Mycoplasmas – Methods of Analysis

2.6.7. MYCOPLASMAS

Where the test for mycoplasmas is prescribed for a master cell bank, for a working cell bank, for a virus seed lot or for control cells, both the culture method and the indicator cell culture method are used. Where the test for mycoplasmas is prescribed for a virus harvest, for a bulk vaccine or for the final lot (batch), the culture method is used. The indicator cell culture method may also be used, where necessary, for screening of media.

CULTURE METHOD

CHOICE OF CULTURE MEDIA

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 of mycoplasmas that may be present in the product to be examined. Liquid media must contain phenol red. The range of media chosen is shown to have satisfactory nutritive properties for at least the organisms shown below. The nutritive properties of each new batch of medium are verified for the appropriate organisms in the list.

• Acholeplasma laidlawii (vaccines for human and veterinary use where an antibiotic has been used during production)
• Mycoplasma gallisepticum (where avian material has been used during production or where the vaccine is intended for use in poultry)
• Mycoplasma hyorhinis (non-avian veterinary vaccines)
• Mycoplasma orale (vaccines for human and veterinary use)
• Mycoplasma pneumoniae (vaccines for human use) or other suitable species of D-glucose fermenter
• Mycoplasma synoviae (where avian material has been used during production or where the vaccine is intended for use in poultry).

The test strains are field isolates having undergone not more than fifteen subcultures and are stored frozen or freeze-dried. After cloning the strains are identified as being of the required species by a suitable method, by comparison with type cultures, for example :

INCUBATION CONDITIONS

Divide inoculated media into two equal parts and incubate one in aerobic conditions and the other in microaerophilic conditions ; for solid media maintain an atmosphere of adequate humidity to prevent desiccation of the surface. For aerobic conditions, incubate in an atmosphere of air containing, for solid media, 5 to 10 per cent of carbon dioxide. For microaerophilic conditions, incubate in an atmosphere of nitrogen containing, for solid media, 5 to 10 per cent of carbon dioxide.

NUTRITIVE PROPERTIES

Carry out the test for nutritive properties for each new batch of medium. Inoculate the chosen media with the appropriate test organisms; use not more than 100 CFU (colony-forming units) per 60 mm plate containing 9 ml of solid medium and not more than 40 CFU per 100 ml container of the corresponding liquid medium; use a separate plate and container for each species of organism. Incubate the media in the conditions that will be used for the test of the product to be examined (aerobically, microaerophilically or both, depending on the requirements of the test organism). The media comply with the test for nutritive properties if there is adequate growth of the test organisms accompanied by an appropriate colour change in liquid media.

INHIBITORY SUBSTANCES

Carry out the test for nutritive properties in the presence of the product to be examined. If growth of the test organisms is notably less than that found in the absence of the product to be examined, the latter contains inhibitory substances that must be neutralised (or their effect otherwise countered, for example, by dilution) before the test for mycoplasmas is carried out. The effectiveness of the neutralisation or other process is checked by repeating the test for inhibitory substances after neutralisation.

TEST FOR MYCOPLASMAS IN THE PRODUCT TO BE EXAMINED

For solid media, use plates 60 mm in diameter and containing 9 ml of medium. Inoculate each of not fewer than two plates of each solid medium with 0.2 ml of the product to be examined and inoculate 10 ml per 100 ml of each liquid medium. Incubate at 35 °C to 38 °C, aerobically and microaerophilically, for 21 days and at the same time incubate an uninoculated 100 ml portion of each liquid medium for use as a control. If any significant pH change occurs on addition of the product to be examined, restore the liquid medium to its original pH value by the addition of a solution of either sodium hydroxide or hydrochloric acid. On the first, second or third day after inoculation subculture each liquid culture by inoculating each of two plates of each solid medium with 0.2 ml and incubating at 35 °C to 38 °C aerobically and microaerophilically for not less than 21 days. Repeat the procedure on the sixth, seventh or eighth day and again on the thirteenth or fourteenth day of the test. Observe the liquid media every 2 or 3 days and if any colour change occurs subculture immediately. Observe solid media once per week.

If the liquid media show bacterial or fungal contamination, repeat the test. If, not earlier than 7 days after inoculation, not more than one plate at each stage of the test is accidentally contaminated with bacteria or fungi, or broken, that plate may be ignored provided that on immediate examination it shows no evidence of mycoplasmal growth. If, at any stage of the test, more than one plate is accidentally contaminated with bacteria or fungi, or broken, the test is invalid and must be repeated.

Include in the test positive controls prepared by inoculating not more than 100 CFU of suitable species such as M. orale and M. pneumoniae.

At the end of the incubation periods, examine all the inoculated solid media microscopically for the presence of mycoplasmas. The product passes the test if growth of mycoplasmas has not occurred in any of the inoculated media. If growth of mycoplasmas has occurred, the test may be repeated once using twice the amount of inoculum, media and plates ; if growth of mycoplasmas does not occur, the product complies with the test. The test is invalid if the positive controls do not show growth of the relevant test organism.

INDICATOR CELL CULTURE METHOD

Cell cultures are stained with a fluorescent dye that binds to DNA. Mycoplasmas are detected by their characteristic particulate or filamentous pattern of fluorescence on the cell surface and, if contamination is heavy, in surrounding areas.

VERIFICATION OF THE SUBSTRATE

Using a Vero cell culture substrate, pretest the procedure using an inoculum of not more than 100 CFU (colony-forming units) of a strain growing readily in liquid or solid medium and demonstrate its ability to detect potential mycoplasma contaminants such as suitable strains of Mycoplasma hyorhinis and Mycoplasma orale. A different cell substrate may be used, for example the production cell line, if it has been demonstrated that it will provide at least equal sensitivity for the detection of potential mycoplasma contaminants.

Test method

Take not less than 1 ml of the product to be examined and use it to inoculate in duplicate, as described under Procedure, indicator cell cultures representing not less than 25 cm2 of cell culture area at confluence.

Include in the test a negative (non-infected) control and two positive mycoplasma controls, such as M. hyorhinis and M. orale. Use an inoculum of not more than 100 CFU for the positive controls.

If for viral suspensions the interpretation of results is affected by marked cytopathic effects, the virus may be neutralised using a specific antiserum that has no inhibitory effects on mycoplasmas or a cell culture substrate that does not allow growth of the virus may be used. To demonstrate the absence of inhibitory effects of serum, carry out the positive control tests in the presence and absence of the antiserum.

Procedure

1. Seed culture at a regular density (2 × 104 to 2 × 105 cells/ml, 4 × 103 to 2.5 × 104 cells/cm2) and incubate
   at 36 ± 1 °C for at least 2 days. Inoculate the product to be examined and incubate for at least 2 days ; make not fewer than one subculture. Grow the last subculture on coverslips in suitable containers or on some other surface suitable for the test procedure. Do not allow the last subculture to reach confluence since this would inhibit staining and impair visualisation of mycoplasmas.
2. Remove and discard the medium.
3. Rinse the monolayer with phosphate buffered saline pH 7.4 R, then with a mixture of equal volumes of phosphate buffered saline pH 7.4 R and a suitable fixing solution and finally with the fixing solution; when bisbenzimide R is used for staining, a freshly prepared mixture of 1 volume of glacial acetic acid R and 3 volumes of methanol R is a suitable fixing solution.
4. Add the fixing solution and allow to stand for 10 min.
5. Remove the fixing solution and discard.
6. If the monolayer is to be stained later, dry it completely. (Particular care is needed for staining of the slides after drying because of artefacts that may be produced.)
7. If the monolayer is to be stained directly, wash off the fixing solution twice with sterile water and discard the wash.
8. Add bisbenzimide working solution R or some other suitable DNA staining agent and allow to stand for 10 min.
9. Remove the stain and rinse the monolayer with water.
10. Mount each coverslip, where applicable, with a drop of a mixture of equal volumes of glycerol R and phosphate-citrate buffer solution pH 5.5 R; blot off surplus mountant from the edge of the coverslip.
11. Examine by epifluorescence (330 nm/380 nm excitation filter, LP 440 nm barrier filter) at 100-400 × magnification or greater.
12. Compare the microscopic appearance of the test cultures with that of the negative and positive controls, examining for extranuclear fluorescence. Mycoplasmas give pinpoints or filaments over the cytoplasm and sometimes in intercellular spaces.

The product to be examined complies with the test if there is no evidence of the presence of mycoplasmas in the test cultures inoculated with it. The test is invalid if the positive controls do not show the presence of the appropriate test organisms.

The following section is published for information.

RECOMMENDED MEDIA FOR THE CULTURE METHOD

The following media are recommended. Other media may be used providing their ability to sustain the growth of mycoplasmas has been demonstrated on each batch in the presence and absence of the product to be examined.

RECOMMENDED MEDIA FOR THE DETECTION OF MYCOPLASMA GALLISEPTICUM

“European Pharmacopoeia,” 5th Edition, Main Volume 5.0, 2005 with Supplements 5.1 and 5.2, pages 149-152, Council of Europe, Strasbourg, 2004, p. 2308.