Procedure for Identification of Bacterial Culture

The following points highlight the four main procedure for identification of bacterial culture. The procedure are: 1. Examination of Solid Media 2. Examination of Liquid Medium 3. Primary Diagnosis 4. Final Identification/Confirmation of Bacteria.

Bacterial Culture: Procedure # 1. Examination of Solid Media:

The medium and colonial appearance of bacterial growth is to be identified. It is useful for provisional identification of bacteria. Study a Gram’s-stained smear of bacteria and observe staining reaction and microscopic morphology of bacteria.

1. Medium:

Usually nutrient agar, blood agar or MacConkey’s agar.

2. Colony morphology (Fig. 6.2):

i. Size:

Pinpoint (streptococcus), pinhead (staph.), large (Klebsiella), moderately large (E. coli), Small (Shigella) etc.

ii. Shape (Fig. 6.2):

Dome (Staph), flat (vibrio), low convex (Salmonella, Shigella), droughtsman (S. pneumoniae), convex (Klebsiella, E.coli) irregular (Bacillus) etc.

iii. Surface (Fig. 6.2):

Rough (Some Neisseria, Bacillus), matt (E. coli), ridged (Pseudomonas) smooth (Salmonella) etc.

iv. Margin (Fig. 6.2):

Entire (Salmonella), irregu­lar (Pseudomonas), crenated (Bacillus), spre­ading (Proteus) etc.

v. Translucency:

Opaque (Staph), transparent (Vibrio), semi-translucent (Salmonella).

vi. Pigment:

Golden yellow (Staph aureus), bluish- green (Pseudomonas), brick-red (Serratia marcescens).

vii. Pigment restricted to colony — Staph, Micrococcus, Serratia.

viii. Pigment diffused in medium — Pseudomonas.

ix. Haemolysis — Beta haemolysis (S. pyogenes), alfa haemolysis (S. pneumoniae).

x. Haemodigestion — Proteus, Pseudomonas etc.

xi. Consistency — Mucoid (Klebsiella), butyrous (Staph), friable (some Neisseriae), firm (Bacillus), Sticky (V. parahaemolyticus).

3. Gram stained smear examination:

(i) Staining reaction:

Gram-positive/Gram-negative/Gram-variable.

(ii) Microscopic morphology (Fig. 6.1) — Note the following:

a. Shape— rod (E. coli etc.), spherical or cocci (Staph etc.), coccobacilli (Brucella etc.), comma (Vibrio).

b. Length/Size— long (E. coli), short (Klebsiella etc.), large (Micrococcus), small (Streptococcus) etc.

c. Width — stout (E. coli, Bacillus), slender (Salmonella) etc.

(iii) Unstained space for spore (Bacillus, Clostri­dium).

(iv) Ends — rounded (E. coli), square cut (bacillus), tapered (diphtheroid).

(v) Sides — parallel, un-parallel and irregular.

(vi) Inclusion granules.

Bacterial Culture: Procedure # 2. Examination of Liquid Medium:

Follow these steps:

i. Identify the liquid medium.

ii. Note the bacterial growth pattern.

iii. Examine the motility and morphology (if possible) in hanging drop preparation.

1. Identification of liquid culture medium:

i. Peptone water:

No tinge of colour. Most of the non-fastidious bacteria are grown and supplied in it.

ii. Glucose in nutrient broth:

All broths show straw/yellowish-blue. Usually growth of Streptococcus is supplied in this medium.

2. Bacterial growth pattern (Fig. 6.3):

i. Uniform turbidity: Example – most of the common gram negative rods.

ii. Uniform turbidity with surface pellicle formation: Examples — Strict aerobes like Vibrio, Pseudo­monas and Bacillus concentrate near the surface.

iii. Deposit at bottom: Heavy bacteria form deposit leaving slight haze above. Example — Strepto­coccus.

3. Hanging drop preparation:

Note:

i. True/active motility or non-propagative brownian movement.

ii. If motile, characteristic of motility (e.g., darting and fish in stream by Vibrio cholerae, tumbling motility by Y. enterocolitica etc.)

iii. Morphology — rods are identifiable but cocci in cluster, discrete or in short chain are difficult to visualize (Fig. 6.4).

Bacterial Culture: Procedure # 3. Primary Diagnosis:

Colony character, medium on which growth appe­ared, pattern of growth in liquid medium, gram stained morphology and motility study generate clues for primary diagnosis and help to formulate scheme for confirmatory or final diagnosis.

Bacterial Culture: Procedure # 4. Final Identification/Confirmation of Bacteria:

This is done by:

1. Biochemical tests:

Sugar fermentation tests, utilisation pattern of amino acids, tests for some enzymes and metabolic pattern, ability to grow on synthetic medium, H₂S production, indole production, MR-VP test, urease produ­ction, P.P.A deaminase test etc.

2. Slide agglutination test using high titre anti- sera.

3. Toxigenicity test or animal pathogenicity test.

4. Molecular method (hybridisation with DNA probe, detection of signature sequence in DNA and in 16S rRNA).