NPCB
MOH
STERILITY TEST
(ST) USP -71
Introduction
Certificate of Analysis
Media Validation
Tests Methods
Method Validation
Definition : The sterility of a product is defined
by the absence of viable and actively
multiplying microorganisms when tested in
specified culture media.
The test is applied to substance, preparations or
articles which, according to the
Pharmacopoeia, are required to be sterile.
Turbidity in the broth media usually
indicates contamination.
□ Test is performed on the end –product and is one
of the quality control tests specified for release of a
batch of sterile product.
Specification and Result
□ Asper British Pharmacopoeia or
USP
□ BP – Appendix XVI A. Sterility
Gowning technique
Specification and Result
□ Asper British Pharmacopoeia or
USP
□ BP – Appendix XVI A. Sterility
Specification and Result
□ Asper British Pharmacopoeia or
USP
□ BP – Appendix XVI A. Sterility
Media types
□ Fluid Thioglycollate medium
(FTM)
□ pH after sterilization: 7.1±0.2.
□ Soybean Casein Digest Medium (SCD
or TSB)
□ pH after sterilization: 7.3±0.2
Store at a temperature between 2° and 25°
TSB V.s FTM
Media for Penicillins or Cephalosporins
Where sterility test media are to be used in the Direct Inoculation of the
Culture Medium method under Test for Sterility of the Product
to be Examined, modify the preparation of Fluid Thioglycollate Medium
and the Soybean–Casein Digest Medium as follows. To the
containers of each medium, transfer aseptically a quantity of ß-lactamase
sufficient to inactivate the amount of antibiotic in the
specimen under test. Determine the quantity of ß-lactamase required to
inactivate the antibiotic by using a ß-lactamase preparation
that has been assayed previously for its penicillin- or cephalosporin-
inactivating power. [ N — Supplemented ß-lactamase media
can also be used in the membrane filtration test.]
Alternatively (in an area completely separate from that used for sterility
testing), confirm that an appropriate amount of ß-
lactamase is incorporated into the medium, following either method under
Method Suitability Test, using less than 100 colony-forming
units (cfu) of Staphylococcus aureus (see Table 2) as the challenge.
Typical microbial growth of the inoculated culture must be
observed as a confirmation that the ß-lactamase concentration is
appropriate.
Prior to test, make sure that:
□ Media is sterile
□ Media supports growth of microorganisms
2 components in Media validation :
□ Media sterility Test
□ Growth Promotion Test
Media sterility
□ Negative Control - may be used to identify a
―false positive test result
□ Incubate
for 14 days prior to use, may be
conducted concurrently with test
30 - 35°C for Fluid Thioglycollate medium (FTM)
20 - 25°C for Soybean Casein Digest Medium
(SCD/TSB)
Acceptance criteria:
□ Should be sterile, no growth observed
– Media Validation (co)
Growth Promotion Test
□ To test the ability of media to support the growth of
micro- organisms
□ The media should be inoculated with <100 cfu of
challenge organisms. The challenge inoculum
should be verified by concurrent viable plate counts
□ Growth promotion challenge organisms should show
clearly visible growth in the test media within 3 days for
bacteria and 5 days for fungi.
– Media Validation (co)
Table 2
Methods are defined in Pharmacopoeia:
□ Membrane Filtration Method
(open or a closed system)
□ Direct Inoculation Method
*When the preparation to be tested has an
antimicrobial effects, these effects must be
reduced or neutralised by adding an
appropriate substance to the specified test
media, to diluents or solvents, or to the
preparation prior to testing.
⚫ Membrane Filtration Method (Open Funnel Method)
⚫ Membrane Filtration Method(Closed System Method)
Direct Inoculation of the culture medium
□ Transfer the preparation directly into the culture medium
□ Volume of the product is not more than 10% of the volume
of the mediu
□ Period : At least 14 days incubation
□ Temperature : 30-35°C for FTM
20-25°C for SCD/TSB
Incubation and Examination
□ All test & sterility control containers – incubated for
at least 14 days (unless microbial contamination
detected earlier)
□ Examine for evidence growth
□ Preparation not readily seen (turbid/cloudy due to
its nature) – after 14 days of incubation transfer
a suitable portion (2-5% of contents) to fresh, same
medium incubate for 7 days
⚫ No evidence of microbial growth is
found.
If turbidity or other evidence of growth is
seen:
□ Streak on solid media
□ Examine the suspected growth
microscopically by Gram stain
□ Identify the isolates, as far as the
genus and preferably species level
⚫ No evidence of microbial growth is
found.
If turbidity or other evidence of growth is
seen:
□ Streak on solid media
□ Examine the suspected growth
microscopically by Gram stain
□ Identify the isolates, as far as the
genus and preferably species level
Validation (bacteriostasis & fungistasis)Test
□ The test should be validated by inoculation with <100
cfu of challenge organism strains to the media/product
container at the beginning of the test incubation period.
□ The challengeinoculum should be verified by concurrent
viable plate counts.
Validation (bacteriostasis & fungistasis)Test
□ The challenge organisms, preferably, should be added
directly to the product prior to membrane filtration or
direct inoculation. If this is not practicable, the
challenge organisms should be added to the last
rinse solution (membrane filtration) or directly to
media containing the product (direct inoculation).
□ Validation done should mimic the test proper in every
detail.
□ Perform a growth promotion test as a positive control.
Incubate all the containers containing medium for not
more than 5 days.
Interpretation of results
□ Challenge organisms should clearly show visible
growth of bacteria within 3 days, and fungi
within 5 days in the test media containing
product.
□ Visually
comparable to that in the control vessel
without product .
Validation (bacteriostasis & fungistasis)Test
□ Ifperformed concurrently with ST should confirmed
validation tests as successful before the results of the
ST are interpreted
□ Validation
to be performed on all new product and
repeated whenever there is a change in the
experimental conditions.
