BIOCHEMICAL TESTS: IDENTIFICATION OF BACTERIAL ISOLATES AND DIFFERENT BACTERIAL SPECIES

Biochemical tests are essential in microbiology for identifying and characterization of microorganisms.

                                                              Image source: chegg.com

Carbohydrate Fermentation Test:

• Microorganisms use carbohydrates differently based on their enzyme components.
• In fermentation, substrates and alcohols undergo anaerobic dissimilation, producing organic acids (e.g., lactic acid, formic acid, or acetic acid).
• Phenol Red indicator detects acid production (red at neutral pH 7, turns yellow at slightly acidic pH 6.8).
• Gas production (e.g., hydrogen or carbon dioxide) can also accompany acid production.
• Procedure:

1. Inoculate a carbohydrate fermentation broth with the test microorganism.
2. Incubate at 37°C for 24 hours.
3. Observe color changes and gas bubble formation.

• Interpretation:
• Positive (yellow color and gas bubble): Fermentation occurred.
• Negative (no change): No fermentation.
• Examples:
• Glucose: Fermented by E. coli and Klebsiella (acid and gas production).
• Lactose: Fermented by S. aureus (acid production only).
• Sucrose: Fermented by S. aureus (acid production only).

• Oxidation-Fermentation Test:

• Semisolid tube medium containing glucose and a pH indicator.
• Acid produced only at the surface (aerobic conditions) indicates oxidative breakdown.
• Acid production throughout the medium (including lower layers) under aerobic conditions indicates fermentative breakdown.
• Procedure:

1. Inoculate semisolid glucose medium.
2. Incubate appropriately.
3. Observe acid production.

• Interpretation:
• Oxidative (acid only at the surface): Aerobic breakdown.
• Fermentative (acid throughout): Fermentation.
• Applications: Differentiating between oxidative and fermentative bacteria.

Commonly used biochemical tests:

• Catalase Test:

• To differentiate between Staphylococci (catalase-positive) and Streptococci (catalase-negative).

             Procedure: Add hydrogen peroxide to the bacterial colony; if bubbles form (indicating oxygen                                  release), the test is positive.

• Oxidase Test:

• Helps identify Neisseria, Pasteurella, Vibrio, and Pseudomonas.
• Procedure: Detects the presence of bacterial cytochrome oxidase using a reagent impregnated on filter paper.

• Indole Test:

• Differentiates gram-negative rods, particularly E. coli.
• Procedure: Tests the ability of an organism to split tryptophan to form indole.

• Coagulase Test:

• Identifies Staphylococcus aureus.
• Procedure: Determines whether the bacterium produces coagulase (positive result) or not.

• Urease Test:

• Helps identify organisms that produce urease (e.g., Proteus, Helicobacter pylori).
• Procedure: Tests the ability to hydrolyze urea.

• Citrate Utilization Test:

• Differentiates members of the Enterobacteriaceae family.
• Procedure: Determines if the bacterium can utilize citrate as a sole carbon source.

• Bile Solubility Test:

• : Differentiates Streptococcus pneumoniae from other alpha-hemolytic streptococci.
• Procedure: Bile or a solution of bile salt rapidly lyses pneumococcal colonies.

• Litmus Milk Decolorization Test:

• Helps identify Enterococcus and some Clostridia.
• Procedure: Detects the ability to metabolize litmus milk.

• Bacitracin Sensitivity Test:

• Differentiates Streptococcus pyogenes (positive) from other beta-hemolytic streptococci (resistant).

• DNase Test:

• Identifies Staphylococcus aureus.
• Procedure: Determines the ability to hydrolyze DNA.

• Lysine Decarboxylase Test:

• Assists in the identification of Salmonella and Shigella.

Remember, these tests provide valuable information for bacterial identification and are essential tools in the microbiology laboratory! 

Reference:

• Aslanzadeh, J., n.d. Biochemical Profile-Based Microbial Identification Systems. Advanced Techniques in Diagnostic Microbiology, pp.84-116. https://link.springer.com/chapter/10.1007/0-387-32892-0_6
• Chauhan, A., Jindal, T. (2020). Biochemical and Molecular Methods for Bacterial Identification. In: Microbiological Methods for Environment, Food and Pharmaceutical Analysis. Springer, Cham. https://doi.org/10.1007/978-3-030-52024-3_10