Sterility
The test for sterility is applied to pharmacopoeial articles that are required according to the
Pharmacopoeia to be sterile. However, a satisfactory result only indicates that no contaminating viable
microorganisms have been found in the sample examined in the conditions of the test. If the number of
microorganisms present in a given amount of the article under examination is large, the probability of
detecting them increases. Very low levels of contamination cannot be detected on the basis of random
sampling of a lot. Moreover, if contamination is not uniform throughout the lot, random sampling
cannot detect contamination with any certainty. Compliance with the test for sterility alone cannot
therefore provide absolute assurance of freedom from microbial contamination. Greater assurance of
sterility must come from reliable manufacturing procedures and compliance with good manufacturing
practices.
The test must be carried out under aseptic conditions designed to avoid accidental contamination of the
product during testing. For achieving these conditions, a grade A laminar air-flow cabinet or an isolator
is recommended. The test environment has to be adapted to the way in which the tests are performed.
Precautions taken for this purpose should not adversely affect any microorganisms, which are to be
revealed in the tests. The working conditions in which the tests are carried out should be monitored
regularly by appropriate sampling of the air and surfaces of the working area and by carrying out
control tests.
The test is designed to reveal the presence of microorganisms in the samples used in the test;
interpretation of the results of testing is based on the assumption that all units of an article or the entire
bulk product or the contents of every container of the filled product in a lot or batch, had they been
tested, would also have given the same results. Since all the units or the bulk or all the containers cannot
be tested, a sufficient number of samples of units or of containers should be examined to give a suitable
degree of confidence in the results of the tests.
No sampling plan for applying the tests to a specified proportion of discrete units selected from a batch
is capable of demonstrating that all of the untested units are in fact sterile. Therefore, in determining the
number of units to be tested, the manufacturer should have regard to the environmental conditions of
manufacture, the volume of preparation per container and other special considerations particular to the
preparation being examined. Table 1 gives guidance on the minimum number of containers
recommended to be tested in relation to the number of items in the batch on the assumption that the
preparation has been manufactured under conditions designed to exclude contamination. If the contents
of each container are of sufficient quantity, they may be divided so that equal appropriate portions are
added to each of the specified media. If any container does not contain sufficient quantity of material to
inoculate each of the specified media, use twice the minimum number of containers indicated in the
table 1.
1
Table 1
---------------------------------------------------------------------------------------------------------------------------* Number of containers in the batch
**Minimum number of
containers recommended
to be tested
---------------------------------------------------------------------------------------------------------------------------1. Parenteral preparations
Not more than 100
containers
10 per cent or 4
containers,
whichever is greater
10 containers
More than 100 but not more
than 500 containers
More than 500 containers
2 per cent or 20
containers,
whichever is less
2 per cent or 10containers,
whichever is less, unless otherwise
justified & authorized.
For large-volume parenterals
2. Ophthalmic and other non-parenteral preparations
Not more than 200
containers
More than 200 containers
5 per cent or 2 containers,
whichever is greater
10 containers
3. Surgical dressings and devices
Catgut, surgical sutures and
other sterile medical devices
for veterinary use
2 per cent or 5 packages,
whichever is greater,
up to a maximum of
20 packages
10 per cent or 4
packages,
whichever is greater
10 packages
Not more than 100
packages
More than 100 but not
more than 500 packages
More than 500 packages
2 per cent or 20 packages,
whichever is less
4. Bulk solids
Up to 4 containers
More than 4 containers but not more
than 50 containers
Each container
20 per cent or 4
containers, whichever is greater
More than 50 containers
2 per cent or 10 Containers,
whichever is greater
* If the batch size is unknown use maximum number of articles prescribed
**If the contents of one container are enough to inoculate the two media, this column gives the number
of containers needed for both the media together.
2
This sampling is, however, applicable at manufactures, not applicable to the inspectors under the Drugs
and Cosmetics Act for drawing statutory samples and for government analyst.
Culture Media
Media for the tests may be prepared as described below, or equivalent commercially available
dehydrated mixtures yielding similar formulations may be used provided that when reconstituted as
directed by the manufacturer, they comply with the growth promotion test. Other media may be used
provided that they have been shown to sustain the growth of a wide range of microorganisms.
The following culture media have been found to be suitable for the test. Fluid thioglycollate medium is
primarily intended for the culture of anaerobic bacteria; however, it will also detect aerobic bacteria.
Soyabean-casein digest medium is suitable for the culture of both fungi and aerobic bacteria.
Fluid Thioglycollate Medium – For use with clear fluid products.
L-Cystine
0.5g
Sodium chloride
2.5 g
Dextrose monohydrate/anhydrous
5.5 g/5.0 g
Granular agar (moisture content
less than 15 per cent, w/w)
0.75 g
Yeast extract (water-soluble)
5.0 g
Pancreatic digest of casein
15.0g
Sodium thioglycollate or
0.5 g
Thioglycollic acid
0.3 ml
Resazurin sodium solution
(0.1 per cent), freshly prepared
1.0 ml
Distilled water to
1000 ml
pH of the medium after sterilisation 7.1 ± 0.2
Mix the ingredients other than the thioglycollate or thioglycollic acid and the resazurin sodium solution,
in the order given above, in a mortar, with thorough grinding. Stir in some heated distilled water,
transfer to a suitable container, add the remainder of the distilled water, and complete the solution by
heating in a boiling water-bath. Dissolve the sodium thioglycollate or thioglycollic acid in the solution
and, if necessary, add 1M sodium hydroxide so that, after sterilisation, the solution will have a pH of 7.1
± 0.2. If filtration is necessary, heat the solution again without boiling and filter while hot through
moistened filter paper. Add the resazurin sodium solution, mix and distribute the medium into suitable
vessels that provide a ratio of surface to depth of medium such that not more than the upper half of the
medium has undergone a colour change indicative of oxygen uptake at the end of the incubation period.
Sterilise in an autoclave at 121º for 20 minutes. If the medium is to be stored, cool promptly to 25º and
store at 2º to 25º, avoiding excess of light. If more than the upper one-third of the medium has acquired
a pink colour, the medium may be restored once by reheating in a water-bath or in free-flowing steam
until the pink colour disappears, and cooling rapidly, taking care to prevent the introduction of nonsterile air into the container. When ready for use, not more than the upper one-tenth of the medium
should have amore than the upper one-tenth of the medium should have a pink colour. Medium more
than 4 weeks old should not be used.
Use fluid thioglycollate medium by incubating it at 30º to 35º.
Alternative Thioglycollate Medium — For use with turbid and viscid products and for devices having
tubes with small lumina.
3
L-Cystine
0.5 g
Sodium chloride
2.5 g
Dextrose monohydrate/anhydrous 5.5 g/5.0 g
Yeast extract (water-soluble)
5.0 g
Pancreatic digest of casein
15.0 g
Sodium thioglycollate or
0.5 g
Thioglycollic acid
0.3 ml
Distilled water to
1000 ml
pH of the medium after sterilisation 7.1 ± 0.2
Heat the ingredients in a suitable container until solution is effected. Mix, add 1M sodium hydroxide, if
necessary, so that, after sterilisation, the medium will have a pH of 7.1 ± 0.2. Filter, if necessary, place
in suitable vessels and sterilise at 121º for 20 minutes. Store at a temperature between 2º and 25º in a
sterile sealed container, unless it is intended for immediate use.
The medium is freshly prepared or heated in a water-bath and allowed to cool just prior to use. It should
not be reheated.
Use alternative thioglycollate medium in a manner that will assure anaerobic conditions for the duration
of the incubation at 30º to 35º.
Soyabean-casein Digest Medium
Pancreatic digest of casein
Papaic digest of soyabean meal
Sodium chloride
Dipotassium hydrogen phosphate
(K2HPO4)
17.0 g
3.0 g
5.0 g
2.5 g
Dextrose monohydrate/anhydrous 2.5 g/2.3 g
Distilled water to
1000 ml
pH of the medium after sterilisation 7.3 ± 0.2
Dissolve the solids in distilled water, warming slightly to effect solution. Cool to room temperature and
add, if necessary, sufficient 1M sodium hydroxide so that after sterilisation the medium will have a pH
of 7.3 ± 0.2. Filter, if necessary, distribute into suitable containers and sterilise in an autoclave at 121º
for 20 minutes.
Use soyabean-casein digest medium by incubating it at 20º to 25º under aerobic conditions.
Media for Penicillins and Cephalosporins
Where sterility test media are to be used in Method B described under Test Procedures modify the
preparation of fluid thioglycollate medium and the soyabean-casein digest medium as follows. To the
containers of each medium, transfer aseptically a quantity of penicillinase sufficient to inactivate the
amount of antibiotic in the sample under test. Determine the appropriate quantity of penicillinase to be
used for this purpose by using a penicillinase preparation that has been assayed previously for its
penicillin- or cephalosporin-inactivating power.
NOTE — Supplemented penicillinase media can also be used in Method A.
Alternatively (in an area completely separate from that used for sterility testing) confirm that the
appropriate quantity of penicillinase is incorporated into the medium, following either method under
Validation of Tests, using less than 100 CFU of Staphylococcus aureus (ATCC 6538) as the challenge.
Typical microbial growth of the inoculated culture must be observed as a confirmation that the
penicillinase concentration is appropriate.
4
Suitability of Media
The media used should comply with the following tests, carried out before or in parallel with the test on
the preparation under examination.
Sterility. Incubate portions of the media for 14 days at the temperatures indicated under each medium.
No growth of microorganisms occurs.
Growth Promotion Test. Test each autoclaved load of each lot of the medium for its growthpromoting qualities using suitable strains of microorganisms indicated in Table 2.
Inoculate duplicate portions of each medium with a small number (not more than 100 CFU) of the
microorganisms specified, using separate portions of the medium for each of the microorganisms and
incubating according to the conditions specified in Table 2.
The media are suitable if a clearly visible growth of the microorganisms occurs. The tests may be
conducted simultaneously with any test for sterility done using the same lot of media. However, such
tests will be considered invalid if the test media show inadequate growth response.
If freshly prepared media are not used within 2 days, they should be stored in the dark, preferably at 2º
to 25º. Finished media, if stored in unsealed containers, may be used for not more than one month
provided they are tested within one week of use.
Validation of Tests. Carry out a test as described under Test Procedures using exactly the same
methods with the following modifications.
----------------------------------------------------------------------------------------------------------------------------Table 2
----------------------------------------------------------------------------------------------------------------------------Medium
Test micro-organism
Incubation
----------------------------------------------Temp(0) Duration
Type of
micro-organism
Fluid Thioglycollate 1. Clostridium sporogens (ATCC1 19404)
2. Staphlococcus aureus( ATCC 6538)
2
3. Pseudomonas aeruginosa (ATCC 9027)
30 to 35
30 to 35
30 to 35
3days
3days
3days
Anaerobic
Aerobic
Aerobic
Alternative
Thioglycollate
1.Bacteroides vulgatus (ATCC 8482)
30 to 35
2. Clostridium sporogenes (ATCC 19404)
30 to 35
2
3. Bacillus subtilis (ATCC 6633; NCIMB 8054) 30 to 35
3days
3days
3days
Anaerobic
Anaerobic
Aerobic
Soyabean-Casein
Digest
1. Aspergillus brasilensis (ATCC 16404)
20 to 25
2. Candida albicans (ATCC 10231;
20 to 25
4
ATCC 2091; NCYC 854)
5days
5days
Aerobic
Aerobic
3. Bacillus subtilis (ATCC6633; NCIMB5 8054) 30 to35
3days
Aerobic
3
1. Available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852,
USA.
2. An alternative micro-organism is Micrococcus luteus (ATCC No. 9341).
3. If a spore-forming organism is desired, use Clostridium sporogenes (ATCC No. 11437) at the
incubation temperatures indicated in the Table.
5
4. Available from National Collection of Yeast Cultures, AFRC Food Research Institute, Colney Lane,
Norwich NR4 7UA, England.
5. Available from National Collection of Industrial and Marine Bacteria Ltd, 23 St Machar Drive,
Aberdeen, AB2 IRY, Scotland.
NOTE — Seed lot culture maintenance techniques (seed-lot systems) should be used so that the viable
micro-organisms used for inoculation are not more than 5 passages removed from the original master
seed-lot.
Membrane Filtration. After transferring the contents of the container or containers to be tested to the
membrane add an inoculum of a small number of viable microorganisms (not more than 100 CFU) to
the final portion of sterile diluent used to rinse the filter.
Direct Inoculation. After transferring the contents of the container or containers to be tested to the
culture medium add an inoculum of a small number of viable microorganisms (not more than 100 CFU)
to the medium.
In both cases use the same microorganisms as those described under Growth Promotion Test. Perform a
growth promotion test as a positive control. Incubate all the containers containing medium for not more
than 5 days.
If clearly visible growth of microorganisms is obtained after the inoculation, visually comparable to that
in the control vessel without product, either the product possesses no antimicrobial activity under the
conditions of the test or such activity has been satisfactorily eliminated. The test for sterility may then
be carried out without further modification.
If clearly visible growth is not obtained in the presence of the product under examination, visually
comparable to that in the control vessels without product, the product possesses antimicrobial activity
that has not been satisfactorily eliminated under the conditions of the test. A suitable sterile neutralising
agent may be used where the preparation under examination has antimicrobial activity. If a neutralising
agent is not available, modify the amounts of the preparation and medium to be used in order to
eliminate antimicrobial activity and repeat the validation test. Where the specified amounts of the
preparation have antimicrobial activity in the medium, increase the quantities of medium so that the
specified quantity of the preparation is sufficiently diluted to prevent inhibition of growth.
This validation is performed
(a) when the test for sterility has to be carried out on a new product,
(b) whenever there is a change in the experimental conditions of the test.
The validation may be performed simultaneously with the test for sterility of the substance or
preparation under examination.
Test Procedures
Either of the following methods, Method A – Membrane Filtration or Method B – Direct Inoculation,
may be followed. Method A is to be preferred where the substance under examination is (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 bacteriostatic and/or fungistatic properties.
Appropriate negative controls are included.
For liquid products where the volume in a container is 100 ml or more, Method A should be used.
Select the number of samples to be tested from Table 1 and use them for the culture medium for
bacteria and the culture medium for fungi.
General. The exterior surface of ampoules and closures of vials and bottles should be cleaned with a
suitable antimicrobial agent and access to the contents should be gained in a suitable aseptic manner. If
6
the contents are packed in a container under vacuum, sterile air should be admitted by means of a
suitable sterile device, such as a needle attached to a syringe barrel filled with non-absorbent cotton.
Method A – Membrane Filtration
The method calls for the routine use of positive and negative controls. A positive control is small
number (not more than 100 CFU) of microorganisms specified in separate portion of each medium.
Apparatus
A suitable unit consists of a closed reservoir and a receptacle between which a properly supported
membrane of appropriate porosity is placed. A membrane generally suitable for sterility testing has a
nominal pore size not greater than 0.45µ and diameter of approximately 50 mm, the effectiveness of
which in retaining microorganisms has been established. Cellulose nitrate filters are used for aqueous,
oily and weakly alcoholic solutions and cellulose acetate filters, for strongly alcoholic solutions.
Preferably assemble and sterilise the entire unit with the membrane in place prior to use. Where the
sample to be tested is an oil, sterilise the membrane separately and, after thorough drying, assemble the
unit using aseptic precautions.
Diluting Fluids
Fluid A. Dissolve 1 g of peptic digest of animal tissue (such as bacteriological peptone) or its
equivalent in water to make 1 litre, filter or centrifuge to clarify, adjust to pH 7.1 ± 0.2, dispense into
flasks in 100-ml quantities and sterilise at 121º for 20 minutes.
NOTE — Where fluid A is to be used in performing the test for sterility on a specimen of the penicillin
or cephalosporin class of antibiotics, aseptically add a quantity of sterile penicillinase to the fluid A to
be used to rinse the membrane(s) sufficient to inactivate any residual antibiotic activity on the
membrane(s) after the solution of the specimen has been filtered.
Fluid B. If the test sample contains lecithin or oil, use fluid A to each litre of which has been added 1
ml of polysorbate 80, adjust to pH 7.1 ± 0.2, dispense into flasks and sterilise at 121º for 20 minutes.
NOTE — A sterile fluid shall not have antibacterial or antifungal properties if it is to be considered
suitable for dissolving, diluting or rinsing a preparation being examined for sterility.
Quantities of Sample to be used
For parenteral preparations. Whenever possible use the whole contents of the container, but in any
case not less than the quantities prescribed in Table 3, diluting where necessary to about 100 ml with a
suitable diluent such as fluid A.
For ophthalmic and other non-parenteral preparations. Take an amount within the range prescribed
in column (A) of Table 4, if necessary, using the contents of more than one container, and mix
thoroughly. For each medium use the amount specified in column (B) of Table 4, taken from the mixed
sample.
Method of Test
For aqueous solutions. Prepare each membrane by aseptically transferring a small quantity (sufficient
to moisten the membrane) of fluid A on to the membrane and filter it. For each medium to be used,
transfer aseptically into two separate membrane filter funnels or to separate sterile pooling vessels prior
to transfer not less than the quantity of the preparation under examination that is prescribed in Table 3
or Table 4. Alternatively, transfer aseptically the combined quantities of the preparation under
examination prescribed in the two media onto one membrane. Draw the liquid rapidly through the filter
with the aid of vacuum. If the solution under examination has antimicrobial properties, wash the
membrane(s) by filtering through it (them) not less than three successive quantities, each of 100 ml, of
sterile fluid A. Do not exceed a washing cycle of 5 times 100 ml per filter, even if it has been
7
demonstrated during validation that such a cycle does not fully eliminate the antimicrobial activity. The
quantities of fluid used should be sufficient to allow growth of a small inoculum of organisms
(approximately 100 CFU) sensitive to the antimicrobial substance in the presence of the residual
inhibitory material on the membrane.
After filtration, aseptically remove the membrane(s) from the holder, transfer the whole membrane or
cut it aseptically into 2 equal parts. Transfer one half to each of two suitable media. Use the same
volume of each medium as in the procedure for
Validation of Tests. Alternatively, transfer the medium onto the membrane in the apparatus. Incubate
the media for not less than 14 days.
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.
For liquids immiscible with aqueous vehicles, and suspensions. Carry out the test described under
For aqueous solutions but add a sufficient quantity of fluid A to the pooled sample to achieve rapid
filtration. Sterile enzyme preparations such as penicillinase or cellulase may be added to fluid A to aid
in dissolving insoluble substances. If the substance being examined contains lecithin, use fluid B for
diluting.
For oils and oily solutions. Filter oils or oily solutions of sufficiently low viscosity without dilution
through a dry membrane. Dilute viscous oils as necessary with a suitable sterile diluent such as
isopropyl myristate that has been shown not to have antimicrobial properties under the conditions of the
test. Allow the oil to penetrate the membrane and filter by applying pressure or by suction, gradually.
Wash the membrane by filtering through it at least three successive quantities,
Table 3
---------------------------------------------------------------------------------------------------------------------------Quantity in each container
Minimum quantity to be used for
of injectable preparation
each culture medium
---------------------------------------------------------------------------------------------------------------------------For liquids
Less than 1 ml
Total contents of a container
1 - 40 ml
Half the contents of a container, but not less than 1 ml
More than 40 ml but not more than 100 ml
20 ml
More than 100 ml
10 per cent of the contents of a 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
The whole contents of each container to provide
not less than 200 mg
For solids
Less than 50 mg
Total contents of a container
50 mg or more but less than 300 mg
Half the contents of a container but not less than 50
mg.
300 mg to 5g
150 mg
more than 5g
500 mg
8
For catgut and other surgical sutures for
veterinary use
3 sections of a strand (each 30 cm long)
For surgical dressings/cotton/gauze (in packages)
100 mg per package
For sutures and other individually packed single
Use materials.
The whole device.
Other Medical devices
The whole device or material, cut into pieces or
disassembled
----------------------------------------------------------------------------------------------------------------------------Table 4
---------------------------------------------------------------------------------------------------------------------------Type of preparation
Quantity to be mixed
(A)
Quantity to be used for
each culture medium
(B)
-------------------------------------------------------------------------------------------------------------------Ophthalmic solutions; other than non-parenteral
liquid preparations
10 to 100 ml
5 to 10 ml
Other preparations; preparations soluble in water
or appropriate solvents; insoluble preparations to
be suspended or emulsified (ointments and creams)
1 to 10 g
0.5 to 1 g
Absorbent cotton
Not less than 1 g*
----------------------------------------------------------------------------------------------------------------------------* One portion
quantities, each of approximately 100 ml, of sterile fluid B or any other suitable sterile diluent.
Complete the test described under
For aqueous solutions, beginning at the words “After filtration,……….”.
For ointments and creams. Dilute ointments in a fatty base and emulsions of the water-in-oil type to
give a fluid concentration of 1 per cent w/v, by heating, if necessary, to not more than 40º with a
suitable sterile diluent such as isopropyl myristate previously rendered sterile by filtration through a
0.22 µm membrane filter that has been shown not to have antimicrobial properties under the conditions
of the test. Filter as rapidly as possible and complete the test as described under For oils and oily
solutions, beginning at the words “Wash the membrane by ………”. In exceptional cases, it may be
necessary to heat the substance to not more than 44º and to use warm solutions for washing the
membrane.
NOTE — For ointments and oils that are insoluble in isopropyl myristate, use Method B.
For soluble solids. For each medium, dissolve not less than the quantity of the substance under
examination, as prescribed in Tables 3 and 4, in a suitable sterile solvent such as fluid A and carry out
the test described under For aqueous solutions using a membrane appropriate to the chosen solvents.
For solids for injection other than antibiotics. Constitute the test articles as directed on the label, and
carry out the test as described under For aqueous solutions or For oils and oily solutions, as applicable.
9
NOTE — If necessary, excess diluent may be added to aid in the constitution and filtration of the
constituted article.
For antibiotic solids, bulks, and blends. Aseptically remove a sufficient quantity of solids from the
appropriate amount of containers prescribed in Table 3, mix to obtain a composite sample, equivalent to
about 6 g of solids, and transfer to a sterile flask. Dissolve in about 200 ml of fluid A, and mix. Carry
out the test as described under For aqueous solutions.
For antibiotics in packages of 5 g or less. From each of 20 containers, aseptically transfer about 300
mg of solids into a sterile flask, dissolve in about 200 ml of fluid A and mix, or constitute as directed on
the label of containers and transfer a quantity of liquid or suspension, equivalent to about 300 mg of
solids into a sterile flask, dissolve in about 200 ml of fluid A, and mix. Carry out the test as described
under For aqueous solutions or For oils and oily solutions, as appropriate.
Devices with Pathways labeled Sterile: Aseptically pass not less than 10 pathway volumes of fluid B
through each of not less than 20 devices tested. Collect the fluid in sterile containers and filter the entire
volume through the membrane filter the entire volume through the membrane filter funnel(s) as
described under For aqueous solution or oil or oily solution.
In the case of sterile, empty syringes, draw sterile diluent into the barrel through the sterile needle, if
attached, or through a sterile needle attached for the purpose of the test and express the contents into a
sterile polling vessel. Proceed as directed above.
For catheters where the inside lumen and outside surface are required to be sterile, either cut them into
pieces such that the medium is in contact with the entire lumen or full the lumen with medium and then
immerse the intact unit.
Method B – Direct Inoculation
Quantities of Sample to be used
The quantity of the substance or preparation under examination to be used for inoculation in the culture
media varies according to the quantity in each container. Follow the directions given in Table 3.
Method of Test
For aqueous solutions and suspensions. Remove the liquid from the test containers with a sterile
pipette or with a sterile syringe or a needle. Transfer the quantity of the preparation under examination
prescribed in Table 4 directly into the culture medium so that the volume of the preparation under
examination is not more than 10 per cent of the volume of the medium, unless otherwise prescribed.
When the quantity in a single container is insufficient to carry out the tests, the combined contents of
two or more containers are to be used to inoculate the media.
If the preparation under examination has antimicrobial activity, carry out the test after neutralising this
with a suitable neutralising substance or by dilution in a sufficient quantity of culture medium. When it
is necessary to use a large volume of the product it may be preferable to use a concentrated culture
medium prepared in such a way that it takes account of the subsequent dilution. Where appropriate, the
concentrated medium may be added directly to the product in its container.
Incubate the inoculated media for not less than 14 days (irrespective of method of sterilization).
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.
For oils and oily solutions. Use media to which has been added a suitable emulsifying agent at a
concentration shown to be appropriate in the validation of the test, for example, polysorbate 80 at a
concentration of 10 g per l and which has been shown not to have any antimicrobial properties under
the conditions of the test. Carry out the test as described under for aqueous solutions and suspensions.
10
During the incubation period shake the cultures gently each day. However, when thioglycollate medium
or other similar medium is used for the detection of anaerobic microorganisms keep shaking or mixing
to a minimum in order to maintain anaerobic conditions.
For ointments and creams. Prepare by diluting to about 1 in 10 by emulsifying with the chosen
emulsifying agent in a suitable sterile diluent such as fluid A. Transfer the diluted product to a medium
not containing an emulsifying agent. (Before use, test the emulsifying agent to ascertain that in the
concentration used it has no significant antimicrobial effects during the time interval for all transfers).
Mix 10 ml of the fluid mixture so obtained with 80 ml of the medium and proceed as directed under For
aqueous solutions and suspensions.
For solids. Transfer the quantity of the preparation under examination to the quantity of medium
specified in Table 4 and mix. Proceed as directed under For aqueous solutions and suspensions.
For surgical dressings and related articles. From each package under examination, aseptically
remove two or more portions of 100 to 500 mg each from the innermost part of the sample. From
individually packaged, single-use materials, aseptically remove the entire article. Immerse the portions
or article in each medium, and proceed as directed under For aqueous solutions and suspensions.
For sterile devices. For articles of such size and shape that permit complete immersion in not more than
1000 ml of the culture medium, for large device , immerse completely those portion of the device that
comes into direct contact with patient test the article, using the appropriate media, and proceed as
directed under For aqueous solutions and suspensions.
For catheters where the inside lumen and outside are required to be sterile, either cut them into pieces or
fill the lumen with medium, and then immerse the intact unit.
Observation and Interpretation of Results
At intervals during the incubation period and at its conclusion, examine the media for macroscopic
evidence of microbial growth. If the material being tested renders the medium turbid so that the
presence or absence of microbial growth cannot be easily determined by visual examination, 14 days
after the beginning of incubation, 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.
If no evidence of microbial growth is found, the preparation under examination complies with the test
for sterility. If evidence of microbial growth is found, the preparation under examination does not
comply with the test for sterility. Do not repeat the test unless it can be clearly shown that the test was
invalid for causes unrelated to the preparation under examination. The test may be considered invalid
only when one or more of the following conditions are fulfilled:
(a) microbial growth is found in the negative controls;
(b) data on microbial monitoring of the sterility testing facility show a fault;
(c) a review of the testing procedure used for the test in question reveals a fault;
(d) after identifying the microorganisms isolated from the containers showing microbial growth, the
growth may be ascribed without any doubt to faults with respect to the materials and/or technique
used in conducting the test procedure.
If the test is declared to be invalid, repeat with the same number of units as in the original test. If no
evidence of microbial growth is found in the repeat test, the preparation under examination complies
with the test for sterility. If microbial growth is found in the repeat test and confirmed microscopically,
the preparation under examination does not comply with the test for sterility.
11