Bacterial Fermentation

Microbial metabolic processes are complex, but they permit the microbiologist to distinguish among microorganisms grown in culture. Many clinical pathogens can be identified by inoculating pure cultures into media that contain one or more specific biochemicals.

The biochemical reactions that take place can then be determined by relatively simple indicator reagents, included in the medium or added to the culture later.

Some bacteria ferment carbohydrates, producing acidic, alcoholic, or gaseous end products.

The nature of products formed in amino acid metabolism also provides information as to the identification of bacterial species.

The production of visible pigments distinguishes certain types of bacteria (pseudomonas, serratia).

Working with pure cultures freshly isolated from clinical specimens, the microbiologist uses a carefully selected battery of special media to identify their outstanding biochemical properties.

Many different species are distinguished on the basis of the carbohydrates they do or do not attack, as well as by the nature of end products formed during fermentation.

Aerobic respiration produces 36-38 ATP per glucose molecule, and is the most efficient form of energy production.

2) Fermentation : uses an organic molecule as a final electron acceptor.

Fermentation is the least efficient means of energy production; it produces only two ATP per glucose molecule.

organic molecule are mainly carbohydrates (glucose, lactose, maltose, sucrose …etc)

Bacteria mainly produce ATP by :

1) Aerobic respiration : is an oxidative process which uses oxygen as a final electron acceptor.

3) Anaerobic respiration : is similar to aerobic respiration, but it uses an inorganic molecule other than oxygen as the final electron acceptor.

The ATP yield per glucose molecule varies, depending on the

final electron acceptor used.

inorganic molecule may be : Fe, Mn, Co, NO3, sulfate or others.

all fermentative bacteria can ferment the simplest sugar

{dextrose (D-glucose)}.

Fermentation of carbohydrates

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.

Fermentation end products:

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:

1) Acid end product2) Acid and gas end product

Detection of acid production is carried out by pH indicator like phenol red which turns to yellow below pH 6.8 (pH decreases by acids production).

Detection of gas production carried out by Durham tube.

Carbohydrate Utilization

Bacteria produce acidic products when they ferment certain carbohydrates. The carbohydrate utilization tests are designed to detect the change in pH which would occur if fermentation of the given carbohydrate occurred.

Acids lower the pH of the medium which will cause the pH indicator (phenol red) to turn yellow.

If the bacteria do not ferment the carbohydrate then the media remains red.

If gas is produced as a by product of fermentation, then the Durham tube will have a bubble in it.

The carbohydrate tests are the: Glucose (Dextrose) test. Lactose test. Sucrose test.

All carbohydrate test media should be inoculated with the transfer loop.

Left tube shows less acid formation than far right tube, but gas is still made.

Center shows no carbohydrate utilization to produce acid or gas.

Right tube shows acid was produced as evidenced by the yellow color, and gas was made (look at the bubble in the Durham tube).

MR-VP Test

Used to differentiate between enteric bacilli (Coliform) as well as indole and citrate test {IMViC tests}.

This test composes from tow portions:1) The MR portion (methyl red) is used to

determine if glucose can be converted to acidic products like lactate and acetate.

2) The VP portion (Voges-Proskauer) is used to determine if glucose can be converted to acetoin.

A)Methyl redSome Coliform 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 occurred.

Principle

B) Voges-Proskauer TestOther 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, 5% alpha-naphthol (vp1) and 40% potassium hydroxide(vp2), 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.

Limitations

Most members of the family Enterobacteriaceae give either a positive MR test or a positive VP test. However, certain organisms such as Proteus mirabilis may give a positive result for both tests.

Non-fermentative bacteria will give –ve results for both tests (pseudomonas).

Read the VP test at 48 hours. Increased incubation may produce acid conditions in the broth that will interfere with the readings of the results.

VP reagents must be added in the order and the amounts specified or a weak-positive or false-negative reaction may occur. A weak-positive reaction may be masked by a copper-like color which may form due to the reaction of KOH and a-naphthol.