STERILITY TESTING

B. K. SAJEEB
Sterility Testing
A sterility test may be defined as a test that critically assesses
whether a sterilized pharmaceutical product is free from
contaminating microorganisms.
“According to Indian Pharmacopoeia (IP, 1996) the sterility testing is
intended for detecting the presence of viable forms of
microorganisms in or on the Pharmacopeial preparations.”
The test is applied to substances, preparations or articles which,
according to the Pharmacopoeia, are required to be sterile. However,
a satisfactory result only indicates that no contaminating
microorganism has been found in the sample examined in the
conditions of the test.
Products which are needed to be sterilized – Injectable, implants,
syringes, bandages, dressing, needles, surgical instruments, and
ophthalmic products.
Objectives of sterility testing –
−For validation of sterilization process.
−To check presence of microorganisms in preparation which are sterile.
−To prevent the issue of contaminated products in market.

Precautions during sterility testing –

−The test for sterility is carried out under aseptic conditions.
−The sterility test must be conducted in specific medium that contains
nutritive material and water, and maintained duly at a favorable
temperature (37 ± 2 °C), the microbes can grow.
−The test environment has to be adapted to the way in which the
sterility test is performed.
−The precautions taken to avoid contamination are such that they do
not affect any microorganisms which are to be revealed in the test.
−The working conditions in which the tests are performed are
monitored regularly by appropriate sampling of the working area and by
carrying out appropriate controls (GMP).
Culture media
Media must initiate and maintain the vigorous growth of small
numbers of aerobic and anaerobic bacteria including spores. It must
have sufficient moisture, adequate pH, adequate nutrients and
suitable redox potential. All the media must be previously assessed
adequately for their nutritive characteristic features i.e., in fertility
tests to ascertain the growth of specified microorganisms. All the
media are duly incubated at the stipulated temperature.
 Classification of Media

For detection of aerobic For detection of anaerobic

A.Peptone broth A.Cooked meat medium (Clostridia)
B.Glucose peptone broth B.Semi fluid meat medium
C.Liver broth

For detection of aerobic and anaerobic

A.Fluid thioglycollate medium
B.Thioglycollate broth medium
C.Corn steep liquor-sodium thioglycollate medium
D.Semi-fluid hydrosulphite medium

For detection of aerobic and fungi

A.Soya-bean casein digest medium
B.Sabouraud medium

N.B.: Sabouraud medium – an acidic medium, contains a rapidly

fermentable carbohydrate like glucose and maltose.
Fluid thioglycollate medium (anaerobic and aerobic)
Fluid thioglycollate medium is a differential medium used primarily
to determine the oxygen requirements of microorganisms. Sodium
thioglycollate in the medium consumes oxygen and permits the
growth of obligate anaerobes. The oxygen concentration at a given
level is indicated by a redox-sensitive dye such as resazurine that
turns pink in the presence of oxygen.
For example to detect the presence of Escherichia coli, Clostridium
genus, Actinomycetes etc.
After sterilization pH of the medium must be maintained within of
7.1 ± 0.2.

Incubation temperature
Fluid thioglycollate medium is to be incubated at 30­35 °C.
 Composition of fluid thioglycollate medium

Ingredients Amount
L-Cystine 0.5 g
Agar, granulated (moisture content not in excess 0.75 g
of 15 %)
Sodium chloride 2.5 g
Glucose monohydrate/anhydrous 5.5/5.0 g
Yeast extract (water soluble) 5.0 g
Pancreatic digest of casein (phosphoprotein) 15.0 g
Sodium thioglycollate/Thioglycollic acid 0.5/0.3 g
Resazurin sodium solution (1 g/l of resazurin 1.0 ml
sodium, a blue dye), freshly prepared
Water upto 1000 ml
Preparation of fluid thioglycollate medium
A.Mix L­cystine, agar, sodium chloride, glucose, water­soluble yeast extract and
pancreatic digest of casein with required amount of water and heat until solution
is affected.
B.Dissolve the sodium thioglycollate or thioglycollic acid in the solution and, if
necessary, add 1 M sodium hydroxide so that, after sterilization, the solution will
have a pH of 7.1 ± 0.2.
C.If filtration is necessary, heat the solution again without boiling and filter while
hot through moistened filter paper.
D.Add the resazurin sodium solution, mix and place the medium in suitable
vessels that prevent the color change indicative of oxygen uptake at the end of
the incubation period.
E.Sterilize using a validated process.
F.If the medium is stored, store at 2­25 °C in a sterile, airtight container.
G.If more than the upper third of the medium has acquired a pink color, the
medium may be restored once by heating the containers in a water­bath or in free­
flowing steam until the pink color disappears and cooling quickly, taking care to
prevent the introduction of non­sterile air into the container.
H.Do not use the medium for a longer storage period than has been validated.
Soya­bean casin digest medium
A general purpose medium used for the cultivation of a wide variety
of microorganisms like aerobic and fungi species e.g. Staphylococcus
aureus, Staphylococcus epidermidis, Candida albicans, Bacillus
subtilis, Neisseria meningitides, etc.
After sterilization pH of the medium must be 7.3 ± 0.2 at 25 ºC.

Incubation temperature
Soya­bean casein digest medium is to be incubated at 20­25 °C.
 Composition of soya­bean casin digest medium
Ingredients Amount
Pancreatic digest of casein 17.0 g
Papaic digest of soyabean meal 3.0 g
Sodium chloride 5.0 g
Dipotassium hydrogen phosphate 2.5 g
Glucose monohydrate/anhydrous 2.5/2.3 g
Water upto 1000 ml
Preparation of soya­bean casin digest medium
A.Dissolve the solids in required amount of water, warming slightly to effect
solution.
B.Cool the solution to room temperature.
C.Add 1 M sodium hydroxide, if necessary, so that after sterilization the
medium will have a pH of 7.3 ± 0.2.
D.Filter, if necessary, to clarify, distribute into suitable vessels and sterilize
using a validated process.
E.Store at 2­25 °C in a sterile well­closed container, unless it is intended for
immediate use.
F.Do not use the medium for a longer storage period than has been validated.
Sabouraud medium
Sabouraud medium is used for the isolation, cultivation, and
maintenance of fungi and yeasts. Sabouraud was formulated by
Raymond Sabouraud (French) in 1892. The pH is adjusted to
approximately 5.6 ± 0.2 at 25 ºC in order to enhance the growth of
fungi. The acidic pH inhibits bacterial growth.
Molds are incubated at room temperature (22 to 25 °C) and yeasts
are incubated at 28 to 30 °C. Dimorphic fungi are incubated at 37 °C.

N.B.: Dimorphic fungi are fungi that can exist as mold/hyphal/filamentous form or as yeast. For example Penicillium marneffei.
 Composition of sabouraud medium

Ingredients Amount
Dextrose (Glucose) 40 g
Peptone 10 g
Agar 15 g
Water upto 1000 ml

Preparation of sabouraud medium

A.Combine all ingredients in deioinized water.
B.Adjust to pH 5.6±0.2 with 1M hydrochloric acid and adjust final volume
to 1 l.
C.Heat to boiling to dissolve the medium completely.
D.Autoclave at 121 °C for 15 minutes.
E.Cool to 45 – 50 °C and pour into petridishes or tubes for slants.
The media used must comply with the following tests that are carried out
before or in parallel with the test on the product to be examined.
Sterility test
Incubate portions of the media for 14 days . No growth of microorganisms
occurs.
Growth promotion test
Test each batch of ready prepared medium and each batch of medium
prepared either from dehydrated medium or from the ingredients.
Inoculate portions of fluid thioglycollate medium with a small number (not
more than 100 CFU) of the following microorganism:
Clostridium sporogenes, Pseudomonas aeruginosa, Staphylococcus aureus.
A separate portion of medium must be used for each of the species of
microorganism.
Inoculate portions of soya-bean casein digest medium with a small number
(not more than 100 CFU) of the following microorganisms, using a separate
portion of medium for each of the following species of microorganism:
Aspergillus niger, Bacillus subtilis, Candida albicans.
Incubate for not more than 3 days in the case of bacteria and not more
than 5 days in the case of fungi. The media are suitable if a clearly visible
growth of the microorganisms occurs.
 Microorganisms used in growth promotion test

Clostridium sporogenes Anaerobic bacteria

Pseudomonas aeruginosa Fluid thioglycollate
Staphylococcus aureus Aerobic bacteria medium

Bacillus subtilis Aerobic bacteria

Candida albicans Soya-bean casein medium
Fungi
Aspergillus niger
Validation test
Carry out a test as described below under test for sterility of the product to
be examined using exactly the same methods mentioned above except for
the following modifications.
Membrane filtration
After transferring the contents of the container or containers to be tested
to the membrane, add inoculums of a small number of viable
microorganisms (not more than 100 CFU) to the final portion of sterile
diluent used to rinse the filter. (pore size 0.45 µm and diameter 47 mm).
Aqueous solution, soluble solid, oily and oily solid, ointment cream.

Fluid A: 1 g of peptic digest of animal tissue or its equivalent in water to make up

the volume upto 1L, filter or centrifuge to clarify, adjust to pH 7.1 ± 0.2, dispense
into flasks in 100 mL quantities, and finally sterilize at 121 °C for 20 minutes.
Fluid B: In a specific instance, when the test sample usually contains either oil or
lecithin, 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 sterilize at 121 °C for 20 minutes.
Membrane filtration method
Direct inoculation
After transferring the contents of the container or containers to be
tested (for catgut and other surgical sutures for veterinary use:
strands) to the culture medium add an inoculum of a small number
of viable microorganisms (not more than 100 CFU) to the medium.
Oily liquid, ointment, cream, catgut and suture, vetenary strands.
In both cases, use the same microorganisms as those described
above under growth promotion test of aerobes, anaerobes and
fungi. 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
incubation, 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.
Direct inoculation
If clearly visible growth is not obtained in the presence of the
product to be tested, 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. Modify the conditions in order to eliminate the
antimicrobial activity and repeat the validation test.
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.
C.The validation may be performed simultaneously with the test for
sterility of the product to be examined.
 Sampling technique
(minimum quantity to be used for each medium)
 Sampling technique
(minimum number of items to be tested)
References
British Pharmacopoeia (2016) volume IV, appendix XVIA, test for
sterility.
WHO, test for sterility, document QAS/11.413 final march, 2012.
Pharmaceutical Microbiology, Ashutosh Kar.
Thank You