STERILITY TESTING

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.
 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.

Methods of sterility testing

Tests for sterility are carried out by two methods:
(a) Membrane Filtration Method (Method A)
(b) Direct Transfer / Inoculation Method. (Method B)
The Membrane Filtration Method is used as the method of choice wherever feasible.

Media used in Sterility Testing

Fluid Thioglycollate Medium (Medium 1) and Soybean-Casein Digest Medium (Medium 2) are the two
media generally used for tests for sterility.

Method of Preparation:

1. 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.
2. Sodium thioglycollate is dissolved in the solution.
3. The specified quantity of Polysorbate 80 is added if this ingredient is to be included.
 4. 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.
5. If the solution is not clear, mixture is heated to boiling and filtered while hot through moistened
filter paper.
6. Resazurin sodium solution is added and mix.

Method of Preparation:

1. The ingredients are mixed in the proportions given above with slight warming.
2. The solution is cooled to room temperature.
3. The specified quantity of Polysorbate 80 is added if this ingredient is to be included.
4. 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.
5. If the solution is not clear it is filtered through moistened filter paper.

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.

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.
Table no-1

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.

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.
 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.

Method of Membrane Filtration

a) an oil,
b) an ointment that can be put into solution,
c) a non bacteriostatic solid not readily soluble in the culture medium
d) a soluble powder or a liquid that possesses inherent bacteristatic and fungistatic properties.
e)For liquid products where the volume > 100ml or more

Precautions:
 A laminar sterile airflow cabinet – to avoid accidental contamination.
 Working conditions monitored regularly by sampling the air and surfaces of the working area.

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. The filter should be a membrane filter disc of cellulose esters or other suitable plastics. Cellulose
nitrate filters are recommended for aqueous, oily and weakly alcoholic solutions and cellulose acetate
filters for strongly alcoholic solutions.

The membrane should be held firmly in a filtration unit which consists of a supporting base for the
membrane, a receptacle for the fluid to be tested, a collecting reservoir for the filtered fluid, and the
necessary tubes or connections. The apparatus is so designed that the solution to be filtered can be
introduced and filtered under aseptic conditions. It permits the aseptic removal of the membrane for
transfer to medium or it is suitable for carrying out the incubation after adding the medium to the
apparatus itself.

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.

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.

For aqueous solution:  Prepare each membrane by aseptically

transferring fluid A + media + preparation
being examined.
 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.
 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 and other membrane in
FTM (30º to 35º).
For liquids immiscible with aqueous vehicles and  Same as above but add sufficient quantity
suspensions: 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.
For oils and oily solutions:  Low viscous, filter without dilution through
dry membrane
 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.
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.
For soluble solids:  Dissolve substance in a suitable sterile
solvent for each medium and carry out
test described under for aqueous
solutions.
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 of Direct Transfer
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.
 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.
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.
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 and proceed same as aqueous
solutions & suspension

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.
 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.

Aseptic technique

Aseptic technique or sterile technique is used to avoid contamination of sterile media and equipment
during cell culture. Sterile technique should always be employed when working with live cell cultures
and reagents/media that will be used for such cultures. This technique involves using flame to kill
contaminating organisms, and a general mode of operation that minimizes exposure of sterile media
and equipment to contaminants.

When working with cultures of living organisms, it is extremely important to maintain the environments
in which cells are cultured and manipulated as free of other organisms as possible. This requires that
exposure of containers of sterilized culture media to outside air should be minimized, and that flame is
used to “re-sterilize” container lids and rims. This means passing rims and lids through the flame
produced by a Bunsen burner in order to kill microorganisms coming in contact with those surfaces.

Sterile technique, in general, is a learned state-of-being, or mantra, where every utilization of any sterile
material comes with the caveat of taking every precaution to ensure it remains as free of contaminants
as possible for as long as possible. Heat is an excellent means of killing microorganisms, and the Bunsen
burner is the sterile technician’s best friend.