Transcript STERILITY TEST - Faculty of Pharmacy, Cairo University

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"
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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
Fluid
hioglycollate
llate
Medium
m
Formulation of "FTM"
Resazurin
Resazurin
Layer
layer
Formulation of "TSB"
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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
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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)