IDENTIFICATION OF BACTERIA
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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.
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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
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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
Bacteriocine typing
Production of bacteriocine
Pneumococcus, H. influenzae Pseudomonas, Klebsiella
Antibiogram typing
Susceptibility to antibiotics
Salm.typhi, Staph. aureus
Resistogram typing
Susceptibility to chemicals such as heavy metals
Salmonella, Klebsiella
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 Botulism Tetanus Plague Tuberculosis Enterotoxins
Guinea pig Mice Rats Mice 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.
