Biochemical tests- MICROBIOLOGY

Carbohydrate Oxidation-Fermentation

Posted on: July 28, 2009

glycolysis

Fermentation can occur in the presence of oxygen or absence of oxygen.

If bacteria utilises carbohydrates for nutrients, there may be 2 end products, a gas and acid.  The substrate formed from the metabolism of carbohydrate is either glucose or lactose.

Even if bacteria releases enzymes that enable to use carbohydrates through fermentation and oxidation, gas may or may not be produced.

FERMENTATION is noted by acid production which can be observed by a colour change in Durham tubes aka  carbohydrate fermentation tube.

Phenol Red indicator is red in neutral or alkaline solution

If acid is present(+), phenol red changes from red –> yellow

If there is a small “space” at the top of the small tube, it means that gas is trapped in the small  inverted tube inside the bigger tube. Therefore, gas is produced from the breakdown of carbohydrates.

durham fermenation tube with gas produced

Alfred.E.Brown. (2007).  Bensons’s microbiological applications: laboratory manual in general microbiology. (10th ed.). New York: Mc Graw Hill.

reaction in fermentation tubes

Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco: Pearson Education.

Look at the 1st picture above, why is it red if gas is produced and it just means fermentation has occured?

Due to prolonged incubation periods(more than 24h), bacteria will begin to grow oxidatively(oxygen dependent) on the peptone contents of the fermentation medium after using up the carbohydrate contents, causing the neutralization of phenol red indicator and turing it red due to NH3 production.

table of durham fermentation

The only organism that has the ability to break down carbohydrate into glucose and lactose is E.coli and it also has the ability to produce gas during fermentation.

For P. aerguinosa, it displayed negative results for all, which means it can’t break down carbohydrate into glucose & lactose.

Another test which is the MRVP test is able to differentiate between organisms that produce large amounts of acid and organisms that only produce neutral content(acetoin)

M-Methyl Red. VP- Voges -Proskauer. Methyl Red is different from Phenol Red that we mention earlier. It has a pH of 4.4-6. Hence, it changes colour at this range.

For MR test,  if  organic acid is produced here, the (+) result is just a no change in colour of Methyl Red. RED remains.

if neutral acetoin is produced here, the (-) result is a change in colour from RED to YELLOW because at pH 7, the indicator will change its colour.Methyl red test

Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco: Pearson Education.

To test for the presence of acetoin, we use VP test where potassium hydroxide and α naphthol are added.

The upper of the medium will turn red. (+) If the medium turns light brown, it is a (-) result.

Please note that production of acetoin is also affected by the duration of incubation, hence, false negative results may be observed.

We can use Koser’s citrate or Simmon’s citrate to test for the ability of bacteria to ferment citrate. When citric acid or sodium citrate is in solution, it loses a proton or sodium ion to form a citrate ion. Bacteria with citrate lyase can break down citrate to form pyruvate. Pyruvate can be further reduced in fermentation.

Purpose of performing the citrate test

  • Tests for the ability of bacteria to convert citrate (an intermediate of the Kreb’s cycle) into oxaloacetate (another intermediate of the Kreb’s cycle)

Contents  of Simmon citrate include

  • sodium citrate as the carbon source
  • monoammonium phosphate as the nitrogen source
  • and bromthymol blue indicator that changes to blue when medium turns alkaline, which means  (+) result.

Why alkaline means citrate is utilised by the bacteria?

When bacteria uses citrate(carbon) and ammonium(nitrogen), medium turns alkaline as ammonia is produced from ammonium.

citrate test

Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco: Pearson Education.

Citrobacter, Enterobacter will display (+) result while E. coli & Klebsiella will display (-) result.

enterbacteriaceae family

After knowing that some bacteria utilises citrate, we can also find out if other products are formed during metabolism. For example, Hydrogen Sulphide.
hydrogen sulphide prod

Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco: Pearson Education.
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