The Enterobacteriaceae family comprises Gram-negative, facultatively anaerobic bacteria that include both pathogenic and non-pathogenic species.
Accurate identification of these organisms is crucial in clinical diagnostics, food safety, and environmental microbiology.
Biochemical tests play a key role in differentiating Enterobacteriaceae based on their metabolic properties.
This article discusses the essential biochemical tests used in laboratories to identify members of this family.
Common Biochemical Tests for Enterobacteriaceae
1. Indole Test
- Principle: Detects the production of indole from tryptophan by the enzyme tryptophanase.
- Reagent: Kovac’s reagent (contains p-dimethylaminobenzaldehyde).
- Procedure:
- Inoculate tryptone broth with the test organism.
- Incubate at 37°C for 24-48 hours.
- Add 5 drops of Kovac’s reagent.
- Interpretation:
- Positive: Red/pink ring at the surface (E. coli, Proteus vulgaris).
- Negative: No color change (Klebsiella, Salmonella).
2. Methyl Red (MR) Test
- Principle: Determines mixed acid fermentation by detecting stable acid end products.
- Reagent: Methyl red indicator (pH-sensitive dye).
- Procedure:
- Inoculate MR-VP broth and incubate at 37°C for 48 hours.
- Add 5-6 drops of methyl red reagent.
- Interpretation:
- Positive: Red color (E. coli, Proteus).
- Negative: Yellow color (Klebsiella, Enterobacter).
3. Voges-Proskauer (VP) Test
- Principle: Detects acetoin production (a precursor to 2,3-butanediol fermentation).
- Reagents: Barritt’s A (α-naphthol) and Barritt’s B (KOH).
- Procedure:
- Inoculate MR-VP broth and incubate at 37°C for 48 hours.
- Add 0.6 mL of Barritt’s A and 0.2 mL of Barritt’s B.
- Shake and observe after 10-15 minutes.
- Interpretation:
- Positive: Red/pink color (Klebsiella, Enterobacter).
- Negative: No color change (E. coli, Salmonella).
4. Citrate Utilization Test
- Principle: Determines if bacteria can use citrate as the sole carbon source.
- Medium: Simmons citrate agar (contains bromothymol blue indicator).
- Procedure:
- Streak the organism on slant citrate agar.
- Incubate at 37°C for 24-48 hours.
- Interpretation:
- Positive: Blue color (alkaline pH, Klebsiella, Enterobacter).
- Negative: No color change (remains green, E. coli, Shigella).
5. Urease Test
- Principle: Detects urease enzyme, which hydrolyzes urea to ammonia and CO₂.
- Medium: Christensen’s urea agar.
- Procedure:
- Inoculate urea agar slant.
- Incubate at 37°C for 24 hours.
- Interpretation:
- Positive: Pink/red color (Proteus, Klebsiella).
- Negative: No color change (E. coli, Salmonella).
6. Triple Sugar Iron (TSI) Agar Test
- Principle: Assesses carbohydrate fermentation (glucose, lactose, sucrose) and H₂S production.
- Medium: TSI agar slant (phenol red indicator).
- Procedure:
- Stab the butt and streak the slant.
- Incubate at 37°C for 18-24 hours.
- Interpretation:
- Acid butt (yellow) / Alkaline slant (red): Glucose fermentation only (Shigella, Salmonella).
- Acid butt and slant (yellow): Lactose/sucrose fermentation (E. coli, Klebsiella).
- Black precipitate: H₂S production (Proteus, Salmonella).
7. Motility Test
- Principle: Detects bacterial motility using semi-solid agar.
- Medium: Motility test medium (0.4% agar).
- Procedure:
- Stab inoculate the medium.
- Incubate at 37°C for 24 hours.
- Interpretation:
- Positive: Diffuse growth radiating from stab line (Proteus, E. coli).
- Negative: Growth only along stab line (Klebsiella, Shigella).
Summary Table of Key Biochemical Reactions
| Organism | Indole | MR | VP | Citrate | Urease | TSI Slant/Butt | H₂S | Motility |
|---|---|---|---|---|---|---|---|---|
| Escherichia coli | + | + | – | – | – | A/A | – | + |
| Klebsiella pneumoniae | – | – | + | + | + | A/A | – | – |
| Proteus mirabilis | – | + | – | V | + | K/A | + | + (Swarming) |
| Salmonella Typhi | – | + | – | + | – | K/A | + | + |
| Shigella flexneri | – | + | – | – | – | K/A | – | – |
Key:
+ = Positive, – = Negative, V = Variable, A = Acid, K = Alkaline
Conclusion
Biochemical tests remain a cornerstone in the identification of Enterobacteriaceae. By analyzing metabolic patterns such as indole production, citrate utilization, and carbohydrate fermentation, microbiologists can accurately differentiate between species like E. coli, Klebsiella, Salmonella, and Shigella.
These tests are cost-effective, reliable, and essential for clinical and environmental microbiology laboratories.
For definitive identification, biochemical tests are often supplemented with molecular methods (PCR, MALDI-TOF MS) in modern diagnostic labs. However, traditional biochemical assays remain indispensable for routine bacterial classification.