Biochemical reactions – Pharmaceutical Microbiology Third Semester PDF Notes

Biochemical reactions

Learning objectives

At the end
of this lecture, the student will be able to:

       Explain the significance of
biochemical reactions in identification of microorganisms

       Explain the principle involved in
each of the biochemical reaction


       Simple, differential and structural
stains, even if combined with cultivation and observation of colony
characteristics, are not sufficient for the identification of bacterial

       For identification, results
of staining and cultivation must be combined with the results from biochemical

       Biochemical tests evaluate the
metabolic properties of an isolate
which are unique for each species

       A combination of biochemical
tests can be used
to determine the biochemical pattern for an isolate

Carbohydrate utilization

Carbohydrates —————–à acid (pH decreases) or acid
and gas

(yellow and bubbles in durham tube)

       Bacteria that ferment a
e produce acid or acid and gas a end products.

       Acids lower the pH of the medium

       Causing the bromocresol purple or
phenol red pH indicator to turn yellow

       Gases (if produced) collect to form
bubbles in the Durham tube

Indole production


Tryptophan ————————à NH3 + pyruvic acid +

Indole + Kovac’s reagent
= red color

acid tryptophan can be broken down by the enzyme tryptophanase to form indole,
pyruvic acid and ammonia as the end products

aldehyde in Kovac’s reagent combines with indole to form a red color on the
surface of agar. Red color represents a positive test for indole production

Methyl red test

Glucose ————————-à pyruvic acid (1 day)

Pyruvic acid  ——————-à  lactic, acetic and formic acids (2-5 days)

Acids + methyl red ———–à 
Red color  

initially produce pyruvic acid from glucose metabolism

that subsequently metaboloze pyruvic acid to other acids lower the pH of the
medium to 4.2

At  this point methyl red turns red

colour represents positive test

Voges-Proskauer test

à pyruvic acid (1 day)

Pyruvic acid ——————à acetoin

+added alpha naphthol + added KOH = red color

initially produce pyruvic acid from glucose metabolism

that subsequently produce neutral end products, such as acetoin are detected
with Voges-Proskauer test

reacts with alpha naphthol and KOH (KOVAC’s reagent) to produce a red colour in
the medium

copper colour represents a negative voges proskauer test

Citrate utilization test


à oxloacetic acid +acetic acid

Oxaloacetic acid —-à pyruvic acid + carbondioxide

Carbondioxide + sodium
ions ——–
à sodium
carbonate (pH increases)

(Blue and growth)

with the enzyme citrase metabolize citrate to produce alkaline end products
that raise the pH of  the medium to 7.6

causes the bromo thymol blue to turn blue

of growth and blue colour represents a positive test.

Starch hydrolysis test


à glucose

Glucose subunits + added grams
iodine ———-
à clear zone around growth

       Bacteria that produce extracellular
enzyme amylase breakdown starch into single subunits of alpha-D-glucose

       Grams iodine reacts with starch to
produce a purple blue color throughout the agar medium

       A clear zone around the bacterial
growth indicates starch hydrolysis

Catalase test

Organism +
added H2O2                ——–
à H2O + O2

       The catalase enzyme breaks down
hydrogen peroxide into water and oxygen

       The oxygen causes bubbles to form
within seconds, indicating a positive test

Gelatin liquefaction test

Gelatin —————à polypeptides / amino acids

       Gelatin is a protein that is
digested by bacterial extracellular enzymes called gelatinases

       If gelatinases are produced, the
medium will not gel when chilled because gelatin has been broken down

       A liquid medium after chilling
represents a positive test for gelatin utilization


       Biochemical reactions evaluate the
metabolic properties of bacteria

       Biochemical reactions along with
staining and growth characteristics is used to identify bacteria

       IMViC – Indole production, Methyl
red, Voges Proskauer and citrate utilization test is ised to characterize
enteric bacteria

       Citrate utilization test identifies
bacteria that metabolized carbohydrates to acid and alcohol

       Catalase – presence of the enzyme catalase

       Gelatin liquefaction – presence of

       Starch hydrolysis – presence of

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