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Guidelines for sterility testing of therapeutic goods

13 September 2006

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Annex II. Composition and preparation of media

Relevant section of the BP/Ph Eur: 'Culture media and incubation temperatures'

This Annex describes methods for preparation and sterilisation of the standard sterility test media (see also Section 6). Commercially available dried media that differ slightly from the specified composition may be used provided the reconstituted medium has been shown to support the growth of aerobic and anaerobic bacteria and fungi, as described in clause 611-615. Alternative media types may be appropriate where the nature of the product or method of manufacture may result in the presence of fastidious organisms (eg, vaccines, blood products).

Inactivators of antimicrobials may be incorporated into culture media or solutions if indicated by validation studies.

If heat labile additives such as serum are included the media may be sterilised by a validated filtration method. Cogent reasons would be required to justify the use of filtration as a method of sterilisation of media that can be terminally sterilised.

Medium 1 (Fluid Thioglycollate Medium)

Composition
Pancreatic Digest of Casein0 15.0 g
Yeast Extract (water-soluble) 5.0 g
Glucose monohydrate/anhydrous 5.5 g/5.0 g
Sodium chloride 2.5 g
L-Cystine 0.5 g
Sodium thioglycollate 0.5 g
0.1% Resazurin Sodium Solution (freshly prepared) 1.0 mL
Granulated Agar (moisture not more than 15%) 0.75 g
Purified Water  1000 mL
Polysorbate 80 (optional) 5.0 mL
pH after sterilisation (measured at room temperature): 7.1± 0.2

Method of preparation

Either

Mix the pancreatic digest of casein, yeast extract, glucose, sodium chloride, L-cystine, agar and water in the proportions specified above and heat until dissolved. Dissolve the sodium thioglycollate in the solution. Add the specified quantity of Polysorbate 80 if this ingredient is to be included. If necessary, add sufficient 1 M sodium hydroxide or 1 M hydrochloric acid so that after the solution is sterilised its pH will be 7.1± 0.2. If the solution is not clear, heat to boiling but do not boil, and filter while hot through moistened filter paper. Add the resazurin sodium solution and mix.

Or

Dissolve a mixture of dehydrated mixture ingredients in the specified proportions, in water. Observe the instructions provided by the manufacturer of the dehydrated mixture to effect solution and to obtain a clear solution of the specified pH. Immediately prior to adjusting the pH, add the specified quantity of Polysorbate 80 if this ingredient is to be included.

Medium 2 (Soybean-Casein Digest Medium)

Composition
Pancreatic Digest of Casein 17.0 g
Papain Digest of Soybean Meal 3.0 g
Glucose monohydrate/anhydrous 2.5 g/2.3 g
Sodium chloride 5.0 g
Dipotassium hydrogen phosphate, K2HPO4 2.5 g
Purified Water 1000 mL
Polysorbate 80 (optional) 5.0 mL
pH after sterilisation (measured at room temperature): 7.3±0.2

Method of preparation

Either

Mix the ingredients, in the proportions specified above, warming slightly to effect solution. Cool the solution to room temperature. Add the specified quantity of Polysorbate 80 if this ingredient is to be included. If necessary, add sufficient 1 M sodium hydroxide or 1M hydrochloric acid so that after the solution is sterilised its pH will be 7.3± 0.2. If the solution is not clear filter through moistened filter paper.

Or

Dissolve a mixture of dehydrated ingredients in water in the amount necessary to obtain the required concentration. Follow the instructions provided by the manufacturer to obtain a clear solution of the specified pH. Just prior to adjusting the  pH, add the specified  quantity of Polysorbate 80 if this ingredient is to be included.

Both media

Filling and containers

Distribute all media into clear colourless glass vessels with external screw threaded necks in the required volumes. Each vessel for Medium 1 should provide a ratio of surface to depth of medium such that when inoculated with a sterile inoculum not more than the upper one-half of the medium becomes pink in colour at the conclusion of the test for sterility. The capacity of each vessel for Medium 2 should be at least twice the volume of the medium placed in it. Close all vessels with aluminium or heat resistant plastic screw-caps.

Sterilisation

Media should be sterilised within four hours of the start of its preparation.

Sterilise the vessels of media by exposing them to saturated steam in an autoclave for a time sufficient to ensure that the whole of the contents of each vessel is maintained at 121°C for 20 minutes. In the process of sterilisation do not prolong the heating up or delay the cooling down of the media unnecessarily. Bring the pressure in the autoclave chamber to atmospheric pressure at such a rate so as not to cause the medium to boil over. Allow the media to cool to about 30°C, either in the autoclave chamber or a sterile environment, then tighten the cap of each vessel. Cooling may be carried out in a controlled clean environment provided that the process has been validated.

Alternative validated terminal sterilisation methods may be used.

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