biochemical tests and physiological tests for various groups of bacteria

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,Plant pathology,
RLBCAU,Jhansi
Submitted by,
Harish J
Ag/Pg/0020/19
M.sc (Agri) Plant pathology,
RLBCAU,Jhansi

2. Biochemical Tests
• Each different species of bacterium has a different molecule of DNA (i.e., DNA with
a unique series of nucleotide bases).
• 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.
• 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 Biochemical tests
• IMVIC
a.Indole test
b.Citrate utilization test
• Sulphate reduction test
• Catalase production test
• Urease test
• Fermentation of carbohydrates
• Gelatin liquefaction test
• Oxidase test
• Starch hydrolysis
• Protein hydrolysis
• KOH solubility test

4. IMVIC
• This test important for coliform group of bacteria.
• The coliform group of bacteria includes all the aerobic and facultatively
aerobic bacteria.
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.

5. 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.

6. Sulphate reduction test
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.

7. Catalase Production 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.

8. 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.
Fermentation of Carbohydrates
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.

9. Starch Hydrolysis:
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.

10. Casein hydrolysis
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.

11. Oxidase test:
Principle:Cytochrome are heam 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 a1,a3 is called cytooxidase or indophenolase or
endophenol oxidase or ferro cytochrome C or oxygen oxido reductase.

12. Protein hydrolysis
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.
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.

13. Gelatin Liquification test
Principal: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 -4oc
for bacteriological use and edible grade of gelatin is used since it is
free of preservatives and inhibitory amount of heavy metals.

14. Lipase test
Lipolytic organisms split off the fatty acid, and the calcium salts of
the fatty acids produce opaque zones around the colonies.

15. Physiological methods
• Temperature
• pH range
• Utilization of carbon source
• NaCl range
• Utilization of nitrogen source
• Relation to Oxygen
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.

16. pH range
An essential part of the description of any actinobacteria 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.

17. 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.

18. Nacl range
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.
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%).