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.

Biochemical Tests
A large number of tests are available which help in identifying the bacteria:
1. Tests for metabolism of carbohydrates and related compounds
- Tests to distinguish between aerobic and anaerobic break down of carbohydrates.
- Tests to show the carbohydrates that can be attacked such as glucose, sucrose, mannitol, lactose etc.
- Tests for specific breakdown products such as MR, VP tests.
- Tests to show ability to utilize substrates such as citrate, malonate etc.
2. Tests for metabolism of proteins and amino acids
- Gelatin liquefaction
- Indole production
- Amino acids decarboxylase tests
- Phenylamine deaminase test
3. Test for metabolism of fats
- Hydrolysis of tributyrin
4. Tests for enzymes
- Catalase test
- Oxidase test
- Urease test
- ONPG test
- Nitrate reduction

Antigenic Characters
The absolute specificity of antigen-antibody reaction is made use of in confirming the identity of an isolate
in the laboratory by treating it with its specific antibody (commonly called as antiserum). Such antisera
may show agglutination of the isolate if they are specific for each other or are homologous.

Fluorescent Microscopy
Antibody molecule labelled with a fluorescent dye is used in direct fluorescent antibody test (FAT) to
detect the antigen. If antigen is present, the antibody gets tagged to it and the fluorescent dye, on
exposure to ultraviolet rays shall emit bright light indicating the presence of homologous organisms.

Typing of Bacteria
Typing techniques for bacteria

Designation Character upon which based Examples

Biotyping Biochemical tests Vibrio cholerae, Salmonella

Phage typing Bacteriophage susceptibility Staph. aureus, Salm. typhi

Serotyping Surface antigens Pneumococcus,

H. influenzae
Bacteriocine typing Production of bacteriocine Pseudomonas, Klebsiella

Antibiogram typing Susceptibility to antibiotics Salm.typhi, Staph. aureus

Resistogram typing Susceptibility to chemicals such Salmonella, Klebsiella

as heavy metals

Plasmid profile Plasmid All bacteria

Animal Pathogenicity
Isolation of certain organisms is facilitated by the inoculation of clinical material into selectively
susceptible animals. However, animal models are more frequently employed for ascertaining whether
isolated strain is pathogenic (toxigenic) or not. Some of these are:
Disease Animal

Diphtheria Guinea pig

Botulism Mice
Tetanus Plague Rats
Tuberculosis Mice
Enterotoxins Guineapig
Rabbit

Antibiotic Sensitivity Determination

Bacteria can be tested in the laboratory to find the most suitable antimicrobial agent. One method is
Stokes' method in which the test strain is tested against standard reference strain for sensitivity to
antibiotics and the results are based on the comparison between the zone of inhibition produced by the
same drug for test and standard strain. The second method is modified Kirby Bauer's method (NCCLS
method) where zone of inhibition produced by antimicrobial agent for a particular strain is measured and
its diameter is compared with the standard figures. Based upon this the strain is pronounced as sensitive,
or resistant to that particular antimicrobial agent.

Molecular Techniques
Recent molecular techniques which have been used for the identification of bacteria include: DNA probes,
polymerase chain reaction (PCR), nucleic acid hybridization and flowcytometry. These tools provide rapid
and sensitive means of diagnosis.