STERILITY TESTING

PHARMACEUTICAL MICROBIOLOGY
SEMESTER V

Dr. Jigar Shah

 Examples for which compliance with the test is
required
• Ready made injections – including solutions and
suspension, both aqueous and oily
• Solids for injection – including a number of materials from
biological sources, e.g. heparin, hyaluronidase, and the
antibiotics
• Eye drops, eye ointments and eye lotions
• Water for injections
• Immunological products – vaccines, antisera, and
diagnostic preparations
• Implants
• Catgut
• Absorbable haemostatics
Every batch of final containers must be tested for sterility, except in
special circumstances, issues are allowed until tests have been passed
Special circumstances are –
•Preparation required in emergency by medical practitioner and
manufacturer has no stock, tests are under process and results take
several days to come, so issue is allowed only when –
(a)Bulk from which the containers are filled has been tested and has
passed, and
(b)Tests on samples from some of the filled containers are set up,
examined daily and, if contamination is detected, the practitioner is
notified at once.
•When the substance is so unstable that it loses appreciable activity if
held until completion of the test. In this case the bulk test is waived.
E.g. Liquid BCG vaccines – freeze dried form
 Tests for Sterility

• The tests for sterility are intended for detecting the

presence of viable forms of micro-organisms in or
on pharmacopoeial preparations.
• The tests must be carried out under conditions
designed to avoid accidental contamination of the
product during the test.
• The working conditions in which the tests are
performed should be monitored regularly by
sampling the air and the surfaces of the working
area and by carrying out control tests.
 Tests for Sterility
• The tests are based upon the principle that if
micro-organisms are placed in a medium which
provides nutritive material and water, and kept at a
favourable temperature, the organisms will grow
and their presence can be indicated by a turbidity
in the originally clear medium.
• Sterility tests can only show that organisms
capable of growing in the test media under the
selected conditions are absent from the fraction of
the batch that has been tested
To obtain reliable results from sterility tests, it is necessary to –
• Take sufficient samples
• To use sensitive culture media
• During testing, need to reduce accidental contamination to a
minimum
• Careful supervision of operatives, regular air sampling and full scale
runs using N. broth
 SAMPLING OF LOTS
• The sampling schedules set out the minimum
number of items to be sampled from each batch (lot)
and the minimum quantity to be tested from each
container, unless otherwise justified and authorised.
• The batch size and conditions of manufacture
should be considered when planning a sampling
regimen. It is assumed that the product has been
manufactured under conditions designed to exclude
contamination.
• A batch of product is defined as a homogeneous
collection of sealed containers prepared in such a
manner that the risk of contamination is the same for
each of the units of the batch.
 Important aspects of sampling are:
1) Stages at which samples should be taken:
- For heat sterilized products, the need for tests on the
final containers which is the only stage where they
are relevant.
- For aseptically sterilized products, it is advisable to
test the bulk from which the final containers will be
filled. (i.e. testing at bulk as well final containers)
2) Selection of the samples:
- Samples must be representative of the whole of the
bulk material and the lot of final containers.
- For the bulk, material must be thoroughly mixed
before the sample is taken
- For final containers, the samples must be selected at
random, but-
Organization / Testing stage
Pharmacopoeia Bulk Final container

WHO √ √
Therap Sub X (except in √
Regulations emergency)
USP √ √ (IF bulk passed,
fewer containers)
EP X √
Int Pharm X √
Selection of the samples Continued…
(a) For terminally sterilised products the samples should
be made up from units drawn from various sites
throughout the steriliser load.
- Some of the units should be taken from that place in
the load known to be least accessible to the sterilising
agent and less satisfactory sterilizing conditions are
believed to exist.
- Samples may be taken representatively from across
each load.
Selection of the samples Continued…
b) For aseptically prepared products, the samples should
be taken at regular intervals during the filling
operations in such a way that every filling point is
represented by an approximately equivalent number of
samples.
- Further, the first and last items dispensed at each
filling point and the first item filled after any machine
break-down or change, any non-validated intervention
or interruption, should be included amongst the
samples.
- Also, an identical group of containers has been filled
with the same product from the same bulk lot.
 Selection of the samples Continued…
c) For articles sterilized by continuous process, such as
radiation sterilization, samples are selected from the
total number of similar items subjected to uniform
sterilization durian an appropriate period, which the
U.S.P. suggests should not exceed one day.

3) Sample Size:
Sampling Schedule - Minimum Number of Items to be tested
from each Batch
Minimum Quantity of Product to be tested from each container
in the Sample
 Culture Media
- Sterility test media must initiate and maintain the
vigorous growth of small numbers of –
(a) The aerobic and anaerobic bacteria that can be
cultivated on artificial culture media including –
(i) Common saprophytes,
(ii)Pyogenic cocci and
(iii)Spore bearing bacteria pathogenic to man
(b) The lower fungi, i.e. yeasts and moulds responsible
for spoilage.
- Separate media specifically designed for (i) aerobes,
(ii) anaerobes and (iii) the lower fungi.
 Culture Media…

- Some joint media such as broths can support

growth of aerobes and anaerobes, aerobes and
lower fungi or even all 3 types of organism.
- The WHO report lists a number of media used for
bacterial sterility testing and classifies them as
follows:
- 1) For aerobes: a) peptone broth,
b) glucose peptone broth
- 2) For anaerobes: a) Cooked-meat medium,
b) Semi fluid meat medium,
c) Liver broth
 Culture Media…

- 3) For aerobes and anaerobes: a) Fluid thioglycollate medium,

b) Soyabean-casein digest medium.
 Culture Media…

- Distribute into suitable vessels, sterilize in an autoclave

- Cool to 25º and store at 20º to 30º, avoiding excess of
light
- If more than upper one third has acquired a pink color,
medium may be restored once by reheating in a water-
bath until pink color disappears and cooling rapidly.
- Medium more than 3 weeks old should not be used.
- Use fluid thioglycollate medium by incubating it at 30º -
35º under aerobic conditions.
 Culture Media…

- Use soyabean-casein digest medium by incubating it

at 20º to 25º under aerobic conditions.
- The media used should comply with the following
tests carried out before or in parallel with the test on
the preparation being examined.
- Sterility: Incubate portions of the medium 1 at 30º -
35º and medium 2 at 20º - 25º for not less than 7
days; no growth of microorganisms occurs.
 Culture Media…

- Growth Promotion test: Test each autoclaved load

of each lot of the medium for its growth promoting
qualities by separately inoculating duplicate test
containers of each medium with about 100 viable
micro-organisms of each of strains listed in table 1
and incubating according to the conditions specified.
- The test media are satisfactory if clear evidence of
growth appears in all inoculated media containers
within 7 days.
- If freshly prepared media are not used within 2 days,
store them in dark, preferably at 2º to 25º.
 Culture Media…

- Tests for bacteriostasis and fingistasis:

- Prepare cultures of bacteria and fungi from the
strains of micro-organisms mentioned in table 1.
- Inoculate sterility test media with about 100 viable
microorganisms using volumes of medium listed for
liquids in table 2.
- Add specified portions of examine preparation to the
containers already containing the inoculum and
culture medium.
- Incubate the containers under the conditions listed in
table 1 for not less than 7 days.
 Culture Media…

- If preparation is bacteriostatic and/or fungistatic, use a

neutralizing agent.
- Specified amount of preparation and larger volumes of
medium used to determine the ratio of preparation to
medium in which the growth of organisms is not
adversely affected.
- If the specified amount of preparation is bacteriostatic
in the medium, decrease amount of preparation to find
the maximum amount that does not adversely affect the
growth of test organism in medium.
- For liquids and suspensions, if this amount is less than
1 ml, increase the quantity of medium so that 1 ml is
sufficiently diluted to prevent inhibition of growth.
 Test Procedures
- Method A – Membrane Filtration,
- Method B – Direct Inoculation.
- Method A – for a) an 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 or a liquid that possesses inherent
bacteristatic and fungistatic properties.
- For liquid products where the volume > 100ml or more
 Test Procedures…

- Precautions: A laminar sterile airflow cabinet – to

avoid accidental contamination.
- Working conditions monitored regularly by sampling
the air and surfaces of the working area.
- General Procedures: Antimicrobial agent and needle
attached to a syringe barrel filled with non-absorbent
cotton.
- Method A: a) Apparatus: A suitable unit consists of
a closed reservoir and a receptacle between which a
properly supported membrane of appropriate porosity
is placed. Nominal pore size not > 0.45µm and
diameter of approx. 47mm.
 Test Procedures…

- Diluting fluids:
- Fluid A: Dissolve 1g of peptic digest of animal
tissue in water to make 1 liter, filter / centrifuge,
adjust to pH 7.1±0.2, dispense into flasks in 100-ml
quantities and sterilise at 121º for 20 minutes.

- Fluid B: If the test sample contains lecithin or oil,

use fluid A to each liter of which has been added 1
ml of polysorbate 80, adjust to pH 7.1±0.2, dispense
into flasks in 100-ml quantities and sterilise at 121º
for 20 minutes.
 Test Procedures…
- Method of test:
1) For aqueous solution:
- Prepare each membrane by aseptically transferring
fluid A + media + preparation being examined.
- Alternatively, first, combined quantities of preparation
+ prescribed in the two media.
- Draw the liquid rapidly through filter with aid of
vacuum
- If the solution being examined has antimicrobial
properties, wash the membrane by filtering 100ml of
sterile fluid A, three times, or quantities should be
sufficient to allow growth of small inoculum of
organisms.
 Test Procedures…
- After filtration, aseptically remove membrane from
holder, cut the membrane in half, immerse one half
of membrane in 100ml of soyabean-casein digest
medium and incubate at 20º to 25º for not < 7days.
- Similar, other membrane in FTM (30º to 35º).
2) For liquids immiscible with aqueous vehicles and
suspensions:
- Same as above but add sufficient quantity of fluid A
to achieve rapid filtration
- Sometimes use sterile enzyme preparations such as
penicillinase or cellulase to aid in dissolving
insoluble substances.
- If lecithin is there, use fluid B for diluting.
 Test Procedures…
3) For oils and oily solutions:
- Low viscous, filter without dilution th’gh dry memb.
- Viscous oils – dilute as per needed using sterile
diluent like isopropyl myristate – filter by applying
pressure or suction gradually.
- Wash the membrane at least 3 times sterile fluid B
(100 ml) or to heat not > than 45º and use warm
solutions for washing membrane.

4) For ointments and creams:

- Dilute ointments in a fatty base and emulsions of the
w/o type to give a fluid concentration of 1%w/v, by
heating (if necessary), not > than 40º with a suitable
sterile diluent.
 Test Procedures…

5) For soluble solids:

- Dissolve substance in a suitable sterile solvent for each
medium and carry out test described under for aqueous
solutions.
6) For sterile devices:
- Aseptically pass a sufficient volume of fluid B through
each of not less than 20 devices so that not less than
100ml is recovered from each device.
- Collect fluids in sterile containers and filter the entire
volume through membrane filter funnel, and follow the
test as under for aqueous solutions.
Method B Direct Inoculation
- Quantities of the sample to be used
 Direct Inoculation…
- Method of test
1) For aqueous solutions and suspensions:
Remove liquid from test containers with a sterile pipette
or with a sterile syringe or a needle

Aseptically transfer specified volume of the material from

each container to a vessel of the culture medium

Mix the liquid with the medium but do not aerate

excessively

Incubate the inoculated media for not less than 14 days,

unless otherwise specified in the monograph.
 Direct Inoculation…
- Incubate at 30º to 35º in case of fluid thioglycollate
medium and at 20º to 25º in case of soyabean casein
digest medium.
- If test sample renders the medium turbid, it is difficult
to examine presence or absence of microbial growth
by visual examination.
- Transfer suitable portions of the same medium to
fresh vessels between the third and seventh days after
test is started.
- Continue incubation of transfer vessels for not less
than 7 additional days after the transfer and for a total
of not less than 14 days.
 Direct Inoculation…
2) For oils and oily solutions:
- In media, add 0.1% w/v of (4 –tert -octylphenoxy)
polyethoxyethanol, 1% w/v of polysorbate 80 or
other suitable emulsifying agent, in an appropriate
conc.
- Oil containing cultures should be shaken gently each
day.

3) For ointments:
- Preparation is diluted ten fold in a sterile diluent such
as fluid B or any other aqueous vehicle capable of
dispersing test material homogenously throughout the
fluid mixture.
- Mix 10ml of fluid mixture with 80 ml of the medium
 Direct Inoculation…
4) For solids:
- Mix, and incubate same as 1)

5) For sterile devices:

- For articles of such size and shape as permit complete
imersion in not more than 1000ml of culture medium test
the intact article, using the appropriate media and
incubate same as 1).
 Direct Inoculation…

- For transfusion or infusion assemblies or where the

size of an item makes immersion impracticable, flush
the lumen of each of 20 units with a sufficient quantity
of fluid thioglycollate medium and soyabean casein
medium to yield a recovery of not less than 15ml of
each medium, and incubate with not less than 100 ml
of each of two media.
- Where the presence of the specimen being tested,
interferes with test because of bacteriostatic action,
rinse the article thoroughly with minimum amount of
fluid A. Recover the rinsed fluid and test as described
for sterile devices under method A.
 Observation and Interpretation of Results
- At interval during incubation period, and at its
conclusion, examine media for macroscopic evidence of
microbial growth.
- If no evidence of growth is found, the preparation being
examined passes the test for sterility.
- If evidence of microbial growth is found, reserve the
containers, and it is demonstrated that causes not due to
preparation, hence the tests for sterility are invalid and
may be recommenced.
- Perform a retest using the same number of samples,
volumes to be tested and the media – if no evidence of
microbial growth is then found, the preparation being
examined passes the test for sterility.
 Observation and Interpretation of Results…

- If evidence of microbial growth is found, isolate and

identify the organisms.
- If they are not readily distinguishable from those
growing in the containers reserved in the first test, the
preparation being examined fails the test for sterility.
- If they are readily distinguishable from those growing
in the containers reserved in the first test, perform a
second retest using the twice number of samples.
- If no evidence of microbial growth is found in second
retest, the preparation being examined passes the test
for sterility.
- If evidence of growth of any micro-organisms is found
in second retest, the preparation being examined fails
the test for sterility.
 CONTROL TESTS
• The results of sterility tests relied upon either
negative result (sterility of the sample) or positive
result (contamination of the result)
• A negative result could also be due to –
1) Inability of the broth to support microbial growth. The
causes of this
a) Inadequate formulation
b) Accidental omission of an ingredient
c) Overheating during preparation and sterilization
d) In anaerobic media, failure to boil off oxygen from
excessively oxygenated containers
 CONTROL TESTS
2) Inhibition of Contaminants by a substance added in
the test. This could be-
a) The sample itself
b) A neutralising agent destroy the antibacterial effect
of an ingredient of the sample
• A positive result could be caused by-
1) Lack of sterility of medium
2) Accidental contamination during testing

• Therefore, control tests are essential to show that

these factors are most unlikely to be the explanation
of the result.
 NEGATIVE CONTROLS
• Here, no growth is expected.
(a) A container of medium from each batch used for the test is
incubated at the same time as the test containers
• This control serves three purposes –
(i) It confirms that the medium is sterile
(ii) It shows the oxidation-reduction qualities of indicator
containing anaerobic media are satisfactory.
(iii) It serves as a standard with which the corresponding test
container can be compared during and after incubation.
(b) Any substance, other than the sample, added to a test tube
should be proved sterile by incubating suitable amounts in
appropriate media.
 POSITIVE CONTROLS (FERTILITY TESTS)
• In this, growth is expected
(a) The sensitivity of media must be confirmed to check that it
has not been affected by any error in preparation or storage.
• Each type of medium is inoculated with an appropriate
organism and after incubation under suitable conditions is
examined for growth.
• Europ Pharm suggests – S. aureus as aerobe
- Plectridium sphenoides as anaerobe (spore containing
m.org.)
- Candida albicans as the yeast
 POSITIVE CONTROLS (FERTILITY TESTS)
(b) The medium must be shown capable of supporting the
growth of small numbers of bacteria in the presence of
sample.
• Organisms are added to containers in which there are exactly
the same quantities of the same inclusions as in the tests
• The EP requires at least two tubes for each type of organism.
• The bacteria are incubated at 30 to 32˚C for 7 days and the
yeast at 22 to 25˚C for 2 days.
3) Controls to check working conditions and operators’
technique
• Used at regular and frequent intervals to confirm that the risk
of accidental contamination is low
 POSITIVE CONTROLS (FERTILITY TESTS)

a) General air sampling: High quality of the air supply to

the room is being maintained
b) Air sampling at each working space: Settling plates at
and near the working place help to detect poor technique
and excessive movement
c) Dummy runs: Tests are performed with materials known
to be sterile, e.g. ampoules or bottles of Water for
Injections or Sodium Chloride have been sterilized for
longer times and at higher temperature
• The operators should not be aware that the run is a
control because this might encourage the exceptional care.
 Modifications in filtration technique
• Davies and Fishburn pointed out several drawbacks in
official method of sterility testing like
1) Tremendous efforts required to discover which
medicaments inhibitory and to find suitable neutralizing
agents
2) Loss of sensitivity of medium due to high dilution is used
to overcome inhibitory effects
• Recommended a different technique where sterile
bacteria proof asbestos cellulose filter pad was used. It is
used to retain organisms on a pad and bactericide and
inhibitory medicaments are filtered by washing with
sterile solvent.
 Modifications in filtration technique
• Sykes and Hooper developed a new method in which pad
was replaced by a membrane filter.
• Advantages
- Membrane are so thin that retention of inhibitory substances
is very small
- Quicker filtration
- For oil, not need to dissolve them in an organic solvent first.
Oils pass through easily and quickly
- The light peroleum may adversely affect the viability of
some organisms.
 Advantages of Filtration Technique
• Wide application – can be used for solutions, soluble solid,
insoluble solids, oils, ointments, syringes like articles
• Very large volume can be tested with one pad
• Small volume of broth is required than direct inoculation
into culture media
• Apply to substances for which no satisfactory inactivators
are known
• Some strong antibacterial agents like mercurials and
quaternary ammonium compounds, inactivated by treatment
with appropriate neutralising solution.
• Subculturing is often eliminated
 Disadvantages of Filtration Technique

• The possibility of adsorption of sufficient medicament

• Highly skilled staff and exceptionally good aseptic
techniques are necessary.
• A vertually sterile environment is essential
• A room with laminar air flow and conventional asepsis
laboratory is needed.