IDENTIFICATION OF BACTERIA

The accurate identification, in an overwhelming number of cases, can be

accomplished by a series of procedures which are:
 Isolation in pure form
 Morphology of bacterial colony
 Morphology and staining reaction ·
 Biochemical tests
 Antigenic characters
 Fluorescent microscopy
 Typing of bacteria
 Animal pathogenicity
 Antibiotic sensitivity determination ·
 Molecular techniques

Isolation in Pure Form

Studies on the biochemical, antigenic and other characters of bacteria can be done
only if the organism is available in pure form. For the isolation of organism in pure
form following techniques are available:
Plating on Solid Culture Media: Clinical sample is streaked onto a solid medium
(nutrient agar, blood agar or MacConkey agar) in such a way so as to ensure isolated
discrete colonies. A colony is presumed to be a pure culture since all the bacteria in it
are descendents of a single bacterium.
Use of Selective, Enrichment or Indicator Media: A selective medium such as
blood tellurite agar is used to obtain pure growth of diphtheria bacilli because
majority of other bacteria will not grow on this medium. Similarly, growth of
Salmonella is favoured by enrichment medium such as selenite F. In indicator media
some bacteria shall present with an altered visible appearance of colonies and the
same can be picked up and sub-cultured to procure a pure culture, e.g. Escherichia
coli and Klebsiella grow as pink coloured (lactose fermenting) colonies on MacConkey
agar as compared to Salmonella and Shigella colonies which do not ferment lactose
and hence are pale yellow in appearance.
Use of Selective Growth Conditions: Most important example of this is the
growth of anaerobic bacteria which will not take place in an environment having
oxygen.
Animal Inoculation: Some laboratory animals are selectively susceptible to some
bacteria and use of this property is made to obtain pure growth of bacteria.

Morphology of Bacterial Colony

The following characters of the colonies are noted as these are produced by different
bacteria:
 Size (diameter in mm)
 Outline (circular, entire, wavy, indented)
 Elevation (flat, raised, low convex, dome shaped) ·
 Translucency (clear and transparent, opaque, tras Ius cent)
 Colour (colourless, white, yellow, black, pink)
 Changes in medium (haemolysis)
 Mucoid
 Adherence to medium
  Surface : glistening or dull
 Consistency : butyrous, brittle etc.
 Odour : Some bacteria have distinctive odour

Morphology and Staining Reaction

The colonies are subjected to basic staining with Gram stain and their motility is
checked by making a 'hanging drop' or wet preparation. These steps show the
morphology and motility of bacteria which are of great help in identifying them.