333
A n t o n i e van L e e u w e n h o e k 45 ( 1 9 7 9 )
NETHERLANDS SOCIETY MEETING AT ROTTERDAM SECTION
FOR
FOR MICROBIOLOGY O N 30 N O V E M B E R 1979
PHARMACEUTICAL
MICROBIOLOGY
Sterility testing V. F . M . RIJNIERSE
Organon International B. V., Oss, The Netherlands When the result of a sterility test indicates that none of the tested samples shows growth, then there is still a finite chance that some of the units that were not tested, were nevertheless not sterile. This chance depends on the number of samples tested, provided that the infected units were uniformly spread over the entire lot. Besides the fact that the minimum number of samples that has to be tested according to the different pharmacopoeias (e.g. the U.S.P. and the E.P.) is not the same, the number of samples is always so low that only a good process control can give the assurance of a reliable quality. The sterility test is actually only a check on gross errors in the production process. Two types of controls are necessary to assure that the results of the test are reliable: to prevent false negative results, a check on the growth-promoting properties of the media is necessary, as well as a check on the growth-inhibiting properties of the tested preparation, both by inoculating with a limited number of test organisms; on the other hand false positive results can be detected by the simultaneous testing of blanks, preferably in such a way that the analyst does not know that he is testing a blank. Two test methodologies are currently in use: the direct inoculation technique and the membrane filter technique. When using the direct inoculation the whole or a part of the contents of a unit under test is divided over two media: one promoting the growth of aerobic and anaerobic bacteria (e.g. Fluid thioglycolate incubated at 30-35 °C) and the second promoting the growth of yeasts, moulds and aerobic bacteria (e.g. Soy-bean Casein Digest incubated at 20 25 °C). After an incubation period varying between 7 and 14 days a final reading is made. Factors that may influence the result of the test are the presence of preservatives, antibiotics or other growth-inhibiting substances. When testing emulsions or suspensions which produce a turbid medium preventing the observation of growth subculturing into a fresh medium after 3-7 days may be necessary. A disadvantage of the method of direct inoculation is the relatively large change of secondary infections. Another method (extremely useful for the testing of clear aqueous and oily solutions) is the membrane filter technique, 0.45 micron filters being used. The filter is aseptically removed from the filter holder, divided into two pieces that are incubated in the two media for the prescribed period. This technique requires a considerable aseptic handling, consequently leading to a fair chance of secondary infection. This can for the greater part be prevented by the use of a closed system. The samples are pumped via a peristaltic pump provided with sterile pump tubes over two filters. The influence of growth-inhibiting substances is eliminated by washing the filters with a suitable solvent before the filter holders are filled with the media; then they are incubated. When the membrane filter technique is performed according to this last method the chance of secondary infections is about 0,1 ~ and certainly lower than when using the direct inoculation technique. In the proposals for USP XX and the E.P., preference for the membrane filtration technique has been expressed.
A n t o n i e van L e e u w e n h o e k 45 ( 1 9 7 9 )
NETHERLANDS SOCIETY MEETING AT ROTTERDAM SECTION
FOR
FOR MICROBIOLOGY O N 30 N O V E M B E R 1979
PHARMACEUTICAL
MICROBIOLOGY
Sterility testing V. F . M . RIJNIERSE
Organon International B. V., Oss, The Netherlands When the result of a sterility test indicates that none of the tested samples shows growth, then there is still a finite chance that some of the units that were not tested, were nevertheless not sterile. This chance depends on the number of samples tested, provided that the infected units were uniformly spread over the entire lot. Besides the fact that the minimum number of samples that has to be tested according to the different pharmacopoeias (e.g. the U.S.P. and the E.P.) is not the same, the number of samples is always so low that only a good process control can give the assurance of a reliable quality. The sterility test is actually only a check on gross errors in the production process. Two types of controls are necessary to assure that the results of the test are reliable: to prevent false negative results, a check on the growth-promoting properties of the media is necessary, as well as a check on the growth-inhibiting properties of the tested preparation, both by inoculating with a limited number of test organisms; on the other hand false positive results can be detected by the simultaneous testing of blanks, preferably in such a way that the analyst does not know that he is testing a blank. Two test methodologies are currently in use: the direct inoculation technique and the membrane filter technique. When using the direct inoculation the whole or a part of the contents of a unit under test is divided over two media: one promoting the growth of aerobic and anaerobic bacteria (e.g. Fluid thioglycolate incubated at 30-35 °C) and the second promoting the growth of yeasts, moulds and aerobic bacteria (e.g. Soy-bean Casein Digest incubated at 20 25 °C). After an incubation period varying between 7 and 14 days a final reading is made. Factors that may influence the result of the test are the presence of preservatives, antibiotics or other growth-inhibiting substances. When testing emulsions or suspensions which produce a turbid medium preventing the observation of growth subculturing into a fresh medium after 3-7 days may be necessary. A disadvantage of the method of direct inoculation is the relatively large change of secondary infections. Another method (extremely useful for the testing of clear aqueous and oily solutions) is the membrane filter technique, 0.45 micron filters being used. The filter is aseptically removed from the filter holder, divided into two pieces that are incubated in the two media for the prescribed period. This technique requires a considerable aseptic handling, consequently leading to a fair chance of secondary infection. This can for the greater part be prevented by the use of a closed system. The samples are pumped via a peristaltic pump provided with sterile pump tubes over two filters. The influence of growth-inhibiting substances is eliminated by washing the filters with a suitable solvent before the filter holders are filled with the media; then they are incubated. When the membrane filter technique is performed according to this last method the chance of secondary infections is about 0,1 ~ and certainly lower than when using the direct inoculation technique. In the proposals for USP XX and the E.P., preference for the membrane filtration technique has been expressed.
