2. 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.

3. 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.
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.

4. Bacteria mainly produce ATP by :
Aerobic respiration : is an oxidative process which uses oxygen as a final electron acceptor.
Aerobic respiration produces ATP per glucose molecule, and is the most efficient form of energy production.
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)

5. 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)}.

6. 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.

7. 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:
Acid end product
Acid and gas end product

8. 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.

9. 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.

10. 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).

12. MR-VP Test
Used to differentiate between enteric bacilli (Coliform) as well as indole and citrate test {IMViC tests}.
Some of these pathways produce unstable acidic products which quickly convert to neutral compounds. Some organisms use the butylene glycol pathway, which produces neutral end products, including acetoin and 2,3-butanediol.
Other organisms use the mixed acid pathway, which produces acidic end products such as lactic, acetic, and formic acid. These acidic end products are stable and will remain acidic.

13. Principle A)Methyl red
Some 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.

14. B) Voges-Proskauer Test
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.

15. Barritt‘s reagents, 5% alpha-naphthol (vp1) and 40% potassium hydroxide(vp2), are added to a 24 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.

16. Limitations
Non-fermentative bacteria will give –ve results for both tests (Pseudomonas).
Ensure that the reagents are added in the correct sequence; if added first, KOH can react with the peptone in the medium to yield a salmon-pink colour that could be misinterpreted as a positive result.
VP test performed on cultures after 72 hours of incubation may yield weakly positive or false negative results.