Sterility Test in Microbiology: Sterility tests can be carried out by using the following methods:
- Method A: Membrane Filtration.
- Method B: Direct Inoculation.
Method A: Membrane Filtration
This method is to be preferred where the substances being examined are (a) an oil, (b) an ointment that can be put into a solution, (c) a non-bacteriostatic solid not readily soluble in the culture medium, and (d) a soluble powder or a liquid that possesses inherent bacteriostatic and fungistatic properties. For liquid products where the volume in the container is 100 ml or more, only Method A should be employed.
This method needs good skill and special knowledge, and it also calls for the routine use of positive and negative controls. A positive control is a small number (not more than 100 CFU) of microorganisms specified in a separate portion of each medium.
Apparatus: The sterility test apparatus consists of a closed reservoir and a container to collect the filtrate, between which a properly supported membrane of appropriate porosity is placed. A membrane generally suitable for sterility testing has nominal porosity of 0.45 µm, a diameter of about 50 mm, and a flow rate of 55-75 ml of water/minute at a pressure of 70 mm of mercury. Cellulose nitrate filters are used for aqueous, oily, and weakly alcoholic solutions, and cellulose acetate filters, are for strongly alcoholic solutions. The complete unit should be free from microorganisms including the membrane, and operation should be carried out aseptically. Preferably assemble and sterilize the entire unit with the membrane in place before use.
Generally, two types of dilution fluids are used:
1. Fluid A: Dissolve 1 gm of peptic digest of animal tissue (such as bacteriological peptone) in one liter of water. Filter and adjust the pH to 7.1 ± 0.2. Dispense fluid into the flasks (100 ml) and sterilize at 121 oC for 20 minutes.
2. Fluid B: If the test sample contains lecithin or oil, use fluid A, for each liter of which has been added 1 ml of polysorbate 80. Adjust pH to 7.1 ± 0.2 and sterilize at 121 oC for 20 minutes using an autoclave.
Quantities of sample to be used:
(i) For parenteral preparations: Whenever possible use the whole contents of the container but not less than the quantities prescribed in Table.1, if necessary, dilute to about 100 ml with a suitable diluent (e.g. fluid A).
(ii) For ophthalmic and other non-injectable preparations: Take an amount within the range prescribed in column I of Table.2, if necessary, use the contents of more than one container and mix thoroughly. For each medium use the amount specified in column II of Table.2, taken from the mixed sample.
Table 1: Quantities of injectable preparations used for sterility testing
Table 2: Quantities of ophthalmic and other non-injectable preparations for sterility testing
Method of test:
(a) For aqueous solutions: Transfer aseptically the quantities of the preparation being examined prescribed in the two media onto one membrane. Draw the liquid rapidly through the filter with the aid of a vacuum. The membrane is removed aseptically and cuts into two, one part is then immersed in 100 ml of soyabean casein digest medium and incubated at 20 to 25 oC for not less than 14 days. Similarly, the other part is immersed in 100 ml of liquid thioglycollate medium and incubated at 30 to 35 oC for not less than 14 days.
(b) For liquids immiscible with aqueous vehicles and suspensions: Carry out the test described, for aqueous solutions but add a sufficient quantity of fluid ‘A’ to the pooled sample to achieve rapid filtration.
(c) For oils and oil solutions: Filter oils or oily solutions of sufficiently low viscosity without dilution through a dry membrane. Dilute viscous oils are necessary with a suitable diluent.
(d) For ointments and creams: Dilute ointments in a fatty base and emulsions of the water-in-oil type to give a fluid concentration of 1% w/v, by heating, if necessary, to not more than 40 oC with a suitable sterile diluent such as isopropyl myristate. If ointments and oils are insoluble in isopropyl myristate then use Method B.
(e) For soluble solids: Dissolve not less than the quantity of the substance being examined as prescribed in Tables 1 and 2, in a suitable sterile solvent such as fluid ‘A’.
(f) For solids for injection other than antibiotics: Constitute the test articles as directed on the label and carry out the test as described for aqueous solutions or oils and oily solutions.
(g) For antibiotic solids, bulks, and blends: Aseptically remove a sufficient quantity of solids from the mix to obtain a composite sample, equivalent to about 6 g of solids, and transfer to a sterile flask. Dissolve in about 200 ml of fluid A, and mix.
(h) For antibiotics in packages of 5 g or less: From each of 20 containers, aseptically transfer about 300 mg of solids into a sterile flask, dissolve in about 200 ml of fluid A, and mix.
(f) For sterile devices: Aseptically passes a sufficient volume of fluid ‘B’ through the 20 devices to recover not less than 100 ml from each device. Collect the fluids in sterile containers and filter the entire volume through a membrane filter. In the case of sterile, empty syringes, draw sterile diluent into the barrel through the sterile needle, if attached or through a sterile needle attached for the test, and express the contents into a sterile polling vessel. For catheters where the inside lumen and outside surface are required to be sterile, either cut them into pieces such that the medium is in contact with the entire lumen or fill the lumen with medium and then immerse the intact unit.
Method B: Direct Innoculation
Quantities of sample to be used: The quantity of the substance or preparation being examined which is to be used for inoculation in the culture media (Fig.1) varies according to the quantity in each container.
Method of test:
(a) For aqueous solutions and suspensions: Remove the liquid from the test containers with a sterile pipette or syringe. Transfer the quantity of the preparation under examination (Table.1) directly into the culture medium so that the volume of the preparation under examination is not more than 10% of the volume of the medium unless otherwise prescribed. When the quantity in a single container is
insufficient to carry out the tests, the combined contents of two or more containers are to be used to inoculate the media. The inoculated medium is incubated for 14 days at 30 to 35 oC in the case of fluid thioglycollate medium and at 20 to 25 oC in the case of soybean-casein digest medium. If the preparation under examination has antimicrobial activity, carry out the test after neutralizing this with a suitable neutralizing substance or by dilution in a sufficient quantity of culture medium.
(b) For oils and oily solutions: Use media to which have been added 0.1%, w/v of (4-tert-octylphenoxy) polyethoxyethanol or 1% w/v of polysorbate 80 or other suitable emulsifying agents, in an appropriate concentration and proceed with the test.
(c) For ointments and creams: Prepare by diluting to ten-fold by emulsifying with the chosen emulsifying agent in a suitable sterile diluent such as fluid ‘A’. Transfer the diluted product to a medium not containing an emulsifying agent.
(d) For solids: Transfer the required quantity of the material to the medium.
(e) For surgical dressings and related articles: Aseptically remove two or more portions of 100 to 500 mg each from the innermost part of the sample or package under examination. From individually packaged, single-use materials, aseptically remove the entire article. Immerse the portions or articles in each medium and proceed with the test.
(f) For sterile devices: Immerse the device in 1000 ml of culture medium. If immersion is impracticable, flush the lumen of 20 units with the fluid thioglycollate medium and soybean-casein digest medium separately and recover 15 ml of each medium. Incubate with not less than 100 ml of each medium. For catheters where the inside lumen and outside surface are required to be sterile, either cut them into pieces such that the medium is in contact with the entire lumen or fill the lumen with medium and then immerse the intact unit.
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