List of Biochemical Tests For Identification of Bacteria

sterility test of pharmaceuticl product

List of Biochemical Tests For Identification of Bacteria

List of Biochemical Tests

Large scale and careful in depth investigations carried out on a host of fermentation procedures using different types of substrates.

What are the biochemical tests?

Exclusively dependent upon a broad spectrum of biochemical tests ultimately lead to the production of ethanol by yeast, acetyl methyl carbinol, lactic acid, acetic acid, ethanol by E.coli, acetone plus CO2, citric acid (Krebs Cycle), and CO2+H₂.

The most vital and important and abundantly employed list of biochemical tests are as described below with appropriate explanations whenever required in the course of the prevalent discussion:

List of Biochemical Tests For Identification of Bacteria

  1. Carbohydrate (Sugar) Fermentation
  2. Litmus Milk
  3. Indole Production
  4. Methyl Red Test [MR-Test]
  5. Voges-Proskauer Test [VP-Test]
  6. Citrate Utilization
  7. Nitrate Reduction
  8. Ammonia Production
  9. Urease Test
  10. Production of Hydrogen Sulphide H2S
  11. Reduction of Methylene Blue
  12. Production of Catalase (Tube Catalase Test)
  13. Oxidase Reaction
  14. Egg-Yolk Reaction
  15. Growth in Presence of Potassium Cyanide (KCN)
  16. Composite Media
list of biochemical tests

Biochemical Test Name

What are the biochemical tests in Microbiology?

(1) Carbohydrate (Sugar) Fermentation

The carbohydrate fermentation is generally tested in a sugar media.
Thus, the generation of acid is determined by a possible change in the colouration of the subsequent medium either to pink or to red, and the resulting gaseous products obtained gets suitably collected in a strategically placed Durham’s tube.

(2) Litmus Milk

In this specific occasion there may not be any change in the medium, or acid/alkali could be produced thereby allowing for clotting of milk, and peptonization or saponification may take place greatly.

The resulting clot i.e., coagulation of the milk protein (viz., casein) could face a disruption by virtue of the gas evolved (usually termed as stormy fermentation).

(3) Indole Production

In actual practice the indole production is normally tested in a peptone water culture after an interval of 48 or 96 hours incubation at 37 °C; whereby the generation of indole from the amino acid tryptophan is duly ascertained as given below:

When Kovak’s Reagent, 0.5 mL, is added carefully and shaken gently for a while, it yields a red colouration thereby indicating a positive reaction i.e., indole production.

(4) Methyl Red Test [MR-Test]

The MR-test is frequently used to carry out the detection for the production of acid in the course of fermentation of glucose, besides maintaining pH below 4.5 in an old culture medium methyl red in quantity ratio 4.2 (red) to 6.3 (yellow).


list of biochemical tests for identification of bacteria

5 drops of methyl red solution [0.04% (w/v)] are added into the culture in glucose-phosphate medium that had been already incubated at 30°C for 5 days, Then it is mixed well, and observation is noted instantly

Appearance of red colour (acidic) gives a positive test, whereas yellow colour represents a negative test.

(5) Voges-Proskauer Test [VP-Test]

The underlying principle of the VP-Test exclusively rests upon the production of acetyl methyl carbinol from pyruvic acid via an intermediate stage in its strategic conversion to form 2, 3-butylene glycol i.e., [CH3CH-(OH)CH(OH)CH3].

However, it has been duly observed that in the presence of alkali and atmospheric oxygen (O2) the relatively small quantum of acetyl methyl carbinol present in the medium gets oxidized.

To form corresponding diacetyl derivative that subsequently interacts with the peptone content in the culture broth to produce a distinct red colouration.


The VP-Test may be easily performed by the careful addition of 0.6 mL of a 5% (w/v) solution of α-naphthol in ethanol and 0.2 mL solution of 40% (w/v) potassium hydroxide to 1 ml of glucose phosphate medium culture of the following organism already incubated suitably at 30 °C for a time period of 5 days or at 37 °C for period of 2 days.

Thus, we have two reactions;

a. Positive Reaction : indicated by the appearance of a pink colouration in just 2-5 minutes, that ultimately gets deepened either to magenta or crimson red in about 30 minutes duration.

b. Negative Reaction: Designated by the appearance of a colourless solution up to 30 minutes. Importantly, the development of any traces of pink colouration must be ignored completely.

(6) Citrate Utilization

In practical coarse, Koser’s citrate medium having citric acid acts as the primary source of carbon.

Evidently, the ability as well as the efficacy for the citrate utilization (i.e., the prevailing substrate) is adequately indicated by the production of reasonably measurable turbidity in the medium.

Note: The characteristic list of biochemical tests which are indole, Methyl Red, Voges-Proskauer test, and citrate are quite useful in the proper and precise identification of Gram-negative (gram -ve) microorganisms.

Hence, these tests are frequently referred to by the Single ‘IMV IC’ Tests.
Kovak’s Reagent consists of:

10 grams p-Dimethyl amino benzaldehyde; 150 mL Amyl or Isoamyl alcohol and 50 mL concentrated Hydrochloric Acid.

It is always prepared in small quantities and duly stored in a refrigerator (5-10 °C)

As a matter of choice another primary physiological difference that may be abused specifically associate to the consequent ‘growth temperature’.

It has been suitably determined that at 44 °C only A.aerogenes must show growth particularly, whereas E.coli will not show any growth.

Therefore, the specific incubation at 44 °C shall be able to make a clear cut distinction between these two microorganisms which is invariably known as the Eijkman (E) test.

The menomic that is aiding the memory is IMVEC, wherein I stand for Indole, M stand for methyl red, V stands for voges-Proskauer, E stands for Eijkman and C stands for citrate

Summarily, therefore, the apparent behaviour of the said two microorganisms may be stated as below, whereby a sort of comparison between E.coli and Aerobic aerogenes has been recorded.

(7) Nitrate Reduction

The nitrate reduction test is performed after allowing the particular bacterium to grow for Five days at 37° C in a culture broth having potassium nitrate [1% (w/v)].

The test reagent contains a mixture of equal volumes of the solutions of sulphanilic acid (C6H7NO3S) and α-naphthylamine (C10H9N) in acetic acid (CH3COOH) carefully mixed just before use.

Now, 0.1 ml of the test reagent is suitably added to the culture medium.
The results of the test may be inferred as given under;

list of biochemical tests names

Positive Reaction: Development of a red colouration within a short span of a few minutes confirms a positive reaction.

Negative Reaction: The absolute absence of the above mentioned red colouration indicates a negative reaction.

Importance: The nitrate reduction test shows particularly the presence of the enzyme nitrate reductase that helps to reduce nitrate (NO3) to nitrite (NO2).

(8) Ammonia Production

The ammonia production test is generally performed by adding very carefully the Nessler’s Reagent into a peptone water culture grown carefully for 5 days at 37°C.

The inferences of this test may be drawn as stated under;
Positive Test: Appearance of a Brown colour;
Negative Test: Appearance of faint Yellow colour.

(9) Urease Test

The test is generally carried out in Christensen’s Urea-Agar medium or it is carried out in Christensen’s urease medium.


The slope is inoculated profusely and incubated at 37°C.
The slope is duly examined at intervals of 4 hours and 24 hours incubation. The test must not be taken as negative till after a duration of 4 days after incubation.


The urease positive cultures give rise to a distinct purple-pink colouration.
The exact mechanism may be explained by virtue of the fact that urease producing microorganisms largely help in the conversion of urea to ammonia (gas) which is particularly responsible for the desired colouration.

(10) Production of Hydrogen Sulphide (H2S)

Importantly, there are several Sulphur-containing amino acids e.g., cystine, cysteine, methionine that may decompose certain organisms to yield H2S (gas) amongst the products of microbial degradation.

In this particular instance lead acetate [Pb(CH3CO)2] is duly incorporated into the culture media which eventually gets turned into either black or brown due to the formation of PbS as given below:

Pb(CH3CO)2 (Lead Acetate) + H2S (HydrogenLead) ⎯→ PbS↓ (Lead sulphide (Black) + 2CH3COOH (Acetic Acid)


The organisms are grown in culture tubes. In actual practice a filter-paper strip soaked in a lead acetate solution [10% (w/v) freshly prepared] is strategically inserted between the cotton plug and the empty-space in the culture tube.

Result: The gradual browning of the filter paper strip rightly confirms the H2S-production.

(11) Reduction of Methylene Blue

The reduction of one drop of the aqueous methylene blue reagent (1% (w/v) which is added into the broth culture and then incubated at 37°C.

The results are as indicated below:
Strongly positive: exhibited by complete decolourization
Weakly positive: displayed by green colouration.

(12) Production of Catalase [Tube catalase Test]

In this specific test a loopful (either a wooden applicator stick or a nichrome wire loop) H2O2 i.e., hydrogen peroxide (3%) is placed meticulously right upon the colonies grown on the nutrient agar medium.

The catalase production is indicated by the prompt effervescence of oxygen (O2) due to the fact that the enzyme catalase aids in the conversion of H2O2 into water and oxygen bubbles (in the form of effervescence).

Importance: It has the unique means of differentiation between Streptococcus (catalase negative) from Staphylococcus (catalase positive).

Caution: Such culture media that particularly contain blood as an integral component are definitely not appropriate for the ‘tube catalase test’ because the blood itself contains the enzyme catalase

(13) Oxidase Reaction

The underlying principle of the ‘oxidase reaction’ is exclusively by virtue of an enzyme known as cytochrome oxidase that particularly catalyzes oxidation of reduced cytochrome by oxygen.

Biochemical test Procedure:

A solution of tetramethyl p-phenylene diamine dihydrochloride in concentration of about 1.0 to 1.5% (w/v)] is then poured slowly as well as carefully over the colonies.

The result is duly indicated by the oxidase positive colonies turning into maroon-purple-black in a span of 10 to 30 minutes.

Kovacs’s Method: Alternatively, the ‘oxidase reaction’ may also be performed by Kovacs’s method.

In this method a part of filter paper is sufficiently moistened with a few drops of 1% (w/v) solution of tetra methyl polyphenylene diamine dihydrochloride.

By the help of a sterilized wooden applicator the actual growth from an agar medium is carefully smeared onto the exposed surface of the said strip of filter paper.

Thus, a positive test is clearly indicated by the specific development of a purple colouration almost punctually (within 10 seconds).

Importance: The obvious importance of the ‘oxidase reaction’ is judiciously employed to obtain a clear cut differentiation/separation of the enteric from the pseudomonas.
Example: Pseudomonas aeruginosa : Positive Test.
Escherichia coli: Negative Test.

(14) Egg-Yolk Reaction

It has been duly demonstrated and proved that all such organisms which essentially and specifically produce the enzyme lecithinase e.g., Clostridium perfringens, on being carefully grown on a solid egg-yolk medium, gives rise to well-defined colonies usually surrounded by a zone of clearing.

(15) Growth in Presence of Potassium Cyanide (KCN)

Occasionally, buffered liquid-culture medium containing KCN in a final concentration of approximately 1/13,000 (i.e., 7.69 × 10–5) is employed critically to identify certain KCN tolerant enteric bacilli.

(16) Composite Media

In the topic of ‘Biochemical Tests’ the important role of composite media is gaining legal or lawful recognition for the specific identification of biological isolates.

Advantages: The various cardinal advantages of the so called composite media are as described under:

It is used as economical and convenient culture media.

A single composite medium graphically indicates various characteristic properties of the bacterium (under investigation) that might have required the important usage of various individual cultural media.

Examples: The two most commonly employed ‘composite media’ are as described under :

(a) Triple Sugar Iron Medium (TSI-Medium):

It shows ‘composite medium’ that particularly indicates whether a bacterium under consideration:

⦁ ferments glucose exclusively,

⦁ ferments either, lactose or sucrose,

⦁ gas formation occurs or not, and

⦁ indicates production of H2S gas.

In actual practice, TSI-medium is distributed in various tubes along with a butt and a slant.

After having subjected them to proper inoculation under perfect aseptic conditions one may draw the following inferences:

Red slant + Yellow butt. Indicates that all sugars molecules which are glucose, lactose, and sucrose are suitably fermented.

⦁ Appearance of bubbles in the butt—shows production of gas, and

⦁ Blackening of the medium—displays evolution of H2S gas in the TSI-Agar Reaction.

Importance : The most spectacular and major advantages of the TSI-medium is to predominantly facilitate the preliminary identification of the Gram-negative Bacilli.

(b) Test for Amino Acid Decarboxylation:

The particular biochemical test necessarily involves the decarboxylases (which are arginine, lysine, ornithine) and the process of decarboxylation of the amino acids always gives rise to the following release of amine and CO2.

In reality, this specific test is alone used for the identification of enteric bacteria.

As a result, there are certain other tests as well, which are fermentation of organic acids, hydrolysis of sodium hippurate (C9H8NNAO3), and oxidation of gluconate which are used few times to carry out the identification of particular critical organisms.

Now, with the emergence of always increasing wisdom and knowledge concern to the excess of metabolic processes in the growth of various microorganisms, the number of reliable tests is also increasing.

Biochemical Tests for Identification of Bacterial Isolates : After having accomplished the microscopic and the critical growth characteristic features of a pure culture of organisms are duly examined, highly accurate and particular ‘biochemical tests’ may be carried out to identify or distinguish them exactly.

Based on the survey of literature and original evidences from numerous researches carried out one may experience certain ‘biochemical tests’ generally employed by most clinical microbiologists in the proper and methodical diagnosis of organism from the patients sample.

What are biochemical test for gram positive bacteria?

Biochemical Tests For Identification of Bacteria Table

Specific Biochemical Tests Carried out by Clinical Microbiologists for the Critical Diagnosis of Microorganisms Derived from the Patient’s Sample Direct

Sr.noSpecific Biochemical TestArticulated InterferenceApplication in Laboratory Procedures
1Fermentation of
Gas (CO2) and/or acid are generated
in the course of fermentation (i.e.,
fermentative growth) along with sug-
ars or sugar alcohols.
Specific sugars upon fermentation
invariably ascertain to differentiate
clearly not only the enteric bacteria
but also other species or genera.
2Hydrolysis of casein
(i.e. milk products)
Aids in the detection for the presence
of caseinase, an enzyme capable of
hydrolyzing exclusively the milk pro-
tein casein. Microorganisms which
make use of ‘casein’ mostly appear as
colonies surrounded by a clear zone.
Distinctly utilized to cultivate and
also differentiate the aerobic
actinomycetes entirely based upon
the casein utilization, such as :
Streptomyces uses casein whereas
Nocardia fails to do so.
3Catalase ActivityThe very presence of ‘catalase’ is de-
tected that solely helps in the conver-
sion of hydrogen peroxide to water
Clearly differentiates between
Streptococcus (–) from
Staphylococcus (+) ; and also Ba-
cillus (+) from Clostridium (–).
4Utilization of citrateWhen ‘citrate’ gets consumed as an
exclusive ‘source of carbon’, it gives
rise to an ultimate alkalinization of
the medium.
Employed solely in the due classifi-
cation of ‘enteric microorganisms,
for instance : Klebsiella (+),
Enterobacter (+), Salmonella (+),
Escherichia (–), and Edward siella
5Caogulase ActivityCritically detects the presence of the
enzyme ‘coagulase’ that causes
plasma to clot.
Categorically differentiates
Staphylococcus aureus (+) from S.
epidermidis (–).
6Decarboxylases (e.g. Arginine, Ornithine, Lysine).Decarboxylation of the cited amino
Classification of enteric microor-
ganisms is accomplished aptly
7Hydrolysis of EsculinHelps in the detection of cleavage of
a glycosidic linkage.
Solely employed for the differentia-
tion of S. aureus, Streptococcus
mitis, and others (–) from S.bovis,
S. mutans, and enterococci (+).
8liquefaction of gelatinEssentially detects whether or not a
microorganism can give rise to
proteases which in turn either carry
out the hydrolysis of gelatin or help
in the liquefaction of solid gelatin
medium (culture).
Identification of clostridium
Flavobacterium, Pseudomonas, and
Serratia are ascertained.
9Liberation of Hydrogen
Sulphide (H2S)
Detects the production of H2S from
the S-containing amino acid cysteine
due to cysteine desulphurase en-
Distinctly vital and important in
the precise identification of
Edward siella, Proteus, and
10Indole ; Methyl Red ;
Voges-Proskauer ;
Citrate [IMViC]
Detects the generation of ‘indole’
from the amino acid tryptophan. Me-
thyl red serves as a pH indicator so
as to confirm the presence of acid
produced by the bacterium. The
Voges-Proskauer Test* (VP-Test)
helps in the detection of the produc-
tion of acetoin. The citrate Test**
establishes whether or not the bacte-
rium is capable of utilizing sodium
citrate as an ‘exclusive’ source of
Extensively employed to separate
Escherichia (showing MR+ ; VP– ;
indole + 😉 from Enterobacter (hav-
ing MR– ; VP+ ; indole– 😉 and
Klebsiella pneumoniae (having
MR– ; VP– ; and indole– 😉 ; besides
being to characterize the various
members belonging to the genus Ba-
11Hydrolysis of Lipid
Helps in the detection, for the pres-
ence of ‘lipase’ that eventually causes
cleavage of lipids into the correspond-
ing simple fatty acids and glycerol.
Used in the distinct separation of
12Nitrate Reduction
Helps to detect whether a bacterium
is capable of using nitrate as an ‘elec-
tron acceptor’.
Employed in the identification of
enteric microorganisms specifically
that are found to be invariably (+).
13Oxidase Activity
Aids in the detection of cytochrome
oxidase which is capable of reducing
oxygen (O2), besides the artificial
electron acceptors.
Extremely vital and important to
carry out the distinction of Neisseria
and Moraxella spp.(+) from
Acinetobacter (–) and enteries (all
–) from pseudomonads (+).
14Hydrolysis of Starch
Detects the presence of the enzyme
amylase, that particularly hydrolyzes
Employed solely to identify typical
starch hydrolyzers, e.g., Bacillus
15Urease Activity
Helps to detect the enzyme, Urease,
that cleaves urea into ammonia
(NH3), and carbon dioxide (CO2).
Largely used to distinguish the or-
ganisms Proteus, Providencia
rettgere, and Klebsiella pneumoniae
(+) from Salmonella, Shigella and
Escherichia (–).

Biochemical Tests Chart

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