• Sterilization is the process of complete removal of microorganisms
from any surface or product.
• Sterility hence can be defined as “Complete freedom from
• The tests for sterility are conducted to make sure there is complete
absence of viable forms of microorganisms on a pharmaceutical
• The products which are to be strictly supervised for absence of the
microorganisms are those who come directly in contact with the
systemic circulation e.g. ophthalmic preparations, parenteral
injections, implants, bandages, surgical dressings, needles, surgical
• The test for sterility has to be conducted in aseptic environment so as to
avoid the contamination of the product.
• A pyrogen can be defined as “Fever Producing Substance”, which
induces “Fever (Hyperpyrexia)”.
• Bacterial endotoxins are the most common type of “Exogenous
• Bacteria, viruses, malaria parasites and fungi on entering in the body
releases certain chemicals that act as Pyrogen.
• During the sterilization process these microorganisms die but presence of
pyrogens in the products may cause a febrile reaction.
• Test for sterility ensures absence of microorganisms while the
Pyrogen test ensures absence of pyrogens in the pharmaceutical
• The lipopolysaccharide (LPS) from gram negative bacterias is a very
common example of pyrogen.
• The common pyrogen tests are,
• LAL Test (Limulus Amoebecyte Lysate Test).
• Sham Test (Rabbit Test).
Test for Sterility.
• A viable microorganism when provided with favorable conditions and
nutrients shows growth indicating its presence in the product.
• The probability of detecting the microbes in a product increases with
the number of the testings done.
• The external surfaces of the ampoules and vials or the containers
should be cleaned with the suitable antimicrobial substance and test be
carried out in an aseptic environment in order to avoid any contamination
of microbes in the products under testing.
• Following culture media are used for conducting the sterility tests,
1. Fluid Thioglycollate Medium (FTM).
2. Alternative Thioglycollate Medium (ATM).
3. Soybean Casein Digest Medium (SCDM).
1. Fluid Thioglycollate Medium (FTM)
• For use with Clear fluid products.
• Mainly intended for “Anaerobic Bacteria” but can also detect Aerobic
• It is an indicator culture medium as it contains “Resazurin” a dye which
becomes pink on contact with oxygen.
• While using not more than one tenth of the upper portion of the medium
should have pink color.
• If more than one third of the medium is looking pink (Showing presence of
oxygen) it can be simply restored by reheating in the water bath until color
• pH: 7.1 ± 0.2.
• Sterilization: Autoclaving @ 121℃ for 20 mins.
2. Alternative Thioglycollate Medium (ATM):
• For use with turbid or viscous products and devices with small layers.
• It is an anaerobic medium i.e. made for cultivation of anaerobes and hence
anaerobic conditions must be followed while preparing and using.
• pH: 7.1 ± 0.2.
• Sterilization: Autoclaving @ 121℃ for 20 mins.
3. Soybean Casein Digest Medium (SCDM):
• Suitable for cultivation of both fungi and aerobic
• pH: 7.3 ± 0.2.
• Sterilization: Autoclaving @ 121℃ for 20 mins.
Sr No. Component FTM ATM SCDM
1 L-Cysteine 0.5 gm 0.5 gm --
2 Sodium CHloride 2.5 gm 5.5 gm 5. gm
3 Dextrose (Monohydrate or
5.5 gm / 5.0 gm 5.5 gm / 5.0 gm 2.5 gm / 2.3 gm
4 Yeast Extract (Water Soluble) 5.0 gm 5.0 gm --
5 Pancreatic Digest of Casein 15.0 gm 15.0 gm 17.0 gm
6 Sodium Thioglycollate /
0.5 gm / 0. ml 0.5 gm / 0.3 ml --
7 Papic digest of Soybean meal -- -- 3.0 gm
8 Dipotassium Hydrogen
-- -- 2.5 gm
9 Resazurin Sodium 1.0 ml -- --
10 Distilled water upto 1000 ml 1000 ml 1000 ml
11 pH after Sterilization by
Autoclave @ 121℃ for 20
7.1 ± 0.2 7.1 ± 0.2 7.3 ± 0.2
Table 2.1: Composition of the Culture Medias used for Sterility Test.
Tests for Culture Media:
• The media to be used in sterility testings should pass the following tests,
1. Sterility of Media.
2. Growth Promotion Test.
3. Test for Bacteriostatic and Fungistatic.
1. Sterility of Media:
• Incubate the portions of FTM and ATM @ 30-35℃ and SCDM at 20-25℃ and
incubate them for not less than 14 days.
• The presence of microbial growth fails the test, while absence of growth passes
2.Growth Promotion Test:
• This test is used to check the capability of the medium to promote the bacterial
• Medium sample from each autoclaved medium container is taken and inoculated with
about 100 test organisms of each “Test Strain” and incubated as per the requirements
of the given strain.
• The sample passess the test if it shows satisfactory growth of the organisms.
• The test is considered failed on inadequate growth of the microbes in the sample.
Medium Test Microorganisms Incubation Parameters
Temperature Duration in Days Conditions
FTM 1.Clostridium Sporogenes 30-35℃ 3 Anaerobic
2.Staphylococcus aureus 30-35℃ 3 Aerobic
3.Pseudomonas aeruginosa. 30-35℃ 3 Aerobic
ATM 1.Bacteroides vulgatus 30-35℃ 3 Anaerobic
2.Clostridium Sporogenes. 30-35℃ 3 Anaerobic
3.Bacillus subtilis. 30-35℃ 3 Aerobic
SCDM 1.Aspergillus brasiliensis 20-25℃ 5 Aerobic
2.Candida albicans 20-25℃ 5 Aerobic
3.Bacillus subtilis. 30-35℃ 3 Aerobic
Table 2.2: List of Test Microorganisms for the Growth Promotion Test of Media.
3) Test for Bacteriostatic and Fungistatic:
• Prepare cultures of the test bacterias and fungi.
• Inoculate the required volume of culture media with 100 viable microorganisms.
• Add the required amount of the preparation under test to the half of the
containers containing medium and microbes.
• The half of the containers are kept as control.
• After specified time the growth of microbes is visually compared in test and control
• If the test preparation is found to be bacteriostatic or fungistatic it can be neutralized
using “Neutralising agents” as specified.
Table 2.2: List of Neutralizing substances for antimicrobial compounds.
Sr No. Antimicrobial
1 Penicillin Penicillinase
2 Streptomycin Streptomycin
3 Cephalosporins Cephalosporinase.
4 Sulfonamides P Amino benzoic Acid
Sterility Test Methods.
• Sterility tests can be carried out using one of the following
1. Methods A: Membrane Filtration.
2. Method B: Direct Inoculation.
1. Method A: Membrane Filtration.
• This method is preferred when substance to be tested 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 inherent bacteriostatic and fungistatic properties.
• For liquid products where the volume > 100ml or more
• A laminar sterile airflow cabinet – to avoid accidental contamination.
• Working conditions monitored regularly by sampling the air and surfaces of the working area.
• 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.
• Flow rate is adjusted to 55-75 ml of water / minute.
• Pressure 70 mm of Hg.
• Cellulose nitrate filter for aqueous, oily and weakly alcoholic solutions.
• Cellulose acetate solution for strong alcoholic solutions.
• Dilution 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
• Quantities of sample to be used:
Table 3.1 Quantities of sample to be used:
Preparation Quantity in each container
of the preparation
Minimum quantity to be used for each culture medium
Liquids Less than 1 ml. Total contents of container.
1-40 ml Half of the container but not less than 1 ml.
40 ml to 100 ml. 20 ml
More than 100 ml 10% of the container but not less than 20 ml.
Antibiotic liquids 1 ml
Other soluble preparations The whole contents of each container to provide not less than 200mg.
Insoluble preparations ointments & creams. The whole contents of each container to provide not less than 200mg.
Solids Less than 50mg Total container.
50 mg - 300 mg Half of the container but not less than 50 mg.
300 mg - 5g 150 mg
More than 5 gm 500 mg
Catgut and other surgical sutures for veterinary use. Three sections of a strand each 30 cm long
Surgical dressings/ cotton/ gauze (In packages) 100 mg per package
Sutures and other individually packed single use material. Whole device
Other medical devices Whole device or material cut into pieces or diassembled
Method of test:
1) For aqueous solution: -
• Prepare each membrane by aseptically transferring fluid A + media + preparation being
• Alternatively, first, combined quantities of preparation + prescribed in the two media.
• Draw the liquid rapidly through a 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.
• Similar, other membranes 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.
3) For oils and oily solutions:
• Low viscous, filter without dilution through 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.
5) For soluble solids:
• Dissolve substance in a suitable sterile solvent for each medium and carry out test described
under 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 a membrane filter funnel,
and follow the test as under for aqueous solutions.
2. Method B Direct Inoculation
•The quantities of substance or preparation being examined
which is to be used for inoculation for culture media varies
according to quantity in each container.
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.
• Incubate at 30º to 35º in case of fluid thioglycollate medium and at 20º to 25º in
case of soyabean casein digest medium.
• If a 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 the 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
2) For oils and oily solutions:
• In media, add 0.1% w/v of (4 –tert -octylphenoxy) polyethoxy ethanol, 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 tenfold in a sterile diluent such as fluid B or any other aqueous vehicle
capable of dispersing test material homogeneously throughout the fluid mixture.
• Mix 10ml of fluid mixture with 80 ml of the medium and proceed the same as aqueous
solutions & suspension.
4) For solids: -
• Transfer the required quantity of the material to the medium.
5) For sterile devices: -
• For articles of such size and shape as permit complete immersion in not more than
1000ml of culture medium test the intact article, using the appropriate media and
incubate same as 1).
• 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 the 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
• If evidence of microbial growth is found, reserve the containers, and it is demonstrated
that it is not due to preparation, hence the tests for sterility are invalid and may be
• 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
• 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 twice the number of samples.
• If no evidence of microbial growth is found in the 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.
1. Rabbit Test.
• For this test, three healthy rabbits are selected each weighing at least 1.5
• No rabbit should be selected if:
• 1. It has a normal temperature greater than 49.8°C.
• 2. It was used in a positive test during the last two weeks.
• The pyrogen testing is performed in an air-conditioned room.
• The food and water is withheld from the rabbit overnight.
• A clinical thermometer is inserted in the rectum of each rabbit to a depth of
not less than 7.5 cm.
• Two readings of the temperature of rabbit in normal conditions should be taken
at the interval of half an hour before the start of the test and the mean of both
should be calculated to determine the initial temperature.
• The equipment, injectors and needles used in the test should be pyrogen-
• These should be washed with water for injection and then heated at 260°C for
• The injection is warmed to 38°C before injecting to the rabbits.
• 0.5 to 1.0 ml per kg dose should be injected through the ear vein.
• Six readings of temperature are recorded at an interval of half an hour.
• Pyrogen Test Results:
• The response of each rabbit is detected by the difference of initial temperature and the
highest temperature recorded.
• The response of all three rabbits gives the sum of responses and can be concluded as:
• i) If the sum of responses does not exceed 1.4°C and any rabbit shows the response less than 0.6° C,
the product passes the test.
• ii) If the sum of responses is greater than 1.4 °C or any rabbit shows the response 0.6 or greater,
continue the test using 5 rabbits.
• iii) If the test is done using 5 rabbits, then if the sum of responses of all 5 rabbits is greater than
3.7°C and the individual response of not more than three rabbits is greater than 0.6°C, the product
passes the test.
2. LAL Test.
• Also called “Limulus Amoebocyte Lysate Test” and “Bacterial Endotoxin
• The LAL test reagent is prepared by lysing amoebocyte blood cells from
• American Horseshoe crab Limulus polyphemus and other species like
Tachyleus tridentus, Tachypleus gigas and Carcinoscropius rotundicauda.
• The horseshoe crab has blue colored blood.
• The amebocyte blood cells of horseshoe crab show defensive clotting
in presence of bacterial endotoxins.
• When a test material containing a pyrogen is added to the LAL reagent
it forms turbidity or a gel consistency indicating presence of
• The test is 100 times more sensitive than the rabbit test.
• LAL reagent is available commercially and is a popular alternative to