Sterility testing is required for all products labeled as sterile to ensure they have been effectively sterilized. Tests are conducted using specific culture media and procedures to detect any viable bacteria, fungi, or yeasts. The USP outlines sterility testing methods for various pharmaceutical products and devices, including membrane filtration and direct inoculation. Interpretation of results involves incubating samples and checking for any microbial growth over time, with growth indicating test failure.

1. Sterility testing
• All products labeled sterile must pass
the sterility test as they have ben
subjected to an effective process of
sterilization as per
• BP recommends or as specified in the
International Pharmacopoeia and USP
• These tests are suitable to reveal the
presence of viable forms of bacteria,
fungai and yeasts in a pharmaceutical
products or devices

2.  Extraneous microorganisms should be
excluded throughout the test procedure
and period
 The sterility testing of human and
veterinary products is conducted by
specific procedures
 Test is based on assumptions that
MOs grow on the provided culture
medium
 Limitations (different organism have
different nutritional requirements,
temp. for growth, spores take more
time to grow)

3. Antimicrobial precautions
 To avoid any accidental contamination use
Laminar Flow Hood
 Already present Microorganisms on the
product must be killed
 Working area should be monitored
periodically both
 Air
 Surface

4.  TheUSP sterility tests for various
pharmaceutical products and
devices are as follows:

5. Culture media
 Culture media should give an early and copious
(abundant) growth
 Different types of culture media for aerobes,
anaerobes and fungi are given in official books
 Media may be prepared as stated in official books or
dehydrated mixtures may be procured and used
after reconstitution as directed by the manufacturer
 USP (media containing L-cystein & thioglycolate
both for aerobe and anaerobes, and modified
Sabouraud liquid medium for moulds and yeast) e.g
Fluid thioglycolate medium and Soybean-casein
digest medium

6. Tests for culture media
¢ Sterility; incubate for bacteria and fungi, and check
growth in 7 days
¢ Nutritive properties; inoculate with 100 viable
microorganisms of each type separately, incubate. An
early and copious growth indicate the nutritional
properties of the medium
¢ Effectiveness of media/ antimicrobial activity;
prepare 2 containers of media (15, 40, 80 mL) for each
MO, add preparation and inoculate with MOs. Prepare
another set without preparation. Incubate, equal growth
in all indicate preparation has no AM activity

7.  AM activity is there need to be
eliminated by
 Suitable sterile inactivating agent
 Dilution either the product or increasing
the media
 Filtration

8. Tests for culture media
 If freshly prepared media are not used
within 2 days, store them in the dark,
preferably 2 – 25oC
 Finished media may be stored in unsealed
containers for 10 days, provided that they
are tested weekly for growth promotion
 If stored in sealed containers, media may
be used for one year, provided that they
are tested for growth promotion every 3
months

9. Procedure
 Opening of containers
 Clean the exterior of ampoules and closures
with an antimicrobial agent, and make access
to contents in a suitable manner
 If packed under vacuum admit sterile air
 Sampling
 Test 20 units in each medium
 If contents are sufficient, may be divided so
that portions are added to two specified media

10. Methods for sterility testing
¢ Membrane filtration (for aqueous, alcoholic,
oily and other preparation that are miscible)
¢ Direct inoculation (for concentrated
preparation)

11. Method I (Membrane
filtration)
 Procedure; Filters made up of esters or
mixtures of esters and cellulose, pore size
0.45µm and diameter 50mm are usually
used.
 Adjust the sterilized filter assembly and filter
under aseptic conditions.
 Transfer membrane either directly or cut
into pieces to add in culture media and
incubate.
(for bacteria 20-25oC, for fungi 30-35oC for 7
days)

12. Pre-treatment of dosage forms
before membrane filtration
 Aqueous solutions; (i) moisten membrane with meat
or casein peptone solution (ii) dilute sample to 100ml
and filter immediately (iii) incubate for 14 days.
 Soluble solids; dissolve specific qty in meat or casein
peptone solution, proceed as above.
 Oils and oily solutions; (i) low viscous filter through
dry membrane (ii) dilute viscous preparations with
isopropyl myristate (iii) filter using pressure or suction
(iv) wash with diluent and complete as above.
 Ointments and creams; fatty bases are heated to
45oC & diluted by IM filter rapidly, proceed as oily
solutions.

13. Method II (Direct inoculation)
 Dilute liquids 10 fold and solids 100
folds using diluent, oily preparations
use emulsifying agents.
 Use larger volume for AM activity
elimination or for concentrated
solutions.
 Incubate for 14 days, shake oily
preparations during incubation but not
to anaerobes.

14. Interpretation of results
 During or at the end of incubation if no growth, test
pass.
 If growth is observed, preserve it and repeat the test.
 If no growth test pass.
 If growth occurs, compare with first, if non
distinguishable, then test fails
 If growth is distinguishable repeat second time taking
twice the samples.
 If no growth test pass, if growth occurs test fails.
 How and qty of sample to be taken, given in table I, II,
III of BP.

15. Tests for ophthalmic
preparations
 Clarity
 Heavy metal particles
 Active content determination
 pH
 Sterility
 Retention time