LAB: USING BIOCHEMICAL TESTING TO IDENTIFY BACTERIA

A. STARCH HYDROLYSIS

DISCUSSION

Starch is a polysaccharide which appears as a branched polymer of the simple sugar glucose. This means that starch is really a series of glucose molecules hooked together to form a long chain. Additional glucose molecules then branch off of this chain as shown below.

GLU|

( ---GLU-GLU-GLU-GLU-GLU-GLU-GLU--- )n

Some bacteria are capable of using starch as a source of carbohydrate but in order to do this, they must first hydrolyze or break down the starch so it may enter the cell. The bacterium secretes an exoenzyme which hydrolyzes the starch by breaking the bonds between the glucose molecules. This enzyme is called a diastase.

( ---GLU / GLU / GLU / GLU / GLU / GLU / GLU--- )naction of diastase

The glucose can then enter the bacterium and be used for metabolism.

MEDIUM

Starch agar (one plate)

ORGANISMS

Trypticase Soy broth cultures of Bacillus subtilis and Escherichia coli.

PROCEDURE (to be done in pairs)

1. Using a marker, draw a line on the bottom of a Starch agar plate so as to divide the plate in half. Label one half B. subtilis and the other half E. coli.

2. Make a single streak line with the appropriate organism on the corresponding half of the plate.

3. Incubate at 37°C until the next lab period.

4. Next period, iodine will be added to see if the starch remains in the agar or has been hydrolyzed by the exoenzyme diastase. Iodine reacts with starch to

produce a dark brown or blue/black color. If starch has been hydrolyzed there will be a clear zone around the bacterial growth because the starch is no longer in the agar to react with the iodine. If starch has not been hydrolyzed, the agar will remain a dark brown or blue/black color.

B. PROTEIN HYDROLYSIS

DISCUSSION

Proteins are made up of various amino acids linked together in long chains by means of peptide bonds. Many bacteria can hydrolyze a variety of proteins into peptides (short chains of amino acids) and eventually into individual amino acids. They can then use these amino acids to synthesize their own proteins and other cellular molecules or to obtain energy. The hydrolysis of protein is termed proteolysis and the enzyme involved is called a protease. In this exercise we will test for bacterial hydrolysis of the protein casein, the protein that gives milk its white, opaque appearance.

MEDIUM

Skim Milk agar (one plate)

ORGANISMS

Trypticase Soy broth cultures of Bacillus subtilis and Escherichia coli.

PROCEDURE (to be done in pairs)

1. Divide the Skim Milk agar plate in half and inoculate one half with Bacillus subtilis and the other half with Escherichia coli as done above with the above starch agar plate.

2. Incubate at 37°C until the next lab period. If casein is hydrolyzed, there will be a clear zone around the bacterial growth. If casein is not hydrolyzed, the agar will remain white and opaque.

 

C. FERMENTATION OF CARBOHYDRATES

DISCUSSION

Carbohydrates are complex chemical substrates which serve as energy sources when broken down by bacteria and other cells. They are composed of carbon, hydrogen, and oxygen (with hydrogen and oxygen being in the same ratio as water; [CH2O]) and are usually classed as either sugars or starches.

Facultative anaerobic and anaerobic bacteria are capable of fermentation, an anaerobic process during which carbohydrates are broken down for energy production. A wide variety of carbohydrates may be fermented by various bacteria in order to obtain energy and the types of carbohydrates which are fermented by a specific organism can serve as a diagnostic tool for the identification of that organism.

We can detect whether a specific carbohydrate is fermented by looking for common end products of fermentation. When carbohydrates are fermented as a result of bacterial enzymes, the following fermentation end products may be produced:

1. acid end products, or 2. acid and gas end products.

In order to test for these fermentation products, you inoculate and incubate tubes of media containing a single carbohydrate (such as lactose or maltose), a pH indicator (such as phenol red) and (optional) a Durham tube (a small inverted tube to detect gas production). If the particular carbohydrate is fermented by the bacterium, acid end products will be produced which lowers the pH, causing the pH indicator to change color (phenol red turns yellow to clear. If gas is produced along with the acid, it collects in the Durham tube as a gas bubble. If the carbohydrate is not fermented, no acid or gas will be produced and the phenol red will remain red.

MEDIA

2 tubes of Phenol Red Lactose broth

ORGANISMS

Trypticase Soy agar cultures of Bacillus subtilis, and Escherichia coli

PROCEDURE (to be done in pairs)

1. Label each tube with the name of the sugar in the tube and the name of the bacterium you are growing.

2. Inoculate one Phenol Red Lactose broth tube with Bacillus subtilis.

3. Inoculate a second Phenol Red Lactose broth tube with Escherichia coli.

4. Incubate all tubes at 37°C until next lab period.

 

D. INDOLE PRODUCTION

DISCUSSION

Sometimes we look for the production of products produced by only a few bacteria. As an example, some bacteria use the enzyme tryptophanase to convert the amino acid tryptophan into molecules of indole, pyruvic acid and ammonia. Since only a few bacteria contain tryptophanase, the formation of indole from a tryptophan substrate can be another useful diagnostic tool for the identification of an organism. Indole production is a key test for the identification of Escherichia coli.

By adding Kovac's reagent to the medium after incubation we can determine if indole was produced. Kovac's reagent will react with the indole and turn red. If indole is not produced the Kovac's reagent remains yellow.

MEDIUM

Two tubes of Indole medium. This medium contains the amino acid tryptophan. It can be used to detect indole production.

ORGANISMS

Trypticase Soy broth cultures of Bacillus subtilis, and Escherichia coli

PROCEDURE (to be done in pairs)

1. Stab one tryptone broth medium tube with Escherichia coli.

2. Stab a second tryptone broth medium tube with Bacillus subtilis .

3 . Incubate at 37°C until the next lab period.

4. Next lab period add Kovac's reagent to each tube to detect indole production.

E. DIFFERENTIAL MEDIA FOR GRAM NEGATIVE AND GRAM POSITIVE BACTERIA

DISCUSSION

Eosin-methylene blue agar is selective for gram-negative bacteria against gram-positive bacteria.  In addition, EMB agar is useful in isolation and differentiation of the various gram-negative bacilli and enteric bacilli, generally known as coliforms and fecal coliforms respectively.  The bacteria which ferment lactose in the medium form colored colonies, while those that do not ferment lactose appear as colorless colonies.  EMB agar is used in water quality tests to distinguish coliforms and fecal coliforms that signal possible pathogenic microorganism contamination in water samples.  EMB agar is also used to differentiate the organisms in the colon-typhoid-dysentery group:  Escherichia coli colonies grow with a metallic sheen with a dark center, Aerobacter aerogenes colonies have a brown center, and non-lactose-fermenting gram-negative bacteria appear pink.

MEDIUM

One plate of EMB medium. This medium contains eosin and methylene blue and agar. It can be used to differentiate Gram (+) and Gram (-) bacteria.

ORGANISMS

Trypticase Soy agar cultures of Bacillus subtilis, and Escherichia coli

PROCEDURE (to be done in pairs)

1. Divide the EMB agar plate in half and inoculate one half with Bacillus subtilis and the other half with Escherichia coli as done above with the above starch agar plate.

2. Incubate at 37°C until the next lab period.

F. UTILIZATION OF CITRATE AS SOLE CARBON SOURCE

DISCUSSION

The citrate test is commonly employed as part of a group of tests, the IMViC tests, that distinguish between members of the Enterobacteriaceae family based on their metabolic by-products (1, 2, 4). In the most common formulation, citrate is the sole source of carbon in the Simmons citrate medium while inorganic ammonium salt (NH4H2PO4) is the sole fixed nitrogen source (3, 4, 7, 8). When an organic acid such as citrate is used as a carbon and energy source, alkaline carbonates and bicarbonates ultimately are produced (5, 7). The visible presence of growth on the medium and the change in pH indicator color due to the increased pH are the signs that an organism can import citrate and use it as a sole carbon and energy source; such organisms are considered to be citrate positive. Citrate, a Krebs cycle (i.e., TCA cycle or citric acid cycle) intermediate, is generated by many bacteria; however, utilization of exogenous citrate requires the presence of citrate transport proteins (permeases) (7). Upon uptake by the cell, citrate is cleaved by citrate lyase to oxaloacetate and acetate. The oxaloacetate is then metabolized to pyruvate and CO2.

citrate = oxaloacetate + acetate

oxalacetate = pyruvate + CO2

Further metabolic breakdown is dependent upon the pH of the medium. Under alkaline conditions, pyruvate is metabolized to acetate and formate.

pyruvate = acetate + formate

At pH 7.0 and below, lactate and acetoin are also produced.

pyruvate = acetate + lactate + CO2

pyruvate = acetoin + CO2

The carbon dioxide that is released will subsequently react with water and the sodium ion in the medium to produce sodium carbonate, an alkaline compound that will raise the pH. In addition, ammonium hydroxide is produced when the ammonium salts in the medium are used as the sole nitrogen source. The bromothymol blue pH indicator is a deep forest green at neutral pH. With an increase in medium pH to above 7.6, bromothymol blue changes to blue. Although uncommon, natural E. coli variants that are citrate positive have been isolated. Citrate-negative strains of E. aerogenes have also been found.

MEDIUM

Two slant tubes of Simmons citrate medium. This medium contains citrate as sole carbon source. No sugars.

ORGANISMS

Trypticase Soy agar cultures of Bacillus subtilis, and Escherichia coli

PROCEDURE (to be done in pairs)

1. Streak surface of one tube with Escherichia coli.

2. Streak surface of a second tube with Bacillus subtilis.

3. Incubate at 37°C until the next lab period.