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U.S. Department of Health and Human Services

CFR - Code of Federal Regulations Title 21

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Help | More About 21CFR
[Code of Federal Regulations]
[Title 21, Volume 7]
[Revised as of April 1, 2013]
[CITE: 21CFR610]





TITLE 21--FOOD AND DRUGS
CHAPTER I--FOOD AND DRUG ADMINISTRATION
DEPARTMENT OF HEALTH AND HUMAN SERVICES
SUBCHAPTER F--BIOLOGICS
 
PART 610GENERAL BIOLOGICAL PRODUCTS STANDARDS
 

Subpart D--Mycoplasma

Sec. 610.30 Test forMycoplasma.

Except as provided otherwise in this subchapter, prior to clarification or filtration in the case of live virus vaccines produced from in vitro living cell cultures, and prior to inactivation in the case of inactivated virus vaccines produced from such living cell cultures, each virus harvest pool and control fluid pool shall be tested for the presence ofMycoplasma, as follows:

Samples of the virus for this test shall be stored either (1) between 2 and 8 deg. C for no longer than 24 hours, or (2) at -20 deg. C or lower if stored for longer than 24 hours. The test shall be performed on samples of the viral harvest pool and on control fluid pool obtained at the time of viral harvest, as follows: No less than 2.0 ml. of each sample shall be inoculated in evenly distributed amounts over the surface of no less than 10 plates of at least two agar media. No less than 1.0 ml. of sample shall be inoculated into each of four tubes containing 10 ml. of a semisolid broth medium. The media shall be such as have been shown to be capable of detecting knownMycoplasma and each test shall include control cultures of at least two known strains ofMycoplasma, one of which must beM. pneumoniae. One half of the plates and two tubes of broth shall be incubated aerobically at 36 deg. C +/-1 deg. C and the remaining plates and tubes shall be incubated anaerobically at 36 deg. C +/-1 deg. C in an environment of 5-10 percent CO2in N2. Aerobic incubation shall be for a period of no less than 14 days and the broth in the two tubes shall be tested after 3 days and 14 days, at which times 0.5 ml. of broth from each of the two tubes shall be combined and subinoculated on to no less than 4 additional plates and incubated aerobically. Anaerobic incubation shall be for no less than 14 days and the broth in the two tubes shall be tested after 3 days and 14 days, at which times 0.5 ml. of broth from each of the two tubes shall be combined and subinoculated onto no less than four additional plates and incubated anaerobically. All inoculated plates shall be incubated for no less than 14 days, at which time observation for growth ofMycoplasma shall be made at a magnification of no less than 300*. If the Dienes Methylene Blue-Azure dye or an equivalent staining procedure is used, no less than a one square cm. plug of the agar shall be excised from the inoculated area and examined for the presence ofMycoplasma. The presence of theMycoplasma shall be determined by comparison of the growth obtained from the test samples with that of the control cultures, with respect to typical colonial and microscopic morphology. The virus pool is satisfactory for vaccine manufacture if none of the tests on the samples show evidence of the presence ofMycoplasma.

[38 FR 32056, Nov. 20, 1973, as amended at 63 FR 16685, Apr. 6, 1998]

Authority: 21 U.S.C. 321, 331, 351, 352, 353, 355, 360, 360c, 360d, 360h, 360i, 371, 372, 374, 381; 42 U.S.C. 216, 262, 263, 263a, 264.
Source: 38 FR 32056, Nov. 20, 1973, unless otherwise noted.

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