Testing for Mycoplasma by Culture Isolation

Cook Book Volume 12

Fundamental Techniques in Cell Culture Laboratory Handbook-2nd Edition

Flow Scheme for Detection of Mycoplasma by Culture


Detection of mycoplasma by culture 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 (see figure 1).

Figure 1 Typical “fried egg colonies” Mycoplasma pneumoniae (magnification x400)

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  • 70% (v/v) isopropanol in sterile water
  • Mycoplasma plates (in 5cm petri dishes)
  • Mycoplasma horse serum broths (in 1.8ml aliquots)
  • M. orale (NCTC* 10112)
  • M. pneumoniae (NCTC* 10119)

*Available from the National Collection of Type Cultures (NCTC)

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  • Personal protective equipment (sterile gloves, laboratory coat, safety visor)
  • Waterbath set to 37°C
  • Microbiological safety cabinet at appropriate containment level
  • Incubator set at 37°C
  • Anaerobic jar system

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  1. Inoculate 2 agar plates with 0.1ml of test sample.
  2. Inoculate an agar plate with 100 cfu of each control organism.
  3. Leave 1 agar plate un-inoculated as a negative control.
  4. Inoculate 1 broth with 0.2 ml of test sample.
  5. Inoculate a broth with 100 cfu of each control organism.
  6. Leave 1 broth un-inoculated as a negative control.
  7. Incubate agar plates anaerobically for 14 days at 37°C.
  8. Incubate broths aerobically for 14 days at 37°C.
  9. 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.
  10. Observe agar plates after 14 days incubation at x400 magnifi cation using an inverted microscope for the presence of mycoplasma colonies (see Figure 1).

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.


  1. Mycoplasma colonies have a typical colony formation commonly described as “fried egg” due to the opaque granular central zone of growth penetrating the agar surrounded by a flat translucent peripheral zone on the surface.
  2. Mycoplasma pneumoniae is a potential pathogen and must be handled in a class 2 microbiological safety cabinet operating to ACDP Category 2 Conditions.
  3. This test procedure should be carried out in a microbiology laboratory away from the cell culture laboratory.
  4. ECACC recommends that samples are tested for mycoplasma using at least two detection methods (e.g. indirect DNA stain and culture isolation) for a more reliable result. This is due to the varying detection sensitivities of the methods for different species of mycoplasma.
  5. ECACC also offers a mycoplasma screening PCR assay which can return a result within 24 hours.

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