STERILITY TEST It’s a quality control test for any sterile product, required for all articles or substances to be introduced into raw tissue (injections and ophthalmics). Sterility testing attempts to reveal the presence or absence of viable micro-organisms in a sample number of containers taken from a batch of product. Sterility test is a final control on the sterilization process (i.e. the test indicates that a sterilization process is effective or not). The test shows that samples tested were free from living bacteria and fungi but not viruses.

Methods of Sterility Test Membrane filtration Direct transfer

Direct transfer method  Culture media for sterility test must be: Capable of initiating and maintaining the vigorous growth of a small number of organisms. Sterile. There is no one medium which could detect both bacteria and fungi.

The USP recommends two culture media for sterility testing: a) Fluid Thioglycolate Medium (FTM) for detection of both aerobic and anaerobic bacteria. b) Trypticase Soya Broth (TSB) (Fluid soya bean casein digest medium) primarily intended for the culture of both fungi and highly aerobic bacteria. Direct transfer method

Formulation of "FTM" Yeast extract …… nutrient (growth factor) Pancreatic digest of casein………nutrient (nitrogen source) Dextrose……...nutrient (carbon source) L-cysteine antioxidant, supports growth of clostridia & neutralizes metal preservatives Sod. thioglycolate……...antioxidant & neutralizes metal preservatives. Agar (0.075%)…… viscosity inducer to reduce oxygen diffusion Resazurin sod……... redox indicator (pink at high O 2 tension in upper third of container and yellow at low O 2 tension in lower 2/3 of the container). Distilled water pH after sterilization 7.1  0.1

Formulation of "FTM"

Pancreatic digest of casein nutrient Papaic digest of soya bean nutrient Dextrose (high %) nutrient Sod. chloride isotonic agent Dibasic potassium phosphate buffer Distilled Water pH after sterilization 7.3  0.2 Formulation of "TSB"

Suitable quantity of the preparation to be examined is transferred directly into the appropriate culture medium. Volume of the product is not more than 10% of the volume of the medium. Suitable method for aqueous solutions, oily liquids, ointments an creams. Direct transfer method

Procedure: The container is cleaned with suitable antimicrobial agent and aseptically opened to avoid contamination and hence false positive result. The specified volume of the sample is withdrawn (1 ml). Aseptically, one half is added to "FTM" and the other half is added to "TSB". "FTM" is incubated at o C (suitable for bacteria). "TSB" is incubated at o C (suitable for fungi). Incubation time is up to 14 days according to USP, to assure the sterility of the product. Direct transfer method

Interpretation of results If no growth is observed after the incubation period the batch passes the test. If visible growth is observed the test is repeated. If again growth is observed the material fails the test. It is recommended to identify the contaminant and trace it (by staining for bacterial contaminant) to know the source of contamination in a sterilization process.

Direct simple stain procedure 1. Prepare a heat fixed bacterial smear. 2. Leave to cool. 3. Cover the film with crystal violet. 4. Leave for 30 sec. 5. Wash gently with water. 6. Blot dry between 2 filter papers. 7. Add oil and examine using oil immersion lens.

Staphylococcus aureus Shape: cocci Size: small Arrangement: Bunches (or Clusters) Color: violet

Rods Shape: Rods Size: Small Arrangement: Single Color: violet

Candida albicans Shape: Oval Size: Large Arrangement: Single Color: violet