sterility testing

1. 1
BY
Dr. Suman Pattanayak
Associate Professor
Department of Pharma Analysis & QA.
Vijaya Institute of Pharmaceutical Sciences for Women
B. Pharm IV year / II Sem
DRA, IPR & Patents

2. A sterility test is essentially a test which assesses
whether a sterilized pharmaceutical or medical
product is free from contaminating microorganisms
by incubation of either the whole or a part of that
product with a nutrient medium.
Destructive test and is of questionable suitability for
testing large, expensive or delicate products or
equipment.
Random sampling should be applied to products that
have been processed and filled aseptically. With
products sterilized in their final containers, samples
should be taken from the potentially coolest

3. A sterility test is intended to
demonstrate that no viable organisms are
present, but failure to detect them could
simply be a consequence of the use of
unsuitable media or inappropriate cultural
conditions.
To be certain that no organisms are
present it would be necessary to use a
universal culture medium suitable for the
growth of any possible contaminant and to
incubate the sample under an infinite
variety of conditions.

4. Clearly, no such medium or
combination of media are available and, in
practice , only media capable of supporting
non-fastidious bacteria, yeasts and moulds
are employed.
The sterility test does have an important
application in monitoring the microbiological
quality of filter-sterilized, aseptically filled
products and does offer a final check on
terminally sterilized articles

5. There are three alternative methods available
when conducting sterility tests.
1.The direct inoculation procedure involves
introducing test samples directly into nutrient
media. (European Pharmacopoeia)
1.Fluid thioglycollate medium
or
Fluid mercaptoacetate Medium
2. Soyabean casein digest medium
or
Tryptone soya broth

6. Composition: w/v
Pancreatic Digest of Casein 15.0 g
Yeast Extract (water-soluble) 5.0 g
Glucose monohydrate/anhydrous 5.5 g/5.0 g
Sodium chloride 2.5 g
L- Cystine 0.5 g
Sodium thioglycollate 0.5 g
0.1% Resazurin Sodium Solution (freshly
prepared)
1.0 mL
Granulated Agar (moisture not more than 15%) 0.75 g
Purified Water 1000 mL
Polysorbate 80 5.0 mL
pH after sterilization (measured at room
temperature): 7.1± 0.2 particularly suitable for the
cultivation of anaerobic organisms (incubation temperature
30–35°C)
Medium 1 (Fluid Thioglycollate Medium)

7. Medium 2 (Soybean-Casein Digest
Medium)
Composition w/v
Pancreatic Digest of Casein 17.0 g
Pepsin Digest of Soybean Meal 3.0 g
Glucose monohydrate/anhydrous 2.5 g /2.3 g
Sodium chloride 5.0 g
Di potassium hydrogen phosphate 2.5 g
Purified Water 1000 mL
Polysorbate 80- 5.0 mL
pH after sterilization (measured at room temperature):
7.3±0.2
which will support the growth of both aerobic
bacteria (incubation temperature 30–35°C) and fungi
(incubation temperature 20–25°C)

8. Recommended by most pharmacopoeias, the method by
which the great majority of products are examined.
membrane filter (pore size 0.45 μm); any
microorganism present being retained on the surface of
the filter. After washing in situ, the filter is divided
aseptically and portions are transferred to suitable
culture media which are then incubated at the
appropriate temperature for the required period of time.
Water-soluble solids can be dissolved in a suitable
diluent and processed in this Way oil-soluble products
may be dissolved in a suitable solvent, e.g. isopropyl
myristate.

9. precaution against accidental
contamination, product testing must
be carried out under conditions of
strict asepsis using, for example, a
laminar airflow cabinet to provide a
suitable environment.

10. 3. A sensitive method for detecting low
levels of contamination in intravenous infusion
fluids involves the addition of a concentrated
culture medium to the fluid in its original
container, such that the resultant mixture is
equivalent to single strength culture medium. In
this way, sampling of the entire volume is
achieved.
precaution against accidental
contamination, product testing must be carried
out under conditions of strict asepsis using, for
example, a laminar airflow cabinet to provide a
suitable environment.

11. sampling of air and surfaces and carrying out tests
using samples' known’ to be sterile (negative controls).
samples that have been subjected to a very
reliable sterilization process, e.g. radiation, or samples
that have been subjected to a sterilization procedure
more than once
In order to minimize the risk of introducing
contaminants from the surroundings or from the
operator during the test itself, isolators are often
employed which physically separate the operator from
the materials under test.

13. preservative, its activity must be nullified in some
way during sterility testing so that an inhibitory
action in preventing the growth of any
contaminating microorganisms is overcome.
This is achieved by the following methods.
Specific inactivation
An appropriate inactivating (neutralizing) agent is
incorporated into the culture media. The
inactivating agent must be non-toxic to
microorganisms

14. Inhibitory agents Inactivating agents
Phenols, cresols None (dilution)
Alcohols None (dilution)
Parabens Dilution and Tween
Mercury compounds -SH compounds
Quaternary ammonium Lecithin + Lubrol W;
compounds Lecithin + Tween (Letheen)
Benzylpenicillin † b-Lactamase from Bacillus
cereus
Ampicillin
Other antibiotics† None (membrane
filtration)
Sulphonamides p-Aminobenzoic acid
*Neutralizing agents.

15. The antimicrobial agent is diluted in the
culture medium to a level at which it ceases to
have any activity, for example phenols, cresols
and alcohols.
Membrane filtration (antibiotic-no specific
in activators are available)
Basically, a solution of the product is
filtered through a hydrophobic- edged membrane
filter that will retain any contaminating
microorganisms. The membrane is washed in situ
to remove any traces of antibiotic adhering to the
membrane and is then transferred to appropriate
culture media.
Dilution

16. Positive controls
small numbers of suitable
microorganisms to grow in media in the
presence of the sample is measured.
The microorganism used for positive
control tests with a product containing an
antimicrobial agent must.
indicates a satisfactory inactivation
To be sensitive to that agent

17. In practice, a positive control (medium with added test
sample) and a negative control (medium without it) are
inoculated simultaneously-not necessary to do both.
All the controls may be conducted either before,or in
parallel with, the test itself, providing that the same batches of
media are used for both.
If the controls are carried out in parallel with the tests and
one of the controls gives an unexpected result, the test for sterility
may be declared invalid, and, when the problem is resolved, the
test may be repeated.
Specific cases
the sterility testing of parenteral products, ophthalmic and other
non-injectable preparations, catgut and surgical sutures will be
found in the European Pharmacopoeia. These procedures cannot
conveniently be applied to items like surgical dressings and medical
devices because they are too big.

18. In such cases the most convenient approach is to immerse the
whole object in culture medium in a sterile flexible bag, but
care must be taken to ensure that the liquid penetrates to all
parts and surfaces of the material
Sampling
A sterility test attempts to infer the state (sterile or non-
sterile) of a batch from the results of an examination of part of
a batch.
p -the proportion of infected containers in a batch.
q- the proportion of non-infected containers in a batch
Eqn……p + q = 1 or q = 1 - p.

19. Suppose also that a sample of two items is taken
from a large batch containing 10% infected containers.
The probability of a single item taken at
random being infected is p = 0.1 (10% = 0.1),
whereas the probability of such an item being noninfected
is given by q = 1 - p = 0.9. The probability
of both items being infected is p2 = 0.01, and of both
items being non-infected, q2 = (1– p)2 = 0.81. The
probability of obtaining one infected item and one
non-infected item is 1 – (0.01 + 0.81) = 0.18 = 2pq.

20. In a sterility test involving a sample size of n
containers,
the probability p of obtaining n consecutive
‘steriles’ is given by qn = (1 - p)n. Values for various
levels of p (i.e. proportion of infected containers in a
batch) with a constant sample size are given in

21. Re-tests
Under certain circumstances a sterility test may be repeated, but
the only justification for repeating the test is unequivocal evidence
that the first test was invalid; a re-test cannot be viewed as a second
opportunity for the batch to pass when it has failed the first time.
Circumstances that may justify a re-test would include, for
example, failure of the air filtration system in the testing facility
which might have permitted airborne contaminants to enter the
product or media during testing, non-sterility of the media used for
testing, or evidence that contamination arose during testing from
the operating personnel or a source other than the sample under
The Sample size increases, the probability of the batch being passed
as sterile decreases. the correct conclusion to be drawn from a
satisfactory test result is that the batch has passed the sterility test
not that the batch
is sterile.

22. Tests for Sterility
Tests for sterility are carried out by two methods:
(a) Membrane Filtration Method
(b) Direct Transfer / Inoculation Method.
The Membrane Filtration Method is used as the method of
choice wherever feasible.
Fluid Thioglycollate Medium (Medium 1) and Soybean-
Casein Digest Medium (Medium 2) are the two media
generally used for tests for sterility.
Media used in Sterility Testing

23. Method of Preparation: The pancreatic digest of
casein, yeast extract, glucose, sodium chloride, L-
cystine, agar and water are mixed in the proportions
given above and heat until dissolved. Sodium
thioglycollate is dissolved in the solution. The
specified quantity of Polysorbate 80 is added if this
ingredient is to be included. If necessary, 1 M sodium
hydroxide or 1 M hydrochloric acid is added so that
after the solution is sterilized its pH will be 7.1± 0.2. If
the solution is not clear, mixture is heated to boiling
and filtered while hot through moistened filter paper.
Resazurin sodium solution is added and mix.

24. Method of Preparation: The ingredients are mixed in the
proportions given above with slight warming. The solution is
cooled to room temperature. The specified quantity of
Polysorbate 80 is added if this ingredient is to be included. If
necessary, sufficient 1 M sodium hydroxide or 1M
hydrochloric acid so that after the solution is sterilized its
pH will be 7.3± 0.2. If the solution is not clear it is filtered
through moistened filter paper.
Method of Membrane Filtration
Procedure
The filter should be a membrane filter disc of cellulose esters
or other suitable plastics, having a nominal average pore
diameter not exceeding 0.45 μm.

25. Cellulose nitrate filters are recommended for aqueous,
oily and weakly alcoholic solutions and cellulose
acetate filters for strongly alcoholic solutions. The
entire unit should be sterilized by appropriate means
with the membrane filter and sterile airways in place.
The method of sterilization should not be deleterious
to the membrane, eg, weaken it or change the
nominal average pore diameter. The sterile airways
should provide free access to the sterilizing agent.
After sterilization, the apparatus should be free of
leaks to the atmosphere except through the sterile
airways.

26. Method of Direct Transfer
Procedures
Liquids and soluble or dispersible solids:
Appropriate quantities of the preparation to be examined are added
directly into Medium 1 and Medium 2. Approximately equal
quantities of the preparation should be added to each vessel of
medium. The test vessels of Medium 1 is incubated at 30 - 35°C and
the vessels of Medium 2 is incubated at 20- 25°C.
The volume of Medium 1 should be such that the air space above
the medium in the container is minimized. The volume of Medium
2 should be such that sufficient air space is left above the medium
to provide conditions that permit the growth of obligate aerobes.
Unless otherwise prescribed, in no case should the volume of
material under test be greater than 10% of the volume of the
medium alone, i.e, 90% medium and 10% product. If a large volume
of product is to be tested it may be preferable to use concentrated
media, prepared so as to take the subsequent dilution into account.

27. Where appropriate the concentrated medium may be added
directly to the product in its container. Wherever possible solid
articles such as devices should be tested by immersion in or filling
with culture media. Immerse all parts of each article in sufficient
medium contained in one vessel to completely cover all parts. The
volume of Medium 1 should be such that the air space above the
medium in the container is minimized. The volume of Medium 2
should be such that sufficient air space is left above the medium to
provide conditions that permit the growth of obligate aerobes.
Place half the articles into Medium 1 and the remaining half into
Medium 2. Incubate the test vessels of Medium 1 at 30 - 35°C and
the vessels of Medium 2 at 20 - 25°C.

28. Ointments and oily preparations: Ointments and oily
preparations may be tested by the method of Direct Transfer if
testing by the method of Membrane Filtration is not feasible, i.e.
when a suitable solvent is not available
Incubation and examination of sterility tests: All test vessels
of Medium 1 are incubated at 30 - 35°C. The vessels of Medium
2 are incubated at 20 - 25°C. All test and control vessels, other
than the subcultured vessels referred to below, must be
incubated for at least 14 days unless microbial contamination
is detected at an earlier time.
If turbidity, precipitate, or other evidence of microbial growth
during incubation is seen: the suspected growth is examined
microscopically by Gram stain; attempts are made to grow single
colonies using appropriate microbiological methods; colonies of
each type of micro-organism present are examined for colonial
morphology and cellular morphology by Gram stain; attempts are
made to identify the isolates, as far as the genus, and preferably

29. Interpretation of the test results: If microbial growth is not
evident in any of the vessels inoculated with the product, the
sample tested complies with the test for sterility, if microbial
growth is evident the product does not comply with the test for
sterility unless it can be clearly demonstrated that the test was
invalid for causes unrelated to the product being examined. If the
test is declared to be invalid it may be repeated with the same
number of units as in the original test. If there is no evidence of
growth in any vessels inoculated with the product during the repeat
test the product passes the test for sterility. This interpretation
applies even if growth occurs in negative product control vessels. If
there is evidence of growth in the test vessels the product fails the
test for sterility. Further testing is not permitted under any
circumstances

30. Evaluation of Sterilization Method
Sterile products possess several unique properties, such as freedom
from microorganism, pyrogens, particulates and high standards of
purity and quality. This ultimate goal in the manufacture of sterile
products can be attained by evaluation of sterilization procedure.
The sterilization processes are likely to be subjected to the most
detailed and complex validation procedures.
The judgment of sterility has relied on official sterility test. A
validated manufacturing procedure is one which has been proved
to do what it purports to do. The proof of evaluation is obtained
through the collection and evaluation of data, preferably beginning,
from the process development phase and continuing through the
production phase. Evaluation of processing includes equipments,
process, personnel, material etc.

31. The principle involve in the evaluation of sterilization process
are:
i. To build sterility into product.
ii. Perform a maximum level of probability.
iii. Establish specification and performance characteristic.
iv. To provide greater assurance of support of the result.
v. Specific methodology, process and equipment.
vi. Final product testing using validated analytical method and
vii. Verification, calibration and maintenance of equipments used
in the processes.

32. Evaluation of sterilization methods are done to ensure that
the product produce by design process should be of best
quality. The process control and finished product testing
alone are not sufficient to assure product quality. When
testing a specified portion of the total product and if the
specified portion passes the test of sterility, it cannot assure
that the total product is sterile.
Evaluation of sterilization methods provides a high degree of
assurance which indicates a specific process will consistently
produce a product that will meets it predetermined
specifications and quality assurance. So this action proves
that any procedure, process, equipments, material activity or
system actually leads to the expected result and produce
quality product. This concept of evaluation has been
expended to encompass a wide range of activities from
analytical methods used for quality control of drug substance
and drug products.