Biochemical Tests

As we observed previously, bacterial colonies can differ greatly in their morphologies.  These differences can help us in identifying different species of bacteria.  Likewise, bacterial species differ in their cellular morphologies and staining properties.  Again, these differences can be used to aid in identifying different species.  Finally, as we observed previously, we can use selective and/or differential media to aid in identifying bacterial species.  Generally, selective and differential media rely on some structural or metabolic property of the species that is preferentially selected.

We also have observed, however, that most of these tests are not extremely specific.  Gram staining, for instance, can allow us to distinguish Gram positive from Gram negative organisms and rod-shaped organisms from coccus-shaped organisms, but does not allow us to make a more specific identification.  Likewise, a selective and differential medium like MacConkey allows us to identify Gram negative, lactose fermenting organisms, but does not allow us to positively identify what specific Gram negative, lactose fermenting organism we are examining.  To aid in the more definitive identification of bacteria, microbiologists have developed a series of biochemical tests that can be used to differentiate even closely related organisms.  These various tests were designed to identify various metabolic properties of different bacterial species.  More importantly, these tests, in conjunction with a dichotomous tree, can lead to the unambiguous identification of an organism.  Today, we will examine the usefulness of several common biochemical tests.

Because clinical microbiologists often must identify bacteria quickly and accurately, a number of companies produce rapid identification systems.  These systems allow one to quickly perform approximately 20 biochemical tests on a sample.  These systems also contain an easy to read chart that allows one to quickly identify an unknown isolate based on the color changes that occur in the various tests. 


Each group will receive trypyic soy agar plates containing:

Escherichia coli
Salmonella typhimurium

Pseudomonas aeruginosa

Streptococcus pyogenes

Staphylococcus aureus


Metabolic reactions that occur in the presence of water and oxygen often result in the formation of hydrogen peroxide (H2O2).  This compound is toxic to cells.  Therefore, most organisms that can grow in the presence of oxygen possess catalase, an enzyme that converts hydrogen peroxide to water and oxygen.

2H2O2 + catalase --> 2H2O + O2

To test for catalase, place a drop of hydrogen peroxide on a microscope slide.  Using a sterile needle, remove a colony from a tryptic soy agar plate.  Mix the bacteria with the hydrogen peroxide and observe.  Test Staphylococcus aureus and Streptococcus pyogenes.


Cytochrome oxidase C is an enzyme that reduces (adds electrons to) oxygen.  This enzyme, therefore, is an oxygen reductase.  Cytochrome oxidase is the last step in the electron transfer system in most aerobic organisms.  It transfers electrons from the electron transport chain to oxygen, forming water in the process.  Not all aerobic organism, though, possess cytochrome oxidase.  Some species possess alternative electron transport molecules.  Because this enzyme is present in some, but not all bacteria, microbiologists have developed a rapid means of testing for the presence of oxidase.

We will test E. coli, S. typhimurium, and P. aeurinosa for the presence of oxidase.  Hold an oxidase reagent dropper upright between your thumb and forefinger.  Squeeze the tube to break a glass ampule inside the plastic tube.  Turn the dropper upside down and apply a few drops of reagent to a strip of filter paper.  With a sterile needle, pick a colony of bacteria and streak it on the reagent-soaked filter paper.  If oxidase is present, the reagent will undergo a chemical reaction resulting in a violet or purple color change.


As we have discussed in class, bacteria can catabolize many different organic substances. Some bacteria possess the enzyme tryptophanase, which converts the amino acid tryptophan to pyruvate and indole through a deamination reaction. The pyruvate, then, can be used in fermentation or respiration reactions. Production of indole is used as a test to differentiate tryptophanase positive and tryptophanase negative organisms.

We will test E. coli and S. typhimurium for the presence of tryptophanase. Add several colonies of bacteria to a tube containing 1mL of indole broth (0.03% tryptophan, 0.1% peptone, and 0.5% dipotassium phosphate). Incubate at 37 degrees C for 2 hours. Squeeze an indole reagent dropper to break the glass ampule inside the plastic tube. Invert the dropper and add the contents to the bacterial culture. If indole is present, it will react with the p-dimethylaminobenzaldehyde present in the reagent to produce a red ring at the surface of the broth. This reaction should occur within 30 seconds.

Triple sugar iron medium

The large intestine of humans contains a large number of microorganisms.  There are estimates, in fact, that human fecal material normally contains more than 1011 bacteria per gram.  Most of these organisms are non-pathogenic and some even benefit us by, for instance, manufacturing vitamin K.  The most common genera present in the human large intestine include Bacteriodes, Bifidobacterium, Enterococcus, Lactobacillus, Escherichia, Enterobacter, Citrobacter, and Proteus.   As a whole, these bacteria often are referred to as enterics.  More appropriately, the enterics are bacteria in the family Enterobacteriaceae (Gram negative, rod-shaped, facultative anaerobes).

While most microorganisms found in the large intestine are non-pathogenic, a number of gastrointestinal diseases are caused by bacteria.  Most often, these bacteria are transmitted via ingestion of contaminated food or water, a transmission mechanism referred to as fecal-oral spread.  Because of these pathogens, several media have been developed to differentiate the intestinal bacteria.  MacConkey medium differentiates lactose fermenters from lactose non-fermenters.  Gram negative, rod-shaped lactose fermenters are referred to as coliform bacteria and, generally, are non-pathogenic.  Triple sugar iron medium often is used to further characterize intestinal microflora.  This medium contains:

0.1% glucose

1.0% lactose

1.0% sucrose

0.02% ferrous sulfate

phenol red

nutrient agar

The phenol red is a pH indicator.  If the medium becomes acidic, then the phenol red turns yellow.  If the medium becomes alkaline, then the phenol red turns purple.  If an organism can only ferment glucose, then the medium initially will turn yellow.  Because there is so little glucose in the medium, however, the bacteria quickly will exhaust the glucose supply and begin to oxidize amino acids for energy.  The oxidation of amino acids produces ammonia as a by-product.  The ammonia will cause an increase in pH and a return to a red or purple color on the surface of the slant.  Therefore, organisms that can only ferment glucose will produce a slant with a red surface and yellow butt.  If the organism being tested can ferment lactose and sucrose, then the entire tube will turn, and remain, yellow.  As we discussed in class, many metabolic reactions result in the production of gas as a by-product.  The production of gas can be detected in TSI slants by the presence of bubbles within the agar.  Furthermore, if the produced gas is H2S, it will react with the ferrous sulfate to produce ferrous sulfide, a black precipitate.

Each group will receive four slants of TSI medium.  One tube will be a negative control.  The other three slants will be used to investigate the properties of E. coli, S. typhimurium, and P. aeruginosa.

With a sterile needle, pick an isolated colony from the TSA plate.  Stab the needle to the bottom of the slant, then streak the surface of the slant.  Incubate overnight at 37 degrees C and record your results.  Repeat for all three species.