Eosin Methylene Blue Agar

-Selective and Differential Media

SELECTIVE: Eosin Y and Methylene Blue inhibit growth of Gram-positive Bacteria; Allows Growth of Gram
Negative
DIFFERENTIAL : Differentiates Lactose fermenting, Gram-negative bacteria from Non-Lactose fermenting,
Gram-negative bacteria in the Family Enterobacteriaceae
(Positive Results yield dark purple to black; green for E.coli; Negative results colorless or Normal
color of bacteria)
Mannitol Salt Agar
-Selective and Differential Media
SELECTIVE: High concentration of salt (7.5%) selects for members of the genus Staphylococcus (Gram-Positive),
since they can tolerate high saline levels.
DIFFERENTIAL: MSA also contains the sugar mannitol and the pH indicator phenol red. If an organism can
ferment mannitol, an acidic byproduct is formed that will cause the phenol red in the agar to turn yellow.
MacConkey Agar
-Selective and Differential Media
SELECTIVE: Bile salts and crystal violet inhibit the growth of Gram positive organisms. Allow Growth of Gram-
Negative Bacteria
DIFFERENTIAL: Lactose provides a source of fermentable carbohydrate, allowing for differentiation.
Neutral red is a pH indicator that turns red at a pH below 6.8 and is colorless at any pH greater than 6.8.
Organisms that ferment lactose and thereby produce an acidic environment will appear pink because of
the neutral red turning red. Non-fermenters will produce normally-colored or colorless colonies.
 TRIBUTYRIN AGAR
-Differential Media; Used in LIPASE TEST
-DIFFERENTIAL: Tributyrin agar is a differential medium that tests the ability of an organism to produce an
exoenzyme, called lipase, that hydrolyzes tributyrin oil/glycerol tributyrate (tributyrin)
-Positive Results: When an organism produces lipase and breaks down the tributyrin, a clear halo surrounds
the areas where the lipase-producing organism has grown.
 Tube 1: No fermentation. The pH indicator remains purple. There can still be growth due to the use of amino
acids as sources of energy (usually by respiration).

Tubes 2A and 2B: Fermentation with the production of acid (yellow color) but no gas. A slight amount of
acid is seen in tube 2A, but fermentation is still recorded for this tube.

Tubes 3A and 3B: Fermentation with the production of acid (yellow color) and insoluble gas (bubble in
Durham tube). Tube 3B shows an alkaline reaction on top; this is simply due to deamination of amino acids
whose alkaline reaction has not been over-neutralized by the acid diffusing through the tube from fermentation.
SUGAR FERMENTATION BROTH
-DIFFERENTIAL; Used to Test for Carbohydrate Fermentation
-Phenol red broth is a test is differential for gram negative bacteria.
When the organism ferments carbohydrates, acidic organic by products (Lactic acid, formic acid or acetic acid)
is accumulated which turns the medium into yellow color with reduction in the pH (acidic).
- The inverted Durham tubes will detect the presence of gas.
-The degradation of peptones in the broth may result in the production of alkaline end products, which will
change the broth color to pink often at the top of the tube.
 When Simmons Citrate agar is inoculated withSalmonella When Simmons Citrate agar is inoculated with Escherichia coli ,
typhimurium , the medium turns royal blue. This is a positive result the medium remains green. This is a negative result for the
for the citrate test. citrate test.
SIMMONS CITRATE AGAR

-DIFFERENTIAL; Used for Citrate Test

-Organisms which can utilize citrate as their sole carbon source use the enzyme citrase or citrate-permease to
transport the citrate into the cell.
-These organisms also convert the ammonium dihydrogen phosphate to ammonia and ammonium hydroxide,
which creates an alkaline environment in the medium.
-At pH 7.5 or above, bromthymol blue turns royal blue. At a neutral pH, bromthymol blue is green, as
evidenced by the uninoculated media.
-If the medium turns blue, the organism is citrate positive. If there is no color change, the organism is citrate
negative. Some citrate negative organisms may grow weakly on the surface of the slant, but they will not
produce a color change.
The Serratia marcescens on the left is positive for gelatinase production, as The Salmonella typhimurium on the right is negative, as
evidenced by the liquidation of the media. evidenced by the solidity of the media.
NB + Gelatin
-DIFFERENTIAL; tests the ability of an organism to produce an exoenzyme, called gelatinase, that hydrolyzes
gelatin.
-When gelatin is at a temperature below 32C (or within a few degrees thereof), it is a semisolid material. At
temperatures above 32C, it is a viscous liquid.
-Gelatinase allows the organisms that produce it to break down gelatin into smaller polypeptides, peptides,
and amino acids that can cross the cell membrane and be utilized by the organism.
-When gelatin is broken down, it can no longer solidify. If an organism can break down gelatin, the areas
where the organism has grown will remain liquid even if the gelatin is refrigerated.
 LYSINE IRON AGAR
- Dextrose is an energy source. L-Lysine is the substrate used to detect lysine
decarboxylase and lysine deaminase enzymes. Ferric Ammonium Citrate is an indicator of hydrogen sulfide
production. Sodium Thiosulfate is added as a source of inorganic sulfur. Bromcresol Purple, a pH indicator, is
yellow at or below pH 5.2 and purple at or above pH 6.8. Agar is the solidifying agent.
Casease Test
Skim milk agar is a differential medium that tests the ability of an organism to produce an exoenzyme, called
casease, that hydrolyzes casein. Casein forms an opaque suspension in milk that makes the milk appear white.
Casease allows the organisms that produce it to break down casein into smaller polypeptides, peptides, and
amino acids that can cross the cell membrane and be utilized by the organism.
When casein is broken down into these component molecules, it is no longer white. If an organism can break
down casein, a clear halo will appear around the areas where the organism has grown.
Thioglycollate broth (Fluid Thioglycollate Medium)
-is a medium designed to test the aerotolerance of bacteria. Along with nutrients to support bacterial growth,
it contains sodium thioglycollate, thioglycollic acid, L-cystine, methylene blue, and 0.05% agar. The sodium
thioglycollate, thioglycollic acid, and L-cystine reduce the oxygen to water. Methylene blue is an indicator that
is colorless in an anaerobic environment and greenish-blue in the presence of oxygen. The agar helps retard
oxygen diffusion and helps maintain the stratification of organisms growing in different layers of the broth.
Oxygen is driven out of the broth by autoclaving, but as the broths sit at room temperature, oxygen begins to
diffuse back into the tube. This is evidenced by the small layer of blue-green at the top of the broth.
Obligate aerobes will only grow in this oxygen-rich top layer.
Obligate anaerobes will only grow in the lower areas of the tube.
Microaerophiles will grow in a thin layer below the richly-oxygenated layer.
Facultative or aerotolerant anaerobes can grow throughout the medium but will primarily grow in the middle
of the tube, between the oxygen-rich and oxygen-free zones.
TRYPTONE BROTH

The amino acid tryptophan can be broken down by enzyme tryptophanase to form indole, pyruvic
acid and ammonia as end products. Tryptophanase differentiates indole-positive enterics, such as
E. coli and P.vulgaris from indole-negative enterics, such as S. marcescens.

*Media and Reagents:*

SIM with tryptophan and Kovac's reagent.

*Expected Results:*
Positive test : Kovac's reagent combines with indole and turns the surface red.
Negative test: No red color or copper color development
 Triple Sugar Iron Agar
Triple sugar iron agar (TSI) is a differential medium that contains lactose, sucrose, a small amount of glucose
(dextrose), ferrous sulfate, and the pH indicator phenol red. It is used to differentiate enterics based on the
ability to reduce sulfur and ferment carbohydrates.
As with the phenol red fermentation broths, if an organism can ferment any of the three sugars present in the
medium, the medium will turn yellow. If an organism can only ferment dextrose, the small amount of dextrose in
the medium is used by the organism within the first ten hours of incubation. After that time, the reaction that
produced acid reverts in the aerobic areas of the slant, and the medium in those areas turns red, indicating
alkaline conditions. The anaerobic areas of the slant, such as the butt, will not revert to an alkaline state, and
they will remain yellow. This happens with Salmonella and Shigella.
NOTE:
SIM medium should be read after an incubation of only 24 hours because a longer incubation time can cause a
false negative. Vigorous fermenters such as Escherichia coli and Entrobacter cloacae will ferment all the
available sugars and then begin using the amino acids. This will produce amine groups and cause the medium
to turn alkaline.
If an organism can reduce sulfur, the hydrogen sulfide gas which is produced will react with the iron to form iron
sulfide, which appears as a black precipitate. If the precipitate is formed, it can mask any acid/alkaline results.
Sulfur reduction requires an acidic environment, so if the black precipitate is present, some fermentation took
place. If the butt of the slant is obscured by the precipitate, look at the top of the slant to determine if the
organism could ferment only dextrose (red), or if it could ferment either lactose and/or sucrose (yellow).
If the fermentation produced gas, you may see fissures in the medium, or the entire slant may be raised above
the bottom of the test tube.
 Salmonella typhimuriumferments glucose
Enterobacter cloacaeexhibits fermenation Staphylococcus aureusexhibits acidic
& reduces sulfur.
of glucose and gas production but no sulfur fermentation.
reduction.

This would be read K/A,G. This would be read A/A.

Would be read K/A, H2S.
 Bacillus megateriumfermented sugars but
Micrococcus luteus uses the amino acids Enterobacter aerogenesfermented the
didn't grow in the anaerobic area of the
and does not grow in the butt of the slant. sugars but turned to the amino acids.
butt.
This would be read K/NC. This would be read as K/A.
This would be read A/NC.

Results (slant/butt) Symbol Interpretation

Glucose fermentation only; Peptone
Red/yellow K/A
catabolized
Glucose and lactose and/or sucrose
Yellow/yellow A/A
fermentation
Red/red K/K No fermentation; Peptone catabolized
Red/no color change K/NC No fermentation; Peptone used aerobically
Glucose and lactose and/or sucrose
Yellow/yellow with bubbles A/A,G
fermentation; Gas produced
Red/yellow with bubbles K/A,G Glucose fermentation only; Gas produced
Red/yellow with bubbles and Glucose fermentation only; Gas produced;
K/A,G, H2S
black precipitate H2S produced
Red/yellow with black
K/A, H2S Glucose fermentation only; H2S produced
precipitate
Yellow/yellow with black Glucose and lactose and/or sucrose
A/A, H2S
precipitate fermentation; H2S produced
No change/no change NC/NC No fermentation

A=acid production; K=alkaline reaction; G=gas production; H2S=sulfur reduction