1. STERILITY TEST FOR PARENTRAL
PRODUCTS

2. • DEFINITION:
• Sterility Testing: It is a testing procedure
applied to products intended to be sterile
before marketing, to check that these
products are free from all living
microorganisms.

3. • Sterility tests are performed on random
samples from the batch and must be carried
out under aseptic conditions in order to avoid
accidental contamination of the product
during the test using, for example, a laminar
air flow cabinet.

4. • Growth of microorganisms, if present in
samples under test, is usually checked by
incubating over specified culture Media at
specified temperature for specified time.

5. Methods of Sterility Test

6. Direct Inoculation of Culture Media
• This test is performed by direct transferring of
the test sample into the culture media.
• However, in case of large volumes such as
intravenous fluids, a concentrated medium
may be added directly to the preparation in its
container.

7. Membrane Filtration Method
• The membrane filtration method requires the
test sample to first pass aseptically through a
membrane filter capable of retaining
microorganisms.
• The filter is rinsed and then the membrane is
transferred into the appropriate test medium.

8. CULTURE MEDIA USED FOR STERILITY TESTING

9. • Fluid Thioglycollate Medium
Primarily intended for culturing anaerobic
bacteria. However, it also detects aerobic
bacteria.

10. • Soybean–Casein Digest Medium:
Suitable for culturing both fungi and aerobic
bacteria.

11. • Tryptic Soya Broth
• A nutritious medium that supports the
growth of a wide variety of microorganisms,
especially common aerobic and facultatively
anaerobic bacteria.

12. Media for Penicillins or Cephalosporins
• If sterility testing of samples having Penicillins
or Cephalosporins is to be performed, then
the above mentioned media are treated with
β-lactamase sufficient to inactivate the
amount of antibiotic in the sample under test.

13. Suitability Tests for Media

14. Sterility
The sterility behavior of each culture
medium is confirmed by incubating a portion of
the media at the specified incubation
temperature for 14 days.
No growth of microorganisms should occur.

15. Growth promotion test of Aerobes, Anaerobes
and Fungi
Each lot of the medium is checked for growth promotion of
micro-organisms.
Strains to be tested
• Aerobic micro-organisms:
Staphylococcus aureus, Pseudomonas
aeruginosa.
• Anaerobic micro-organisms:
Clostridium sporogenes
• Fungi:
Candida albicans, Aspergillus niger.

16. • Inoculate portions of Fluid Thioglycollate
Medium with a small number of the stated
aerobic and anaerobic micro-organisms using
a separate portion of medium for each of the
species of microorganism.
• Incubate for not more than 3 days.

17. • Inoculate portions of Soybean–Casein Digest
Medium with a small number of the stated Fungi
using a separate portion of medium for each of the
following species of microorganism.
• Incubate for not more than 5 days.
• The Medias are suitable if a clearly visible growth of
the microorganisms occurs.

18. Storage
If prepared media are stored in unsealed
containers, they can be used for one month and
if stored in tight containers, the media can be
used for one year.

19. Minimum quantity and Number of Articles to
Be Tested
• Unless otherwise specified in the individual
monograph, test the minimum quantity and number
of articles specified.

20. Quantity per Container Minimum Quantity to be Used
Liquids (other than antibiotics)
Less than 1 mL The whole contents of each container
1–40 mL Half the contents of each container, but not less than 1 mL
40-100 mL 20 mL
>100 mL 10% of the contents of the container, but not less than 20 mL
Antibiotic liquids 1 mL
Other preparations soluble in water or in isopropyl myristate The whole contents of each container to provide not less than 200 mg
Insoluble preparations, creams, and ointments to be suspended or
emulsified
Use the contents of each container to provide not less than 200 mg
Solids
Less than 50 mg The whole contents of each container
50 - 300 mg Half the contents of each container, but not less than 50 mg
300 mg–5 g 150 mg
> 5 g 500 mg

21. Number of Items in the Batch Minimum Number of Items to be Tested for Each Medium
Parenteral preparations
< 100 containers 10% or 4 containers, whichever is the greater
100 - 500 containers 10 containers
> 500 containers 2% or 20 containers, whichever is less
For large-volume parenterals 2% or 10 containers, whichever is less
Antibiotic solids
Pharmacy bulk packages (<5 g) 20 containers
Pharmacy bulk packages (> 5 g) 6 containers
Ophthalmic and other noninjectable preparations
Not more than 200 containers 5% or 2 containers, whichever is the greater
More than 200 containers 10 containers

22. Membrane Filtration Method
• Use membrane filters having a nominal pore size not
greater than 0.45 µm whose effectiveness to retain
microorganisms has been established.
• Cellulose nitrate filters are used for aqueous, oily and
weakly alcoholic solutions.
• Cellulose acetate filters are used for strongly alcoholic
solutions. Specially adapted filters may be needed for
certain products (e.g., for antibiotics).
• The filtration apparatus and membrane are sterilized by
appropriate means.

23. Procedure
Aqueous solutions
 Transfer the contents of the containers to be tested to the membrane,
if necessary, after diluting with the chosen sterile diluent.
 Filter immediately. If the product has antimicrobial properties, wash
the membrane not less than 3 times by filtering through it each time
the volume of the chosen sterile diluents.
 Transfer the whole membrane to the culture medium or cut it
aseptically into two equal parts, and transfer one half to each of two
suitable media. Incubate the media for not less than 14 days.

24. • For soluble solids (other than antibiotics).
• Use quantity of the product prescribed in
Tables 1 and 2 for each medium, dissolved in a
suitable solvent and proceed as described
above for Aqueous Solutions.

25. For oils and oily solutions
• Low viscosity solutions may be filtered without
dilution through a dry membrane. Viscous oils may
be diluted as necessary with a suitable sterile diluent
such as isopropyl myristate. Filter the oily solutions
by applying the pressure or suction gradually.
• Wash the membrane at least 3 times by filtering
about 100 mL of a suitable sterile solution. Transfer
the membrane to the culture medium as described
above for Aqueous Solutions and incubate at the
same temperatures and for the same times.

26. For ointments and creams
• Use quantity of the product prescribed in Tables 1
and 2 for each medium.
• Ointments in a fatty base and emulsions of the
water-in-oil type may be diluted to 1% in
isopropyl myristate as described above, by
heating, if necessary, to not more than 40 ºC.
Filter as rapidly as possible, and proceed as
described above for Oils and Oily Solutions

27. For solids for injection other than antibiotics
• Constitute the test articles as directed on the label,
and proceed as directed for Aqueous Solutions or
Oils and Oily Solutions, whichever applies.

28. Direct Inoculation of the Culture Medium
Transfer the quantity of the preparation to be examined
prescribed in Tables 1 and 2 directly into the culture
medium so that the volume of the product is not more
than 10% of the volume of the medium, unless
otherwise prescribed.

29. • If the product to be examined has antimicrobial
activity, carry out the test after neutralizing this
with a suitable neutralizing substance.
• For large volume products, it may be preferable
to use a concentrated culture medium that
undergoes subsequent dilution with the product.
Where appropriate, the concentrated medium
may be added directly to the product in its
container.

30. Procedure
For oily liquids
• Use media to which have been added a
suitable emulsifying agent e.g., polysorbate 80
at a concentration of 10 g per L.

31. For ointments and creams
• Prepare by diluting to about 1 : 10 with the chosen
emulsifying agent in a suitable sterile diluent.
• Transfer the diluted product to a culture medium
not containing an emulsifying agent and incubate for
not less than 14 days.

32. For solids
Use quantity of the product prescribed in Tables 1 and
2 for each medium. Transfer the material so obtained
to 200 mL of Fluid Thioglycollate Medium, and mix.
Similarly, transfer the same quantity to 200 mL of
Soybean–Casein Digest Medium, and mix. Proceed as
directed previously.

33. OBSERVATION AND INTERPRETATION OF RESULTS
• The culture media is examined throughout the
incubation period at intervals for macroscopic
evidence of microbial growth.
• If the medium becomes turbid then after 14 days
transfer portions (each not less than 1 mL) of the
medium to fresh vessels of the same medium, and
then incubate the original and transfer vessels for
not less than 4 days.

34. • 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, unless there has been a fault in the test
conditions.

35. • If the test is declared to be invalid, it is repeated
with the same number of units as in the original
test.
• If no evidence of microbial growth is found in the
repeat test, the product examined complies with
the test for sterility. If microbial growth is found
in the repeat test, the product examined does not
comply with the test for sterility.