Identification Of Bacteria And Microorganisms – Method

Identification Of Bacteria

After isolation of bacteria in pure culture from a specimen, it has to be identified of bacteria. The following studies are necessary to characterise a bacterial strain and help in the identification of bacteria.

  • Morphology of bacterial colony
  • Growth in liquid media
  • Staining
  • Hanging drop preparation
  • Biochemical tests
  • Antigenic structure
  • Typing methods
  • Pathogenicity tests
  • Antibiotic sensitivity tests


Morphology Of Bacterial Colony

The following features of the colony are studied.

  • Size
  • Shape
  • Surface
  • Elevation
  • Edge
  • Opacity
  • Colour
  • Haemolysis
  • Consistency

Growth in Liquid Media

In liquid medium bacterial growth may appear in the following forms:

  • Uniform turbidity
  • Deposit at bottom
  • Surface pellicle formation


Staining methods are employed to examine smears prepared from the bacterial colony or liquid culture, staining is helpful in the identification of microorganisms and bacteria.

Commonly used stains are as follows for identification of bacteria:-

  1. Gram’s staining
  2. Albert’s staining
  3. Ziehl-Neelsen (ZN) staining

Hanging Drop Preparation

Helps to differentiate motile bacteria from non-motile ones.

Biochemical Tests

Some of the widely used biochemical tests are described below.

Sugar Fermentation

It determines the ability of an organism to ferment a specific carbohydrate (sugar) incorporated in a medium producing acid or acid with gas.


  • Positive-Pinkish red (acidic)
  • Negative-Yellow to colourless (alkaline)

Indole Production

It determines the ability of organs to decompose amino acid tryptophan into indole. Tryptophan is decomposed an enzyme tryptophanase produced by certain bacteria help in bacterial identification.


  • Indole positive – A red coloured ring near the surface of the medium, Example-Escherichia coli.
  • Indole negative -Yellow coloured ring near the surface of the medium, Example-Klebsiella species.

Urease Production

It determines the ability of an organism to produce an enzyme urease which splits urea to ammonia. Ammonia makes the medium alkaline and thus phenol red indicator changes to pink-red in colour.


  • Positive -Pink colour, Example-Klebsiella Species.
  • Negative -Pale yellow colour, Example-Escherichia coli.

Citrate Utilisation Test

This test detects the ability of an organism to utilise citrate as the sole source of carbon for its growth, with resulting alkalinity.


Simmon’s Citrate Medium

  • Positive -Growth with an intens├ęthe blue colour on the slant. The blue colour is due to the alkaline pH which results from the utilization of citrate. Bromothymol blue (indicator) is blue in alkaline conditions, Example-Klebsiella Species.
  • Negative-No growth with no change in colour (green) Example-Escherichia coli.


  • Positive-Immediate bubbling O2 formed), Example-Staphylococcus Species
  • Negative-No bubbling (no O, formed), e.g., Streptococcus Species.

Oxidase Test

It determines the presence of an enzyme cytochrome oxidase which catalyses the oxidation of reduced cytochrome by molecular oxygen. The freshly prepared solution of 1% tetramethyl paraphenylenediamine dihydrochloride (Oxidase reagent) is used for the identification of bacteria. A filter paper strip, soaked in the oxidase reagent is smeared with the test organism.


  • Positive-Deep purple colour within 10 seconds, Example-Pseudomonas Species.
  • Negative-No colour change, Example-All members of Enterobacteriaceae.

Phenylalanine Deaminase Test

It determines the ability of an organism to minute phenylalanine to phenyl pyruvic acid (PPA). This test is also commonly called a PPA test major for bacterial identification.


  • Positive -Green colour, Example Proteus Species.
  • Negative-No colour change, Example all members of Enterobacteriaceae.

Methyl Red (MR) Test

This test detects the production of sufficient acid during fermentation of glucose by bacteria and sustained maintenance of a pH.


  • Positive-Red colour, Example, Escherichia coli.
  • Negative-Yellow colour, Example Klebsiella species.

Voges-Proskauer (VP) Test or Acetoin Production Test

In the presence of alkali and atmospheric Oxygen, acetoin is oxidised to diacetyl, which reacts with a-naphthol to give red colour.


Example-Klebsiella species. Negative-Colourless for 30 minutes, Example-Escherichia coli
Indole, MR, VP and citrate tests are commonly referred to by the sigla ‘IMVIC’ tests.

10. Triple-Sugar Iron (TSI) Agar

It determines the ability of an organism to attack specific carbohydrate incorporated in a growth medium, with or without the production of gas, along with the determination of possible hydrogen sulphide (H, S) production.


Yellow colour(acidic)-Fermentation of carbohydrate.

Red colour (alkaline)-No Fermentation Bubbles in the butt-Gas production Blackening of the medium.

Antigenic Structure

Biochemically identified organisms are further confirmed by agglutination or precipitation reaction. Slide agglutination test using specific antisera is a commonly performed test for identification of bacterial antigen.

Typing Methods

It is useful for epidemiological studies. It helps in intraspecies differentiation and help in bacteria identification. Bacteriophage typing and bacteriocin typing are some examples of typing methods.

Pathogenicity Test

Guinea pigs, rabbits and mice are mostly used. These animals may be injected by subcutaneous, intramuscular intraperitoneal, intravenous or intracerebral routes depending upon the organism to be tested. Postmortem findings and cultural characteristics help in the identification of the bacterial organism.

Antibiotic Sensitivity Tests 

The isolated bacterium is subjected to antibiotic sensitivity tests invitro for selecting the appropriate antibiotic for therapeutic use.

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