Biochemical Tests for Bacteria and Fungi

Biochemical Tests for Bacteria

Biochemical tests help to identify bacteria by evaluating their metabolic properties, enzymatic activity, and reaction to specific chemicals. These tests are essential for distinguishing species, particularly those with similar morphological characteristics.

Here’s a more detailed explanation of the key biochemical tests used in bacteriology:

1. Gram Staining

• • Purpose: Differentiates bacteria into Gram-positive and Gram-negative based on the structure of their cell walls, specifically the thickness of the peptidoglycan layer.
  • Procedure:
    1. Crystal Violet Staining: All cells are stained purple.
    2. Iodine Treatment: Iodine forms a complex with crystal violet.
    3. Decolorization: Alcohol or acetone is applied. Gram-negative cells lose the purple stain due to their thin peptidoglycan layer, while Gram-positive cells retain the color.
    4. Counterstaining: Safranin is used to stain Gram-negative cells red or pink.
  • Results:

• • • Gram-positive: Stains purple (e.g., Staphylococcus aureus).
    • Gram-negative: Stains pink (e.g., Escherichia coli).

2. Catalase Test

• • Purpose: Determines the presence of catalase, an enzyme that breaks down hydrogen peroxide (H₂O₂) into water and oxygen.
  • Procedure: A small bacterial colony with hydrogen peroxide is added to a slide.
  • Results:
    • Catalase-positive: Bubbling or effervescence due to oxygen release (e.g., Staphylococcus species).
    • Catalase-negative: No bubbling (e.g., Streptococcus species).

3. Coagulase Test

• • Purpose: Detects the enzyme coagulase, which can clot plasma. Certain bacteria, notably Staphylococcus aureus produce coagulase.
  • Procedure:
    1. Slide test: A drop of plasma is mixed with bacterial cells, and clumping indicates the presence of coagulase.
    2. Tube test: Bacteria are incubated in plasma and observed for clot formation.
  • Results:

• • • Coagulase-positive: Clot formation (e.g., Staphylococcus aureus).
    • Coagulase-negative: No clot (e.g., Staphylococcus epidermidis).

4. Oxidase Test

• • Purpose: Identifies bacteria that produce cytochrome c oxidase, which is involved in the electron transport chain.
  • Procedure: A few drops of oxidase reagent (e.g., tetramethyl-p-phenylenediamine) are added to a bacterial colony or filter paper.
  • Results:
    • Oxidase-positive: A purple color appears within 10 seconds (e.g., Pseudomonas aeruginosa).
    • Oxidase-negative: No color change (e.g., Escherichia coli).

5. Indole Test

• • Purpose: Tests the ability of bacteria to break down tryptophan into indole using the enzyme tryptophanase.
  • Procedure: Bacteria are incubated in tryptone broth, and Kovac’s reagent is added.
  • Results:
    • Indole-positive: Red or pink layer forms (e.g., Escherichia coli).
    • Indole-negative: No color change (e.g., Klebsiella species).

6. Methyl Red (MR) and Voges-Proskauer (VP) Tests

• • Purpose: These tests differentiate enteric bacteria based on their end products from glucose fermentation.
  • Methyl Red (MR) Test:
    • Purpose: Detects acidic end products from glucose fermentation.
    • Procedure: Add methyl red reagent to the culture.
    • Results:
      • Positive: Red color (e.g., Escherichia coli).
      • Negative: Yellow (e.g., Enterobacter).
  • Voges-Proskauer (VP) Test:
    • Purpose: Detects the production of acetoin, an intermediate in the fermentation of glucose.
    • Procedure: Add Barritt’s reagent (alpha-naphthol and potassium hydroxide) to the culture.
    • Results:
      • Positive: Red color (e.g., Enterobacter species).
      • Negative: No color change (e.g., Escherichia coli).

7. Urease Test

• • Purpose: Tests for the presence of the enzyme urease, which hydrolyzes urea to ammonia and carbon dioxide.
  • Procedure: Inoculate the bacteria into urea broth. The medium contains phenol red, which turns pink at alkaline pH (ammonia production).
  • Results:
    • Urease-positive: Pink color (e.g., Helicobacter pylori).
    • Urease-negative: No color change (e.g., Escherichia coli).

8. Carbohydrate Fermentation Tests

• • Purpose: Determines whether a bacterium can ferment specific carbohydrates (e.g., glucose, lactose, sucrose) and produce acid or gas as byproducts.
  • Procedure: Bacteria are inoculated into carbohydrate fermentation broths containing a pH indicator (e.g., phenol red) and a Durham tube to detect gas production.
  • Results:
    • Acid production: The broth turns yellow.
    • Gas production: Bubbles in the Durham tube.

9. Triple Sugar Iron (TSI) Test

• • Purpose: Differentiates enteric bacteria based on glucose, sucrose, and lactose fermentation, as well as the production of hydrogen sulfide (H2S).
  • Procedure: Bacteria are inoculated into a TSI agar slant. The slant is observed for color changes and H2S production.
  • Results:
    • Yellow butt and slant: Fermentation of glucose and lactose or sucrose.
    • Black precipitate: H2S production (e.g., Salmonella species).
    • Red slant and yellow butt: Only glucose fermentation.

 

---

Biochemical Tests for Fungi

For fungi, the identification is generally based on morphology, growth patterns, and specific metabolic reactions. Many biochemical tests for fungi focus on their ability to assimilate nutrients or produce specific enzymes. Here’s a detailed look at some common biochemical tests for fungi:

1. Germ Tube Test

• • Purpose: Used primarily to identify Candida albicans from other Candida species.
  • Procedure: Inoculate yeast in serum at 37°C and incubate for 2-3 hours.
  • Results:
    • Candida albicans: Germ tubes (small projections from the yeast cell).
    • Other Candida species: No germ tube formation.

2. Chlamydospore Formation

• • Purpose: Identifies yeasts that form chlamydospores (thick-walled, asexual spores).
  • Procedure: Grow yeast on Cornmeal agar and incubate.
  • Results:
    • Candida albicans: Chlamydospores form under certain conditions.
    • Other Candida species: May not form chlamydospores.

3. Sugar Assimilation Tests

• • Purpose: Identifies fungi by their ability to assimilate different sugars.
  • Procedure: Fungi are inoculated onto media containing a variety of sugars.
  • Results: Fungi that can use the sugar as a carbon source will grow, whereas others will not. This can help identify species based on their sugar utilization profile.

4. Urease Test for Yeasts

• • Purpose: Identifies Cryptococcus neoformans and other urease-producing fungi.
  • Procedure: Inoculate yeast culture into a urea broth or slant and observe for color change.
  • Results:
    • Urease-positive: Pink color (e.g., Cryptococcus neoformans).
    • Urease-negative: No color change.

5. Biochemical Identification Systems

• • API 20C (Yeast Identification) and VITEK 2 (Yeast and Moulds) are automated systems that identify fungi based on metabolic and enzymatic profiles.

6. Potassium Hydroxide (KOH) Preparation

• • Purpose: A simple test for detecting fungal elements in clinical specimens like skin scrapings or sputum.
  • Procedure: Add a drop of KOH to the specimen on a slide, heat briefly, and observe for fungal elements under the microscope.
  • Results: Fungal hyphae and spores will appear after clearing of the tissue.