Detection of mycoplasma by culture isolation methods is the reference method of detection and has a theoretical level of detection of 1 colony-forming unit (cfu). However, there are some strains of mycoplasma that are non-cultivable (certain strains of Mycoplasma hyorhinis). The method is suitable for the detection of mycoplasma in both cell cultures and cell culture reagents and results are obtained within 4 weeks. Mycoplasma colonies observed on agar plates have a ‘fried egg’ appearance.
- 70% (v/v) alcohol in sterile water (793213)
- Mycoplasma plates
- Mycoplasma horse serum broths (M0535)
- M. orale (NCTC* 10112)
- M. pneumoniae (NCTC* 10119)
* Available from the National Collection of Type Cultures (NCTC)
- Personal protective equipment (sterile gloves, laboratory coat, safety visor)
- Waterbath set to 37 °C
- Microbiological safety cabinet at appropriate containment level
- Incubator set at 36 °C
- Anaerobic jar system
- Inoculate 2 agar plates with 0.2mL of test sample.
- Inoculate 2 plates with 100 cfu of each control organism.
- Leave 2 plates un-inoculated as a negative control.
- Inoculate 1 broth with 10mL of test sample.
- Inoculate a broth with 100 cfu of each control organism.
- Leave 1 broth un-inoculated as a negative control.
- Incubate agar plates anaerobically for 14 days at 36 °C.
- Incubate broths aerobically for 14 days at 36 °C.
- Between days 3 - 7 and 10 - 14 of incubation, subculture 0.1 mL of test broth onto an agar plate and incubate plate anaerobically as above.
- Observe agar plates after 14 days incubation at x4 magnification using an inverted microscope for the presence of mycoplasma colonies (Figure 9).
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 color 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.