magnification of the objective lens increases, the working distance (distance between
the object on slide and the objective lens, when in focus) decreases.
o Condenser directs light towards the objective lens in bright field microscopy
In dark field microscopy the condenser directs light at oblique angles away from the
objective lens in a manner that allows only objects in the field of view to redirect or
scatter light into the objective lens. This causes objects to appear white on a dark field.
The iris diaphragm (lever located in the condenser) adjusts the diameter of the cone of
light so that it just fills the objective lens.
As you close down the diaphragm:
1. The light intensity decreases
2. Contrast improves
3. Depth of field increases
4. Limit resolution (with oil immersion lens)
Resolution (also called the Resolving Power):
Expressed as d
d = the smallest distance between two objects which can be seen as separate
d = the diameter of the smallest resolvable object
d = / 2 NA
= wavelength of light
NA = numerical aperture
To improve resolution, lower d
o d can be decreased by lowering or increasing the NA.

BACTERIAL MORPHOLOGY

Light microscopy reveals three principle forms of microorganism:


o More or less spherical organisms:
COCCI
o Cylindrical organisms:
BACILLI
o Spiral shaped:
HELICOIDAL

Incompletely separated cocci may appear in a number of different patterns depending upon the plane in
which they divide and how they remain attached:
o Diplococci (pairs) divide in one plane
o Streptococci (chains) divide in one plane
o Tetracocci (tetrads) divide in two planes
o Staphylococci (clusters) divide in three planes irregularly
o Sarcinae (cuboidal packets) divide in three planes regularly

Bacilli can appear in a number of different cylindrical shapes:


o Coccobacilli are very short and almost appears spherical, but they are just slightly longer in one
direction then the other.
o Fusiform bacilli are tapered at the ends, appearing as football like in shape.
o Filamentous bacillary forms grow in long threads.

STAINING PROCEDURES
Most microorganisms are difficult to see using light microscopy due to their size and the lack of contrast
between the cell and the environment. The contrast is improved with the help of dyes. Dyes are organic compounds
containing a chromophore with affinity for cellular material.
Types of Dyes:
Cationic (basic dyes, positively charged chromophore): methylene blue, crystal violet
Anionic (acidic dyes, negatively charged chromophore): acid fuchsin, Congo red, Nigrosin
Fat Soluble (no charge): Sudan black stains granules of Poly--OH-butyric acid
Insoluble Dyes (water insoluble): India ink (colloid suspension of carbon particles)
Types of Staining Procedure:
1. Negative Staining
Stains the background and not the cell in bright-field microscopy (not the same thing as dark field
microscopy).
Does not require heat fixation, therefore does not distort size or shape of cells.
Two dyes used:
o Nigrosin a black anionic (negatively) charged dye. The negatively charged dye is repelled by the
negatively charged surface of the bacterial cell.
o India ink an insoluble dye (a colloidal suspension of carbon particles) which does not penetrate the
cell surface.
2. Simple Staining
One dye used to stain all cells the same color. Can be used to tell morphology (shape) and size [although
negative staining is better for size]. Cationic dyes are positively charged and are attracted by ionic forces to
the negatively charged surface of the bacterial cell.
Two commonly used dyes are methylene blue and crystal violet.
3. Differential Staining
Staining procedure, which causes cells to stain differently based on properties of the cell.
Two examples of differential staining:
o
Acid Fast Stain differential stain procedure that causes cells to stain differently based on
characteristics of their cell wall. Acid fast microorganisms have a high wax content in their walls,
which requires the use of steam to allow dye to penetrate the cell. Cells are steamed in the presence of
carbol fuchsin and decolorized with acid alcohol. Cells which are acid fast (microorganisms have a
high wax content in their walls) will not decolorize and remain red, while non-acid fast organism will
readily lose their stain and become colorless. These cells are then counterstained with methylene blue.

Primary stain
Decolorizer
Counterstain
o

Stain
Acid Fast (+) Acid Fast (-)
Carbol fuchsin
red
red
Steam
red
red
Acid alcohol
red
colorless
Methylene blue
red
blue

Two genera of acid fast organisms (all other genera are non-acid fast):
i. Mycobacterium they do not Gram-stain well if mature because of high wax content
within walls. If the colony is young it will appear as Gram-positive tapered rods that
sometimes fragment.

Two important species:


a.
M. tuberculosis: causes tuberculosis
b.
M. leprae: causes leprosy
ii. Nocardia some species stain partially acid fast but a less harsh decolorizer must be
used.

Gram Stain differential stain procedure that causes cells to stain differently based on characteristics
of their cell wall. Gram-positive microorganisms have a higher peptidoglycan and lower lipid content
than Gram-negative microorganisms. Cells are stained with crystal violet, then fixed with iodine
forming a crystal violet-iodine complex within the cell. Ethanol is then added as a decolorizer. Gramnegative cells are easily decolorized because the ethanol dissolves the high lipid cell wall allowing the
crystal violet-iodine complex to readily exit the cell. These cells are then counterstained with Safranin.
Gram-positive cells resist decolorization due to the difference in cell wall consistency retaining the
crystal violet-iodine complex.

Primary stain
Mordant
Decolorizer
Counterstain
4.

Stain
Crystal violet
Iodine
Ethanol
Safranin

Gram (+) Gram (-)


purple
purple
purple
purple
purple
colorless
purple
pink

Structural Staining
Spore Staining some microorganisms produce heat and chemical resistant structured called
endospores or free spores. To stain the spores the cells must be steamed to allow for the dye (malachite
green) to enter the spores. Once the spores are stained, all other microorganisms and vegetative cells can
easily be decolorized with water, while the free spores and endospores retain the malachite green. The
other microorganisms and vegetative cells are then counterstained with Safranin.
o Endospores appear as a green center within a pink sporangium
o Free spores appear as small green oval bodies
o Three genera of spore forming organisms
Bacillus aerobic, Gram-positive rod
Aerobic green = endospore/free spores of Bacillus
Aerobic pink = vegetative/sporangia of Bacillus
Clostridium anaerobic, Gram-positive rod
Anaerobic green = endospore/free spores Clostridium
Anaerobic pink = vegetative/sporangia of Clostridium
Sporosarcina cocci

Primary stain
Decolorizer
Counterstain

Stain
Malachite green
Steam
Water
Safranin

Spores
green
green
green
green

Non Spore
green
green
colorless
pink

MEDIA AND BIOCHEMICAL TESTS


There are five methods of tube media preparation:
1. Pour: 15 20 mL of liquid agar used to pour into a plate
2. Broth: 5 7 mL of liquid media
3. Deep: 5 7 mL of media which has solidified in an upright position
4. Slant: 5 7 mL of media which has solidified at an angled position
5. Fermentation Broth: broth with Durham tube added
Natural media is composed of complex raw materials whose actual chemical composition is unknown.
Example: Nutrient Agar (NA)
Synthetic medias exact chemical composition is known and in many instances is designed for isolation, selection or
differentiation of specific types of microorganisms. Two main categories are:
a) Selective media favors the growth of one type of microorganism over another. This is accomplished by
either inhibiting unwanted microorganisms or enriching by providing conditions which are preferential to
the desired microorganism.
b) Differential media differentiates or distinguishes between different types of microorganisms based on
differences in appearance of growth or color changes.
Selective and/or Differential Media:
1.
2.

3.

4.

Phenylethyl Alcohol Agar (PEA)


o Selects for the growth of Gram-positive microorganisms because phenylethyl alcohol is inhibitory to
the growth of Gram-negative organisms.
DESoxycholate Agar (DES)
o Selects for Gram-negative microorganism because desoxycholate is inhibitory towards the growth of
Gram-positive organisms.
o Differentiates lactose-positive/negative microorganisms:
Lactose fermenters produce acid, which precipitates the bile salts in the media and absorbs the
neutral red dye, therefore appearing red.
Non-fermenters do not do this and do not appear red.
Eosin Methylene Blue Agar (EMB)
o Selects for Gram-negative organisms.
The acid soluble dyes eosin (pink) and methylene blue (blue) inhibit the growth of Grampositive organisms.
o Differentiates lactose-positive/negative microorganisms from the interaction between the fermentation
by-products and the eosin or methylene blue dyes:
Lactose-positive show a color change.
Lactose-negative do not show a color change.
o Can further differentiate lactose-positive fermenters based on the amount of acid produced during
lactose fermentation.
Mixed acid fermenters produce more acid and produce colonies with dark blue-black centers
(center is almost the size of the whole colony) and some microorganisms, like Escherichia
coli, produce a metallic green sheen.
Butanediol fermenters, like Enterobacter, produce less acid so that the colonies have pale
pink to lavender centers. The centers are only a small part of the colony (i.e. bulls-eye
colonies), and will not have a metallic green sheen.
MacConkey Agar
o Selects for Gram-negative organisms.
Crystal violet and bile salts inhibit growth of Gram-positive organisms.
o Differentiates lactose-positive/negative microorganisms from the interaction between the fermentation
by-products and neutral red indicator

5.

Lactose fermenters produce acid, which precipitates the bile salts in the media and absorbs the
neutral red dye, therefore appearing red/pink.
Non-fermenters do not do this and do not appear red/pink but will form colorless colonies.

Blood Agar
o Differentiates microorganisms based on their reactions on blood. Shows presence or absence of an
exoenzymes known as hemolysins that can break down hemoglobin in blood.
Gamma () hemolysis: no blood hemolysis, no zone of clearing around the colony.
Beta () hemolysis: complete blood hemolysis and complete clearing around the colony.
Hemoglobin is completely degraded.
Alpha () hemolysis: partial blood hemolysis and partial clearing around colony. Partial
clearing sometimes appears green due to partial reduction of hemoglobin in blood. Greenish
color is due to a partial breakdown product of hemoglobin called biliverdin.

Biochemical Tests:
Tests used to determine physiological characteristics of microorganism, particularly in terms of bacterial enzymes
and the chemistry of bio-oxidation. Biochemical tests on agar plates often (but not always) look for the presence of
exoenzymes which are enzymes secreted by the bacteria into their environment to break down macromolecules,
such as proteins and carbohydrates, into monomeric subunits small enough to be transported into the bacterial cell.
Endoenzymes are enzymes active inside the bacterial cell to metabolize substrates inside the bacterial cell, and can
be detected in both solid media and broths.
1.

2.

3.

4.

Starch Agar
Tests for the presence of the exoenzyme amylase, which hydrolyzes starch to simple sugars.
o
These simple sugars can then be transported inside the cell.

Iodine
is
added to the starch plate and appears blue/black when iodine forms a complex with starch.
o
If amylase is present, starch will be hydrolyzed and the blue/black to purple color will not be observed
o
around the colonies.
Milk Agar
Tests for the presence of the exoenzyme caseinase, which hydrolyzes casein (a predominant protein in
o
milk) into amino acid products.
Casein gives milk its white color so a breakdown in casein causes the milk plate to lose its white color
o
and become clear around the caseinase positive colonies.
Lipase Plate
Tests for the presence of the exoenzyme lipase, which hydrolyzes fat to form glycerol and fatty acids.
o
The production of the fatty acids lowers the pH just enough to produce a dark blue precipitate when a
o
microorganism is lipase positive.
Spirit blue dye is the indicator in this test.
o
Positive test shows an intensification of blue color around the colony.
o
Sugar Fermentation Tubes
Used to determine if a microorganism can ferment a particular sugar.
o
The fermentation tubes contain the sugar of interest (glucose, lactose, or mannitol), a pH indicator
o
(phenol red) and a Durham tube.
If a microorganism is able to ferment the sugar being tested the result of the fermentation will lead to
o
the production of acid, therefore lowering the pH of the solution and causing the liquid to turn yellow
from its original red color.
Some microorganisms also produce gas during fermentation, which is important to know when
o
identifying unknown bacteria. This gas will collect in the Durham tube and appear as a void or bubble
in the inverted tube.
An alkaline reaction can also occur, which is due to the utilization of the peptone in the broth and not
o
the testing sugar, releasing ammonia into the medium. An alkaline reaction is indicated by the
darkening of the red pH indicator color into a hot pink/cerise color.
Yellow = acid

Yellow and gas = acid & gas

Red = negative
Cerise = alkaline
Methyl Red (MR)
HCOOH CO2 + H2
o
Tests for a mixed acid fermenter which produce drastic amounts of acid from the fermentation of
o
sugars.
This acid ultimately results in the lowing of the pH below 5.1, so when the indicator methyl red is
o
added to the culture the methyl red remains red.
Red is the only true indicator of a positive result.

Escherichia is MR positive.
o

5.

6.

7.

8.

9.

Voges-Proskauer (VP)
HCOOH acetyl methyl carbinol (AMC)/acetoin 2,3-butanediol
o
Tests for 2,3-butanediol fermenters which produce less acid and more neutral products than mixed acid
o
fermenters.
Because AMC is easier to detect than 2,3-butanediol, AMC is tested for when determining if a
o
microorganism is a 2,3-butanediol producer.
Barritts reagents, also known as VPI (-naphthol) and VPII (KOH), are added to the test culture.
o
When oxygen is present, KOH (VP II) will react with AMC oxidizing it to diacetyl, which reacts with
o
a guanidine-containing compound in the media to produce a brick red color, indicating that the
microorganism is a 2,3-butanediol producer.
-naphthol (VP I) is used to intensify the red color by catalyzing the oxidation of AMC by

KOH (VPII).
Enterobacter is VP positive.
o
Catalase
Tests for the presence of catalase, which converts hydrogen peroxide to water and oxygen.
o
2 H2O2 2 H2O + O2

Hydrogen peroxide is produced during oxygen utilization and must therefore be eliminated since
o
hydrogen peroxide is toxic to the cell.
Enzyme presence can be tested for by adding 3% H 2O2 to the culture and looking for the production of
o
oxygen bubbles.
Bubble production is a positive result.

Oxidase
Cytochrome c oxidase is an enzyme which can oxidize aromatic amines to form a colored product.
o
The aromatic amine used to test for oxidase is dimethyl-p-phenylenediamine hydrochloride which in
o
the presence of oxidase will turn a dark blue-black color.
Nitrate
Tests for the enzyme nitrate reductase (nitrase), which can reduce nitrate (NO3-) to nitrite (NO2-) in a
o
single step.
NO3- + 2e- + 2H+ NO2- + H2O

Nitrate I (sulfanilic acid) and Nitrate II (dimethyl--naphthylamine) react with NO 2 to produce a brick
o
red color.
This is a positive result.

If nitrate is not used and is residual, then zinc powder can catalyze the reaction instead.
o
A red result after Zn addition will show that nitrate was not used because the organism lacks

nitrase.
No color after addition of reagents, but a red color after Zn, is a negative result.

Some microorganisms have nitrate reductase as well as nitrite reductase and other reductases.
o
Nitrate will be reduced to either ammonia (NH 3) or nitrogen gas (N2) in a multistep process

known as denitrification.
NO2 N2 + NH3 and other products

Reagents I and II followed by Zn addition will not form a red color since both nitrate and nitrite have
o
been consumed.

This is also a positive result.

10. Tryptone (Indole)


Tests for the enzyme tryptophanase, which converts trytophan to indole and pyruvic acid.
o
Tryptophan pyruvic acid + indole

Indole can easily be tested for by adding Kovacs Reagent (p-dimethylaminobenzaldehyde (DMABA)
o
and HCl dissolved in amyl or butyl alcohol).
A positive result is when the Kovacs reagent interacts with indole.
o
11. Urea
Tests for the enzyme urease, which converts urea to ammonia and carbon dioxide.
o
Urea 2 NH3 + CO2

Contains the substrate urea and the pH indicator phenol red.


o
When ammonia is released the pH of the solution increases and once the pH is above 8.1 the phenol
o
red indicator will appear cerise.
A cerise color indicates a positive result for urease.

Proteus
is
urease positive.
o
12. Hydrogen Sulfide Production (H2S)
It tests for the enzyme cysteine desulfurase which removes the sulfur side chain from cysteine to
o
produce H2S, which when in the presence of iron salts (contained in Klingers Iron Agar and SIM
medium) forms a black precipitate.
Cystein H2S + amino acrylic acid imino acid pyruvic acid + NH3

A black color indicates a positive result for cysteine desulfurase.

Proteus
is
H2S positive.
o
13. SIM
Tests for Sulfur reduction (H2S production), presence of Indole, and Motility.
o
H2S positive = black precipitate
o
Black precipitate is formed when the produced H2S combines with iron from ferrous

ammonium sulfate ferric sulfide (FeS)


Indole positive = Kovacs reagent turns red after addition
o
See Tryptone Broth above

Motility positive = growth away from inoculation line (appears as cloudiness in tube)
o
14. Simmons Citrate
Tests for the ability of a microorganism to utilize citrate as the sole carbon source.
o
Media contains ammonia salts (sole nitrogen source), citrate (sole carbon source), and bromothymol
o
blue (indicator).
If a microorganism can use citrate as the sole carbon source the microorganism will grow on the
o
bacterial medium and the media will turn a deep Prussian blue color.
Growth and the appearance of the Prussian blue color are indications of a citrate positive

microorganism.
Metabolism of citrate production results in production of bicarbonate and carbonate, raising

the pH.
15. Phenylalanine (PPA)
Tests for the presence of the enzyme phenylalanase, which removes the amine group (NH 2) from
o
phenylalanine, producing phenylpyruvic acid (PPA) and NH3.
Phenylalanine PPA + NH3

Ferric chloride (FeCl3) is added to the media to test for the presence of PPA.
o
In the presence of PPA, ferric chloride will appear a deep green color.
o
A deep green color is indicative of a positive test for phenylalanase.

16. Litmus Milk


Tests for (a) lactose fermentation, (b) reduction of litmus, (c) presence of caseinase, and the (d)
o
deamination of amino acids to produce NH3.
Litmus milk contains the pH indicator litmus and powdered milk (which contains casein and amino
o
acids). From this mixture, multiple different Litmus Milk results can be obtained:
a) Acid reaction: pink liquid due to drop in pH from the fermentation of lactose.

b) Acid curd reaction: pink solid due to acid production and coagulation of proteins causing the
formation of a solid.
c) Reduction: litmus is reduced and it becomes colorless; the tube appears white since only the
milk remains. Color may remain at top of media where oxygen can oxidize the litmus and
restore its color.
d) Alkaline reaction: appears as a blue liquid that is usually caused when protein breakdown
produces amino acids that are deaminated and release ammonia, increasing the pH in the tube.
e) Peptonization/Proteolysis: clearing of the medium (may be brown or amber) caused by
enzyme caseinase, which breaks down the white protein casein in milk.
More than once reaction can be observed in a single tube:
Example: acid curd reduction looks like acid curd but the tube turns white except for a small

region at the top where oxygen reoxidizes the litmus to the colored (red) form.

17. Gelatin
o
o
o

Tests for the presence of the gelatinase enzyme, which hydrolyzes gelatin to amino acids.
Gelatin is a protein that solidifies at lower temperatures.
This tube is stab inoculated and then placed on ice for several minutes after incubation. After
incubation on ice the media may be:
Liquid, this is a gelatinase-positive result (gelatin is hydrolyzed)

Solid, this is a gelatinase-negative result (gelatin is still present)

*** For the lab midterm you are responsible for knowing everything before this point. PowerPoints, lab lectures,
and whiteboards are all fair game for the test. ***

Other Tests/Media:
18. IMViC
Set of four tests that are used to differentiate between Escherichia coli and Enterobacter aerogenes
o
Indol, Methyl Red, Voges-Proskauer, and Citrate
o

E. coli
E. aerogenes

I
+
-

M
+
-

Vi
+

C
+

19. Motility Media


Tests if the bacteria are motile or not
o
Contains tetrazolium chloride, a growth indicator that turns red in the presence of growing bacteria.
o
Therefore a red color away from the inoculation line is an indicator of growth.
o
Red color is only a growth indicator and indicates that tetrazolium chloride is reduced; the red color
o
does not mean motility. Where the red color appears is an indication of motility, not the appearance of
red.
20. Russels Double Sugar (RDS)
Tests for fermentation of glucose and/or lactose as well as to test for gas production.
o
Glucose and lactose fermentation is determined using a pH indicator (phenol red) which begins red and
o
will turn yellow. If the butt of the tube turns yellow it means glucose is fermented, if the slant turns
yellow it means lactose is fermented.
Contains: 1% lactose and 0.1% glucose

RDS
is
read
slant over butt, meaning that the results from the slant are read first and then the results
o
are read from the butt of the tube.
Example: red slant with yellow butt = glucose-positive, lactose-negative

RDS should ideally be read within 24-48 hours after inoculation. After 48 hours the organism could
o
begin to break down the peptone present in the media, which will raise the pH and cause the slant to
revert back to red. This would be a false negative for lactose fermentation.

10

11

21. Kliglers Iron Agar (KIA)


Tests for the ability to ferment glucose and/or lactose, tests H 2S production, and can also be used to test
o
for gas production.
Contains: 1% lactose and 0.1% glucose

KIA differs from RDS due to the addition of iron salts to the media (see SIM above).

Glucose and/or lactose fermentation is determined using a pH indicator (phenol red) which begins red
o
and will turn yellow in the butt of the tube if glucose is fermented. It will cause the slant to turn yellow
if lactose is able to be fermented.
If the bacteria contains the enzyme cysteine desulfurase, a black precipitant will form from

the reaction of H2S with iron salts.


Gas production can be determined by cracks and/or the lifting of the media off the bottom of

the tube.
KIA is ideally read ~18 hours after inoculation and the lactose reaction should be read from the bottom
o
of the slant as the tip of the slant may revert back to red as the inoculation ages beyond 18-24 hours in
some species.

22. Bismuth Sulfide Agar (BSA)


Starts off as a dull green color.
o
Ferrous sulfite is included in the media as an H2S indicator.
o
Bismuth sulfite and brilliant green dye inhibit growth of Gram-positive and certain coliforms.
o
Salmonella typhi produces a black or very dark brown color.
o
23. SS Agar
This media contains bile salts, sodium citrate, and brilliant green to inhibit Gram-positive and certain
o
coliforms, as well as limits swarming by Proteus.
Sodium thiosulfate and ferric citrate allow detection of H2S.
o
Neutral red dye is included as an indicator of lactose fermentation.
o
Salmonella usually produces a black colony.
o
Shigella produces a colorless colony.
o
All lactose positive colonies appear red.
o
24. Desoxycholate Citrate
Selects for Gram-negative organisms.
o
Differential for lactose fermentation.
o
Desoxycholate and citrate salts inhibit Gram-positive.
o
Acid-soluble neutral red dye serves as indicator of acid produced by lactose fermentation.
o
Some lactose-positive colonies do grow but they will appear red.
o
25. Coagulase
Tests for the enzyme coagulase, which convert fibrinogen to fibrin.
o
Incubate bacteria in small tube of plasma overnight.
o

12

If plasma becomes clumpy and or solidifies, then bacteria are coagulase positive

Test is only valid on Gram-positive staphylococcus-like bacteria since Gram-negative bacteria are able
to provide false positive reactions from a non-coagulase like mechanism.
Phenol Red Mannitol Salt Agar (MSA)
Selects for Staphylococcus due to high salt concentration 7.5%
o
Medium is red, but plate and colonies will turn yellow if organisms are mannitol-positive.
o
Staphylococcus 110 Medium
Contains mannitol and 7.5% NaCl, but lacks phenol red as in MSA plate.
o
Selects for Staphylococcus and allows for development of natural colony pigment formation unlike in
o
MSA.
DNase
Tests for the exoenzyme DNase, which is able to hydrolyze DNA into free nucleotides.
o
Methyl green dye in media complexes with intact DNA in the media. Breakdown of DNA displaces
o
this dye, and forms a clear area.
Zone of clearance around the streak is a positive result for the presence of DNase.
o
m-Staphylococcus Broth
Enriched media containing 10% NaCl, which selects for Staphylococcus since Staphylococcus prefer
o
the higher salt concentration, which inhibits most other organisms
Endo Agar
Selective for Gram-negative. Combination of sodium sulfite and the basic fuchsin inhibits Gramo
positive bacteria.
Differential for lactose fermentation.
o
Basic fuchsin acts as indicator of acid production from lactose fermentation.

Lactose-positive gives red colonies and surrounding medium

Coliforms produce a golden metallic golden sheen.


o
o

26.

27.

28.

29.
30.

13

PHYSICAL-CHEMICAL CONDITIONS AFFECTING BACTERIAL GROWTH


Temperature:
All species have a temperature range for growth, known as the cardinal temperatures.
Minimum: lowest temperature permitting growth
Optimum: best temperature for growth
Maximum: highest temperature permitting growth
Group terms are based on these temperature ranges:
Min
Psychrophiles: requires low temperatures for growth.
Psychrophile
0C
Mesophiles: grow in a moderate temperature range.
Mesophile
20C
Thermophiles: grow in a high temperature range.
Thermophile
45C

Opt
5C
30-37C
55-65C

Max
15-20C
45C
70-90C

pH:
Group terms are based on pH in growth environment.
Acidophiles: require an acidic pH growth range.
Neutrophiles: best growth at pH 6.5 to 7.5.
Alkylophiles/basophiles: require alkaline growth conditions or growth best above pH 7.0.
Oxidation-Reduction Potential:
Oxidation-reduction (OR) potential refers to the presence or absence of oxygen. Oxygen-rich environments remove
net electrons (aerobic), while oxygen-deficient environment have a net increase in electrons (anaerobic). Microbes
require the presence of oxygen, the absence of oxygen, or tolerate both conditions (facultative). In determining this
trait the reducing agent thioglycollate can remove oxygen, except at the top/meniscus. Thus, you can determine the
position of growth in the tube and group the bacteria accordingly. Resazurin is used as a redox indicator: colorless in
reducing conditions and pink when oxidized.
Group terms are based on OR requirements or tolerance.
Strict aerobes: requires oxygen for metabolism and maintenance of cell structure. Bacteria grow only at
the top of the tube.
Facultative species: can grow with or without oxygen. Bacteria grow throughout the tube.
Strict anaerobes: inactivated by oxygen exposure (metabolism is fermentative), cell structure may be
altered by oxygen. Bacteria grow only at the bottom of the tube.
Osmotic Pressure (aw):
The osmotic pressure is produced by an imbalance of free unbound water on either side of the semi-permeable cell
membrane. High solute (salt) concentration (hypertonic) binds water and intracellular water diffuse out to inactivate
the cell. Some species can control the osmotic pressure changes and tolerate the hypertonic state. There are species
that require Na+ ions for stability and require salt in their environment. This test uses NaCl to reduce free water
availability (not bound by solutes).
Group terms are based on the salt concentration required:
Halophile: require higher concentrations of salt to survive and grow. From about 5% to 30% (w/v) salt.
Halotolerant: do not require higher salt concentrations but can tolerate it.
Non-halophile: grow best with 0.5% salt (if that solute is used). Other solutes bind water and their
concentration is important.
Osmophile: tolerates osmotic pressure produced when other solutes (besides salt) are in high concentration
in their growth environment.
Ultraviolet (UV) Irradiation:
Unlike temperature, pH, osmotic pressure and OR potential, UV irradiation is not in every environment. DNA in the
cell absorbs at 260nm (UV wavelength) maximally, causing damage (especially thymine dimers). Tolerant species
can correct the damage.
Group terms:
UVR: ultraviolet resistant.
UVS: ultraviolet sensitive and lethal.

14

15

GRAM POSITIVE PYOGENIC COCCI


Medical Microbiology is primarily concerned with the isolation and identification of pathogenic organisms. One
of the most frequently encountered types of pathogenic bacteria are the Gram-positive cocci. Of these bacteria the
two genera we will focus on are Staphylococcus and Streptococcus.
Staphylococcus:

Found in nasal membranes, the hair follicles, the skin, and perineum.

Most strains are penicillin resistant, which can cause epidemiology problems since 90% of
healthcare workers carry Staphylococcus.

Divide in multiple planes and there appears as irregular clusters microscopically.

Three important Staphylococcus species are: S. aureus, S. epidermidis, and S. saprophyticus.


o
Of these only S. aureus has the ability to coagulate plasma.
Streptococcus:
Found in pharynx, on surfaces of the teeth, saliva, shin, colon, rectum, and vagina.
Divide in only one plain and therefore appear as chains of cocci.
Streptococci of greatest medical significance are: S. pyogenes, S.pneumoniae, and S. agalactiae.
Isolation and Identification of Staphylococcus and Streptococcus:
Day 1:
Swab nose (N), throat (T), and a fomite (F).
Streak swab onto Blood Agar plate as shown.
Place swabs into m-Staphylococcus Broth.
Day 2:
Staphylococcus:
o Use the three m-Staphylococcus Broths to inoculate two Staphylococcus Medium 110
(SM110) and two Mannitol Salt Agar (MSA) plates.
o Select a beta-hemolytic Staphylococcus from the Blood Agar (BA) plate and inoculate a
third SM 110 and MSA plate.
Streptococcus:
o Identify an alpha- and a beta-hemolytic Streptococcus and a beta-hemolytic
Staphylococcus and streak each onto a new Blood Agar (BA) plate
Day 3:
Staphylococcus:
o Using the SM110 and MSA plates from last lab to identify three presumptive Staphylococcus
colonies by their growth on the SM110 and MSA plates as well as the cluster formation of the
Gram-positive cocci.
Inoculate each of these three colonies onto/into coagulase, DNase, and nitrate broth
and perform a catalase test on each (record these results, as well as any mannitolpositive results).
Into the 4th coagulase, DNase, and nitrate broth inoculate with the provided
Staphylococcus culture.
This will be your positive control.

Streptococcus:
o Using the Blood Agar plates from last lab identify three alpha- or beta-hemolytic
Streptococcus by identifying the chain formation of the Gram-positive cocci.
Inoculate each of these three colonies into a Nitrate Broth and perform a catalase test
on each (record these results as well as the hemolytic results from the Blood Agar)
Into the 4th Nitrate Broth inoculate with the provided Streptococcus culture.

16

This will be your positive control.


Day 4:
Staphylococcus:
o Examine the results of the Nitrate Broth, DNase, and Coagulase inoculations made last
period and identify the species of Staphylococcus.
Streptococcus:s
o Examine the results of the Nitrate Broth inoculations made last lab and identify the
species of Streptococcus.

17

GRAM NEGATIVE INTESTINAL PATHOGENS


Gram-negative intestinal pathogens are a major concern for public health since the two main
pathogens, Salmonella and Shigella, have the ability to cause enteric fevers, food poisoning, dysentery, and
even typhoid fever. Salmonella has over 2200 serotypes including Salmonella typhi, the cause of typhoid
fever, and Shigella contains many serotypes, some of which are the main cause of human dysentery.
Public Health Laboratories routinely test for the presence of the Gram-negative pathogens by the
isolation and identification of Salmonella and Shigella from feces. This makes isolation and identification
difficult due to the presence of Escherichia, Proteus, Enterobactor, Pseudomonas, and Clostridium in feces
(there are actually between 300 to 1000 different species of bacteria in the gut, however, only 30 to 40 of
those species make up the majority of the bacteria found in the gut). In this lab we will isolate and identify
Salmonella from a feces-like mixture of bacteria.
Some identifying traits of Salmonella are: Gram-negative rod, lactose-negative, motility-positive, H2Spositive, urease-negative, and the formation of small colonies on agar.
Day 1:

Perform isolation streaks of the Salmonella containing mixture onto each of the
selective/differential media provided.

From the five selective/differential plates select seven isolated colonies that are presumptive
for being Salmonella based on their appearance and inoculate each into a SIM, Urea, and KIA
media.
o Colonies to be selected:
EMB: lactose-negative bacteria (colonies do not change color)
DES Citrate: lactose-negative bacteria (colonies do not change color)
BGA: lactose-negative (colonies appear pink/white surrounded by red media)
SS Agar: black colonies
BSA: black colonies

Determine the identity of each of the selected colonies.

Day 2:

Day 3:

18

STANDARD PLATE COUNT


To determine a bacterial population count (the number of organisms that are present in a given unit
of volume) several methods are available, the one of the simplest method being the standard plate count.
The standard plate count is accomplished by diluting the culture sample and growing the bacteria up on
Nutrient Agar. Counting the number of colonies that grow on Nutrient Agar determines the number of
bacteria in the diluted sample. To determine the bacterial population count of the original culture, one takes
the bacterial count in the diluted sample and multiples by the dilution factor.
Advantages of the Standard Plate Count over other Bacterial Plate Count Methods is the fact that it
has a very basic principle and technique that requires very minimal amount of equipment but still provides
excellent results. Disadvantages of the Standard Plate Count are that this method can only count bacteria
which are able to develop under the growing conditions provided. Thermophiles or dead colonies cannot be
counted using this procedure.
Rules for Standard Plate Count:
1. Pick the plate that contains between 30 300 bacterial colonies to count.
2. Calculate all the dilution factors (DF) between the counted plate and the original culture.
a. DF = amount added / (amount added + amount already there)
DF = A/(A+B)
b. DF for plating = amounted added / 1ml standard
Plated DF = A/1ml
3. When going from the counted plate back towards the original culture multiple the plate count by the
inverse of all the dilution factors.
Example:
1. Select plate with 32 colonies
2. Plated dilution = 1ml/1ml = 1
All other dilutions = 1/(1+99) = 1/100
3. Bacterial plate count = 32 x 1 x 100 x
100 x 100 = 3.2 x 107 bacteria/ml

19

MYCOLOGY
Terminology:
Mycology is the study of fungi (p) (fungus, s).
Myco- or -mycete indicates fungi or similarity to fungi.
Cellular Traits of Fungi:
They are nonmotile eukaryotes that possess nuclei and organelles.
Fungi seem plant-like but are not plants; they do not possess chloroplasts and are not
photosynthetic.
Large cells ( 10m), making them visible using low magnification (10X objective).
Contain a simple cell wall of chitin (polysaccharide), not similar to the complex peptidoglycan of
bacterial cells, or cellulose cell wall of plants.
Fungi are absorptive heterotrophs, they release exoenzymes into the environment and then
absorb and digest the nutrients.
Most are saprophytes that decompose dead and decaying organic matter; few are parasites of
plants, animals and humans.
Most fungi are aerobic; few are facultative or aerotolerant anaerobes.
Informally divided into:
a. Filamentous molds that create multicellular hyphae. It also includes macrofungi which
produce fleshy reproductive structures (puffballs, mushrooms, and shelf fungi).
b. Unicellular yeast that reproduce by budding.
c. Dimorphic fungi which have both mold (25C) and yeast (37C) life-cycle stages.
Cultural Traits of Fungi:
Fungi tolerate a broad range of pH and osmotic pressures.
Have a narrower tolerance of temperature and redox conditions.
Media selective for fungi will have an acidic pH between 4.8 to 5.0.
Able to metabolize complex substrates, including woody wastes such as cellulose, lignin, tannins,
and hemicelluloses.
o Fungi are critical for decomposition of forest litter and dead trees (saprophytes).
o Yeast form bacteria-like colonies.
Fungal Infections:
Fungal infections are called mycoses (p)/mycosis (s).
Superficial mycosis: topical (skin, hair, nails) infection.
Cutaneous mycosis: infection of the dermis.
o Tinea infections (ringworm, jock itch)
o Candida infections (vaginitis, thrush)
o Subcutaneous mycosis
o Deep seated/systemic mycosis includes respiratory or deep tissue infections (very serious
conditions).
o Coccidiomycosis
o Aspergillosis
Properties of Unicellular Fungi (yeast):
Grow as solitary cells and reproduce by budding.
Distinguished according to the presence or lack of a capsule, size and shape of the cell and method
of formation of the daughter cell.

20

Some yeast can produce pseudohyphae.

Properties of Filamentous Fungi (molds):


Characterized by the development of hyphae (p) are individual fungal filaments (hypha, s).
o Septated hyphae: contain cross-walls forming multiple compartments in the hypha.
o Nonseptate hyphae: each individual hypha is a single, uninterrupted compartment.
o Mycelium: colonial mass of all the hyphae.
o Some molds also produce a sac-like cell known as sporangia.
o Many fungi (such as Mucor spp.) form complementary (+) and (-) mating types, both of
which are required for sexual reproduction.
o Those that form fleshy reproductive structures (mushroom, puffballs, and shelf fungi) are
known as macrofungi.
o A portion of the mycelium grows into the nutrient supply, and is referred to as the
substrate mycelium.
o Part of the mycelium projects upward to produce and spread sexual and asexual spores.
These are known as aerial mycelium.
o Blastospores are asexual spores produced by budding.
Group Terms for Fungi:
Their grouping is based on sexual reproduction and sexual spores, as well as in the presence of cross walls
in the hyphae.
Sexual spores occur after meiosis:
Zygospores: spores located in a zygosporangia, unenclosed spores.
Ascospores: spores located in an ascus (sac-like) cell.
Basidiospore: spores located in a basidia (a microscopic, spore forming structure).
Asexual spores are formed in the sporangium after mitosis and cytoplasmic cleavage:
Sporangiospores: enclosed in sporangium sac.
Conidiospores: unenclosed, exposed spores, forms conidia at the end of hyphae.
I.

Zygomycetes
Vegetative hyphae filaments are branched and nonseptate.
Zygote undergoes meiosis and produce a sexual zygospores.
Can also produce sporangiospores (asexual spores) in a sporangium located on the apical end of
the hyphae.
Includes Mucor and Rhizopus spp.

II. Ascomycetes
Contain septate hyphae.
Produce a haploid ascospores through meiosis in a sac called ascus after mating.
May fuse to form another generation of diploid vegetative cells.
Can also produce asexual conida.
a) Single-cell yeasts:
o Reproduce asexually by binary fission and budding.
o Reproduce sexually by mating. Cell is now an ascus containing many small ascospores.
o Yeasts often produce pseudohyphae by repeated, sequential budding to produce a
filament-like extension of the cell.
o Includes Sacchromyces cervisiae and Schizosaccaromyces octosporus.
b) Multi-cell filamentous molds:
o Sexual mating types.
o Septate vegetative hyphae.
o Asexual conidiospores in chains.
o Includes Penicillium and Asperigillus.

21

III. Basidiomycetes
Septate hyphae that are compacted into large fruiting bodies (mushrooms, shelf fungi, puffballs,
etc).
IV. Deuteromycetes
Also known as fungi imperfecti.
Nearly indistinguishable from septate, conidiospore-forming ascomycetes.
This group is has never been scientifically proven to have a sexual reproductive stage.
Sexual reproduction in this phyla is still considered possible, just unproven.

22

BACTERIAL EXAMINATION OF WATER


Water is routinely examined for fecal contamination, to ensure sanitary water supplies. Fecal
contamination is identified by finding coliforms in the water. A coliform is defined as a facultative anaerobe
that ferments lactose to produce gas, and is a Gram-negative, non-spore-forming rod. Two bacteria that fit
this description are Escherichia coli and Enterobacter aerogenes. Since E. coli and E. aerogenes both fit
the description of coliforms and E. coli is a better indicator of sewage contamination than E. aerogenes, the
IMViC tests must be performed.
Qualitative Tests:
1. Presumptive Test (Day 1):
o A series of 9-12 tubes of lactose broth used to identify if there are any bacteria in the water that
are lactose fermenting gas producing. These tests are also used to determine the most probable
number (MPN) of coliforms present per 100 ml of water.
The MPN is determined by observing the number of lactose broths tubes that are
positive for gas (positive result = 10% or more of Durham tube filled with gas) and
comparing those number to the MPN Determination Table.
2. Confirmed Test (Day 2):
o EMB or Endo Agar plates are inoculated from gas positive lactose broths. On EMB agar,
coliforms produce small colonies with dark centers. On Endo agar, coliforms produce reddish
colonies. These results indicate that the identified colonies are Gram-negative lactose-positive
bacteria. On both EMB and Endo agars metallic sheen is also a result of Gram-negative
lactose-positive bacteria. This step is necessary because in real-world samples you will have a
mix of bacteria and you will need to select for a lactose-positive colony to further tests to
determine if it is E.coli that caused the positive gas reaction or something else.
3. Completed Test (Day 3):
o Lactose-positive Gram-negative colonies are selected and inoculated into lactose broth and
onto Nutrient Agar slants. If gas is produced in the lactose broth and staining reveals that the
bacteria are Gram-negative rods and non-spore-forming, then the water is determined to be
positive for the presence of coliforms.
4. IMViC:
o To confirm that the positive coliform test is due to E. coli and not E. aerogenes, the IMViC set
of tests must be conducted.
Membrane Filter Method:
Method of examining water for bacterial contamination by passing the water through a filter. The
filter captures all the bacteria (does not retain viruses) in the water but allows the water to pass.
The filter is then placed into a differential medium to identify if coliforms are present.
o The membrane is incubated at 44.5oC, which is permissive for the growth of E. coli, but
not E. aerogenes.
o mENDO Agar (pink) shows total coliforms by a golden sheen.
o mFc Agar (light blue) shows fecal coliforms as darker blue colonies on the agar.
Membrane Filter method has advantages over the multiple tube method:
o Higher degree of reproducibility.
o Greater sensitivity.
o Shorter time requirement.

23

ANTISEPTIC EVALUATION
1.

2.

3.

Filter Paper Disk Method


o Used to compare antiseptics based on their bacteriostatic properties.
o Relative effectiveness is measured by the size of the zone of inhibition (measured from edge of
plate to edge of inhibition zone) and can be compared quantitatively against other substances.
o Measures the relative effectiveness of three agents (phenol, formaldehyde, and iodine) against
two organisms (Staphylococcus and Pseudomomas).
Kirby-Bauer Method
o Used to compare effectiveness of antiseptics, both antibiotics (made naturally) and drugs (man
made) which is done under a standardized system.
o Methodology standardizes: diffusibility of the agent, size of the inoculum, type of medium, and
many other factors.
o Relative effectiveness is measured by measuring the diameter of the zones of inhibition.
Minimum Inhibitory Concentration (MIC)
o Establish the minimum concentration of an antiseptic needed to inhibit the growth of a test
microorganism.
o MIC procedures:
Make dilutions of the antiseptic needed for testing as shown:

Tube #
Broth
Dilution
Antiseptic
DI Water
Organism

Set up test and control tubes with the following reagents:

1
5.0 mL
1:10
1.0 mL
3.0mL
1.0mL

2
5.0 mL
1:25
1.0 mL
3.0mL
1.0mL

3
5.0 mL
1:50
1.0 mL
3.0mL
1.0mL

4
5.0 mL
1:75
1.0 mL
3.0mL
1.0mL

5
5.0 mL
1:100
1.0 mL
3.0mL
1.0mL

6
5.0 mL
1:10
1.0 mL
4.0mL
-----

7
5.0 mL
--------4.0mL
1.0mL

8
5.0 mL
--------5.0mL
-----

9
5.0 mL
-----------------

Tubes 6-9 are controls.


Tubes 1-5 are the antimicrobial test tubes.
o The MIC is the most dilute (the minimal concentration) tube which prevents
growth of the test organism.

24

UNKNOWNS
Your unknown will contain 2 different organisms from the following list:
Acinetobacter

Alcaligenes

Bacillus

Corynebacterium

Enterobacter

Eschericia

Klebsiella

Lactobacillus

Micrococcus

Moraxella
Mycobacterium
Neisseria
Proteus
Pseudomonas
Salmonella
Shigella
Staphylococcus
Streptococcus

Procedure:
1. Record the number of your unknown
2. Gram-stain your unknown.
Record Grams result, shape, and arrangement of the organisms in your unknown.
3. Streak your unknown mixture onto 2 TSA plates, and 2 BHI plates. Place 1 TSA and BHI plate in
the incubator, and the other set of plates at room temperature.
4. Identify isolated colonies that appear different on the isolation streaks.
Gram-stain to ensure isolation and identification.
5. Transfer each different isolated colony onto 2 TSA and 2 BHI slants.
1 TSA and 1 BHI slant will be your working stock, while 1 TSA and 1 BHI will be your
reserve stock.
When you use a slant for inoculation, discard that slant and reinoculate a fresh slant from
the reserve stock. The old reserve stock will be come your working stock for the next lab,
and the newly inoculated slant will become the reserve stock.
6. Using the following flowcharts, all other materials provided throughout this class, and Bergeys
Manual (Reference Section of the Library) determine the identity of your unknowns.
7. Submit your results using the dichotomous key on Canvas. Include your unknown number. Circle
the organisms you find and then (using a highlighter) highlight the path you took to figure out
your organisms.
Common Errors When Working with Unknowns:
1. Old cultures (older than 18-24 hours) often produce gram variable reactions. It is best to Gram
stain young cultures.
2. Cultures that have not been properly isolated often produce results that do not fit any particular
pattern. To check for purity, Gram stain and/or re-streak for proper isolation.
3. Contamination with outside bacteria is fairly common, remember to use proper aseptic techniques
and rotate your bacterial stock accordingly.
4. Spore stains are best performed on older cultures as spores will not be formed until your organism
is stressed.
5. Oxidase test is best performed on fresh cultures as older organisms can tend to give false negative
results.

25

26

UNDERSTANDING AND INTERPRETING RESULTS IN MEDIA TO DIFFERENTIATE BACTERIAL SPECIES

OBJECTIVE

SIGNIFICANT
COMPONENT
S

Selects for Gram-positive

Phenylethanol
(PEA) inhibits
Gram-negative
growth

MEDIUM

TYP
E

Phenylethanol
Agar (PEA)

Sele
ctive

Eosin Methylene
Blue Agar (EMB)

Sele
ctive
&
Diffe
renti
al

Selective for Gramnegative


Differential for Lactose
fermenters

Desoxycholate
Agar (DES)

Sele
ctive
&
Diffe
renti
al

Blood Agar

Starch Agar

INDICATOR

RESULTS

Ability to grow with PEA

Growth = Gram-positive
No growth = Gram-negative

Lactose, eosin,
and methylene
blue dye

Colonies absorb dyes due to


the low pH caused from
lactose fermentation

Dark colony = lactose fermenter (+),


absorbs MB due to high acid
production
Pink colony = lactose fermenter (+),
absorbs eosin due to low acid
production
No dye absorbed = lactose fermenter
(-)

Selective for Gramnegative


Differential for Lactose
fermenters

Lactose, and
desoxycholate
(detergent)

Red dye is absorbed into


acid producing colony during
lactose fermentation

Red colony = lactose fermenter (+)


Non-red colony = lactose fermenter (-)

Diffe
renti
al

Detects hemolysins by
observing hemolysis

5% sheep red
blood cells
(RBC)

Action on RBC

Alpha hemolysis = partial lysis of RBC


Beta hemolysis = complete lysis of
RBC
Gamma hemolysis = no lysis
of RBC

Diffe
renti
al

Detects amylase which


hydrolyzes starch to
simple sugar

Soluble starch

Iodine is added after growth


Iodine + starch = purple

Colorless around colony = amylase


(+)
Purple all around colony = amylase
(-)

Lipase Agar

Diffe
renti
al

Detects lipase which


hydrolyzes lipids to fatty
acids and glycerol

Corn oil lipid

Neutral blue dye

Blue dye intensifies = hydrolase (+),


color due to fatty acid release lowering
the pH
No
blue intensification = hydrolase (-)

Milk Agar

Diffe
renti
al

Detects caseinase which


hydrolyzes casein, a milk
protein

Milk (containing
opaque casein)

Loss or retention of opacity

Clearing of opaque milk = caseinase


(+)
No clearing of milk
= caseinase (-)

Gelatin

Diffe
renti
al

Detects gelatinase which


hydrolyzes gelatin protein
into AA

Gelatin

Resolidification of gelatin at
lower temperature

Resolidification = gelatinase (-)


No resolidification = gelatinase (+)

Tryptone Broth

Diffe
renti
al

Detects tryptophanase
which hydrolyzes
tryptone to indole Tests
for presence of indole

Tryptone
polypeptide
containing
tryptophan

Kovac's reagent is added


after growth (3-5 drops)

Red top = tryptophanase (+)


Yellow top = tryptophanase (-)

Nitrate Broth

Diffe
renti
al

Detects nitrate reductase


(nitrase) which converts
nitrate to nitrite

Sodium nitrate

After growth add Nitrate I &


II (10 drops each) and zinc
dust
Look for a red complex to
form

Red without Zn = nitrase (+)


Red with Zn = nitrase (-)
Never turns red = nitrase (+)

Motility Media

Diffe
renti
al

Determines if the
bacteria is motile

Tetrazolium
chloride
(growth
indicator)

Tetrazolium chloride:
red=bacteria growth

Red throughout tube = motility (+)


Red only near stab = motility (-)

Litmus Milk Broth

Diffe
renti
al

Detects moderate/heavy
acid from lactose
fermentation, presence of
caseinase and reductase

Lactose,
Litmus, casein
from milk

Azolitmin dye (Litmusoxidized form):


lavender=neutral, pink=acid,
blue=alkaline, no
color=reduced form

5 to 6 reactions can be read in Litmus


Milk (see below)**

**(1) moderate acid production from lactose = pink, liquid in tube; (2) heavy acid production from lactose = pink, hard or soft curds (coagulation of casein by acid);
(3) alkaline products from metabolism = blue, liquid; (4) reduction of litmus dye to colorless in lower half of broth = viewed as whitish from natural milk color when the litmus
dye is no longer in the oxidized colored state. Due to bacterial reductase in milk; (5) loss of opaque casein protein by caseinase = proteolysis (peptonization),
usually seen starting at the top and increases downward; (6) slimy strands suspended in liquid state of medium = ropiness trait produced by capsule-producing bacteria

MEDIUM
Urea Broth

TYPE

OBJECTIVE

Detects urease (degrades urea to


Urea
ammonia and carbon dioxide)
Detect mixed acid fermenters
Differential
Glucose
(heavy acid production)

Phenylalanine

Oxidase

Catalase

INDICATOR
Phenol red: yellow=acid,
red=neutral, cerise=alkaline
Methyl red is added after growth
(3-5 drops), a pH below 5.1=red
VP I & II are added after growth
(10 drops each)
Bromothymol blue: yellow=acid,
green=neutral, blue=alkaline

Differential

Methyl Red
(MR)
VogesDifferential Detect 2,3-butanediol fermenters
Proskauer (VP)
Simmons
Detect use of citrate as sole
Differential
Citrate Slant
carbon source
Phenol Red
Detects fermentation of selected
(PR) Sugar
Differential sugar
Fermentation
Detects production of gas
Kligler's Iron
(KIA) Tube

SIGNIFICANT COMPONENTS

Glucose
Citrate
Sugar (variable) and Durham
tube

Phenol red: yellow=acid,


red=neutral, cerise=alkaline

Iron ions precipitate H2S


Detects fermentation of glucose, Glucose (0.1%), lactose (1.0%)
Phenol red: yellow=acid,
lactose, and H2S production
and iron salts
red=neutral, cerise=alkaline
Detects phenylalanase
(phenylalanine deaminase) which
Ferric chloride is added after
Differential
Phenylalanine (amino acid)
converts phenylananine to
growth (5-10 drops)
phenylpyruvic acid (PPA)
Dimethyl-p-phenylenediamine
Detects oxidase which removes
Dimethyl-p-phenylenediamine
hydrochloride is added after
Differential hydrogen from different
hydrochloride (oxidase reagent) growth, dark red/purple to black =
substrates
oxidized
Detects catalase which converts
3% hydrogen peroxide is added
Differential hydrogen peroxide to water and
after growth
oxygen
Differential

RESULTS
Red to cerise = urease (+)
Yellow to light orange = urease (-)
Dye stays red = mixed acid fermenter (+)
Turns yellow = mixed acid fermenter (-)
Red on top = 2,3-butanediol fermenter (+)
No red color = 2,3-butanediol fermenter (-)
Growth on slant and blue color = (+)
No growth = (-)
Yellow = fermenter (+)
Light orange to red = fermenter (-)
Gas in tube = gas production (+)
Red slant/yellow butt = glucose fermenter
All yellow = glucose & lactose fermenter
Black = H2S production
Green = phenylalanase (+)
No color change of ferric chloride =
phenylalanase (-)
Dark red/purple to black = oxidase (+)
Remain colorless = oxidase (-)
Bubbles = catalase (+)
No bubbles = catalase (-)

CELLULAR STAINING
STAINING

TYPE

Negative

Simple

Simple

Simple

Gram

Differential

Acid Fast

Differential

Spore

Structural

REASON FOR STAINING

PRIMARY
STAIN

Insoluble negative dye stains


the background
Positively charged stain,
negatively charged bacteria

Indian ink or
Nigrosin
Methylene
blue

Differences in cell wall layers

Crystal violet

Differences in waxy content of


cell wall
Heat driven dye is retained by
spores

Carbol
fuchsin
Malachite
green

MORDANT

DECOLORIZE
R

SECONDARY
STAIN

RESULTS
Background stains black, bacteria are
transparent
All bacteria stain blue

Iodine

Ethyl-alcohol

Safranin

(Heat)

Acid-alcohol

Methylene blue

(Heat)

Water

Safranin

Purple = Gram (+)


Red = Gram (-)
Red = Acid-Fast (+)
Blue = Acid-Fast (-)
Green = spores/endospores
Red = all other cellular material