The sterility test can be carried out by the following methods:

Membrane Filtration Method: This method is employed if the test substance is any of the following:

1. An oily preparation
2. An ointment that can be placed into solution
3. A soluble powder or a liquid with antimicrobial properties
4. A solid without antimicrobial properties and is not readily soluble in the culture media
5. For liquid products where the volume in a container is 100 ml or more

Procedure: Membrane filters of pore size not greater than 0.45 μm and diameter of 47 mm, which can retain microorganisms, are employed for the sterility test. The filtration system and the membrane are sterilized and the substances are filtered through the membrane under aseptic conditions.

If the substances have antimicrobial properties, the membrane is washed with three successive 100 ml quantities of sterile solvent.

The membrane is then aseptically cut into two equal halves. One half of the membrane is immersed in 100 ml of soybean–casein digest medium and incubated at 20°C–25°C. The other half of the membrane is immersed in 100 ml of fluid thioglycolate medium and incubated at 30°C–35°C for a time period not less than seven days.

Direct Inoculation Method: The quantity of the test substance to be used in each culture medium is directly transferred or inoculated into the culture media under aseptic conditions. The inoculated liquid is mixed with the medium. If the test substance contains antimicrobial properties, then it is neutralized by adding suitable inactivating substances (e.g., pencillinase in case of penicillin) to the medium. The inoculated media is incubated in soybean–casein digest medium at 20°C–25°C and also in fluid thioglycolate medium at 30°C–35°C. Both the media are incubated for not less than seven days.

Positive Control Test: A positive test is performed to ascertain that the culture media prepared and the environment conditions maintained during the test period favor the microbial growth. The causative test microorganism is streaked over the culture media under aseptic conditions and the method discussed earlier is adopted. At the end of the study, there should be growth or multiplication in the microbial load.

Negative Control Test: A negative test is performed to ascertain the sterility of the test area and the procedure adopted. The sterilized culture media is exposed to the test area and incubated. At the end of the study, there should not be any growth in the culture media, thereby proving the sterility and absence of microorganisms in the working area; an example is the laminar airflow unit.