VRBGA vs VRBDA: Composition, Principle, and Microbial Applications Explained

Introduction

In the field of pharmaceutical, food, and water microbiology, the detection and enumeration of bile-tolerant Gram-negative bacteria is a critical quality control requirement. Two commonly used selective media for this purpose are Violet Red Bile Glucose Agar (VRBGA) and Violet Red Bile Dextrose Agar (VRBDA).

These media are designed to isolate and enumerate Enterobacteriaceae and other Gram-negative intestinal bacteria capable of growing in the presence of bile salts and crystal violet. While both are similar, they differ in their carbohydrate source—glucose or dextrose—and intended use.

1. Principle

VRBGA and VRBDA work on the principle of selective inhibition and differential fermentation of carbohydrates.

• Bile salts and crystal violet inhibit Gram-positive organisms.
• Glucose or dextrose serve as fermentable carbohydrates.
• Neutral red acts as a pH indicator, turning colonies red or pink when acid is produced during fermentation.

This allows the differentiation of fermenters like Enterobacteriaceae from non-fermenters such as Pseudomonas.

2. Composition (per liter of distilled water)

Ingredient	VRBGA (g/L)	VRBDA (g/L)	Function
Peptone	7.0	7.0	Source of nitrogen and growth factors
Yeast Extract	3.0	3.0	Provides vitamins and amino acids
Bile Salts No.3	1.5	1.5	Inhibits Gram-positive bacteria
Sodium Chloride	5.0	5.0	Maintains osmotic balance
Glucose	10.0	-	Fermentable carbohydrate (VRBGA)
Dextrose	-	10.0	Fermentable carbohydrate (VRBDA)
Neutral Red	0.03	0.03	pH indicator
Crystal Violet	0.002	0.002	Suppresses Gram-positive growth
Agar	15.0	15.0	Solidifying agent
Final pH (at 25°C)	7.4 ± 0.2		—

3. Preparation Procedure

1. Suspend 46.53 g of the medium in 1 L of distilled water.  
2. Heat gently with agitation until dissolved completely.  
3. Do not autoclave — overheating destroys selective components.  
4. Cool to 45–50°C and pour into sterile Petri plates.  
5. Allow to solidify and store in a dark refrigerator at 2–8°C.

4. Method of Use (Microbial Enumeration Test)

1. Prepare sample dilutions according to test protocol.  
2. Transfer 1 mL of diluted sample to sterile Petri plates.  
3. Add 15–20 mL of molten VRBGA or VRBDA (45°C).  
4. Mix gently and allow to solidify.  
5. Overlay with 3–4 mL of the same medium.  
6. Incubate at 35–37°C for 18–24 hours.  
7. Count colonies that appear red/pink (0.5–2 mm diameter) as bile-tolerant Gram-negative bacteria.

5. Interpretation of Results

Observation	Interpretation
Red/Pink colonies with precipitation zone	Positive for bile-tolerant, glucose/dextrose fermenters (e.g., E. coli, Enterobacter)
Colorless or no growth	Non-fermenters or inhibited Gram-positive organisms
No colonies	Sample may be sterile or Gram-positive dominated

6. Applications

Pharmaceutical Industry

Used in Microbial Limit Tests (MLT) to detect and enumerate bile-tolerant Gram-negative bacteria in non-sterile pharmaceutical products according to USP, EP, and IP guidelines.

Food and Beverage Testing

Applied to enumerate Enterobacteriaceae in dairy, meat, and processed food products, ensuring safety and hygiene standards.

Water and Environmental Monitoring

Used for routine testing of drinking water and process water to determine microbial contamination levels.

7. Differences Between VRBGA and VRBDA

Feature	VRBGA	VRBDA
Carbohydrate Source	Glucose	Dextrose
Primary Application	Enumeration of Enterobacteriaceae	Detection of bile-tolerant Gram-negative bacteria
Reference Method	ISO 21528-2	Pharmacopoeial Microbial Limit Test
Indicator Reaction	Acid production → Red colonies	Acid production → Red colonies

8. Quality Control Strains

Organism	Expected Reaction
Escherichia coli ATCC 8739	Red/pink colonies with precipitation zone
Enterobacter aerogenes ATCC 13048	Red colonies with opaque center
Pseudomonas aeruginosa ATCC 9027	Inhibited or pale colonies
Staphylococcus aureus ATCC 6538	No growth (inhibited)

9. Key Points to Remember

• Do not autoclave — selective agents are heat-sensitive.
• Always overlay to reduce oxygen exposure during incubation.
• Use freshly prepared media for best selectivity.
• Include control strains to ensure accuracy.

10. Conclusion

Violet Red Bile Glucose Agar (VRBGA) and Violet Red Bile Dextrose Agar (VRBDA) are crucial in microbiology for detecting and enumerating bile-tolerant Gram-negative bacteria. Understanding their principles, composition, and proper use ensures reliable microbial limit testing and quality assurance across pharmaceutical, food, and environmental industries.

💬 About the Author

Siva Sankar is a Pharmaceutical Microbiology Consultant and Auditor with extensive experience in sterility testing, validation, and GMP compliance. He provides consultancy, training, and documentation services for pharmaceutical microbiology and cleanroom practices.

📧 Contact: siva17092@gmail.com
Mobile: 09505626106

📱 Disclaimer: This article is for educational purposes and does not replace your laboratory’s SOPs or regulatory guidance. Always follow validated methods and manufacturer instructions.