Biochemical tests for characterization of bacteria

1. Biochemical tests for Characterization of Bacteria
By,
Dr. Pavan K. J
Assistant professor
P C Jabin Science College
Hubballi. Karnataka

2. IMViC Tests
The IMViC series is a group of four individual tests that are
commonly used to identify bacterial species, especially
coliforms.
A coliform is a gram negative, aerobic anaerobic rod which produces
gas from lactose within 48 hours. The presence of some coliforms
Each of the letters in “IMViC” stands for one of these tests.
“I” is for indole;
“M” is for methyl red;
“V” is for Voges-Proskauer, and
“C” is for citrate,
lowercase “i” is added for the ease of pronunciation.
IMViC is an acronym that stands for four different tests
gas from lactose within 48 hours. The presence of some coliforms
indicate fecal contamination.

3. To obtain the results of these four tests three test tubes are
inoculated
Indole test tryptone broth
Methyl red test methyl red – (MR-VP broth)
Voges-Proskauer test Voges Proskauer broth
Citrate utilization test Simmons citrate agar
IMViC tests are employed in the identification/differentiation of
members of family enterobacteriaceae.members of family enterobacteriaceae.
General procedure for performing IMViC Tests and their
interpretations:
Cultures of any members of enterobacteriaceae have
to grow for 24 to 48 hours at 37°C and the respective tests can
be performed:

4. Indole test
It is performed on sulfide-indole-motility (SIM) medium or in
tryptophan broth, or in motility urease indole (MIU) medium.
Result is read after adding Kovac’s reagent.
The positive result is indicated by the red layer at the top of the
tube after the addition of Kovács reagent.
A negative result is indicated by the lack of color change at the top
of the tube after the addition of Kovács reagent.of the tube after the addition of Kovács reagent.

5. Voges-Proskauer (VP) test:
Methyl red test and Voges-Proskauer test
both are done in methyl red–Voges-
Proskauer (MR-VP) broth, but the reagents
that are added varies according to the test
Methyl Red (MR)Test:
Positive methyl red test are indicated by the
development of red color after the addition
of methyl red reagent.
A negative methyl red test is indicated by no
color change after the addition of methyl red
reagent
Voges-Proskauer (VP) test:
Negative test is indicated by
lack of color change after the
addition of Barritt’s A and
Barritt’s B reagents.
A positive Voges-Proskauer
test is indicated by the
development of red-brown
color after the addition of
Barritt’s A and Barritt’s B
reagents.

6. Citrate utilization test
The test is performed on Simmons citrate agar:
Negative citrate utilization test is indicated by the lack of growth
and color change in the tube
A positive citrate result as indicated by growth and a blue color
change.

7. IMViC Test
1. IMViC tests of Escherichia coli
2.Indole: Positive
3.Methyl-Red: Positive
4.Voges-Proskauer test: Negative
5.Citrate test: Negative
2.IMViC tests of Enterobacter aerogenes
1.Indole: Negative
2.Methyl-Red: Negative
3.Voges-Proskauer test: Positive
4.Citrate test: Positive4.Citrate test: Positive
3.IMViC tests of Proteus vulgaris
1.Indole: Positive
2.Methyl-Red: Positive
3.Voges-Proskauer test: Negative
4.Citrate test: Negative
4.IMViC tests of Citrobacter freundii
1.Indole: Negative
2.Methyl-Red: Positive
3.Voges-Proskauer test: Negative
4.Citrate test: Positive

8. Oxidase test
The oxidase test is used to identify bacteria that produce cytochrome
c oxidase, an enzyme of the bacterial electron transport chain.
When present, the cytochrome c oxidase oxidizes the
reagent (tetramethyl-p-phenylenediamine) to (indophenols) purple
color end product.
When the enzyme is not present, the reagent remains reduced and is
colorless.colorless.

10. Purpose of Oxidase test
Oxidase test is most helpful in screening colonies suspected of being one of
the Enterobacteriaceae (all negative) and in identifying colonies suspected of
belonging to other genera such as Aeromonas, Pseudomonas, Neisseria,
Campylobacter, and Pasteurella (positive).
Test requirements for Oxidase test: Moist filter paper with the substrate (1%
tetramethyl-p-phenylenediamine dihydrochloride), or commercially preparedtetramethyl-p-phenylenediamine dihydrochloride), or commercially prepared
paper disk, wooden wire or platinum wire.
Expected results of Oxidase test
Positive: Development of dark purple color (indophenols) within 10 seconds
Negative: Absence of color

11. Quality Control of Oxidase Test
Bacterial species showing positive and negative reactions should be
run as controls at frequent intervals. The following are suggested:
A. Positive control: Pseudomonas aeruginosa
B. Negative control: Escherichia coli
Procedure of Oxidase test:
Take a filter paper soaked with the substrate tetramethyl-p-
phenylenediamine dihydrochloride
Moisten the paper with a sterile distilled water
 Pick the colony to be tested with wooden or platinum loop and smear Pick the colony to be tested with wooden or platinum loop and smear
in the filter paper
Observe inoculated area of paper for a color change to deep blue or
purple within 10-30 seconds
Precaution to be taken while performing oxidase test:
Do not use Nickel-base alloy wires containing chromium and iron
(nichrome) to pick the colony and make smear as this may give false
positive results
Interpret the results within 10 seconds, timing is critical

12. Oxidase test results
Bacterial genera characterized as oxidase positive
include Neisseria and Pseudomonas etc. Genera of
the Enterobacteriaceae family are characterized as oxidase
negative.
Name of Oxidase positive bacteria are: Mneomoics for Oxidase
Positive Organisms- PVNCH (It’s just an acronym inspired by the
famous mneomonic for Urease Positive organisms-PUNCH)
P: Pseudomonas sppP: Pseudomonas spp
V: Vibrio cholerae
N: Neisseria spp
C: Campylobacter spp
H: Helicobacter spp/ Haemophilus spp.
Aeromonas spp
Alcaligens

13. Gelatin is a protein derived from the animal protein collagen–
component of vertebrate connective tissue.
 It has been used as a solidifying agent in food for a long time.
Gelatin hydrolysis test is a great way to highlight proteolysis by
bacteria
Gelatin hydrolysis test
Principle of Gelatin hydrolysis test
Gelatin hydrolysis test is used to detect the ability of an organismGelatin hydrolysis test is used to detect the ability of an organism
to produce gelatinase (proteolytic enzyme) that liquefy gelatin.
Hydrolysis of gelatin indicates the presence of gelatinases.
This process takes place in two sequential reactions.
In the first reaction, gelatinases degrade gelatin to polypeptides.
Then, the polypeptides are further converted into amino acids.
The bacterial cells can then take up these amino acids and use
them in their metabolic processes.

14. Procedure /Method of Gelatin hydrolysis test
There are several methods for determining gelatinase production, all of which make use
of gelatin as the substrate.
The standard and most commonly employed method is the nutrient gelatin stab method.
Inoculate a heavy inoculum of test bacteria (18- to 24-hour-old) by stabbing 4-5
times (half inch) on the tube containing nutrient gelatin medium.
Incubate the inoculated tube along with an uninoculated medium at 35°C, or at
the test bacterium’s optimal growth temperature, for up to 2 weeks.
Remove the tubes daily from the incubator and place in ice bath or refrigerator
(4°C) for 15-30 minutes (until control is gelled) every day to check for gelatin
liquefaction.
(Gelatin normally liquefies at 28°C and above, so to confirm that liquefaction was
due to gelatinase activity, the tubes are immersed in an ice bath or kept in
refrigerator at 4°C).
Tilt the tubes to observe if gelatin has been hydrolyzed.

15. Expected results:-
Positive: Partial or total liquefaction of the
inoculated tube (uninoculated control medium
must be completely solidified) even after
exposure to cold temperature of ice bath or
refrigerator (4°C)
Negative: Complete solidification of the
inoculated tube even after exposure to cold
temperature of ice bath or refrigerator (4°C)
Common bacteria and their reactions to the
Gelatin Hydrolysis Test:
Above tube: Positive
Below Tube: NegativeCommon bacteria and their reactions to the
gelatin hydrolysis test performed on nutrient
gelatin.
Below Tube: Negative
Species Growth Liquefaction
Bacillus subtilis + +
Clostridium perfringens + +
Escherichia coli + –
Proteus vulgaris + +
Serratia liquefaciens + +
Staphylococcus aureus + +

16. Control organisms
Positive Control: Proteus vulgaris
Negative Control: Enterobacter aerogenes
Uses of Gelatin Hydrolysis test
Gelatin hydrolysis test is helpful in identifying and differentiating species of Bacillus,
Clostridium, Proteus, Pseudomonas, and Serratia.
It distinguishes the gelatinase-positive, pathogenic Staphylococcus aureus from the
gelatinase-negative, non-pathogenic S. epidermidis .
Gram-positive, spore-forming, rodshaped, aerobic or anaerobic bacteria such as Bacillus
anthracis, Bacillus cereus, Bacillus subtilis, Clostridium perfringens and Clostridium
tetani, are also positive for gelatin hydrolysis.
The test can also be used to differentiate genera of gelatinase-producing bacteria
such Serratia and Proteus from other members of the family Enterobacteriaceae.

17. Starch Hydrolysis Test
Starch is a complex polysaccharide found abundantly in plants and usually
deposited in the form of large granules in the cytoplasm of the cell.
Starch consists of 2 components—amylose and amylopectin, which are
present in various amounts.
The amylose consists of D-glucose units linked in a linear fashion by α-1,4
linkages. It has 2 non-reducing ends and a reducing end.linkages. It has 2 non-reducing ends and a reducing end.
Amylopectin is a branched polysaccharide. In these molecules, shorter chains
of glucose units linked by α-1,4 are also joined to each other by α-1,6 linkages.
The major component of starch can be hydrolyzed by a-amylase, which is
present in some bacteria while well known in case of fungi.
The ability to degrade starch is used as a criterion for the determination of
amylase production by a microbe.

18. Objective
To determine the ability of an organism to hydrolyze starch
To differentiate organism based on their α- amylase enzyme activity
Principle
Many bacteria produce extracellular enzymes used to catalyze chemical reactions
outside of the cell. In this manner, nutrient sources, such as starch, that are too large
to be absorbed through the cell membrane can be broken down into smaller
molecules and transported into the cell via diffusion.
In the starch hydrolysis test, the test bacteria are grown on agar plates containing
starch.starch.
If the bacteria have the ability to hydrolyze starch, it does so in the medium,
particularly in the areas surrounding their growth while the rest of the area of the
plate still contain non-hydrolysed starch.
Since no color change occurs in the medium when organisms hydrolyze starch,
iodine solution is added as an indicator to the plate after incubation.
While the non-hydrolysed starch forms dark blue color with iodine, its hydrolyzed
end products do not acquire such dark blue color with iodine.

19. Consequently, transparent clear zones are formed around the colonies that
hydrolyze starch while the rest of the plate show a dark blue coloration as
iodine forms the colored complex with starch.

20. Media:
Starch agar is a simple nutritive medium with starch added. Beef extract and
pancreatic digest of gelatin provide nitrogen, vitamins, carbon and amino acids. Agar
is the solidifying agent and starch is the carbohydrate.
Composition:
Peptic digest of animal tissue 5.000, Sodium chloride 5.000, Yeast extract 1.500,
Beef extract 1.500 Starch, soluble 2.000 Agar 15.000 Final pH ( at 25°C) 7.4±0.2
Method
Using a sterile technique, make a single streak inoculation of organism to be
tested into the centre of labeled plate.tested into the centre of labeled plate.
Incubate the bacterial inoculated plates for 48 hours at 37°C.
Following incubation, flood the surface of the plates with iodine solution with a
dropper for 30 seconds.
Pour off the excess iodine.
Examine for the clear zone around the line of bacterial growth.

21. Expected Results
Positive test:A clear zone around the line of growth after addition of iodine solution
indicates that the organism has hydrolyzed starch.
Negative test:A blue, purple, or black coloration of the medium (depending on the
concentration of iodine).
Uses
It aids in the differentiation of species of
genera Corynebacterium, Clostridium, Bacillus, Bacteroides, Fusobacterium, and
members ofEnterococcus spp.
Limitations
It is recommended that biochemical, immunological, molecular, or mass
spectrometry testing be performed on colonies from pure culture for complete
identification.
Colonies cannot be subcultured from the medium after the addition of Gram’s
iodine due to the oxidative nature of the reagent and the resulting cell death.