1. Sterility Testing for
Parenteral Products
Dr. Prashant L. Pingale
Associate Professor-Pharmaceutics
GES’s Sir Dr. M. S. Gosavi College of Pharmaceutical Education and Research,
Nashik

2. Sterility testing-Purpose
 Sterility testing attempts to reveal the presence or absence of viable
micro-organisms in a sample number of containers taken from batch
of product.
 Based on results obtained from testing the sample a decision is made
as to the sterility of the batch.
2

3. Sterility testing
 Is made after the product exposition to the one of the possible
sterilization procedures.
 Can only provide partial answers to the state of sterility of the
product batch under test.
 Is inadequate as an assurance of sterility for a terminally sterilized
product.
3

4. Major factors of importance
in sterility testing
 The environment in which the test is conducted
 The quality of the culture conditions provided
 The test method
 The sample size
 The sampling procedure
4

5. Environmental conditions
 avoid accidental contamination of the product during the test
 the test is carried out under aseptic conditions
 regular microbiological monitoring should be carried out
5
Culture conditions
 Appropriate conditions for the growth of any surviving
organism should be provided by the culture media selection.

6. Culture conditions
 Factors affecting growth of bacteria
 Phases of bacterial growth
 Culture media for sterility testing
6
Factors affecting growth of bacteria
 Nutrition
 Moisture
 Air
 Temperature
 pH
 Light
 Osmotic pressure
 Growth inhibitors

7. Phases of bacterial growth
 Lag phase (A)
 Log (logarithmic or exponential) phase (B)
 Stationary phase (C)
 Decline (death) phase (D)
7

8. Culture media for sterility testing
 Capable of initiating and maintaining the vigorous growth of a small
number of organisms
 Sterile
 Types of media:
 Fluid thioglycollate medium
 Soya-bean casein digest medium
 other media
8

9. Fluid Thioglycollate Medium
 specific role of some ingredients
 primarily intended for the culture of anaerobic bacteria
 incubation of the media:
 14 days at 30 -35°C
9

10. FTM (Thioglycollate Medium)
 Supports the growth of a large variety of fastidious microorganisms having a wide range of growth requirements.
 The nitrogen, vitamin and carbon sources are provided by Enzymatic Digest of Casein and Yeast Extract.
 Sodium Thioglycollate & L-Cystine- lower the oxidation-reduction potential of the medium by removing oxygen to
maintain a low Eh. By creating an environment with a low Eh, the reducing agents prevent the accumulation of
peroxides that can be toxic to some organisms.
 The sulfhydryl groups (-SH) of these compounds also neutralize the antibacterial effect of mercurial preservatives,
making thioglycollate media useful in testing material containing heavy metals.
 Resazurin is the oxidation indicator. In the oxidized state, resazurin turns pink. In the reduced state resazurin is
colorless.
 Dextrose is included in this formula to enhance organism growth.
 Sodium Chloride maintains the osmotic balance of the medium.
 The requirement for a sealed environment is eliminated with the addition of Agar, which retards dispersion of
CO2, diffusion of oxygen, and reducing substances.
10

11. Soya-bean casein digest medium
 primarily intended for the culture of both fungi and aerobic bacteria
 specific role of some ingredients
 incubation of the media:
 14 days at 20 -25°C
11

12. Soya-bean casein digest medium
 The combination of pancreatic digest of casein and papaic digest of soybean
meal makes this medium nutritious by providing amino acids and long chain
peptides for the growth of microorganisms.
 Natural sugars in soybean promote growth of fastidious organism.
 Dextrose is the fermentable source of carbon and dipotassium hydrogen
phosphate serves as the buffer in the medium.
 Sodium chloride maintains the osmotic balance of the medium.
12

13. Fertility control of the media
 are they suitable for growth of each micro-organism?
 'Growth promotion test for aerobes, anaerobes and fungi' ;
 inoculation of media tubes with a MO
 incubation (T, t)
 the media are suitable if a clearly visible growth of the micro-
organisms occurs
13

14. Effectiveness of the media
under test conditions
 are culture conditions satisfactory in the presence of the product being
examined?
 comparing the rate of onset and the density of growth of inoculated MO
in the presence and absence of the material being examined
 growth control;
14

15. The test method for sterility
of the product
 Membrane filtration
 Direct inoculation of the culture medium
15

16. Membrane filtration
 Appropriate for:
 filterable aqueous preparations
 alcoholic preparations
 oily preparations
 preparations miscible with or soluble in aqueous or oily (solvents with
no antimicrobial effect)
 solutions to be examined must be introduced and filtered under
aseptic conditions
 All steps of this procedure are performed aseptically in a Class 100
Laminar Flow Hood
16

17. Selection of filters for
membrane filtration
 pore size of 0.45 m
 effectiveness established in the retention of micro-organisms
 appropriate composition
 the size of filter discs is about 50 mm in diameter
17

18. The procedure of membrane filtration
 sterilization of filtration system and membrane
 filtration of examined solution under aseptic conditions (suitable volume,
dissolution of solid particles with suitable solvents, dilution if necessary…)
 one of two possible following procedures:
 the membrane is removed, aseptically transferred to container of appropriate
culture medium
 passing the culture media through closed system to the membrane, incubation
in situ in the filtration apparatus (Sartorius, Millipore).
18

19. Direct inoculation of the
culture medium
 suitable quantity of the
preparation to be examined is
transferred directly into the
appropriate culture medium
 volume of the product is not more
than 10% of the volume of the
medium
 suitable method for aqueous
solutions, oily liquids, ointments
an creams
19

20. 20

21. Advantages of the filtration method
 wide applications
 a large volume can be tested with one filter
 smaller volume of culture media is required
 applicable to substances for which no satisfactory
inactivators are known
 neutralization is possible on the filter
 subculturing is often eliminated
 shorter time of incubation compared with direct
inoculation
21

22. Observation and
interpretation of the results
 Examination at time intervals during the incubation period and at
its conclusion
 When the sample passes the test and when fails?
 When the test may be considered as invalid?
 There is low incidence of accidental contamination or false
positive results
22

23. Sampling
 Selection of the samples
 Sample size
23

24. Minimum number of
items to be tested24

25. Instead of the conclusion - Guidelines
for using the test for sterility
 Precautions against microbial contamination
 The level of assurance provided by a satisfactory
result of a test for sterility as applied to the quality of
the batch is a function of:
 The homogeneity of the batch
 The conditions of manufacture
 Efficiency of the adopted sampling plan
25

26. Guidelines …
 In the case of terminally sterilized products: physical proofs,
biologically based and automatically documented, showing
correct treatment through the batch during sterilization are of
greater assurance than the sterility test.
 Products prepared under aseptic conditions: sterility test is the
only available analytical method.
 Only analytical method available to the authorities who have to
examine a specimen of a product for sterility.
26