Hardy Diagnostics HUGO

PPLO (Mycoplasma) MEDIA

Cat. no. G04 PPLO Selective Agar, 15x60mm Plate, 11ml 10 plates/bag
Cat. no. G105 PPLO Selective Agar, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. R80 PPLO Broth without Antibiotics, 13x100mm Tube, 5ml 20 tubes/box
Cat. no. R88 PPLO Selective Broth, 13x100mm Tube, 4ml 20 tubes/box

INTENDED USE

Hardy Diagnostics PPLO Media are recommended for the cultivation and maintenance of Mycoplasma spp. and Ureaplasma spp.

SUMMARY

PPLO, or pleuropneumonia-like organisms, were first recognized in cattle and many are a major cause of respiratory disease; more still are considered normal flora and potential pathogens of the human urogenital tract. (8) PPLO fall into the class Mollicutes and are recognized as commensal or parasitic colonizers of humans, other animals, insects, and plants. Currently, there are over 100 known species in the genus Mycoplasma whose members are restricted to vertebrate hosts. Far fewer Ureaplasma spp. have been discovered, but members of this genus are also known to be commensal or potentially pathogenic to their hosts.

Mycoplasma spp. lack a cell wall and are unaffected by penicillins or other beta-lactam antibiotics that target cell wall synthesis. However, they do possess characteristic cell shapes believed to play a role in their ability to thrive in certain hosts. All Mycoplasma spp. require sterols, usually in the form of cholesterol, for cytoplasmic membrane stability, making them dependent upon their host's biosynthetic capabilities. Their optimal growth temperature usually falls within the range specific to their host. If their host is unable to regulate its own temperature, many are capable of growth at ambient temperatures.

Ureaplasma spp. also lack a cell wall and require cholesterol for growth. Members of this genus are considered part of the normal flora of the human genital tract, but may cause or play a role in non-specific urethritis, infertility, chorioamnionitis, stillbirth, premature birth and, in the perinatal period, pneumonia, chronic lung disease and meningitis in infants. (9) A defining characteristic of this genus is that members are capable of urea hydrolysis.

PPLO Selective Media are highly nutritious due to the addition of beef heart infusion, peptone supplemented with yeast extract and inactivated horse serum. Yeast extract provides diphosphopyridine nucleotides and serum provides cholesterol and a source of protein. The selective agents amphotericin B, polymyxin B, and penicillin are added to inhibit faster growing contaminants. The concentration of agar is slightly reduced in PPLO solid media in order to encourage growth of larger colonies, since surface colonial growth is minimal. (10) PPLO Selective Broth contains all of the aforementioned ingredients, but is lacking in agar. PPLO Broth without Antibiotics lacks agar and selective agents. All PPLO Broth Media contain a phenol red pH indicator to aid in the detection of Mycoplasma growth.

FORMULA

Ingredients per liter:*

Beef Heart Infusion 50.0gm
Peptone 10.0gm
Sodium Chloride 5.0gm
Polymyxin B 50.0mg
Amphotericin B 5.0mg
Penicillin 1,000,000U
Deionized Water 680.0ml
Horse Serum 200.0ml
Yeast Extract Solution 100.0ml

In addition,

PPLO Selective Agar also contains:

Noble Agar 9.0gm

PPLO Selective Broth also contains:

Phenol Red 18.0mg/ml

PPLO Broth without Antibiotics contains a phenol red pH indicator, but lacks polymyxin B, amphotericin B, and penicillin.

Final pH 7.8 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Specimen/swab should be placed in a tightly sealed transport container with sufficient transport medium to prevent drying and taken directly to the laboratory. Dacron, polyester, or calcium alginate swabs are acceptable. Samples should be processed as soon as possible after arrival in the laboratory. If there is to be a delay in culturing, specimens should be refrigerated. For long-term storage, or if specimens cannot be cultured within 24 hours, freeze specimens at -70ºC. Do not freeze at temperatures greater than -70ºC.

PPLO Selective Agar: Inoculate and streak the specimen as soon as possible after receiving in the laboratory. If using a liquid inoculum, add 0.1ml of liquid to the agar surface and distribute evenly by rocking the plate back and forth or spreading the inoculum with a sterile bent glass rod. Alternatively, if the material being cultured is directly from a swab, roll the swab over the agar surface and streak for isolation. Increased recovery may be enhanced by diluting and plating the specimen serially up to 10-3. Diluting the specimen minimizes the effect of bacterial inhibitors on the growing mycoplasma.(12) Agar plates should be taped to restrict dehydration. Incubate plates in 5-10% CO2 at 35ºC. for up to 30 days. Plates may be incubated anaerobically if Mycoplasma buccale, M. faucium, M. orale or M. salivarium is suspected.

Microscopic examination at 40-60X of inverted plates reveals the colony morphology of mycoplasmas. Organisms are recognized by typical tiny "fried egg" colonies or finely granular ("ground glass") colonies with a berry-like appearance that penetrate the agar surface. Colonies can range from 20-300µm. Consult listed references for more information regarding cultivation and isolation of mycoplasmas.(6,8,11,12)

PPLO Broth: Inoculate the specimen as soon as possible after it is received in the laboratory. Inoculate broth with 0.1ml of transport media containing a swab. Alternatively broth may be inoculated at a 1:10 ratio with blood or CSF. Tissue specimens may be inoculated by placing several minced fragments directly into the broth. Increased recovery may be enhanced by diluting and plating the specimen serially up to 10-3. Diluting the specimen minimizes the effect of bacterial inhibitors on the growing mycoplasma.(12) Incubate at 35ºC. for up to 30 days.

For PPLO Selective Broth: Examine tubes daily for a change in pH. A pH shift will cause the medium to change from red to yellow. As soon as a pH shift is noted, subculture the broth to an appropriate agar medium. Consult listed references for more information regarding cultivation and isolation of mycoplasmas.(6,8,11,12)

LIMITATIONS

Occasional breakthrough of bacterial growth may occur on medium. Similarities of L-form bacteria and mycoplasma organisms on the agar medium may cause some confusion because both exhibit "fried egg" colonies that penetrate the agar surface. L-form colonies tend to be larger and demonstrate a rougher surface. Many L-forms will revert back to the bacterial form if passed to a penicillin-free medium.

Ureaplasma urealyticum will be inhibited on this medium due to the pH of the medium.
Increased recovery may be enhanced by diluting and plating the specimen serially up to 10 -3 . Diluting the specimen minimizes the effect of bacterial inhibitors on the growing mycoplasma. (12)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Cat. no. G04, G105, R88:
Mycoplasma pneumoniae
ATCC ® 15531
K 10-21
days
35°C CO 2 ** Color change from red to yellow; growth observed microscopically at 10-21 days
Mycoplasma arginini
ATCC ® 23838
K 10-21
days
35°C Anaerobic Slight turbidity; no color change; growth observed microscopically at 10-21 days
Escherichia coli
ATCC ® 25922
B 24hr 35°C Aerobic Inhibited
Staphylococcus aureus
ATCC ® 25923
B 24hr 35°C Aerobic Inhibited
Candida albicans
ATCC ® 10231
B 24hr 35°C Aerobic Inhibited


Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Cat. no. R80:
Mycoplasma pneumoniae
ATCC ® 15531
K 10-21
days
35°C Aerobic Growth, color change from red to yellow***
Mycoplasma arginini
ATCC ® 23838
K 10-21
days
35°C Aerobic Growth, slight turbidity, no color change.***

Note: Mycoplasma pneumoniae is inoculated by using a swab to place a single streak on the surface.

** Atmosphere of incubation is enriched with 5-10% CO2.

*** Note: PPLO Selective Broth contains a phenol red indicator which changes from red-orange to yellow with the growth of Mycoplasma spp. To verify growth of Mycoplasma spp. in PPLO Broth and Biphasic Media, a plate must be inoculated and examined for growth.

User Quality Control

PHYSICAL APPEARANCE

REFERENCES

1. ASM. 1984. Cumitech 19; Laboratory Diagnosis of Chlamydial and Mycoplasmal Infections, American Society for Microbiology, Washington, D.C.

2. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

3. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

4. Edward, D.G. 1954. The pleuropneumonia group of organisms. A review together with some new observations. Jour. of Gen. Micro.; 10:27-64.

5. Fisher, O. 1995. Enhancement of propagation of Mycoplasma hyopneumoniae by culture in a biphasic medium. Acta Vet. Brno.; 64:243-247.

6. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

7. Hayflick, L. and R.M. Chanock. 1965. Mycoplasma Species of Man. Bacter. Reviews. Vol. 29, No. 2. American Society for Microbiology, Washington, D.C.

8. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

9. Kafetzis, D.A., C.L. Skevaki, V. Skouteri, S. Gavrili, K. Peppa, C. Kostalos, V. Petrochilou, and S. Michalas. 2004. Maternal genital colonization with Ureaplasma urealyticum promotes preterm delivery: association of the respiratory colonization of premature infants with chronic lung disease and increased mortality. Clin. Infect. Diseases.; 39:1113.

10. Klieneberger-Nobel, E. 1962. Pleuropneumonia-Like Organisms (PPLO) Mycoplasmataceae. Academic Press, New York, NY.

11. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

12. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

13. Shklair, I.L., M.A. Mazzarella, R.R. Gutekunst, and E.M. Kiggins. 1962. Isolation and Incidence of Pleuropneumonia-like Organisms from the Human Oral Cavity. Jour. Bact.; 83:785.


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040416gr