Sterility testing direct inoculation

The purpose of a sterility test is to determine the probable sterility of a specific batch. The USP lists the procedural details for sterility testing and the sample sizes required [1], The USP official tests are the direct (or culture tube inoculation) method and the membrane filtration method. 

Validation of membrane filtration and direct inoculation sterility test methods... 

During each working session (i.e., that uninterrupted period of time in which a sample or group of samples is tested) in which sterility testing is carried out, at least ten negative product control containers should be tested. For a direct inoculation test, these controls should be tested when possible at regular intervals during the test session. 

The Test for Sterility may be performed in one of two ways, by direct inoculation (direct transfer) or by membrane filtration. 

The avoidance of false-negatives is addressed at length in the pharmacopeias. Each batch of medium used in the Test for Sterility must have been shown to be capable of supporting the growth of low inocula of a specified array of microorganisms. Each Test for Sterility applicable to each specific product must be validated by repeated demonstration that the viabilities of low inocula of a specified array of microorganisms are not inhibited by product traces contained in the medium (direct inoculation) or on the membrane filter. 

The USP describes two general methods for conducting the test the direct transfer, or direct inoculation, method and the membrane filtration method. As the name indicates, the direct inoculation method involves the aseptic transfer of a sample of test product solution into the sterility test growth medium. To use this method, it must first be demonstrated that the product solution itself does not inhibit the growth of typical indicator microorganisms specified in the USP method. It should be self-evident why it is important to perform testing to negate the chance of product inhibition of possible microbial contaminants, as this is the purpose of the sterility test. The direct inoculation method, while not theoretically complex, requires the utmost technical precision and aseptic manipulation techniques for proper execution. As a consequence of the repetitive motions involved, it is prone to human error. 

A test for sterility is laid down in the European Pharmacopeia for all parenteral products, and two techniques for testing are described, either membrane filtration of the product with subsequent incubation of the filter in suitable culture media (which is the preferred technique), or direct inoculation of the product into the culture medium, followed by incubation at the appropriate temperature for the specified time. Suitable media are described in the European Pharmacopeia although, it is also recognized that other media may be used. In every case, however, it is necessary to demonstrate that the medium is capable of supporting the growth of microorganisms both in the presence and absence of the material to be tested. 

Finally, it is recognized that for short-lived radiopharmaceuticals, the long incubation time of the culture media (7 days for the membrane filtration technique, 14 days for direct inoculation) means the result of the sterility test cannot be available before the product is used. In these situations, the test constitutes a control of production techniques and will give valuable information about their suitability. 

During batch preparation sterility testing can be performed on randomly selected preparations either by direct inoculation or by the filtration method. During extemporaneous preparation dummy solutions should be prepared and sterility tests performed with rapid detection methods such as the bioluminescence test (see Sect. 19.6.5) or the colorimetric detection of CO2 production in culture bottles although commonly used for blood cultures this last method was shown to be adequate for sterility testing of multicomponent admixtures [68]. 

A recommended indicator cell line is the Vero African green monkey kidney cell line, which has a high cytoplasm/nucleus ratio. The indicator cells are added at a concentration of approximately 1 x 10" cells ml to sterile coverslips in tissue culture dishes 10-24 h before inoculation. The test sample should be added at a concentration of approximately 5 x 10 cells ml" to give a semi-confluent mono-layer at the time of observation (at 1 and 3 days post-inoculation of the sample). Slides are prepared in the same way as those prepared by the direct method. 

---