Mycoplasma Agar Base (PPLO Agar Base)

Intended Use

With the addition of enrichment it is recommended for isolation and cultivation of Mycoplasma species (Pleuro pneumonia- like organisms- PPLO).

Composition**

Final pH ( at 25°C): 7.8±0.2

**Formula adjusted, standardized to suit performance parameters

# Equivalent to Beef heart, infusion from

Directions

Suspend 36.0 grams in 700 ml purified / distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C and aseptically add 300 ml Horse serum (RM1239) or 10 vials of HPYT Enrichment Supplement (FD075). Mix well and pour into sterile Petri plates. 25% Ascitic fluid can be used instead of Horse serum.

Principle And Interpretation

PPLO Agar was described by Morton, Smith and Leberman (1). It was used in a study of the growth requirements of Mycoplasma (2), along with the identification and cultivation of this organism (3,4,5). Pivotal information regarding Mycoplasma has been documented by Sabin (6). Hayflick et al have reported the information regarding the cultivation of Mycoplasma (7).

For the cultivation of Mycoplasma the medium ingredients and all the supplements should be free of any toxic substances even in small amounts. HM infusion B from and peptone provide nitrogen and carbon source, long chain amino acids, vitamins, and other essential nutrients. Sodium chloride maintains the osmotic balance of these formulations. Many Mycoplasma require serum for their good growth and also presence of antibiotic is necessary to prevent the growth of contaminating organisms. Mostly the Mycoplasma species are aerobic or facultatively anaerobic but some are microaerophilic. Few are anaerobic saprophytic Mycoplasma which grow best at 22-35°C while pathogenic strains grow at 35°C. Mycoplasma when grow in the agar medium show typical morphology and form colonies below the agar surface and do no grow without serum.

Plates or tubes should be incubated in an atmosphere containing 5-10% carbon dioxide and examined after incubation of 48 hours but they should not be discarded as negative until after incubation for 3 weeks.

PPLO colonies are round with a dense center and a less dense periphery, resembling a “fried egg” on PPLO Agar. Vacuoles, large bodies characteristic of Mycoplasma species are seen in the periphery. Colonies vary in diameter from 10 to 500 microns (0.01-0.5 mm) and penetrate into the medium.

Type of specimen

Clinical samples- Urine sample, urethral swab, sputum sample; pharmaceutical samples

Specimen Collection and Handling

For pharmaceutical samples, follow appropriate techniques for sample collection, processing as per pharmaceutical guidelines (8).

For clinical samples follow appropriate techniques for handling specimens as per established guidelines (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. Since Mycoplasma species are aerobic or facultatively anaerobic but some are microaerophilic, proper incubation should be carried out for optimal recovery.
2. Few are anaerobic saprophytic Mycoplasma which grow best at 22-35°C while pathogenic strains grow at 35°C, hence growth conditions must be maintained.
3. Since the medium is highly enriched care must be taken during innoculation to avoid contamination.

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 free flowing powder

Gelling: Firm, comparable with 1.5% Agar gel

Colour and Clarity of prepared medium: Yellow coloured clear to slightly opalescent gel forms in Petri plates

Reaction: Reaction of 3.6% w/v aqueous solution at 25°C. pH : 7.8±0.2

pH: 7.60-8.00

Cultural Response

Cultural characteristics observed in presence of 10% Carbon dioxide with added,1% Horse serum (RM1239) or 10 vials of HPYT Enrichment Supplement (FD075), after an incubation at 22-35°C for 48 hours.

Storage and Shelf Life

Store below 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 (9,10).

Reference

1. Morton, Smith and Leberman, 1951, Am. J. Syphilis Gonorrh. Veneral Diseases, 35: 361.
2. Morton and Lecce, 1953. J. Bacteriol., 66:646.
3. Chanock, James, Fox, Turner, Mufso and Hayflick, 1962, Soc. Exp. Biol. Med., 110:884.
4. Craven, Wenzel, Calhoun, Hendley, Hamory and Gwaltney, 1976, J. Clin. Microbiol., 4:225.
5. Gregory and Cundy, 1970, Appl. Microbiol., 19:268.
6. Sabin, 1941, Bacteriol. Rev., 5:1, 331.
7. Hayflick and Chanock, 1965, Bacteriol, Rev., 29:185.
8. The United States Pharmacopoeia-National Formulatory (USP-NF), 2022.
9. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
10. 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.