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Protocol：Detection of Mycoplasma by Culture
　　1. Inoculate 2 agar plates with 0.1ml of test sample.
　　2. Inoculate 1 agar plate with 100cfu M. pneumoniae.
　　3. Inoculate 1 agar plate with 100cfu M. orale.
　　4. Leave 1 agar plate un-inoculated as a negative control.
　　5. Inoculate 1 broth with 0.2 ml of test sample.
　　6. Inoculate 1 broth with 100cfu M. pneumoniae.
　　7. Inoculate 1 broth with 100cfu M. orale.
　　8. Leave 1 agar plate un-inoculated as a negative control.
　　9. Incubate agar plates anaerobically for 14 days at 37°C using a gas jar with anaerobic gas
pak and catalyst.
　　10. Incubate broths aerobically for 14 days at 37°C.
　　11. Between 3 and 7 days and 10 and 14 days incubation, subculture 0.1 ml of test broth onto
an agar plate and incubate plate anaerobically as above.
　　12. Observe agar plates after 14 days incubation at x300 magnification using an inverted
microscope for the presence of mycoplasma colonies (see Figure 14).

Criteria for a Valid Result
　　All positive control agar plates and broths show evidence of mycoplasma by typical colony
　　formation on agar plates and usually a colour change in broths.
　　All negative control agar plates and broths show no evidence of mycoplasma.

Criteria for a Positive Result
　　Test agar plates infected with mycoplasma show typical colony formation.

Criteria for a Negative Result
　　The test agar plates show no evidence of mycoplasma.

Notes
　　1. Mycoplasma colonies have a typical colony formation commonly described as “fried egg”
(See Figure 8) due to the opaque granular central zone of growth penetrating the agar
surrounded by a flat translucent peripheral zone on the surface. However in many cases
only the control zone will be visible.
　　2. Positive controls may be included at a concentration to give 100 colony-forming units.
These controls should obviously be handled in a laboratory remote from the main tissue
culture laboratory.
　　3. Control organisms (M. pneumoniae, and M. orale) are available from National Collection of
Type Cultures (UK).
　　4. Mycoplasma pneumoniae is a potential pathogen and must be handled in a class II
microbiological safety cabinet operating to ACDP Category 2 Conditions.
　　5. This test procedure should be carried out in a microbiology laboratory away from the cell
culture laboratory.

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