OBJECTIVES: Students should be able to

1. explain the biological basis of each biochemical reaction

2. perform each biochemical test and interpret the test reactions

3. apply these tests to the identification of an unknown

4. describe some common multiple test and rapid test systems used in bacteria identification

5. compare the advantages and disadvantages of immunological assays and DNA probes

6. explain the polymerase chain reaction and its use in microbiology

Bacteria are among the most diverse organisms with respect to the types of enzymes they contain. You will be using some of the more common biochemical tests in the identification of your unknown. A variety of specialized media are designed to facilitate determining the biochemical reactions of bacteria. Make use of the DIFCO manual for the composition of the media.



Most bacteria obtain energy from the oxidation of organic compounds such as sugars. Strict aerobes oxidize sugars to carbon dioxide and water through the process of aerobic respiration, using a cytochrome electron transport system and molecular oxygen as the final electron acceptor. Many strict anaerobes perform fermentation reactions. They oxidize sugars to organic compounds (acids, alcohols, aldehydes) that act as the final electron acceptor. No cytochrome system is used in fermentations and oxygen is not involved. Fermentation is not as efficient an energy-yielding process as aerobic respiration. Facultative anaerobes possess the enzymes that allow them to perform either respiration or fermentation depending upon the availablility of oxygen.

The types of compounds that an organism can ferment and the end products that are produced are genetically determined. Among the end products commonly generated by fermentation are organic acids and gases such as carbon dioxide and hydrogen. Fermentation test media contain

1) a single carbohydrate for energy production,

2) nonfermentable sources of nitrogen and other nutritional requirements,

3) a pH indicator such as phenol red or brom cresol purple, and

4) a Durham tube (an inverted vial) that collects gas.

A positive fermentation reaction is indicated by a change in the color of the indicator from red (or purple) to yellow (below pH 7) due to the production of acid end products. Gas production, if observed, is also recorded. Uninoculated controls must be run in order to accurately evaluate results.


Inoculate each broth with a loopful of the organism. Incubate for 24 - 48 hours at 37°C. A set of positive control tubes must be inoculated for comparison.

Examine the tubes for growth (+), acid (A) and gas production (G) and compare to the control tubes. Record your results.


1. How do your fermentation results compare with the data in the Difco manual?

Explain any differences.

2. If organisms do not grow in fermentatin media, what should your interpretation be?

Explain why some organisms grow in the media but do not produce acid.

Methyl Red and Voges-Proskauer Tests (MR-VP)

These fermentation tests are used to differentiate between certain intestinal bacteria called coliforms. The MR-VP broth contains dextrose as the carbohydrate source. Some coliforms will ferment the dextrose to acid products that will cause the pH to drop below pH 5. This is called a mixed acid fermentation. After incubation the addition of methyl red, a dye which turns red below pH 4.4, will indicate whether such fermentation has occured. Other coliforms will convert dextrose to less acidic products such as ethanol or butanediol. These bacteria are negative in the methyl red test.

Butanediol fermentation is demonstrated by the Voges-Proskauer test which measures the presence of acetoin (acetyl methyl carbinol), a precursor to butanediol. This test uses the same medium as the methyl red test and both tests are usually performed in parallel. Barritt's reagents, alpha-naphthol and potassium hydroxide, are added to a 48 hour culture and the tube is shaken to aerate the solution. The development of a pink or red color after agitation is a positive reaction for the production of acetoin.


CONTROLS: E. coli (positive for MR; negative for VP); E. aerogenses (positive for VP; negative for MR)

Inoculate the organism. Incubate for 48 hrs or 5 days at 37°C. (A single uninoculated control should be kept.)

Remove 1 ml from each culture to a clean tube for the VP test.

Methyl red test: Add a few drops of methyl red to the original culture tube and mix the contents. Record your results.

Voges-Proskauer test: Add 0.5 ml (~15 drops) of Barritt's reagent A to the tube and mix. Add 0.5 ml of Barritt's reagent B and mix. Aerate the tube by mixing occasionally over a one hour period or until a pink or red color developments. Record your results.


Among the many enzymes that bacteria may produce are exoenzymes (those that are excreted) used to degrade large polymers into smaller compounds. The detection of such enzyme activities is often confirmatory in identification of unknowns. For example, starch digestion results from the action of amylase released into the surrounding medium. The starch is a polysaccharide that cannot pass across the cell membrane. Amylase breaks starch into smaller sugar residues that can enter the cell and be processed by respiration or fermentation. Gelatinase is another exoenzyme. It can cause the liquefaction of media solidified by gelatin (rather than agar). Caseinase is an enzyme that hydrolyzes casein, the major protein component in milk. As a result of proteolysis (breakdown of protein - also called peptonization) by the enzyme, milk incorporated into agar medium loses its characteristic white appearance and becomes transparent. Lipase production is common to bacteria that grow in foods rich in fats such as butter and mayonnaise. This enzyme breaks fats into its components glycerol and fatty acids. Agar which contains lipids prepared from egg yolks is used in identifying lipolytic activity. The agar loses its opacity surrounding growth of a lipase-producing bacterium.

Most enzymes are endoenzymes. They are produced in the cell and catalyze intracellular reactions. Among the kinds of reactions that are used as evidence in identification of unknown bacteria are a) the breakdown of toxic wastes such as hydrogen peroxide or urea, b) the reduction of nitrate or oxygen, c) the degradation of specific amino acids, and d) the utilization of noncarbohydrate carbon sources for growth

Amylase Production

Amylase activity is demonstrated using starch agar, a medium containing starch as the carbohydrate source.

POSITIVE CONTROL: Bacillus subtilits


Streak each organism across a small portion of the agar surface.

Incubate at 37°C for 48 hours.

Cover the surface with iodine. Rotate to distribute the iodine into a thin layer. Do not flood the plate. Record your results.

Iodine will turn blue when it reacts with starch. A clear zone will be seen where starch has been digested.

Catalase production

Catalase is an enzyme that splits hydrogen peroxide into water and oxygen. Hydrogen peroxide is produced as a byproduct of respiration and is lethal if it accumulates in the cell. All respiring organisms therefore must have some mechanism for detoxification. Catalase is one of the common methods. When hydrogen peroxide is added to a colony of catalase-producing bacteria, it is broken down and the oxygen that is produced can be seen as bubbles.


NEGATIVE CONTROL: Streptococcus sp.

Place a few drops of 3% hydrogen peroxide over a colony.

Observe for production of oxygen.


1. How do aerobic organisms that cannot produce catalase detoxify hydrogen peroxide?

2. What happens to anaerobic organisms in the presence of oxygen?

3. Describe when it would be useful to perform a catalase test?

 Oxidase Production

Oxidases are enzymes that catalyze the reduction of oxygen during respiration. For example, in most gram positive bacteria and many gram negative bacteria cytochrome oxidase performs the final step in electron transport, reducing oxygen to water. Other bacteria, such as the Enterobacteriaceae, do not reduce oxygen using this enzyme. Thus detection of cytochrome oxidase is a valuable tool in differentiating among bacteria. The test utilizes a colorless reagent to detect oxidase. This chemical in the presence of oxygen and an oxidase enzyme will form a colored compound.

POSITIVE CONTROL: Ps. aeruginosa (on agar)


Using a sterile swab, transfer a heavy inoculum of the bacteria to a slide. (A platinum loop may be used to transfer organisms but iron in a nichrome loop may interfere with the reaction.) Place the slide on a white paper and add the oxidase reagent.

Observe for a color change. A positive reaction appears pink, then maroon and finally black. Take care to avoid contact with the oxidase reagent.

NOTE: An alternate procedure is performed by placing some oxidase reagent directly on the colony on the agar or on organisms applied to a filter disk.


1. What is the method for reduction of oxygen in Enterobacteriaceae?

2. How is ATP generated as a result of electron transport systems?

Tryptophan hydrolysis (Indole Production)

The ability to degrade amino acids to identifiable end products is often used to differentiate among bacteria. Tryptophan, for example, is hydrolyzed to indole, pyruvic acid and ammonia by tryptophanase. The pyruvic acid can be further metabolized to produce large amounts of energy. The ammonia is available for use in synthesis of new amino acids.

Indole can be detected by reaction with Kovac's reagent (para-dimethylaminobenzaldehyde in alcohol) to produce a red color.

E. coli

Enterobacter aerogenes

Inoculate tryptone broth (1%) with the organism.

Incubate at 37°C for 48 hours.

Add 10 drops of Kovac's reagent. A red color in the alcohol (upper) layer is a positive result.


1. Why is it important not to incubate your cultures for more than 5 days when performing the indole test? What is the purpose of the alcohol layer?

Citrate Utilization

Some bacteria may be able to use organic compounds other than sugars as their sole source of carbon. The ability to metabolize citrate for example is useful for differentiating among Enterobacteriaceae. Simmons Citrate agar is a medium containing citrate as the sole carbon source and ammonium salts as the sole nitrogen source. Organisms that metabolize citrate utilize the ammonium salts releasing ammonia and increasing the pH of the medium. Brom thymol blue is present in the medium as the indicator dye. It is green at neutral pH and deep blue above pH 7.6.

Koser's citrate broth is another medium used to test for citrate utilization. Growth is evidence of a positive reaction.

E. coli

Enterobacter aerogenes

Using a sterile inoculating needle, streak one organism over the surface of the agar slant, then stab the butt. Repeat with the second organism.

Incubate the tubes at 37°C for 48 hours.

Examine for growth. Is there a change in the indicator dye in the agar?


Why does the agar become alkaline when bacteria grow?

Single Media/Multiple Tests

Several media are designed to yield more than one biochemical reaction. Among the more commonly used media in this category are SIM media, Triple Sugar Iron agar (TSI) and Kliger's Iron agar (KIA).

SIM medium derives its name from three reactions: production of hydrogen sulfide from sulfur-containing amino acids, indole production and motility. Check for motility by observing migration of the inoculum from the stab line through the semisolid medium. The liberation of hydrogen sulfide is indicated by a blackening of the medium. Indole production is determined after the addition of indole reagent. The medium is used primarily for differentiation of gram negative enteric bacteria.

Ps. aeruginosa, E. coli and Shigella

Kliger's iron agar (KIA) and triple sugar iron agar (TSI) are widely used in the identification of gram negative bacteria particularly the Enterobacteriaceae. The media are identical except that TSI contains sucrose in addition to the dextrose and lactose found in KIA. The media are poured as slants and are inoculated with a stab to the butt followed by a streak of the slant surface. The bacteria therefore are exposed to both an anaerobic environment (butt) and an aerobic one (slant). Phenol red is present as an indicator. Do not tighten the cap on the tube.

If the bacteria are nonfermenters, such as Pseudomonas, they can grow on the slant by the aerobic degradation of protein components in the medium to alkaline products. The slant and the butt will remain red.

If the bacteria can ferment dextrose, but not sucrose or lactose, acid is produced in the slant and the butt and the medium turns yellow. The dextrose is used up, however, within 12 hours. Bacteria at the surface continue to grow by degrading proteins . By 18 to 24 hours, the alkaline end products cause the medium in the slant to revert to a red color. Such reactions are characteristic of Shigella and other nonlactose fermenters.

If the bacteria can ferment lactose and/or sucrose as well as dextrose, the slant and butt will remain yellow after prolonged incubation. The high concentration of lactose keeps the slant acidic despite the production of alkaline products by protein degradation.

TSI and KIA also contain sodium thiosulfate and ferrous sulfate as indicators of hydrogen sulfide production. Salmonella spp. (dextrose fermenters, lactose nonfermenters) will yield an acid butt with a black percipitate and an alkaline slant.

Ps. aeruginosa, E. coli and Shigella



Bacteria are not readily identified by a single biochemical test. Gram stain reactions and morphology however often give a strong suggestion of an unknown isolates identity. The results of several selected tests may then be sufficient to verify genus and species. Commercially available systems are composed of a panel of tests capable of differentiating among related organisms. These multiple test systems are usually miniaturized, that is, the tests are performed in a small volume.

Some commercial multiple test systems are also designed to reduce the time needed to establish the identity of a microbe. 

Literature from several kits will be available to allow you to understand what components are present and how they aid in the identification of bacteria. Your instructor will discuss these tests with the class.

Immunological assays have been the most rapid methods for specific identification. These tests employ antibody recognizing only one or a few types of bacteria. Recently rapid identification has become available in the form of DNA probes. DNA probes are pieces of nucleic acid that are unique to a particular organism or group of organisms. The genetic material is tagged with a nonradioactive label. If the bacterial isolate contains a sequence of nucleic acid comparable to that of the probe, the label will be present after the test. Because of their specificity, both immunological and genetic tests are usually used to confirm a suspected identification. A negative result can only be used to eliminate the specific bacteria. Amplification tests using specific primers are among the newest entries into the market for sensitive and specific identification tests.