PPLO Agar: Introduction, Principle, Composition, Preparation, Procedure, Colony Morphology, Uses and Keynotes

PPLO Agar: Introduction, Principle, Composition, Preparation, Procedure, Colony Morphology, Uses and Keynotes

Introduction of PPLO Agar

PPLO Agar is recommended for the isolation and cultivation of Mycoplasma species. Mycoplasma is also called  Pleuropneumonia-like organisms. PPLO is the short form of this organism. This medium is for Mycoplasma and therefore it is called PPLO agar. It is also known as Mycoplasma agar.

Principle of PPLO Agar

The culture medium for the cultivation of Mycoplasma should be free of any toxic substances even in small amounts. Beef heart infusion from peptone supplies nitrogen and carbon source, long-chain amino acids, vitamins, and other essential nutrients for the growth of organisms. Sodium chloride keeps the osmotic balance of these formulations. Numerous Mycoplasma species require serum for their good growth. The presence of an antimicrobial agent in the medium is necessary to prevent the growth of contaminating microbes. Mostly the Mycoplasma species are aerobic or facultatively anaerobic. However, some are microaerophilic too. Few are anaerobic saprophytic Mycoplasma that grows best at 22-35°C.

Pathogenic strains grow at 35°C. Mycoplasma when grown in this medium show typical morphology and form colonies below the agar surface. They do not grow without serum. Cultivated plates or tubes should be incubated in an atmosphere containing 5-10% CO2. Examine after incubation of 48 hours but they should not be discarded as negative until after incubation for 3 weeks.

Composition of PPLO Agar

Ingredients  Gms/Litre
HM infusion B from/o Beef heart, infusion from: 250.0
Peptone: 10.0
Sodium chloride: 5.0
Agar: 15.0
Final pH ( at 25°C) 7.8±0.2

Extra-

300 ml horse serum/10 vials of Mycoplasma Enrichment Supplement/25% Ascitic fluid in 700 ml PPLO Agar base

Preparation  of PPLO Agar 

  1. Suspend 36.0 grams in 700 ml purified/distilled or deionized water.
  2. Heat to boiling to dissolve the medium completely.
  3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
  4. After autoclaving,  leave for cooling to 45-50°C.
  5. Aseptically add 300 ml Horse serum/10 vials of Mycoplasma Enrichment Supplement/25% Ascitic fluid.
  6. Mix properly prior to dispensing.
  7. Pour PPLO agar into each plate and leave plates on the sterile surface until the agar has solidified.
  8. Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life of PPLO Agar

  • Store at 2-8ºC  and away from direct light.
  • Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), or contamination.
  • The product is light and temperature sensitive. Protect it from light, excessive heat, moisture, and freezing.

Test Requirements for PPLO Agar

Test procedure (specimen/organism inoculation)

  1. Allow the plates to warm at 37°C  or room temperature.
  2. It makes the agar surface dry before inoculating.
  3. Inoculate and streak the specimen as soon as possible after collection.
  4. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
  5. Streak for isolation with a sterile loop.
  6. Incubate plates aerobically in 10% Carbon dioxide at 22-35°C for 48 hours.
  7. Examine colony characteristics.

Result and  Interpretation of PPLO Agar

Control strains i.e. Escherichia coli ATCC 25922: Good-luxuriant growth  in absence of antibiotic in medium

Mycoplasma bovis ATCC 25523: Good-luxuriant growth with typical fried egg colony

Colony Morphology of PPLO Agar

  • Mycoplasma species: Fried egg colony(round with a dense center and a less dense periphery) having a diameter from 0.01-0.5 mm and penetrating into the medium
  • Mycoplasma bovis ATCC 25523:Good-luxuriant growth with fried egg colony
  • M. gallinarium ATCC 19708: Good-luxuriant growth resembling fried egg colony
  • M. pneumoniae ATCC 15531: Good-luxuriant growth  of fried egg colony
  • Streptococcus pneumoniae ATCC 6303: Good-luxuriant growth with a small colony

Use of PPLO Agar

Isolation and cultivation of Mycoplasma species

Keynotes on PPLO Agar

  1. The Colour and clarity of the prepared PPLO medium are yellow colored clear to slightly opalescent gel forms in Petri plates.
  2.  Mycoplasma species are aerobic as well as facultatively anaerobic.  Even some are microaerophilic too. Thus, proper incubation should be carried out for optimal recovery.
  3. Few are anaerobic saprophytic Mycoplasma. They grow best at 22-35°C. Pathogenic strains grow at 35°C, therefore growth conditions must be maintained.
  4. PPLO agar is highly enriched. Care must be taken during inoculation to avoid contamination.
  5. Morton, Smith, and Leberman described PPLO Agar.
  6. Sabin has documented key information regarding Mycoplasma.
  7. Hayflick et al have reported information related to the cultivation of
    Mycoplasma.

Further Readings

  • Craven, Wenzel, Calhoun, Hendley, Hamory, and Gwaltney, 1976, J. Clin. Microbiol., 4:22
  • Chanock, James, Fox, Turner, Mufso and Hayflick, 1962, Soc. Exp. Biol. Med., 110:884.
  • Gregory and Cundy, 1970, Appl. Microbiol., 19:268.
  • https://himedialabs.com/TD/M266.pdf
  • Sabin, 1941, Bacteriol. Rev., 5:1, 331.
  • Hayflick and Chanock, 1965, Bacteriol, Rev., 29:185
  • Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition
  • 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.
  • Morton, Smith and Leberman, 1951, Am. J. Syphilis Gonorrhea. Venereal Diseases, 35: 361.
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