This document discusses various biochemical and physiological tests that can be used to identify and characterize different groups of bacteria. It provides details on 14 common biochemical tests including IMVIC, citrate utilization, catalase production, urease, carbohydrate fermentation, and gelatin liquefaction. It also describes 6 physiological tests to determine factors like temperature range, pH tolerance, carbon source utilization, and oxygen relationship. The tests examine the enzymatic activities and metabolic pathways of bacteria, which can help identify them at the genus and species level.

1. RANI LAKSHMI BAI CENTRAL AGRICULTURAL UNIVERSITY
JHANSI,UP.
Course:Plant Bacteriology APP503(2+1)
Topic:Biochemical tests and Physiological tests for various groups of Bacteria.
Submitted to,
Dr.P.P.Jambhulkar
HOD,Associate professor,
Plant pathology,
RLBCAU,Jhansi
Submitted by,
Harish J
Ag/Pg/0020/19
M.Sc(Agri)Plant pathology,
RLBCAU,Jhansi .

2. Bacteria may be conveniently grouped into a number of natural assemblages based
characteristics such as cell shape, spore forming capabilities and whether they are aerobic /
anaerobic or Gram positive / Gram negative.The morphological and biochemical method of
identification of bacteria is the classical method of characterization of bacteria. Classical
identification of individual bacterial species in environmental samples typically involves
isolation, laboratory culture and then taxonomic characterization.These pathogenic bacteria are
some time useful because of their ability to produce pigments, enzymes, any chemicals and
antimicrobial compounds. These compounds like primary and secondary metabolites produced
by microbes are useful in food, agricultural, medicinal and industrial purposes.
In India most of the laboratories are depending on the conventional methods to identify and
study the diversity of bacteria. So in the present study diversity of some selected bacteria was
studied by applying both conventional and molecular methods. The conventional methods
includes, phenotypic characterization (colony morphologies, Gram staining, endospore staining
etc.), biochemical characteristics (nutrient requirements - sugars, enzymatic activities, and /or
metabolic activities). But in conventional methods the characteristics are not static and can
change with stress or evolution. So in this regard the selected cultures are subjected to
biochemical and molecular works to withstand prolonged bacterial study.
BiochemicalTests
Biochemical tests that investigate the enzymatic activities of cells are powerful tests in the
identification of bacteria,itwould be begin by examining the cell morphology, colony
morphology, Gram stain reaction, and environment from which the bacterium was isolated. Once
these properties are known, a specific series of tests, defined by a specific flow chart, would be
performed to aid in the identification of the genus and species of the bacterium.
1.Each different species of bacterium has a different molecule of DNA (i.e., DNA with a
unique series of nucleotide bases).
2.Since DNA codes for protein synthesis, then different species of bacteria must, by way
of their unique DNA, be able to synthesize different protein enzymes.
3.Enzymes catalyze all the various chemical reactions of which the organism is capable.
This in turn means that different species of bacteria must carry out different and unique
sets of biochemical reactions.

3. Types of Biochemicaltests
1.IMVIC
a.Indole test
b.Citrate utilization test
c.Metyl red test
d.Voges–Proskauer test
2.Sulphate reduction test
3.Catalase production test
4.Urease test
5.Fermentation of carbohydrates
6.Gelatin liquefaction test
7.Oxidase test
8.Starch hydrolysis
9.Protein hydrolysis
10.KOH solubility test to antibiotics
1.IMVIC
 This test is important for coliform group of bacteria. The coliform group of bacteria
includes all the aerobic and facultatively aerobic bacteria, gram-negative nonsporulating
bacilli that produce acid and gas from the fermentation of lactose.
 The IMViC name stands for the first letter of each test in the series with the lower ‘i’ for
case for pronunciation the IMViC test is designated to differentiate E.coli from
Enterobacter aerogenes1.
A.Indole test
Principle:Tryptophan is an essential amino acid, which is oxidized by some bacteria resulting in
the formation of indole, pynivic acid and ammonia. The indole test is done by inoculating the test
organism into tryptophan broth, which contain tryptophan. The indole which is produced is
detected by adding KOVAC’s reagent which produced cherry red colored ring.
Procedure:Tryptophan broth was prepared according to the given composition. The broth was
dispensed into the tubes and sterilized. The test organisms were inoculated into the tubes and one
was left uninoculated as control. The tubes were inoculated at 37°c for 48h After incubation 1ml
of KOVAC’s reagent was added to all the tubes including control. The tubes were shaken gently
and allowed to stand for 1-2 min. The tubes were observed for formation of cherry red ring.

4. B.Citrate utilization test
Principle:Citrate test is used to differentiate among enteric bacteria on the basis of their ability
to utilize as their soul carbon source. The utilization of citrate depends upon the presence of
enzyme “Citrate Permease” produced by organism that helps its transports into the cell.
Procedure:Prepare the citrate slant according to the given composition and dispensed into the
the tes tube and autoclaved at 121°c for 15lbs.inocciulate the tubes with the given culture and
then inoculate at 37°c for 48 hours. Observe the tubes for colour change from green to blue.
C.Methyl red test:
The test measures the final acidity of a culture in buffered medium containing glucose and
peptose, but the pH value recorded at the time of the test is not necessarily the lowest value
attained during growth. In the early stage of glucose fermentation sufficient acid is produced by
both E.coli and Klebsiella aurogens to turn methyl red indicator orange and red (+ve) but on
further incubation Klebsiella aurogens breaks down the Pyruvic acid and other acids and
produces a reversion in the reaction of the culture towards neutrality; methyl red turns yellow (-
ve).

5. D.Voges–Proskauertestor VP test
A test used to detect acetoin in a bacterial broth culture. The test is performed by adding alpha-
naphthol and potassium hydroxide to the Voges-Proskauer broth which has been inoculated with
bacteria. A cherry red color indicates a positive result, while a yellow-brown color indicates a
negative result.
Procedure: First, add the alpha-naphthol; then, add the potassium hydroxide. A reversal in the
order of the reagents being added may result in a weak-positive or false-negative reaction.
2.Sulphate reduction test
Principle:
Test is done to show the presence of hydrogen sulphide by sulphide reducing bacteria. Principle-
H2S is formed by some bacteria by reduction of sulphure containing amino acids (Cystein),
Cysteine and metheonine or through reduction of inorganic sulphure compounds like
thiosulphates (S2O3-)or sulphates (SO4--) or sulphite (SO3-- ).The H2S production can be
detected by incorporating a heavy metal salt containing ion (Fe++) or lead (Pb++) ion as a H2S
indicator to neutrient culture medium containing cystein and sodiumthiosulphate as the sulphure
substance.H2S a colourless gas when produced react with metal salts (FeSO4) forming visible
insoluble black Ferrous Sulphide precipitates. Production of H2S from systein and Na2S2O3.
Procedure- Prepare the sulphate agar according to given composition . sterilize the Sulphate
agar by autoclaving at 121°c at 15 lbs pressure for 20 minutes. Dispense the sterilized agar into
sterilized test tubes and labeled with respective organism. Stab inocculate the test organism after
solidification of the medium and incubate the tubes at 37°c for 24 hrs and observed for black
coloration at the point of stab.

6. 3.Catalaseproduction test
Principle-The oxidation of flavoproteins invariably result in the formation of Hydrogen peroxide
as one major product. In addition this oxidation (and other oxygenation) produce small quantities
of and even more toxic radical-The superoxide In aerobes and aerotolerant aerobes the
potentially leathal accumulation of oxygen is prevented by the enzyme superoxide dismutase
which catalysis it to hydrogen peroxide and oxygen.
Procedure- Take a drop of reagent on a clean slide and transfer bacterial colonies into it.
Observe the effervescence formation.
4.Urease test
Principle:Urea is a major organic waste production of protein digestion by most vertebrates and
is excreted in urine.Some microorganisms have the ability to produce the enzyme urease. Urease
is a hydrolytic enzyme, which attack the amide linkage liberating Ammonia.
Procedure- Prepare and dispense Urea agar ( basal medium) into tubes and sterilized.Glucose
and phenol red is added to the basal medium and steamed for 1 Hr.Add filtered sterilized urea
solution and mix all contents well and dispense into sterile test tubes.The test organisms are
inoculated and incubated at 37oc for 24-48 Hrs. Colour change is observed.

7. 5.Fermentationof Carbohydrate
Principle- Fermentation can be defined as an energy yielding process that does not involve an
electron transport chain and exogenous terminal electrons acceptors instead it relies on substrate
level. Phasphorylation and an endogenously generated electron acceptor.
Procedure-
 The fermentation medium is prepared and sterilized with the indicator and Durham’s tube
has no air bubbles in them.
 Sugar solution is autoclaved at 10 lbs/Sq inch pressure for 10 minutes and 0.5 ml of the
sugar is added to sterile peptone water.
 Fermentation tubes are inoculated with the test organism.

6.StarchHydrolysis
Principle: Amylase is an exoenzyme that hydrolysis starch, a polysaccharides into
monosaccharide and disaccharides such as glucose.these mono and disaccharide enters into cell
cytoplasm of bacteria through the semi-permeable membrane and their by attacked by
endoenzymes.Starch is a complex carbohydrate composed of glucose molecules linked together
by α-1,4 and α-1,6 glycosidic bonds.
Procedure
Starch agar medium is poured on to the sterile petriplates and allowed to solidify.The test
organism is streaked on the plate and incubated for 48 hrs at 37°c.Plates are flooded with gram’s
iodine and excess iodine is drained off, are examined for the zone of clearance around the growth
for each organism.

8. 7.Caseinhydrolysis
Principle-Casein is a major protein found in milk. It is a macromolecule composed of amino
acid linked together by peptide bond. Some microorganism have the ability to decrease the
protein casein by producing proteolytic exo-enzymes called protease. The process breake down
the peptide bond by introducing water into the molecule liberating the soluble amino acid pool
for use in the synthesis of structural and functional cellular protein.
Procedure
1. Prepared the Casein media.
2. Sterilize the media by autoclaving
3. Then dispense the media in sterilized petriplates and after solidification streake the test
organism at the
centre of the plate.
4. Incubate the plate at 37°c for 24 hrs.
5. After incubation the plates are observed for hollow and clear zone around the colonies.
8. GelatinLiquification test
Principle:Gelatin is a protein derived form collagen,which is insoluble in cold water but
insoluble in cold water but soluble in hot water and form gel on cooling it. gelatin is liquid at
room temperature that solidify on cooling up to the temperature of -4°c for bacteriological use
and edible grade of gelatin is used since it is free of preservatives and inhibitory amount of heavy
metals.

9. Procedure
 Prepare the media according to the given composition and sterilize after pouring into the
test tubes.
 Inoculate the test organism into the sterilized test tubes and left one uninocculated as
control.
 Incubate the tube at 37°c for 24-48 hrs.
 After incubation kept the tubes in an ice bath for 30 min.
 Note down the results depending on the condition wheather gelatin is in the liquid state
or in the solid form.
9.Oxidase test
Principle-Cytochrome are heamo containing catalytic enzyme which are tightly bound to
(prokaryotic cells) cells of plasma membrane.they are concerned with later stages of biochemical
oxidation.They act as electron or H2 carries of biological oxidation by virtue of their availability
valence by heam ions i.e., they can undergo reduction and oxidation . During period of great
activity they are reduced. Cytochrome C is more abundant and freely soluble in water.
Cytochrome C does not react directly with O2 but reduced form will be oxidized by cytooxidase
with which it is closely associated. Cyto a,a3 is called cytooxidase. or indophenolase or
endophenol oxidase or ferro cytochrome C or oxygen oxido reductase.
Procedure:Place 1-2 drops of 1 % oxidase reagent on 6 cm square piece of whatman
filter paper. Transferr a small colony of test organism using a loop onto soaked filter
paper and observe for purple colour development. A positive test is indicated by
development of purple colour in 5-10 seconds.

10. 10.Proteinhydrolysis
Principle:Proteins are made up of various amino acids linked together in long chains by means
of peptide bonds. Many bacteria can hydrolyze a variety of proteins into peptides (short chains of
amino acids) and eventually into individual amino acids. They can then use these amino acids to
synthesize their own proteins and other cellular molecules or to obtain energy. The hydrolysis of
protein is termed proteolysis and the enzyme involved is called a protease.
11.Nitrate reduction
Many organisms can respire anaerobically by using NO3- as a terminal electron acceptor
for an electron transport system (nitrate respiration or dissimulator nitrate reduction).Nitrate
broth is used to determine the ability of an organism to reduce nitrate (NO3) to nitrite
(NO2) using the enzyme nitrate reductase. It also tests the ability of organisms to perform
nitrification on nitrate and nitrite to produce molecular nitrogen.
12. KOH Solubility test:
The purpose of the potassium hydroxide test (KOH test) is to identify gram negative bacteria.
KOH dissolves the thin layer of peptidoglycan of the cell walls of gram negative bacteria, but
does not affect gram positive cell walls. Disintergration of gram negative cell walls lyses the cell
and release its contents, including the DNA. The DNA will make the solution very viscous and
the solution will stick to the plastic loop when touched. Gram positive bacteria will not be
affected by KOH, because they have thicker peptidoglycan layer in the cell wall. Thus, the cells
will not be lysed, the DNA not released and no viscosity will be observed.

11. 13. Lipase test
Lipolytic organisms split off the fatty acid, and the calcium salts of the fatty acids
produce opaque zones around the colonies.
14.Susceptibility to antibiotics
Antibiotic sensitivity is the susceptibility of bacteria to antibiotics. Antibiotic susceptibility
testing (AST) is usually carried out to determine which antibiotic will be most successful in
treating a bacterial infection in organism. Testing for antibiotic sensitivity is often done by
the Kirby-Bauer method. wafers containing antibiotics are placed on an appropriate agar
plate where actinobacteria have been placed, and the plate is left to incubate. If an antibiotic
stops the actinobacteria from growing or kills the actinobacteria, there will be an area around the
wafer where the bacteria have not grown enough to be visible. This is called a zone of
inhibition.

12. PHYSIOLOGICAL METHODS
1 Temperature
2.pH range
3.Utilization of carbon source
4.NaCl range
5.Utilization of nitrogen source
6.Relation to Oxygen
1.Temperature range
Incubate cultures at a range of temperatures; using constant temperature incubators or
water baths, measure the growth response of the actinobacteria. The tested temperature
range is usually from 0°C to 75°C. In general, temperature experiments employ solid
medium instead of broth in order to better observe. The basal medium is Bennett's
medium or YIM38 medium or nutrient medium.If the temperature is above 100°C, use
screw-cap culture tubes or screw-cap glass bottles and seal the screw caps to prevent the
evaporation of the medium. Incubate the cultures in commercial water baths filled with
dimethyl silicone oil. If the temperature is below 0°C, use an ethylene glycol water bath.
2.pH range
An essential part of the description of any bacteria is the range of pH values at which it
can grow, as well as the optimal pH for growth. Measure growth responses from a
standarisedinoculum using basic medium (Bennett's medium or YIM38 medium or
nutrientmedium) at various pH values. Liquid medium is to be used for pH tests, to measure
thegrowth responses turbidimetrically. The selection of buffer is critical. A buffer should be
usedin most media to maintain a stable pH for growth of the test strain. Buffers are most
effectiveat their pKa values and should be chosen with this in mind.Some buffers such as citrate,
succinate, or glycine may be metabolized by the test organism.Some buffers such as citrate,
succinate, or glycine may be metabolized by thetest organism. Others may be toxic. Sometimes,
a combination of buffers may be helpful.Certain buffers (Good buffer) are non-metabolizable,
non-toxic, have low reactivity with metalions, and have other desirable features.Phosphate salts
are most commonly used becausethey are effective in the growth range of most bacteria.

13. 3.Ultilization of Carbon source
Utilization of carbon source tests usually uses turbidimetric method. Use a chemically
defined basal medium that lacks a carbon source, but otherwise is suitable for growth of
the actinobacteria being tested. The basal medium is Pridham and Gottlieb carbon
utilization medium [8]. Add carbon sources to a concentration (sugar alcohols 0.5–1%,
others 0.10.2%). After growth has occurred, measure the growth response turbidimetrically
with a spectrophotometer.Use liquid medium to avoid the influence of agar. Thermolabile
carbon sources should be sterilized by filtration (filter sterilize 10% solution through
bacteriological filter) or ether sterilization (weigh an appropriate amount of the dry carbon
source and spread in a pre-sterilized Erlenmeyer flask fitted with a loose cotton plug. Add
sufficient acetone-free ethyl ether to cover the carbohydrate. Allow ether to evaporate at
room temperature under a ventilated fume hood overnight or longer. When all ether has
evaporated, add sterile distilled water aseptically to make a 10% w/v solution of the
carbon source). Controls required for the test: No carbon source (negative control); D-
glucose (positive control). For marine actinobacteria, instead of distilled water, use a
synthetic seawater and sterilize the media by filtration.
4.Naclrange
Salt tolerance experiments mainly test the tolerance ability of the organism to NaCl and
other salts, and determine the optimum concentration for growth. Inoculate liquid media
containing a range of NaCl (usually 0–30%, W/V, or relative molar concentrations)
concentrations and measure the growth response turbidimetrically. Bennett's medium or
YIM38 medium or nutrient medium can be used as basal medium.
5.Utilization of nitrogen source
Use the turbidimetric method to test the utilization of nitrogen source, especially sole
nitrogen sources. Basal medium that omit nitrogen source but include a suitable carbon
source are used. Add nitrogen source to a concentration (usually 0.5%).Use liquid medium to
avoid the influence of agar. Thermolabile nitrogen sources should be sterilized by filtration or
ether sterilization. Controls required for the test: negative control (no carbon source).