Biochemical tests for identification of bacteria

• Bacteria utilize various kinds of nutrients in a variety of ways.

Different bacterial species have different sets of enzymes and
therefore have different metabolic pathways. Since these
characteristics of each species may be different, biochemical
characters are used as the means of identifying and classifying them.
• Following are the biochemical tests are used in bacteriology.
Catalase test :
• Principle: Catalase enzyme acts on H2O2 and split it into H2O and
O2. This enzyme is present in Staphylococcus but absent in
Streptococcus and so catalase test is used to distinguish between
Staphylococcus and Streptococcus.
• Method:
Organisms are grown on plain agar. One drop of 3% H2O2 is placed
on a clean slide and a little amount of culture is mixed with platinum
loop.
Result:
Immediately after mixing, the gas bubbles are seen on a slide due to O2
production if catalase test is positive. If test is negative no gas bubbles are
seen.
Staphylococcus : Catalase positive
Streptococcus : Catalase negative
Coagulase test:
• Principle: Coagulase enzyme acts on human or rabbit plasma and clots the
plasma. This enzyme is present in pathogenic Staphylococci while absent in non-
pathogenic Staphylococci. So, this test is used to differentiate pathogenic and
non-pathogenic Staphylococci.
• Method:
The rabbit plasma is diluted 1:10 or 1:15 in normal saline and 0.5ml of diluted
plasma is taken in a small test tube. Add 1-2 drops of young (18-24 hours old)
broth culture of Staphylococcus in it. The test tube is kept in incubator at 37 °C.
The tube is examined at 1 hour, 4 hours and 24 hours interval for plasma clot.
Result:
• Plasma is clotted : Coagulase positive i.e. pathogenic
Staphylococcus
• Plasma is not clotted : Coagulase negative i.e. non-pathogenic
Staphylococcus
IMViC tests:
• This is a group of tests which includes Indole, M.R., V.P. and Citrate
tests. These four tests are very useful in differentiating Gram negative
bacteria particularly those of enterobacteriaceae family.
Indole test:
• Principle: Bacteria utilize various amino acids as their food. Amino
acid tryptophan, may be utilized by certain bacteria by producing
indole as its by product. Indole can be detected in the medium by
colourimetric test on addition of suitable reagents.
• Method:
To a 48 hrs. old culture in peptone/tryptone water, the. Then 1ml of
xylol is added to trap the indole. Then 0.5ml of indole reagent is
added.
Result:
• If indole is produced by the bacterial culture, then red or pink
coloured ring is formed at the top.
Methyl Red test:
• Principle: When some organisms are cultivated in media containing glucose, they
act on glucose and produce organic acids by fermentation. Due to acid
production, pH of culture become acidic (pH 4.5) and is maintained. In such
culture when methyl red indicator is added, culture shows positive reactions.
• Method:
The culture is inoculated in to glucose phosphate peptone water (GPPW)
containing glucose, phosphate (K2HPO4) and peptone and the tube is kept in
incubator for two days. Then two drops of methyl red indicator is added in the
tube.
Result:
• Red colour : MR positive
• Yellow colour : MR negative
Voges-Proskauer test (V.P. test): (Barritt’s
method):
• Principle: Some organisms produce acetyl methyl carbinol from
glucose. This metabolic product is oxidized to diacetyl in presence of
alkali and combines with guanidine group of the reagent to produce
pink colour.
• Method:
To a 2-5 days old culture in GPPW (Glucose phosphate peptone
water) 0.6 ml of 5% alpha naphthol solution and 0.2 ml of 40% KOH
solution are added. Shake and slope the tube. Examine after 25 min.
and 1 hour.
Result:
• Pink colour : V.P. positive
• Yellow colour : V.P. negative
Citrate test:
• Principle: This test is used to test the ability of certain organisms to
utilize citrate as a source of carbon and energy. In this medium no
natural organic food like peptone or meat extract is added.
• Method:
A loopful of colony from solid culture is inoculated in a tube of
simmon’s citrate agar and with this an inoculated tube is kept as a
control. The tubes are kept in incubator up to 7 days at 37 °C.
Result:
• If citrate is utilized by bacteria, there is blue colouration in
medium due to bacterial growth. If citrate is not utilized, then
there is no blue colouration.
Differentiation of enteric bacteria by IMViC
tests:

Genus Indole M.R. V.P. Citrate

Escherichia + + - -

Enterobacter - - + +

Klebsiella - - + +

Salmonella - + - +

Proteus + + - +
Urease test:
• Principle: It is used for the detection of proteus and to differentiate
these organisms from other enteric species. It may also be used for
any species of bacteria that is able to hydrolize urea. The organisms
able to hydrolize urea, produce ammonia and thus alkalinity due to
which media become red/pink..
• Method:
Inoculate heavily a slant of a Christensens’s urea agar and incubate at
37 °C, examine after 4 hours and daily for 5 days.
Result:
• Red colour : Urea positive
Oxidase test:
• Principle: There are a number of oxidases which can utilize oxygen
directly. They contain copper or iron as a part of their molecule. The
ability to produce this enzyme is characteristic of only a few bacteria
e.g. Neisseria and Pseudomonas. All the enterobacteria are negative
for oxidase test.
• Method (Plate method):
Culture grown on a agar plate is covered with the oxidase reagent(1%
para-amino-dimethyl-aniline-monochloride) and after one to two
minutes, excess reagent is poured off (the test can also be performed
on a filter paper).
Result:
• In a positive case, within a few minutes, the colonies change to
pink, red and gradually turn to black.
Gelatin liquefaction test :
• Principle: Gelatin is a protein which is liquefied by gelatinase
enzyme produced some bacteria. Liquefied gelatine can not form a
gel. This criteria helps in identification of certain bacteria.
• Method:
Culture is inoculated to gelatine stab medium and incubated for 48
hours along with the uninoculated control. The tubes are placed in a
refrigerator till the medium of uninoculated tube solidifies.
Result:
• If the gelatine does not solidify, it indicates a positive test.
Nitrate reduction test:
• Principle: This test determines the reduction of nitrate to nitrite, ammonia or
free nitrogen . Presence of nitrite in the culture medium is considered as positive.
In the absce of nitrite , it is difficult to say whether nitrate reduction has taken
place or not. This can be ascertained by testing for the presence of unreduced
nitrate by adding powdered zinc to the culture medium.
• Method:
To 5ml nitrate broth culture, 1ml each of nitrate reduction test solution A
(Sulphanilic acid –acetic acid mixture) and solution B (alpha naphthylamine –
acetic acid mixture) is added and the tube shaken.
Result:
• Red colour indicates a positive test i.e., presence of nitrite .
• If red colour does not develop then on addition of zinc powder of pink
colour develops then it indicates negative test till absence of pink colour
indicates positive test i.e., nitrite has been reduced beyond nitrite to
ammonia or free nitrogen.
Sugar fermentation test:
• Principle: Most of the bacteria have got enzymes which act on
carbohydrates and produce organic acids and gas. A bacterial species can
act on certain sugars which helps in their identification. In this way various
carbohydrates like glucose, lactose, maltose, mannitol, xylose, arabinose,
dulcitol etc. are used in differentiation of various species within a genus.
• Method:
0.5-1gm of respective sugar sdded in 100ml of peptone water and pH
indicator is added (Andrade’s indicator) to find out acid production. To find
out gas production, durham’s tube is kept at the bottom of the tube 2-3
drops of liquid culture is added in sugar medium and the inoculated sugar
tubes are kept in incubator for 24 hours and examined up to 7 days.
Result:
• Acid production is seen by production of yellow colour. Gas production, gas
bubbles in durham’s tube.
Motility tests:

• The majority of bacteria are motile by means of flagella. Motility can

be temperature-dependent and some bacteria tend to be motile at
ambient temperatures but not at 37°C.
• Two main methods are used to demonstrate motility.
 Method 1:
• Direct microscopy using a young broth culture (2-4 hours' incubation) of the bacterium
incubated at room temperature. A 'hanging-drop' preparation is made by placing a drop of
the broth culture in the centre of a clean cover-slip and then inverting it over a
transparent plastic or glass ring (about 5 mm deep) fixed to a microscope slide. The
preparation is brought into focus under low- power and then examined with the high-
power dry objective with reduced illumination.
• Result: The bacterium is motile if individual cells are moving towards and away from other
cells. Brownian movement is a constant and random jiggling of all bacterial cells and other
small particles and must not be mistaken for true motility. If the bacterium appears to be
non-motile by direct microscopy, then this negative result should be checked by the
inoculation of motility media.
Method 2:
• Semi-solid motility media are available commercially. Tetrazolium
salts can be added to these media to aid in the detection of motility.
Before autoclaving the motility medium, 0.05 g of 2,3,5-
triphenyltetrazolium chloride (TTC) is added per litre of medium.
• Result: Tetrazolium salts are colourless but as the bacterium grows
the dye is incorporated into the bacterial cells where it is reduced to
an insoluble red pigment, formazan. The red colour forms only in the
area of medium where the bacterium is growing.
 Oxidation-fermentation (0-F) test:

• This test is used to determine the oxidative or fermentative metabolism of a

carbohydrate by the bacterium. The medium is semi-solid and usually contains
glucose as the test sugar and bromothymol blue as the pH indicator. Bacteria that
can metabolise glucose under either aerobic or anaerobic conditions are
facultative anaerobes and in this test are said to be fermentative. The aerobes that
require atmospheric oxygen for growth and metabolism are called oxidative.
• Result: The uninoculated medium (pH 7.1) is green and if acid is produced by the
bacterium, as a result of glucose utilization, the medium becomes yellow (pH 6.0).
Some bacteria are unreactive in the conventional O-F medium, either because they
are unable to grow in the basal medium or because they cannot attack glucose.