Rogosa Agar, Modified

Intended Use

Recommended for the selective cultivation of Lactobacilli from food and clinical samples.

Composition**

Final pH after addition of glacial acetic acid( at 25°C): 6.2±0.1

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 74.40 grams (the equivalent weight of dehydrated medium per litre) in 1000 ml purified / distilled water. Heat to boiling to dissolve the medium completely. Adjust the pH to about 6.2 at about 50°C with glacial acetic acid (approximately 1.32 ml) and mix thoroughly. Heat to 95°C for 3 mins. DO NOT AUTOCLAVE. Cool to 45-50°C. Mix well and pour into sterile Petri plates.

Principle And Interpretation

Rogosa Agar is primarily a selective medium for the cultivation of Lactobacillus (1). High acetate concentration and low pH effectively suppress other bacteria, but also many strains of other lactic acid bacteria. The modification of the pH to 6.2 instead of 5.5 alters the selectivity of the medium for the whole group of lactic acid bacteria (2,3).

Tryptone, yeast extract provide nitrogenous compounds, sulphur, trace elements and vitamin B complex, essential for growth of Lactobacilli. Glucose acts as fermentable carbohydrate. Polysorbate 80 is the source of fatty acids. Ammonium citrate and sodium acetate inhibit moulds, Streptococci and many other organisms. Monopotassium phosphate provides buffering capability. Magnesium sulphate, manganese sulphate and ferrous sulphate are sources of inorganic ions. Low pH of the medium and addition of acetic acid makes the medium selective for Lactobacilli, inhibiting other bacterial flora (4). It is recommended that the plates should be incubated at 30°C for 5 days or at 37°C for 3 days in an atmosphere of 95% hydrogen and 5% carbon dioxide (5). High acetate concentration and acidic pH suppress many strains of other lactic acid bacteria.

Type of specimen

Clinical samples - Dental carries, Food samples.

Specimen Collection and Handling

For clinical samples follow appropriate techniques for handling specimens as per established guidelines (6,7).

For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (8,9,10).

After use, contaminated materials must be sterilized by autoclaving before discarding.

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. The bacteria sustaining low pH, may grow on this media.
2. Individual organisms differ in their growth requirement and may show variable growth patterns on the medium.
3. 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.

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: Cream to yellow homogeneous soft lumps which can be easily broken down to powder form.

Gelling: Firm, comparable with 1.5% Agar gel

Colour and Clarity of prepared medium: Light yellow coloured opalescent gel forms in Petri plates

Reaction: Reaction of 7.44% w/v aqueous solution with glacial acetic acid at 25°C. pH : 6.2±0.1

pH: 6.10-6.30

Cultural Response: Cultural characteristics observed in presence of 5% Carbon dioxide (CO₂) and 95% H2 after an incubation at 35-37°C for 40-48 hours.

Key : (*) Corresponding WDCM numbers.

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 2-8°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 (6,7).

Reference

1. Rogosa, J.,Mitchell J.A. and Wiseman,R.F.(1951)A selective medium for the isolation and enumeration of oral and fecal lactobacilli J.Bacteriol.62,132-133.
2. ISO (1984) Drafts reports.Enumeration of Lactobacteriaceae in meat and meat products.ISO/TC 34/SC 6/WG 15, no.3 and no.5. International Organization for Standardization,Geneva.
3. Reuter,G. (1985)Elective and selective media for lactic acid bacteria .Int.J.Food Microbiol.2,55-68.
4. MacFaddin J. F., 1985, Media for Isolation-Cultivation-Identification- Maintenance of Medical Bacteria, Vol. I, Williams and Wilkins, Baltimore. Md.
5. Sharpe M. L. (Ed.), 1960, Lab-Practice, 9(4): 223.
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.
8. American Public Health Association, Standard Methods for the Examination of Dairy Products, 1978, 14th Ed., Washington D.C.
9. Salfinger Y., and Tortorello M.L. Fifth (Ed.), 2015, Compendium of Methods for the Microbiological Examination of Foods, 5th Ed., American Public Health Association, Washington, D.C.
10. Wehr H. M. and Frank J. H., 2004, Standard Methods for the Microbiological Examination of Dairy Products, 17th Ed., APHA Inc., Washington, D.C.