Staining Reactions:
Staining
is a simple basic technique that is used to identify microorganisms. Simple
staining is used to study the morphology of all microorganisms (Fig 1).
The
simple stain uses the basic dyes such as Methylene blue or basic fuschin. The
strong negative charge of the bacterial cell will strongly bind with the
positive charged basic dyes and will impart its colour to all bacteria.
Gram
staining is a differential staining technique that imparts different colours to
different bacteria or bacterial structures. Usually it differentiates bacteria
into two groups; gram positive and gram negative. The primary stain Crystal
violet and mordent Iodine form a strong CVI complex all bacteria. Gram positive
cells due to their thick peptidoglycan layer will retain the CVI complex even
after it is subjected to decolourization with acetone or alcohol. Hence the
counter stain Safranin has no action on gram positive cells. But in the case of
gram negative, the thin peptidoglycan layer and more lipid contents in the cell
wall will easily make them susceptible to the action of decolorizer and hence
CVI complex is easily washed out and hence the gram negative cells will the
colour of counter stain Safranin. Hence after the gram staining, the gram
positive cells appear as purple and gram negative cells appear as pink (Fig 2).
The study of morphological features and staining characteristics help in
the preliminary identification of the isolate.
Biochemical reactions:
Gram
negative enteric bacilli play an important role in the contamination of food.
Hence they are the main causative agents of intestinal infection. Gram negative
family includes Shigella, Salmonella, Proteus,
Klebsiella,Escherichia,Enterobacter etc. Usually four tests are used for
differentiation of the various members of Enterobactericeae. They are Indole
test,Methyl red test, Voges proskauer test and Citrate test; collectively known
as IMViC series of reactions.
Indole test:
Indole
tests looks for the presence or absence of tryptophanase enzyme production of
the bacteria. If the enzyme is present, it will degrade the aminoacid
tryptophan in the media and will produce Indole, ammonia and pyruvic acid.
Indole will react with Kovac's reagent to produce a cherry red complex, which
indicates a positive indole test. The absence of red color is indicative of
tryptophan hydrolysis due to the lack of tryptophanse enzyme.
This test detects the ability of microorganism
to ferment glucose and to produce acidic end products. Enteric organism
produces pyruvic acid from glucose metabolism. Some enteric will then use the
mixed acid pathway to metabolize pyruvic acid to other acidic products such as
lactic acid, acetic acid and formic acids. This will reduce the pH of the
media. Methyl red is a pH indicator which is red at the acidic pH (below 4.4)
and yellow at alkaline pH (above 7). The formation of red color after the
addition of Methyl red reagent indicates the accumulation of acidic end
products in the medium and is an indicative of positive test
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