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Mannitol Salt Agar (MSA) acid into gaseous byproducts.

This is a
test commonly used when trying to
identify Gram-negative enteric bacteria,
This type of medium is both selective
all of which are glucose fermenters but
and differential. The MSA will select for
only some of which produce gas.
organisms such as Staphylococcus
species which can live in areas of high
Like MSA, this medium also contains
salt concentration
the pH indicator, phenol red. If an
organism is capable of fermenting the
The differential ingredient in MSA is the
sugar glucose, then acidic byproducts
sugar mannitol. Organisms capable of
are formed and the pH indicator turns
using mannitol as a food source will
yellow. Escherichia coli is capable of
produce acidic byproducts of
fermenting glucose as are Proteus
fermentation that will lower the pH of the
mirabilis (far right) and Shigella
media. The acidity of the media will
dysenteriae (far left). Pseudomonas
cause the pH indicator, phenol red, to
aeruginosa(center) is a nonfermenter.
turn yellow. Staphylococcus aureus is
capable of fermenting mannitol
The end product of glycolysis is
while Staphylococcus epidermidis is not
pyruvate. Organisms that are capable of
converting pyruvate to formic acid and
formic acid to H2 (g) and CO2 (g), via the
action of the enzyme formic hydrogen
lyase, emit gas. This gas is trapped in
the Durham tube and appears as a
bubble at the top of the
tube. Escherichia coli and Proteus
mirabilis (far right) are both gas
producers. Notice that Shigella
dysenteriae (far left) ferments glucose
but does not produce gas.

*Note - broth tubes can be made
containing sugars other than glucose
(e.g. lactose and mannitol). Because
the same pH indicator (phenol red) is
also used in these fermentation tubes,
the same results are considered
Glucose broth with positive (e.g. a lactose broth tube that
turns yellow after incubation has been
Durham tubes inoculated with an organism that can
ferment lactose).
This is a differential medium. It tests an
organism's ability to ferment the sugar
glucose as well as its ability to convert
the end product of glycolysis, pyruvic

uncertain.and then immediately reduced to some other. However. this is indicative of reduction of nitrate to nitrogen gas.has not been converted to NO2. This further testing includes the addition of sulfanilic acid (often called nitrate I) and dimethyl-alpha-napthalamine (nitrate II). If no red color forms upon addition of nitrate I and II. Zinc will convert any remaining NO3. undetectable form of nitrogen (also a positive result). Durham tube. This is considered a positive result. If instead. this indicates a called nitrate reductase). In order to determine which of the preceding is the case.was converted to NO2. Nitrate Broth If the nitrate broth turns red (tubes This is a differential medium. this indicates that either the NO3. this color indicates of reducing nitrate (NO3-) to nitrite (NO2-) a positive result.(a negative result). If there is no color important in the identification of both change in the tube after the addition of Gram-positive and Gram-negative nitrate I and nitrate II. In the case of nonfermenters.to NO2. the tube or other nitrogenous compounds via the turns red (tube pictured on the left) after action of the enzyme nitratase (also the addition of Zn. It is used pictured in the center) after nitrate I and to determine if an organism is capable nitrate II are added. then it will react with nitrate I and nitrate II to form a red compound. If no color change occurs upon addition of zinc then this means that the NO3. .and then was converted to some other undetectable form of nitrogen (a positive result). these tubes are first on the right) after the addition of Zn this inspected for the presence of gas in the indicates a positive test. If the tube is colorless (picture After incubation.and form the red pigment (a verified negative result).thus allowing nitrate I and nitrate II to react with the NO2. If nitrite is present in the media. This test is negative result. in many cases gas is produced by fermentation and further testing is necessary to determine if reduction of nitrate has occurred. or that NO3.was converted to NO2. the result is species. elemental zinc is added to the broth.

and form bright pinky-red colonies (plate the Micrococcus spp. crystal violet which inhibit the growth of Gram- positive bacteria. to oxygen gas clearly indicate a catalase turn a bright pinky-red color. Fermentation of this sugar results in an acidic pH and The bubbles resulting from production of causes the pH indicator.Catalase Test This test is used to identify organisms that produce the enzyme. catalase. agar is commonly used to differentiate TheStreptococcus and Enterococcus between the Enterobacteriaceae. fermentation such as Escherichia coli. Thus positive result. differential. neutral red. The differential ingredient is lactose. MacConkey positive. spp. The sample on the right organisms capable of lactose below is catalase positive. The Staphylococcus spp. The selective ingredients are the bile salts and the dye. . are catalase negative. are catalase pictured on the left here). This MacConkey agar enzyme detoxifies hydrogen peroxide by breaking it down into water and oxygen This medium is both selective and gas.

This is a positive result (the tube on the iodine must be added to the agar.6-glucosidase. In order to interpret (bromthymol blue) from green to blue. Klebsiella iodine reacts with the starch to form a pneumoniae and Proteus mirabilis are dark brown color. bacteria acid. In organisms and amylopectin molecules.6- hydrolyzed into pyruvic acid and CO2. Organism on left is positive for lactose fermentation and that on the right is Starch hydrolysis test negative. It is often the genera Clostridium and Bacillus. Thus. determine if an organism can use citrate Often used to differentiate species from as its sole carbon source. CO2 is produced. If glucosidase into the extracellular space. This test is used to identify bacteria that can hydrolyze starch (amylose and Simmon’s Citrate Agar amylopectin) using the enzymes - This is a defined medium used to amylase and oligo-1. the enzyme citrase hydrolyzes bacterial cell wall. The oxaloacetic acid is then must secrete-amylase and oligo-1. The right is citrate positive). Escherichia coli and Shigella dysenteriae are citrate negative. examples of citrate positive organisms. the results of the starch hydrolysis test. Na2CO3). these capable of utilizing citrate as a carbon organisms can not pass through the source. used to differentiate between members Because of the large size of amylose of Enterobacteriaceae. In order to use these citrate into oxaoloacetic acid and acetic starches as a carbon source.g. which can then enter directly into the The alkaline pH turns the pH indicator glycolytic pathway. it reacts with These enzymes break the starch components of the medium to produce molecules into smaller glucose subunits an alkaline compound (e. hydrolysis of the .

The organism shown on the right is negative for starch hydrolysis. .starch will create a clear zone around the bacterial growth. Bacillus subtilis is positive for starch hydrolysis (pictured below on the left).