113.26—Detection of viable bacteria and fungi except in live vaccine.
Each serial and subserial of biological product except live vaccines shall be tested as prescribed in this section unless otherwise specified by the Administrator. When cell lines, primary cells, or ingredients of animal origin used in the preparation of a biological product are required to be free of viable bacteria and fungi, they shall also be tested as prescribed in this section.
(1)
Fluid Thioglycollate Medium with 0.5 percent beef extract shall be used to test for bacteria in biological products containing clostridial toxoids, bacterins, and bacterin-toxoids.
(2)
Fluid Thioglycollate Medium with or without 0.5 percent beef extract shall be used to test for bacteria in biological products other than clostridial toxoids, bacterins, and bacterin-toxoids.
(3)
Soybean-Casein Digest Medium shall be used to test biological products for fungi; provided, that Fluid Thioglycollate Medium without beef extract shall be substituted when testing biological products containing mercurial preservatives.
(1)
Ten test vessels shall be used for each of two media selected in accordance with paragraph (a)(1), (a)(2), or (a)(3) of this section. Each test vessel shall contain sufficient medium to negate the bacteriostatic or fungistatic activity in the inoculum as determined in § 113.25(d).
(i)
When completed product is tested, 10 final container samples from each serial and each subserial shall be tested. One ml from each sample shall be inoculated into a corresponding individual test vessel of culture medium: Provided, That, if each final container sample contains less than 2 ml, one-half of the contents shall be used as inoculum for each test vessel.
(ii)
When cell lines, primary cells, or ingredients of animal origin are tested, at least a 20 ml test sample from each lot shall be tested. One ml shall be inoculated into each test vessel of medium.
(3)
Incubation shall be for an observation period of 14 days at 30 °to 35 °C. to test for bacteria and 14 days at 20 °to 25 °C. to test for fungi.
(4)
If the inoculum renders the medium turbid so that the absence of growth cannot be determined by visual examination, subcultures shall be made on the seventh to eleventh day from biological products prepared from clostridial toxoids, bacterins, and bacterin-toxoids and the third to seventh day for other biological products. Portions of the turbid medium in amounts of not less than 1.0 ml. shall be transferred to 20 to 25 ml. of fresh medium, and incubated the balance of the 14-day period.
(c)
Examine the contents of all test vessels for macroscopic microbial growth during the incubation period. When demonstrated by adequate controls to be invalid, the test may be repeated. For each set of test vessels representing a serial or subserial in a valid test, the following rules shall apply:
(1)
If no growth is found in any test vessel, the serial or subserial meets the requirements of the test.
(2)
If growth is found in any test vessel, one retest to rule out faulty technique may be conducted using 20 unopened final container samples.
(3)
If growth is found in any test vessel of the final test, the serial, subserial, or ingredients to be used in the preparation of a biological product, as the case may be, is unsatisfactory.