Mannitol Salt Agar

Mannitol Salt Agar (MSA) is a selective and differential medium. The high concentration of salt
(7.5%) selects for members of the genus Staphylococcus, since they can tolerate high saline levels.
Organisms from other genera may grow, but they typically grow very weakly.
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. Most
pathogenic staphylococci, such as Staphylococcus aureus, will ferment mannitol.  Most non-
pathogenic staphylococci will not ferment mannitol.

The Staphylococcus aureus ferments mannitol and turns the medium yellow. 

The Serratia marcescens does not grow because of the high salt content.

Phenylethyl Alcohol Agar

Phenylethyl alcohol agar (PEA) is a selective medium used to cultivate Gram positive organisms.
The active ingredient, phenylethyl alcohol, inhibits or markedly reduces growth of Gram negative
organisms by interfering with DNA synthesis. PEA also prevents Proteus species from swarming
across the surface of the agar.
 Staphylococcus aureus, a Gram positive organism, grows on this PEA plate while Serratia
marcescens, a Gram negative organism, does not.

Eosin Methylene Blue Agar

Eosin methylene blue agar (EMB) is a selective and differential medium used to isolate fecal
coliforms. Eosin Y and methylene blue are pH indicator dyes which combine to form a dark purple
precipitate at low pH; they also serve to inhibit the growth of most Gram positive organisms. Sucrose
and lactose serve as fermentable carbohydrate sources which encourage the growth of fecal
coliforms and provide a means of differentiating them.
Vigorous fermenters of lactose or sucrose will produce quantities of acid sufficient to form the dark
purple dye complex. The growth of these organisms will appear dark purple to black.  Escherichia
coli, a vigorous fermenter, often produces a green metallic sheen. Slow or weak fermenters will
produce mucoid pink colonies.  Normally-colored or colorless colonies indicate that the organism
ferments neither lactose nor sucrose and is not a fecal coliform.

Escherichia coli often produces a metallic green sheen on EMB.

Hektoen Enteric Agar

Hektoen Enteric Agar (HE) is a selective and differential medium designed to isolate and
differentiate members of the species Salmonella and Shigella from other Enterobacteriaceae.  Bile
salts and the dyes bromthymol blue and acid fuchsin inihibit the growth of most Gram positive
organisms.  Lactose, sucrose, and salicin provide fermentable carbohydrates to encourage the
growth and differentiation of enterics. Sodium thiosulfate provides a source of sulfur. Ferric
ammonium citrate provides a source of iron to allow production of hydrogen sulfide from sodium
thiosulfate, which provides a source of sulfur. Ferric ammonium citrate also allows the visualiztion of
hydrogen sulfide production by reacting with hydrogen sulfide gas to form a black precipitate. 
Enterics that ferment one or more of the carbohydrates will produce yellow to salmon-colored
colonies. Non-fermenters will produce blue-green colonies. Organisms that reduce sulfur to
hydrogen sulfide will produce black colonies or blue-green colonies with a black center. 

Klebsiella pneumoniae ferments lactose and produces salmon-colored growth.  Micrococcus

luteus does not grow.  The white specks are crystals formed by a component of the media that
precipitated out of solution when the plates were cooled.  They look somewhat like a contaminant,
but they do not interfere with the performance of the media.

MacConkey Agar
MacConkey Agar (MAC) is a selective and differential medium designed to isolate and differentiate
enterics based on their ability to ferment lactose.  Bile salts and crystal violet inhibit the growth of
Gram positive organisms. 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.  Bile salts may also precipitate out of the media surrounding
the growth of fermenters because of the change in pH.  Non-fermenters will produce normally-
colored or colorless colonies.
 Klebsiella pneumoniae ferments lactose and produces pink colonies on MAC. Micrococcus
luteus does not grow in the presence of bile salts and crystal violet.

Phenol Red Broth

Phenol Red Broth is a general-purpose differential test medium typically used to differentiate gram
negative enteric bacteria.  It contains peptone, phenol red (a pH indicator), a Durham tube, and one
carbohydrate. 
We use three different kinds of phenol red broths. One contains glucose; one contains lactose, and
the last contains sucrose.  The objective of the exercise is to determine which organisms can utilize
each sugar.
Phenol red is a pH indicator which turns yellow below a pH of 6.8 and fuchsia above a pH of 7.4. If
the organism is able to utilize the carbohydrate, an acid by-product is created, which turns the media
yellow.  If the organism is unable to utilize the carbohydrate but does use the peptone, the by-
product is ammonia, which raises the pH of the media and turns it fuchsia.
When the organism is able to use the carbohydrate, a gas by-product may be produced. If it is, an
air bubble will be trapped inside the Durham tube.  If the organism is unable to utilize the
carbohydrate, gas will not be produced, and no air bubble will be formed.
More in-depth information on the biochemical pathways involved in carbohydrate fermentation can
be found in your lab manual.

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.
 Gelatinase Test
Nutrient gelatin is a differential medium that tests the ability of an organism to produce an
exoenzyme, called gelatinase, that hydrolyzes gelatin.
Gelatin is commonly known as a component of gelled salads and some desserts, but it's actually a
protein derived from connective tissue. When gelatin is at a temperature below 32°C (or within a few
degrees thereof), it is a semisolid material.  At temperatures above 32°C, 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.

Lipase Test
In our lab, the only lipase test we perform is the tributyrin agar test.
Tributyrin agar is a differential medium that tests the ability of an organism to produce an
exoenzyme, called lipase, that hydrolyzes tributyrin oil.  Lipases break down lipids (fats). Tributyrin
oil is a type of lipid called a triglyceride. Other lipase tests use different fat sources such as corn oil,
olive oil, peanut oil, egg yolk, and soybean oil.
Lipase allows the organisms that produce it to break down lipids into smaller fragments.
Triglycerides are composed of glycerol and three fatty acids.  These get broken apart and may be
converted into a variety of end-products that can be used by the cell in energy production or other
processes.
Tributyrin oil forms an opaque suspension in the agar. When an organism produces lipase and
breaks down the tributyrin, a clear halo surrounds the areas where the lipase-producing organism
has grown.
 Starch Hydrolysis
Starch agar is a differential medium that tests the ability of an organism to produce certain
exoenzymes, including a-amylase and oligo-1,6-glucosidase, that hydrolyze starch.  Starch
molecules are too large to enter the bacterial cell, so some bacteria secrete exoenzymes to degrade
starch into subunits that can then be utilized by the organism. 
Starch agar is a simple nutritive medium with starch added.  Since no color change occurs in the
medium when organisms hydrolyze starch, we add iodine to the plate after incubation. Iodine turns
blue, purple, or black (depending on the concentration of iodine) in the presence of starch. A clearing
around the bacterial growth indicates that the organism has hydrolyzed starch.
 MR-VP Tests
Methyl Red (MR) and Voges-Proskauer (VP) broth is used as a part of the IMViC tests as the
medium in which both the Methyl Red and Voges-Prosakuer tests can be performed. It is a simple
broth that contains peptone, buffers, and dextrose or glucose.
Different bacteria convert dextrose and glucose to pyruvate using different metabolic pathways.
Some of these pathways produce unstable acidic products which quickly convert to neutral
compounds. Some organisms use the butylene glycol pathway, which produces neutral end
products, including acetoin and 2,3-butanediol. Other organisms use the mixed acid pathway, which
produces acidic end products such as lactic, acetic, and formic acid. These acidic end products are
stable and will remain acidic.
The Methyl Red test involves adding the pH indicator methyl red to an inoculated tube of MR-VP
broth. If the organism uses the mixed acid fermentation pathway and produces stable acidic end-
products, the acids will overcome the buffers in the medium and produce an acidic environment in
the medium. When methyl red is added, if acidic end products are present, the methyl red will stay
red.
NOTE: Methyl red differs from Phenol red (which is used in the fermentation test and the MSA
plates) in that it is yellow at pH 6.2 and above and red at pH 4.4 and below. Phenol red turns yellow
below a pH of 6.8. If you get these two pH indicators confused, you will have a difficult time
interpreting test results.
The VP test detects organisms that utilize the butylene glycol pathway and produce acetoin. When
the VP reagents are added to MR-VP broth that has been inoculated with an organism that uses the
butylene glycol pathway, the acetoin end product is oxidized in the presence of potassium hydroxide
(KOH) to diacetyl. Creatine is also present in the reagent as a catalyst. Diacetyl then reacts to
produce a red color. Therefore, red is a positive result. If, after the reagents have been added, a
copper color is present, the result is negative.
The MR and VP tests are particularly useful in the identification of the Enterobacteriaceae.
 Citrate Test
Simmons citrate agar tests the ability of organisms to utilize citrate as a carbon source. Simmons
citrate agar contains sodium citrate as the sole source of carbon, ammonium dihydrogen phosphate
as the sole source of nitrogen, other nutrients, and the pH indicator bromthymol blue. This test is
part of the IMViC tests and is helpful in differentiating the Enterobacteriaceae .
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.

SIM Medium
SIM medium is a combination differential medium that tests three different parameters, which are
represented by the three letters in the name:
Sulfur Reduction
Indole Production
Motility
The sulfur reduction test is useful in differentiating enteric organisms.  The indole test is a
component of the IMViC series of tests, which is used for differentiating the Enterobacteriaceae. 
The motility test is useful for testing a wide variety of organisms.  As a whole, the SIM test is
primarily useful for differentiating Salmonella and Shigella.
SIM medium contains nutrients, iron, and sodium thiosulfate. One of the nutrients is peptone, which
contains amino acids, including tryptophan.
If an organism can reduce sulfur to hydrogen sulfide, the hydrogen sulfide will combine with the iron
to form ferric sulfide, which is a black precipitate. If there is any blackening of the medium, it
indicates the reduction of sulfur and is a positive result.
The sulfur and motility test results should be determined before you perform the indole test.
Some bacteria possess the ability to produce the enzyme tryptophanase, which hydrolyzes
tryptophan.  The end products of this hydrolyzation are indole, pyruvic acid, and ammonia, by way of
deamination. The Kovac’s reagent that you add to the SIM medium to test for indole contains
hydrochloric acid, p-dimethylaminobenzaldehyde (DMABA), and n-amyl alcohol. DMABA reacts with
indole to produce a red quinoidal compound.  If the reagent turns red, the indole test is positive.

Nitrate Reduction Test

Nitrate broth is used to determine the ability of an organism to reduce nitrate (NO3) to nitrite (NO2)
using the enzyme nitrate reductase.  It also tests the ability of organisms to perform nitrification on
nitrate and nitrite to produce molecular nitrogen.
Nitrate broth contains nutrients and potassium nitrate as a source of nitrate.
After incubating the nitrate broth, add a dropperful of sulfanilic acid and α-naphthylamine. If the
organism has reduced nitrate to nitrite, the nitrites in the medium will form nitrous acid.  When
sulfanilic acid is added, it will react with the nitrous acid to produce diazotized sulfanilic acid.  This
reacts with the α-naphthylamine to form a red-colored compound.  Therefore, if the medium turns
red after the addition of the nitrate reagents, it is considered a positive result for nitrate reduction.
If the medium does not turn red after the addition of the reagents, it can mean that the organism was
unable to reduce the nitrate, or it could mean that the organism was able to denitrify the nitrate or
nitrite to produce ammonia or molecular nitrogen. Therefore, another step is needed in the test.
If the medium does not turn red after the addition of the nitrate reagents, add a small amount of
powdered zinc.  Be careful, as powdered zinc is hazardous! If the tube turns red after the addition
of the zinc, it means that unreduced nitrate was present. Therefore, a red color on the second step is
a negative result. The addition of the zinc reduced the nitrate to nitrite, and the nitrite in the medium
formed nitrous acid, which reacted with sulfanilic acid. The diazotized sulfanilic acid that was thereby
produced reacted with the α-naphthylamine to create the red complex.
If the medium does not turn red after the addition of the zinc powder, then the result is called a
positive complete. If no red color forms, there was no nitrate to reduce. Since there was no nitrite
present in the medium, either, that means that denitrification took place and ammonia or molecular
nitrogen were formed.

Urease Test
Urease broth is a differential medium that tests the ability of an organism to produce an exoenzyme,
called urease, that hydrolyzes urea to ammonia and carbon dioxide.  The broth contains two pH
buffers, urea, a very small amount of nutrients for the bacteria, and the pH indicator phenol red. 
Phenol red turns yellow in an acidic environment and fuchsia in an alkaline environment.  If the urea
in the broth is degraded and ammonia is produced, an alkaline environment is created, and the
media turns pink.
Many enterics can hydrolyze urea; however, only a few can degrade urea rapidly.  These are known
as “rapid urease-positive” organisms.  Members of the genus Proteus are included among these
organisms.
Urea broth is formulated to test for rapid urease-positive organisms.  The restrictive amount of
nutrients coupled with the use of pH buffers prevent all but rapid urease-positive organisms from
producing enough ammonia to turn the phenol red pink.

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.