BTGN (Bile-Tolerant Gram-Negative bacteria) refers to a group of Gram-negative bacteria that can grow in the presence of bile salts. It is mainly used as an indicator of hygiene and contamination, especially in pharmaceutical, food, and microbiological testing.
- Sample Preparation
Prepare a 1 in 10 dilution using a minimum of 1 g of the product to be tested.
Use Soybean–Casein Digest Broth as the diluent.
Mix the sample well and incubate at 20°C to 25°C for 2 to 5 hours to resuscitate any bacteria present without allowing significant multiplication. - Test for Absence of Bile-Tolerant Gram-Negative Bacteria
Use the equivalent of 1 g of the product (prepared as described above) to inoculate Enterobacteria Enrichment Broth(EEB)
Incubate at 30°C to 35°C for 24 to 48 hours.
Subculture on plates of Violet Red Bile Glucose Agar.
Incubate again at 30°C to 35°C for 18 to 24 hours.
Interpretation: The product passes the test if no colonies are observed on the plates.
Quantitative Test for Bile-Tolerant Gram-Negative Bacteria
- Selection and Subculture
*Inoculate suitable amounts of Enterobacteria Enrichment Broth with different dilutions of thesample to represent 0.1 g, 0.01 g, and 0.001 g (or 0.1 mL, 0.01 mL, and 0.001 mL) of the product.
*Incubate at 30°C to 35°C for 24 to 48 hours.
*Subculture each enriched sample onto Violet Red Bile Glucose Agar plates.
*Incubate again at 30°C to 35°C for 18 to 24 hours.
- Interpretation
*Growth of colonies indicates a positive result. - *Record the smallest quantity of product that shows a positive result and the largest quantity that shows a negative result.
