Agar Medium F (Crystal Violet, Neutral Red, Bile Agar with Glucose)

Intended use

Agar Medium F is recommended for detection and enumeration of Enterobacteria in accordance with British Pharmacopoeia.

Composition**

Final pH (at 25°C): 7.4±0.2

**Formula adjusted, standardized to suit performance parameters

$ - Equivalent to pancreatic digest of gelatin

Directions

Suspend 50.12 grams (the equivalent weight of dehydrated medium per litre) in 1000 ml purified /distilled water. Heat to boiling to dissolve the medium completely. DO NOT HEAT IN AN AUTOCLAVE. Cool to 45-50°C. Mix well and pour into sterile Petri plates.

Principle And Interpretation

It is selective medium recommended for detection of Enterobacteriaceae species as recommended by British Pharmacopoeia and is also cited as Agar medium F (1). Mossel et al (2,3,4) added glucose to the medium observing improved detection of coliforms. Incubation can be carried out at different temperatures and incubation time depending upon the group of Enterobacteriaceae to be recovered (5).

Gelatin and yeast extract provide nitrogenous and carbonaceous compounds, long chain amino acids, vitamins and other nutrients essential for bacterial metabolism. This media is selective due to presence of the inhibitors; bile salts and crystal violet. Crystal violet inhibits gram-positive organisms especially Staphylococci. Neutral red indicator helps to detect lactose monohydrate and glucose monohydrate fermentation. Lactose and glucose fermenting strains grow as red or pink and may be surrounded by a zone of acid precipitated bile. Sodium chloride maintains the osmotic equilibrium in the medium. The red colour is due to absorption of neutral red and a subsequent colour change of the dye when the pH of medium falls below 6.8.

Type of specimen

Pharmaceutical samples

Specimen Collection and Handling

For pharmaceutical samples, follow appropriate techniques for handling specimens as per established guidelines (1). After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions

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 specimens. Saftey guidelines may be referred in individual safety data sheets.

Limitations

Further biochemical tests must be carried out for confirmation.

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 After heating, reaction of 5.01% w/v aqueous solution. pH : 7.4±0.2

pH 7.20-7.60

Growth Promotion Test Growth Promotion is carried out in accordance with British Pharmacopoeia and cultural characteristics are observed after an incubation at 35-37°C for 18-24 hours.

Cultural Response

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 inorder 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. Use before expiry date on the label. 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 (6,7).

Reference

1. The British Pharmacopoeia 2008, The Stationery Office. British Pharmacopoeia.
2. Mossel D.A.A., Mengerink W.H.J. & Scholts H.H., 1962, J. Bacteriol, 84 : 381.
3. Mossel D.A.A. et al, 1978, Lab. practice, 27 No. 12 : 1049
4. Mossel D.A.A. et al, 1979, Food Protect., 42 : 470.
5. Mossel D.A.A. et al, 1986, J. Appl. Bact., 60 : 289
6. Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition.
7. Jorgensen,J.H., Pfaller , M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.