The test is carried out using a sufficient number of both solid and liquid media to ensure growth in the chosen incubation conditions of small numbers (approximately 100 colony-forming units, cfu; or 100 color-changing units, ccu) of Mycoplasmas that may be present in the test article/material. Liquid media must contain phenol red. The range of media chosen is shown to have satisfactory nutritive properties for at least the microorganisms shown in Quality Control Test Strain Organisms (below). The nutritive properties of each new batch of medium are verified for the appropriate microorganisms in the list. When testing for Mycoplasmas include in each test at least two known Mycoplasma species or strains (listed in Quality Control Test Strain Organisms) as positive controls, one of which should be a dextrose fermenter (i.e., M. pneumoniae or equivalent species and strain) and one of which should be an arginine hydrolyzer (i.e., M. orale or equivalent species and strain). Only when testing insect cell lines should one include a Spiroplasma control strain (e.g., S. citri ATCC 29747, S. melliferum ATCC 29416, or equivalent species and strains). Additionally, these strains may be a little more fastidious in their nutritional requirements. They require lower incubation temperatures (as do insect cell lines).

Positive control cultures should be not more than 15 passages from isolation. Mycoplasma species or strains suitable for use are listed below:

The test strains may be field isolates that have undergone a limited number of subcultures (not more than 15), are stored frozen (20 or lower) or freeze-dried, and are identified as being of the required species by comparison with type cultures, for example, those shown in Table 1.

Incubate liquid media in tightly stoppered containers at 36 ± 1. Incubate solid media in microaerophilic conditions (hydrogen atmosphere containing < 0.5% oxygen and/or nitrogen containing 5%–10% carbon dioxide in nitrogen). Sufficient humidity should be available to prevent desiccation of the agar surface at 36 ± 1.

Carry out the test for nutritive properties for each new batch of medium. Inoculate the chosen media with the appropriate test microorganisms; use not more than 100 cfu per plate containing at least 9 mL of solid media and per 100-mL container of liquid medium; use a separate plate and container for each species of microorganism. Incubate the media and make subcultures from 0.2 mL of liquid medium to solid medium at the specified intervals (see below under Test for Mycoplasma in the Test Article/Material). The solid medium complies with the test if a count within a 0.5-log unit range of the inoculate amount is found for each test microorganism. The liquid medium complies with the test if growth is found on agar plates subcultured from the broth, for at least 1 subculture for each test microorganism. The use of a microscope at 100× or greater may be helpful.

The test for inhibitory substances is carried out once for a given product and is repeated whenever there is a change in production method that may affect the detection of Mycoplasma. To demonstrate absence of inhibitory substances, carry out the test for nutritive properties in the presence and absence of the test article/material. If growth of a test microorganism occurs more than 1 subculture sooner in the absence of the test article/material than in its presence, inhibitory substances are present. The same is true if plates directly inoculated with the test article/material are not within a 0.5-log unit range of the number of colonies of those inoculated without the test article/material. In both cases, inhibitory substances must be neutralized or their effect otherwise countered, by an appropriate method, for example, by passage in substrates not containing inhibitors or dilution in a larger volume of medium, before the test. If dilution is used, larger medium volumes may be used or the inoculums' volume may be divided among several 100-mL flasks. The effectiveness of the neutralization or other process is checked by repeating the test for inhibitory substances after neutralization.

Inoculate no less than 10 mL of the test article/material per 100 mL of each liquid medium. If a significant pH change occurs upon the addition of the test article/material, the liquid medium is restored to its original pH value by the addition of a sterile solution of either sodium hydroxide or hydrochloric acid. Inoculate 0.2 mL of the test article/material on each plate of each solid medium. Incubate liquid media for 20–21 days. Incubate solid media for not less than 14 days, except those plates corresponding to the 20–21 day subculture, which are incubated for 7 days. Concurrently, incubate an uninoculated 100-mL portion of each liquid medium and agar plate, as a negative control. On days 2–4 after inoculation, subculture each liquid medium by inoculating 0.2 mL on at least 1 plate of each solid medium. Repeat the procedure between days 6 and 8, again between days 13 and 15, and again between days 19 and 21 of the test. Observe the liquid media every 2 or 3 days and if a color change occurs, subculture. If a liquid medium shows bacterial or fungal contamination, the test is invalid. The test is valid if at least 1 plate per medium and per inoculation day can be read. Include in the test positive controls prepared by inoculation of not more than 100 cfu of at least 1 test microorganism on agar medium or into broth medium. Where the test for Mycoplasmas is carried out regularly, it is recommended to use the test microorganisms in regular rotation. The test microorganisms used are those listed under Choice of Media. Incubate broths and plates in a humidified atmosphere with microaerophilic conditions (5%–10% CO2).

At the end of the prescribed incubation period, examine all inoculated solid media for the presence of Mycoplasma colonies. The product complies with the test if growth of typical Mycoplasma colonies has not occurred. The product does not comply with the test if growth of typical Mycoplasma colonies has occurred on any of the solid media. The test is invalid if 1 or more of the positive controls do not show growth of Mycoplasmas on at least 1 subculture plate. The test is invalid if 1 or more of the negative controls show growth of Mycoplasmas. If suspect colonies are observed, use a suitable validated method to determine whether they are due to Mycoplasmas.

The following media are recommended. Other media may be used, provided they meet the criteria given in the sections Choice of Culture Media, Incubation Conditions, Nutritive Properties, and Inhibitory Substances.

Use Vero cells or equivalent cell culture (for example, the production cell line) that is equivalent in effectiveness for detecting Mycoplasmas. Test the effectiveness of the cells to be used by applying the procedure shown below and inoculating not more than 100 cfu or ccu microorganisms of suitable reference strains of M. hyorhinis and M. orale. The cells are suitable if both reference strains are detected. The indicator cells must be subcultured without an antibiotic before use in the test.

The product to be examined complies with the test if fluorescence typical of Mycoplasmas is not present. The test is invalid if the positive controls do not show fluorescence typical of Mycoplasmas. The test is invalid if the negative controls show fluorescence typical of Mycoplasmas.