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

Introduction

•       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

                                                      Tryptophanase

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

Citrase

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

Amylase

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

Summary

•       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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