STERILITY TEST 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 batch of product. Based on results obtained from testing the sample a decision is made as to the sterility of the batch. The test shows that samples tested were free from living bacteria and fungi but not viruses. 

Methods of Sterility Test Membrane filtration Direct inoculation of the culture medium

Direct inoculation of the culture medium 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.

Direct inoculation of the culture medium 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 aerobic bacteria .

Formulation of "FTM" Yeast extract ..........……......... nutrient (growth factor) Pancreatic digest of casein………nutrient (nitrogen source) Dextrose……...nutrient (carbon source) and antioxidant 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 O2 tension in upper third of container and yellow at low O2 tension in lower 2/3 of the container). Distilled water pH after sterilization 7.1  0.1

Formulation of "FTM"

pH after sterilization 7.3  0.1 Formulation of "TSB" Pancreatic digest of casein.................... nutrient Papaic digest of soya bean ................... nutrient Dextrose................................................... nutrient Sod. chloride................................. isotonic agent Dibasic potassium phosphate....................buffer Distilled Water pH after sterilization 7.3  0.1

Direct inoculation of the culture medium 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 inoculation of the culture medium 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 30-35oC (suitable for bacteria). "TSB" is incubated at 20-25oC (suitable for fungi). Incubation time is up to 14 days according to USP, to assure the sterility of the product.

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).

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

Staphylococcus aureus Shape: cocci Size: small Arrangement: Clusters, bunches Color: violet

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

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

Aspergillus niger (wet mount) Head (sporangium) Spores Mycelium (sporangiophore)