The test is designed to reveal the presence of contamination with live microorganisms in antibiotics intended for parenteral administration or for other sterile applications.

Test conditions

The test should be carried out under aseptic conditions in an area as free from contamination as it is possible to achieve by the use of disinfecting agents, germicidal lamps, and air filters. Germicidal lamps and disinfecting aerosols should not be used during actual testing operations. The test manipulations should be carried out in a filtered air environment or under a laminar flow hood, with operators dressed in sterilized, static-free clothing, including head and foot wear. The air pressure in the testing room should be greater than that of the exterior area. The performance of the laminar flow hood should be monitored by particulate count, settle plates, or slit-sampling devices, and the performance of the filters and germicidal lamps checked routinely.

Membrane filtration apparatus

A suitable unit consists of a closed reservoir and a receptacle, separated by a properly supported membrane of appropriate porosity. Membranes generally suitable for sterility testing have a nominal porosity of 0.45 μm, a diameter of approximately 47 mm, and a flow rate of 55-75 ml of water per minute at a pressure of 90 kPa (700 mm Hg). The entire unit should preferably be assembled and sterilized with the membrane in place, prior to use. If each entire membrane is to be cultured, at least 2 filter units should be set up.

All air entering the filtering unit should be passed through an air-filter capable of removing microorganisms.

Sampling

Take the sample in such a manner as to be representative of the material to be tested. The amount taken should be sufficient to perform the tests and any repeat tests that may be required. The sampling should be carried out in such a way as to maintain intact the sterility of the material.

Culture media

The culture media used for sterility tests for bacteria and fungi should be capable of supporting the growth of a wide variety of microorganisms, with both aerobic and anaerobic growth characteristics, including the types found in the environment of the manufacturing operations. More than one culture medium will generally be needed to fulfil these criteria. The media that usually give satisfactory results are fluid mercaptoacetate (thioglycolate) medium (culture medium Cm4) and soybean-casein digest medium (culture medium Cm5). Any other media that are used, however, should have at least demonstrably equivalent growth-supporting properties.

For testing the growth-supporting properties of each culture medium, strains of microorganisms should be used with exacting nutritive and aerobic-anaerobic requirements, in an inoculum containing only a small number of organisms (less than 100). The media should be incubated at the temperatures at which they will be used in the sterility test and growth should be evident after 24 hours.

Each lot of dehydrated medium obtained from a specialized manufacturer or each lot of the medium prepared entirely in the laboratory should be tested for its growth-supporting properties, since not every lot may support the growth of microorganisms to the desired extent. Differences may be caused by the occasional presence of unsatisfactory components in a particular lot, or by the destruction of certain components by overheating or oversterilization of the medium.

Recommended procedures

Membrane filtration test procedure

Aseptically transfer a suitable amount of the solid test material (0.3-6 g depending on the size of the container) into a sterile flask containing about 200 ml of peptone (1 g/l) TS1, stopper the flask and swirl to effect rapid dissolution. If the test material dissolves slowly or if the resulting solution will not filter rapidly, the volume of the solvent may be increased to not more than 400 ml. Immediately after the test material has dissolved, aseptically filter the solution, with the aid of reduced pressure, through the membrane filter previously moistened with sterile water or with peptone (1 g/l) TS1. To speed up the process of filtration, the solution may be filtered using two filtering units simultaneously. To remove the residual antibiotic from the membrane, wash it with sufficient peptone (1 g/l) TS1 to which, if so indicated in the monograph (in the case of penicillin and cephalosporin antibiotics), sufficient penicillinase TS has been added.

Upon completion of the filtration, divide the membrane aseptically into two approximately equal parts. Transfer one part of the membrane into a culture vessel (a test-tube is suitable) containing 50-100 ml of culture medium Cm4 (fluid mercaptoacetate (thioglycolate) medium) and the other part of the membrane into another culture vessel containing 50-100 ml of culture medium Cm5 (soybean-casein digest medium).

A control test should be carried out at appropriate intervals to demonstrate that residual antibiotic activity is being reduced by the above described washing procedure to below the level that allows growth of an inoculum containing 50-100 microorganisms susceptible to the antibiotic tested.

Direct test procedure

Depending on the size of the container take from 1-10 portions, each 0.3 g of the test material, and aseptically transfer them into individual sterile vessels (test-tubes are suitable) containing 50-100 ml of culture medium Cm6 (fluid mercaptoacetate (thioglycolate) medium with penicillinase). Transfer portions of similar size to another set of individual sterile vessels containing 50-100 ml of culture medium Cm7 (soybean-casein digest medium with penicillinase).

Check the ability of the penicillinase contained in the medium to inactivate all the penicillin in the test material by adding to one vessel containing culture medium Cm6 the amount of material taken from one container under test. Next add 1.0 ml of a dilution containing 50-100 microorganisms of a suitable strain (Staphylococcus aureus, ATCC 6538-P is suitable) in culture medium Cm4. Typical microbial growth must be observable after 24 hours of incubation at 30-32 °C.

Incubation

Incubate for 7 days the vessels containing fluid mercaptoacetate (thioglycolate) media (culture media Cm4 and Cm6) at 30-32 °C and the vessels containing soybean-casein digest media (media Cm5 and Cm7) at 22-25 °C. In the direct test procedure, gently agitate the vessels at least once a day or until complete dissolution occurs.

If other culture media are used, the incubation temperature and the period of incubation may have to be appropriately modified.

Examine inoculated culture vessels at regular intervals and on the last day of incubation for evidence of microbial growth. If such growth is observed it should be confirmed by microscopic examination. It is desirable that the incubation apparatus be equipped with a continuous temperature-recording device.

Interpretation of test results

If no evidence of growth is found in any of the culture vessels, except in the positive growth control, the material meets the requirements of the test. If, however, evidence of growth is found, a repeat test may be performed. If no evidence of growth is then found in any of the culture vessels, except in the positive growth control, the material meets the requirement of the test. The material fails to pass the test if growth occurs in the repeat test.

The distinction between failure of the product to pass the test and a possible invalidity of the test procedure requires the competent judgement of an expert.