DetectionofMycoplasmabyCulture

Aim
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 14).

Materials

• 70% ethanol in water (Prod. No. R8382)

• Mycoplasma Pig Agar plates (in 5cm petri dishes)

• Mycoplasma Pig Agar broths (in 1.8ml aliquots)

• M. orale NCTC* 10112

• M. pneumoniae NCTC* 10119

Equipment

• Personal protective equipment (sterile gloves, laboratory coat, safety visor)

• Waterbath set to 37ºC

• Microbiological safety cabinet at appropriate containment level

• CO₂ Incubator set at 32ºC

• Gas Jar (Gallenkamp)

• Gas Pak Anaerobic System (Gallenkamp)

• Gas Pak Catalyst (Gallenkamp)

• Gas Pak Anaerobic Indicator (Gallenkamp)

Procedure

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 37ºC using a gas jar with anaerobic gas pak and catalyst.

10. Incubate broths aerobically for 14 days at 37ºC.

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 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.

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.