STERILISATION was defined in the United States Pharmacopaeia (XIIIth Revision) as "the destruction of all living organisms and their spores in, or their removal from, materials". The word is similarly defined else- where, for example, in the British Pharmaceutical Codex 1954 and the Japanese Phar-macopaia 1951. By inference sterility is the state of being free from living organisms and a sterile product is one entirely free from living organisms of all types. This concept is simple enough but un-fortunately it is unreal, being incapable of experimental verification.
A product is generally regarded as sterile because it has been subjected to a process believed to de-stroy or remove all micro-organisms and may therefore be expected to pass any sterility tests speci-fied by national pharmacopaeias or other authorities. Practical experience however has shown that neither exposure to a process of sterilisation nor passing sterility tests can give absolute certainty of sterility, in the sense of complete absence of living organisms. The most that can be claimed is a probability that the product is sterile although that probability may be very high as, for example, when a needle has been heated until it is red hot or saturated steam under pressure has been properly applied.
The designation sterile is therefore to a certain extent arbitrary and official restrictions are generally placed on its use.
Studies were conducted to evaluate the effects of media, incubation temperature and duration on the detection of bacteria and fungal growth using British, United States and Brazilian compendial sterility test methods. 5 to 50 CFU of nine different microorganisms (including both anaerobic and aerobic bacteria and molds) were used to contaminate test units containing various growth media (sobean-casein digest, thioglycollate, Sabourand and Causen broths). Test units were incubated at temperatures ranging from 12 to 42 degrees C for 1 to 28 days. Inoculations were conducted accord-ing to compendial procedures. Optimal detection conditions were obtained at 22 to 32 degrees C over 14 days using soybean casein digest and thioglycollate broths.
The sterility test is generally recognized as a flawed test for its stated purpose. Since the original de-scription in 1932, this test has generated controversy as to its role in product quality testing and in terms of means to improve the assay. As early as 1956 Bryce published an article describing the two critical limitations of this test (1). He put forward that the test was limited in that it can only recog-nize organisms able to grow under the conditions of the test, and that the sample size is so restricted that it provides only a gross estimate of the state of “sterility” of the product lot. Other concerns about the sterility test (e.g., choice of sample size, choice of media, time and temperature of incuba-tion) were extensively reviewed in an article by Bowman (2). There have been several changes in the compendial sterility test since that time, culminating in the internationally harmonized test (3). How-ever, the two basic problems outlined in 1956 by Bryce remain today.
Isolators in the pharmaceutical environment are used to produce and test sterile drug products with a minimized risk of microbiological contamination from the surrounding. Isolators realize a separated aseptic core practically free of microorganisms, what offers maximized protection for production and testing. For the realization of such an isolated aseptic core, considerations in microbiology play a crucial role in the validation, maintenance and control of the asepsis of an isolator.
Isolators should be run with an adequately developed decontamination cycle to achieve a desired level of microbial reduction in the isolator chamber prior production or testing. After achievement of aseptic conditions by decontamination, the maintenance of the asepsis in the isolator chamber is controlled by the applied overpressure and other control mechanisms in place. A well-designed chamber, adequate maintenance and an established monitoring program play a decisive role in keep-ing an isolator under control. However, microbiological control starts much earlier in the conceptual design of the isolator with a well-accessible and well-designed chamber which is good to maintain and with surfaces and materials easy to clean and decontaminate. The development and qualifica-tion of an adequate decontamination cycle, using biological indicators of Geobacillus stearother-mophilus [1,2] is essential, and should be verified for every routine cycle by a defined physical and microbiological monitoring program. Physical environmental monitoring gives indirect information on the microbiological status of the isolator, as some of the detected particles could be microorgan-isms (however, up to now no correlation is known on this matter). More significant is the microbio-logical environmental monitoring with nutrient media. Relevant for microbiological control is also the differential pressure of the isolator, as ingress of microorganisms from the surrounding is possible in case of downfall of the pressure to (or under) the level of the environment. Therefore, production isolators should be located in a microbiologically controlled environment (Zone C or D) to minimize these scenarios (and also to minimize the bioload of the material prior to decontamination) . Oth-er physical parameters liketemperature, humidity and air velocity do not have an impact on the mi-crobiological status, if they are in normal working range.