For pharmaceuticals as per IP

1. SHITAL TRIVEDI
ASSISTANT PROFESSOR,
Department of pharmaceutical technology,
L.J.INSTITUTE OF PHARMACY
Subject: Pharmaceutical Microbiology
SEMESTER 5
STERILITY TESTING

2. Sterility testing ( As per IP)
• The test for sterility is applied to pharmacopoeial articles/products that are required according
to the Pharmacopoeia to be sterile.
• Satisfactory result only indicates that no contaminating viable microorganisms have been found
in the sample examined in the conditions of the test .
• The test must be carried out under aseptic conditions to avoid accidental contamination of the
product during testing. Grade A laminar air flow cabinet or an isolator is recommended.
Source: Google images

3. Minimum no. of containers recommended to be tested as per
the batch size.

5. Culture media for Sterility Testing
Fluid Thioglycollate medium
For anaerobic bacteria, will
also detect aerobic bacteria.
For use with clear fluid
products
Incubation temp. 30-35 0C
Alternative Fluid Thioglycollate
medium.
For turbid & viscous products
and for devices having tubes
with small lumina.
Incubation temp. 30-35 0C
Soyabean-casein digest
media.
Suitable for both fungi &
aerobic bacteria.
Incubation temp. 20-25 0C
Media for
Penicillins &
Cephalosporins

6. Suitability of Culture media
• Media should comply with the following tests, carried out before or in parallel with sterility testing.
1. Sterility test (negative control):
• Incubate portions of the media for 14 days at the temperature specified for each medium.
• If no growth of microorganisms, then the media is suitable for sterility testing.
2. Growth Promotion Test.
• Each lot of medium for its growth-promoting qualities using suitable stains of microorganisms.
(Table 2)
• Inoculate duplicate portion of each medium with small no. of the microorganisms (not more than
100 CFU), using separate portion of each medium for each of the microorganisms.
• Incubate according to the conditions specified in Table 2.
• If a clear visible growth of microorganisms, then the media is suitable for sterility testing.

7. Growth promotion Test:

8. Validation of tests:
• Required…
1. When the test of sterility has to be carried out on a new product.
2. Whenever there is a change in the experimental conditions of the test.
• Modification of method A & Method B.
• Growth promotion test as positive control.
• Incubation for not more than 5 days.
• Visual comparison of growth of vessels under validation tests and with control vessel.
• If visually comparable growth then the product under examination is having no antimicrobial
activity or the antimicrobial activity is successfully eliminated.
• If clear visible growth is not observed when visually compared with control…product is having
antimicrobial activity and that is to be neutralised by adding suitable sterile neutralising agent
or by sufficient dilution of product with medium.

9. Sterility Testing Procedures/Methods
Membrane Filtration
This method is preferred when product is a)
Oil, b) An ointment that can be put into
solution, c) A non-bacteriostatic solid not
readily soluble in the culture medium, and
d) A soluble powder/a liquid with
bacteriostatic &/or fungistatic properties. e)
when volume of liquid product/container is
of 100 ml or more.
Direct Inoculation
For containers with small volume.
For aqueous solutions & suspensions,
oils, oily solutions, ointments, creams,
surgical dressings, sterile devices.
For ointments & oils that are insoluble
in Isopropyl myristate

10. Membrane Filtration Test Procedure:
• Method involves positive and negative control.
• Apparatus: A suitable unit consists of a closed reservoir and a receptacle between which a
properly supported membrane of appropriate porosity is placed.
• Membrane:
 Nominal pore size not greater than 0.45 micron & diameter of approximately 50 mm.
 Cellulose nitrate filters- for aqueous, oily and weakly alcoholic solutions
Cellulose acetate filters- for strongly alcoholic solutions.

11. Method A: Membrane Filtration: Test Procedure
Observe the containers of media periodically during the 14 days of incubation.
If the test specimen is positive before 14 days of incubation , further incubation is not necessary.
After filtration, aseptically remove the membrane from the holder, transfer the whole membrane or cut it aseptically into two
equal parts. Transfer one half to each of the two suitable media.
Incubate the media for not less than 14 days.
Draw the liquid rapidly through filter with the aid of vacuum. If the solution under examination has antimicrobial properties,
wash the membrane(s) by filtering through it not less than 3 successive quantities, each of 100 ml, of sterile fluid A. Don’t
exceed a washing cycle of 5 times per filter.
Alternatively, transfer aseptically the combined quantities of the preparation prescribed in the two media onto one membrane.
Prepare each membrane by aseptically transferring a small quantity ( sufficient to moisten the membrane) of Fluid A on to the
membrane & filter it. transfer aseptically the quantity of preparation under examination that is prescribed in Table 3 & 4.
Sterilise entire assembly with membrane in place prior to use.
If sample is an oil, sterilise the membrane separately and after thorough drying, assemble the unit using aseptic precautions.

12. Method B: Direct Inoculation: Test Procedure
Observe the containers of media periodically during the 14 days of incubation.
If the test specimen is positive before 14 days of incubation , further incubation is not
necessary.
Incubate the media for not less than 14 days.
When the quantity in a single container is insufficient to carry out the test, the combined
contents of 2 or more containers are to be used to inoculate the media. When it is necessary to
use large volume of the product it may be preferable to use a concentrated medium.
Transfer the quantity ( as per table 3 & 4) of the preparation under examination directly into
the culture medium so that the volume of the preparation under examination is not more
than 10% of the volume of medium.
Remove the liquid from the test containers with a sterile pipette/syringe/needle.

13. Membrane Filtration Direct Inoculation
Source: Google images

14. Diluting Fluids:
Fluid A:
1 g of peptic digest of animal tissue in water
to make 1 litre. pH 7.1 ± 0.2, sterilise at 121
0C for 20 minutes.
If sample is penicillin, addition of suitable
quantity of sterile penicillinase.
Fluid B:
1 ml of Polysorbate 80 to each litre of Fluid
A. pH7.1 ± 0.2, sterilise at 121 0C for 20
minutes.
For sample containing Lecithin/Oil

15. Quantities of sample to be used

16. Quantities of sample to be used

17. Observation & Interpretation of Results of Sterility Testing.
At intervals during/End of Incubation period of 14 days.
If evidence of
microbial
growth
The
preparation
under
examination
doesn’t comply
the test for
sterility.
If no evidence
of microbial
growth
The
preparation
under
examination
complies the
test for
sterility.
If material renders
medium turbid.
Transfer portions( each not less than 1 ml ) of
the medium to fresh vessel of same medium.
Incubate both original and transfer vessels for
not less than 4 days.
If evidence of growth…The
preparation under
examination doesn’t comply
the test for sterility.
If no evidence of
growth…The preparation
under examination
complies the test for
sterility.

18. Invalid only under following conditions.
1) Growth in negative control.
2) Sterility testing facility – faulty data of microbial monitoring.
3) Faulty Testing procedure
4) Fault in material &/ or technique used for testing procedure.
Repeat test
If no evidence of growth---The
preparation under examination
complies the test for sterility.
If evidence of growth….The
preparation under
examination doesn’t comply
the test for sterility.

19. Reference:
• Indian Pharmacopoeia 2018, Eighth Edition, volume 4, page no. 60-66, Published by The Indian
Pharmacopoeia Commission, Ghaziabad.
Thank You