Violet Red Bile Agar

Intended use

Recommended for selective isolation, detection and enumeration of coli-aerogenes bacteria in water, milk other dairy, food products and clinical samples. The composition and performance criteria of this medium are as per the specifications laid down in ISO 4832: 2010 and ISO 11133 :2014, Amd. 2: 2020 (E)

Composition**

ISO specification :

Crystal violet neutral red bile lactose (VRBL) agar

M049- Violet Red Bile Agar

**Formula adjusted, standardized to suit performance parameters

$ - Equivalent to Enzymatic digest of animal tissues

Directions

Suspend 41.53 gram in 1000 ml purified/distilled water. Heat to boiling to dissolve the medium completely. DO NOT AUTOCLAVE. Cool to 45-50°C and immediately pour into sterile Petri plates containing the inoculum. If desired, the medium can be sterilized by autoclaving at 15 lbs pressure (121°C) for 15 minutes.

Principle And Interpretation

The coliform group consists of several genera of bacteria belonging to the family Enterobacteriaceae. The historical definition of this group has been based on the method used for detection i.e. lactose fermentation. This group is defined as all aerobic and facultative anaerobic, gram-negative, non-spore-forming rod shaped bacteria that ferment lactose with gas and acid formation within 48 hour at 35°C (1,2). Examination of foods, ingredients and raw materials, for the presence of marker groups such as coliforms is the one of the common tests.

Violet Red Bile Agar, a modification of MacConkey's original formulation (1) is used for the enumeration of coli-aerogenes bacterial group. It relies on the use of the selective inhibitory components crystals violet and bile salts and the indicator system lactose, and neutral red. Thus, the growth of many unwanted organisms is suppressed, while tentative identification of sought bacteria can be made. Organisms, which rapidly attack lactose, produce purple colonies surrounded by purple halos. Non-fermenters or late lactose-fermenters produce pale colonies with greenish zones (3). VRBA is recommended by APHA (4,5). Selectivity of VRBA can be increased by incubation under anaerobic conditions and/or at elevated temperature, i.e. equal to or above 42°C (6-8). It is also recommended by ISO (9,10).

Peptone and yeast extract serve as sources of carbon, nitrogen, vitamins and other essential growth nutrients. Lactose is the fermentable carbohydrate, utilization of which leads to the production of acids. Neutral red indicator detects the acidity so formed. Crystal violet and bile salts mixture help to inhibit the accompanying gram-positive and unrelated flora. Sodium chloride maintains the osmotic equilibrium. Violet Red Bile Agar is not completely specific for enteric; other accompanying bacteria may give the same reaction. Further biochemical tests are necessary for positive identification (5).

Type of specimen

Clinical samples - Stool; Food and dairy samples; Water samples

Specimen Collection and Handling:

For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (3-5,9,10).

Warning and Precautions :

In Vitro diagnostic Use. For professional use only. Read the label before opening the container. Wear protective gloves/ protective clothing/eye protection/ face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling clinical specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations :

1. Individual organisms differ in their growth requirement and may show variable growth patterns on the medium.
2. Each lot of the medium has been tested for the organisms specified on the COA. It is recommended to users to validate the medium for any specific microorganism other than mentioned in the COA based on the user's unique requirement.
3. Further biochemical tests must be carried out for complete identification.

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance Light yellow to pink homogeneous free flowing powder

Gelling Firm, comparable with 1.5% Agar gel.

Colour and Clarity of prepared medium Reddish purple coloured clear to slightly opalescent gel forms in Petri plates.

Reaction Reaction of 4.15% w/v aqueous solution at 25°C. pH : 7.4±0.2

pH 7.20-7.60

Cultural Response Productivity: Cultural characteristics observed after an incubation at 30 ± 1°C for 24 ± 2 hours. Recovery rate is considered as 100% for bacteria growth on Reference Medium - Tryptone Soya Agar

Selectivity: Cultural characteristics observed after an incubation at 30 ± 1°C for 24 ± 2 hours.

Specificity: Cultural characteristics observed after an incubation at 30 ± 1°C for 24 ± 2 hours.

Key: (*) Corresponding WDCM numbers.

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 20-30°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition. Seal the container tightly after use. Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (12,13).

References

1. MacConkey A., 1905, J. Hyg., 5, 333-3.
2. Wehr H. M. and Frank J. H., 2004, Standard Methods for the Microbiological Examination of Dairy Products, 17th Ed., APHA Inc., Washington, D.C.
3. Corry J. E. L., Curtis G. D. W. and Baird R. M., (Ed.), 1995, Culture Media for Food Microbiology, Vol. 34, Progress in Industrial Microbiology, Elsevier, Amsterdam.
4. American Public Health Association, Standard Methods for the Examination of Dairy Products, 1978, 14th Ed., Washington D.C.
5. Salfinger Y., and Tortorello M.L., 2015, Compendium of Methods for the Microbiological Examination of Foods, 5th Ed., American Public Health Association, Washington, D.C.
6. Mossel D. A. A. and Vega C. L., 1973, Hlth. Lab. Sci., 11:3
7. Mossel D. A. A., Eclderink I., Koopmans M. and Van Rossem F., 1979, Food Protect., 42: 4.
8. Mossel D. A. A. et al, 1986, J. Appl. Bacteriol., 60:2
9. Microbiology of food,animal feeding stuffs and water- Preparation, production,storage and performance culture media, EN ISO 11133:2014/Amd. 2 :2020 (Ε).
10. Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of coliforms — Colony-count technique ISO 4832 :2010
11. Lipps WC, Braun-Howland EB, Baxter TE, eds. Standard methods for the Examination of Water and Wastewater, 24th ed. Washington DC:APHA Press; 2023.
12. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Editio
13. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter,(2015) S.S and Warnock., D.W.Manual of Clinical Microbiology, 11th Edition. Vol. 1