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
• 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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