Sterility Testing
Contents
• Need for sterility testing
• Methods – direct inoculation and
membrane filtration
• Positive and negative controls
Learning objectives
At the
end of this lecture, student will be able to:
• Explain the need for sterility
testing
• List the pharmacopoeial methods for
sterility testing
• Outline the principle involved in
the methods used for sterility testing
• Justify the need for positive and
negative controls during sterility testing
Sterility testing
• Sterility testing examines samples
of the final product for the presence of microorganisms
• Should be applied to all products
that are designated as sterile
• A satisfactory result only indicates
that no contaminating microorganism has been found in the sample examined in
the conditions of the test
Culture media
1. Fluid
thioglycollate medium
– For the culture of aerobic and
anaerobic bacteria
– pH after sterilization 6.9 to 7.3
– To be incubated at 30–35 °C
2. Soya-bean
casein digest medium
– Suitable for the culture of both
fungi and aerobic bacteria
– pH after sterilization 7.1 to 7.5
– To be incubated at 20–25 °C
Sterility
check of media
(Negative control)
• Incubate portions of the media for
14 days. No growth of should occur
Growth
promotion test of aerobes, anaerobes and fungi (Positive control)
• Inoculate each media using test
organism
• Incubate under the specified
conditions
• The media are suitable if a clearly
visible growth of the microorganisms occurs
Aerobic
bacteria
• Staphylococcus aureus ATCC 6538, CIP 4.83, NCTC 10788,
NCIMB 9518, NBRC 13276
• Bacillus subtilis ATCC 6633, CIP 52.62, NCIMB 8054,
NBRC 3134
• Pseudomonas aeruginosa ATCC 9027, NCIMB 8626, CIP 82.118,
NBRC 13275
Anaerobic
bacterium
• Clostridium sporogenes ATCC 19404, CIP 79.3, NCTC 532 or
ATCC 11437, NBRC 14293
Fungi
• Candida albicans ATCC 10231, IP 48.72, NCPF 3179,
NBRC 1594
• Aspergillus brasiliensis ATCC 16404, IP 1431.83, IMI 149007,
NBRC 9455
• ATCC– American Type culture collection, USA
• CIP– Collection de l’Institut Pasteur,
France
• NCTC– National collection of type cultures,
England
• NCIMB– National Collection of Industrial and Marine
Bacteria, National Collections of Industrial, Food and Marine Bacteria, UK
,Scotland
• NBRC – National Biological Resource Centre, Japan
• IMI- CABI GRC (Strain numbers: IMI): The Genetic
Resource Collection, CABI Bioscience UK Centre UK
Sterility testing methods
- Direct inoculation methods
• Involves introducing test samples
directly into nutrient media
- Membrane filtration method
• Involves filtration of fluids
through a sterile membrane filter
• Microorganism present being retained
on the surface of the filter
• Portions of the filter are
transferred to suitable culture media
Sterility testing of
pharmaceutical products
Direct inoculation method
If the
product to be examined 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
Oily
liquids
• Use media to which have been added a
suitable emulsifying agent at a concentration shown to be appropriate in the
method suitability of the test, for example polysorbate 80 at a concentration
of 10 g/L
Ointments
and creams
• Prepare by diluting to about 1 in 10
by emulsifying with the chosen emulsifying agent in a suitable sterile diluent
such as peptone(1 g/L). Transfer the diluted product to a medium not containing
an emulsifying agent
• Incubate the inoculated media for
not less than 14 days
• Observe the cultures several times during
the incubation period
• Shake cultures containing oily
products gently each day
• When fluid thioglycollate medium is
used for the detection of anaerobic microorganisms keep shaking or mixing to a
minimum in order to maintain anaerobic conditions
• Examination of the media for macroscopic evidence of
microbial growth
• If no evidence of microbial growth is found, the product to be
examined complies with the test for sterility
• If evidence of microbial growth is found the product to be examined
does not comply with the test for sterility
The test
may be considered invalid if
• A review of the testing procedure
used during the test in question reveals a fault
• The data of the microbiological
monitoring of the sterility testing facility show a fault
• Microbial growth is found in the
negative controls
Sterility testing of
pharmaceutical products
Membrane filtration method
Membrane
filtration is used for
• Filterable aqueous preparations,
• Alcoholic or oily preparations
• Preparations miscible with or
soluble in aqueous or oily solvents
(Provided
these solvents do not have an antimicrobial effect in the conditions of the
test)
Controls used during sterility
testing
Positive
controls
• To show that microorganisms will
actually grow under the conditions of the test
• The media is inoculated with test
organism and
incubated along with test samples
Negative
control
• To ascertain the sterility of the
media
• Media without sample or test
organism should be
incubated
Summary
• Sterility testing examines samples
of the final product for the presence of microorganisms and applied to all
products that are designated as sterile
• Media used –
Fluid thioglycollate medium (aerobic and anaerobic) Soya-bean casein
digest medium (for fungi)
• Methods – direct inoculation and membrane filtration
• Positive control – To show that microorganisms will
actually grow under the conditions of the test
• Negative control -To ascertain the sterility of the
media
