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PPLO Agar 500g



Intended Use

PPLO (Mycoplasma) agars and broths, when supplemented with nutritive enrichments, are used for isolating and cultivating Mycoplasma. Mycoplasma Broth Base (Frey) is used for the cultivation of avian mycoplasmas.

Summary and Explanation

Members of the class Mollicutes, Mycoplasma was first recognized from a case of pleuropneumonia in a cow.1 The organism was designated “pleuropneumonia-like organism,” or PPLO.1 Although some species are normal human respiratory tract flora, M. pneumoniae is a major cause of respiratory disease (primary atypical pneumonia, sometimes called “walking pneumonia”).1 M. hominis, M. genitalium and Ureaplasma urealyticum are important colonizers (and possible pathogens) of the human genital tract.1
PPLO (Mycoplasma) Agar was described by Morton, Smith and Leberman.2 It was used in a study of the growth requirements of Mycoplasma,3 along with the identification and cultivation of this organism.4-6

Principles of the Procedure

Meat digests, peptones, beef extract and yeast extract provide the nitrogen, vitamins, amino acids and carbon in these media. Sodium chloride maintains the osmotic balance of these formulations. Agar, the solidifying agent, is used in PPLO (Mycoplasma) Agar at a concentration slightly reduced from usual to ensure formation of the largest possible colonies because the organisms grow into the agar with only slight surface growth.13
The base media are supplemented with Mycoplasma Supplement or Mycoplasma Enrichment w/o Penicillin because Mycoplasma spp. are fastidious in their growth requirements.14
Mycoplasma Supplement contains fresh yeast extract and horse serum. Yeast extract provides the preformed nucleic acid precursors that are required by Mycoplasma spp.14 Horse serum supplies cholesterol, a growth stimulant.14
Mycoplasma Enrichment without Penicillin is a selective enrichment containing the inhibitor thallium acetate, to which a penicillin of choice (penicillin G or a broad-spectrum semisynthetic penicillin) can be added at the time of use to make it selective against gram-positive and gram-negative bacteria.

User Quality Control

Identity Specifications
PPLO Agar (Mycoplasma Agar)
Dehydrated Appearance: Beige, homogeneous, free-flowing.
Solution:                        3.5% solution, soluble in purified water upon
                                     boiling. Solution is medium amber, slightly
Prepared Appearance:     Enriched with 30% Mycoplasma Supplement–
                                     Light to medium amber, slightly opalescent.
Reaction of 3.5%
Solution at 25°C:            pH 7.8 ± 0.2
Cultural Response
PPLO Agar or PPLO Broth
Prepare the medium per label directions. Inoculate agar plates with 0.1 mL of serial dilutions of the test organisms. Incubate plates under 5-10% CO2 at 35 ± 2°C for up to 7 days. Inoculate tubes of broth with 1.0 mL of serial dilutions of the test organisms and incubate under 5-10% CO2 at 35 ± 2°C for up to 7 days, then subculture (0.1 mL) to plates of the agar medium and incubate under 5-10% CO2 at 35 ± 2°C for up to 7 days. Daily examine plates microscopically for growth. 

Mycoplasma arginini 23243 Good
Mycoplasma bovis 25523 Good
Mycoplasma gallinarum 19708 Good


Approximate Formula* Per Liter
Beef Heart, Infusion from 50 g...................................... 6.0 g
Peptone..................................................................... 10.0 g
Sodium Chloride.......................................................... 5.0 g
Agar.......................................................................... 14.0 g
PPLO Broth
Consists of the the same ingredients without the agar.
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

PPLO Broth
1. PPLO Agar: Suspend 35 g of the powder in 700 mL of purified water. Mix thoroughly. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
PPLO Broth: Dissolve 21 g of the powder in 700 mL of purified water. Mix thoroughly.
2. Autoclave at 121°C for 15 minutes. Cool medium to 50- 60°C.
3. Aseptically add 300 mL Difco Mycoplasma Supplement to the medium. Mix well.
4. Add selective agents if desired (i.e., thallium acetate or penicillin).
5. Test samples of the finished product for performance using stable, typical control cultures.


Inoculate the surface of plates containing the complete medium by adding drops of liquid inoculum or by a swab-inoculation technique. Incubate plates at 35 ± 2°C for up to 21 days in a moist atmosphere containing 5-10% carbon dioxide or anaerobically if the presence of M. buccale, M. faucium, M. orale or M. salivarium is suspected.13
Test material, either solid or liquid, should be directly inoculated into the broth medium. For preparation of stock organism suspensions, a block of agar culture can be added to the broth. Following incubation at 35 ± 2°C in a moist aerobic atmosphere containing 5-10% carbon dioxide or anaerobically, if appropriate, 13 for various lengths of time, subculture aliquots of the broth to PPLO (Mycoplasma) Agar plates for visualization of typical colonies. The broth usually does not become turbid enough to confirm the presence of growth.
For a complete discussion of the isolation and identification of Mycoplasma spp. from clinical specimens, refer to appropriate procedures outlined in the references.13-15

Expected Results

PPLO colonies are round with a dense center and a less dense periphery, giving a “fried egg” appearance on PPLO (Mycoplasma) Agar. Vacuoles, large bodies characteristic of Mycoplasma spp., are seen in the periphery. Colonies vary in diameter from 10 to 500 microns (0.01-0.5 mm) and penetrate into the medium.
After subculture to plates of PPLO (Mycoplasma) Agar, positive broth cultures produce colonies exhibiting the typical morphology; i.e., “fried egg” appearance.

Limitations of the Procedure

Thallium acetate can partially inhibit some mycoplasmas.13

*Store at 2-8° C.

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Celebrity Endorsements

1. Baron, Peterson and Finegold. 1994. Bailey & Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc. St. Louis, Mo.

2. Morton, Smith and Leberman. 1951. Am. J. Syphilis Gonorrh. 35:361.

3. Morton and Lecce. 1953. J. Bacteriol. 66:646.

4. Chanock, James, Fox, Turner, Mufso and Hayflick. 1962. Soc. Exp. Biol. Med. 110:884.

5. Craven, Wenzel, Calhoun, Hendley, Hamory and Gwaltney. 1976. J. Clin. Microbiol. 4:225.

6. Gregory and Cundy. 1970. Appl. Microbiol. 19:268.

7. Adler and Da Massa. 1967. Appl. Microbiol. 15:245.

8. Leland, Lapworth, Jones and French. 1982. J. Clin. Microbiol. 16:709.

9. Frey, Hanson and Anderson. 1968. Am. J. Vet. Res. 29:2163.

10. Hayflick. 1965. Tex. Rep. Biol. Med. 23:285.

11. Chanock, Hayflick and Barile. 1962. Proc. Nat. Acad. Science 48:41.

12. Hayflick. 1968. Personal communication.

13. Kenny. 1985. In Lennette, Balows, Hausler and Shadomy (ed.). Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C.

14. Waites and Taylor-Robinson. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.). Manual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C.

15. Isenberg (ed.). 1992. Clinical microbiology procedures handbook, vol. 1. American Society for Microbiology, Washington, D.C.

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