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 isolates

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

       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

     (red, purple)                             (yellow and bubbles in durham tube)

       Bacteria that ferment a carbohydrate 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

Indole + Kovac’s reagent = red color

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

       The 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  

       Enterics initially produce pyruvic acid from glucose metabolism

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

       At  this point methyl red turns red

       Red colour represents positive test

Voges-Proskauer test

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

Pyruvic acid ------------------à acetoin

Acetoin +added alpha naphthol + added KOH = red color           

       Enterics initially produce pyruvic acid from glucose metabolism

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

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

       A copper colour represents a negative voges proskauer test

Citrate utilization test


Citrate ---------------------------------------à oxloacetic acid +acetic acid

Oxaloacetic acid ----à pyruvic acid + carbondioxide

Carbondioxide + sodium ions --------à sodium carbonate (pH increases)  

                                                                    (Blue and growth)

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

       This causes the bromo thymol blue to turn blue

       Presence of growth and blue colour represents a positive test.

Starch hydrolysis test


Starch --------------------------------------------à glucose subunits

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 gelatinase

       Starch hydrolysis – presence of amylase

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