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
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
