Identification of unknown bacterial cultures

The first and most important step in Medical microbiology is the identification of the disease causing organism.  Taxonomy is the science devoted to identify, name and classify organisms.  Classification is the separation of organisms into distinct groups whereas; identification is the determination of correct place of an organism in a previously established plan of classification.  Bergey’s Manual has been the internationally accepted reference for the identification of bacteria since 1923.  Bergey’s Manual of Systemic Bacteriology arranges related bacteria into 33 groups called sections rather than into classical taxonomic groupings of phylum, class, order and family.  The inter relationship of the organisms in each section is based on characteristics such as morphology, staining reactions, nutrition, cultural characteristics, physiology, cellular chemistry and biochemical test results for specific metabolic end products. 

When an unknown bacterium is isolated in the laboratory in the pure form from a sample such as food, water, soil, air, blood or tissue,   it is usually identified by a combination of informations gathered about the organism.  These include the cultural characteristics on agar and in broth; microscopic observations like morphology and arrangement of cells; gram stain and other staining reactions; the absence or presence of motility; and biochemical tests.  The information to be collected for the identification of an unknown bacterium is given in Table 1. 

Table 1. Information to be gathered for the identification of an unknown bacterium

1.  Morphological

   Shape              :Spheres, short rods, long rods, filaments, commas, spirals

   Arrangement   :Single, pairs, chains, clusters

   Capsules          :Present or absent

   Gram stain       :Positive or negative

   Spore stain       :Non spore former or spore former (central, sub terminal or                                                              terminal spore)

   Motility           : Motile or non motile

2.  Cultural characteristics

     On Nutrient agar plate        

     Colonies          :           Golden, yellow, white, glistening

     Growth            :           Absent (0), slight (+), moderate (2+), abundant (3+)

     Form                :           Circular, irregular, rhizoid

     Margins           :           Entire, lobate, undulate, serrate, filamentous

     Elevation         :           Flat, raised, convex, umbonate

     Density            :           Opaque, translucent

    

     In Nutrient broth media

     Surface growth           :           Ring, pellicle, none

     Turbidity                     :           Heavy, slight, none

     Sediment                     :           Abundant, scanty, none, granular, flocculent

     On Special media

     a) Differential media

     Egs:    

    Mc Conkey agar          :Inhibit G +^ve bacteria; differentiate   lactose  fermenters and non                lactose fermenters

   Mannitol salt agar :Promote halopiles like Staphylococcus, inhibit the non halophiles

Eosin Methylene Blue (EMB) agar :Inhibit G +^ve bacteria; differentiate  enteric lactose fermenters  and non fermenters as well as identify colon bacillus, E. coli

     b) Enriched media

     Eg:

  Blood agar :Differentiate hemolytic microorganisms like   Streptococcus spp. from non                 hemolytic species

3.  Biochemical tests

     Tests                                                   Results

     a) Carbohydrate fermentation

                 Glucose                       :Red to yellow; with or without gas production

                 Maltose                       :Red to yellow; with or without gas production

                 Mannitol                      :Red to yellow; with or without gas production

                 Sucrose                        :Red to yellow; with or without gas production

                 Lactose                        :Red to yellow; with or without gas production

     b)         IMViC tests

                 Indole production test            :Formation of red layer

                 Methyl-red (MR) test             :Presence of red color

                 Voges - Proskauer (VP) test   :Deep rose (pink) color

                 Citrate utilization test             :Color change of media from green to blue

     c)    Urease test   :Red to deep pink

     d)   Nitrate reduction test :Red color

     e)   Catalase activity  :Bubbles on the release of free oxygen gas (O₂)

     f)   Oxidase test :Development of pink, then maroon and finally  black coloration on the                      bacterial colonies

     g) Triple Sugar Iron (TSI) test  :  Alkaline (red) or/ acidic (yellow) color change in  slant and              butt region; with or without gas production

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