Chapter 8 1

STERILITY TESTING OF
PHARMACEUTICALS
 The test for sterility is intended for detecting the presence of viable forms of microorganisms in
pharmaceutical preparation. A number of products are necessary to be sterilized such as ophthalmic 2
preparation, injections, infusions, implants, syringes, bandages, dressings, needles, surgical instrument
etc. It must be checked for the contamination of microorganism (bacteria & fungi). It is carried out in
aseptic area to avoid accidental contamination of the product during the test. The working is routinely
monitored by sampling the air and surface of the working area.

The test for sterility is based upon principle that if microorganisms are placed on the medium which
provide nutritive material and water and kept at a favorable temperature, the microorganism will grow
& their presence of the microorganism can be indicated by turbidity in the originally clear medium.
The probability of detecting viable microorganism in test for sterility increases with the
microorganism present in given amount of preparation being examined and varies accordingly to the
species of the microorganisms present.
Following medias are used for test of sterility
• Fluid thioglycollate medium- It is used with fluid alternative.
• Alternative thioglycollate medium - It is used with turbid or viscid preparation and for device having
tubes with small lining.
• Soybean casein digest medium. All the media are adjusted to pH 7.1 + 0.2 and sterilized at 1210Cfor 20
minutes.
 
Sterility of media – incubate portion of fluid thioglycollate medium /alternate thioglycollate 3
medium at 30-350 C and soybean casein digest medium at 20 -250 C for less than 7 days and
observe the presence of growth of microorganism in a sterile media
Growth promotion test- test each autoclave lead of media for its growth promoting qualities by
separating inoculating duplicate test container of each of the strains in table.
Test for bacteriostates and tungistates (sample to be tested for sterility) 
Prepare cultures of bacteria and fungi from scheme of microorganism mentioned in table.
Incubate sterility test media with about 100 viable organism using volume of media. Listed in
table. Add portion of preparation.
Test microorganism for growth promotion test of medium:
4
Medium Test Incubation
microorganism Temp. Conditions

1] Fluid  
thioglyolate   30-350C Aerobic
- Bacillus subtilis
 
- Candida albicans
- Bactericides
vulgatus

2] Alternative - Bactericides 30-350C

thioglylolate vulgatus   Anaerobic
 
3] Soya bean casein - Bacillus Subtilis 20-250C Aerobic
digest
-Candida albicans
 Quantity of test samples for test of Sterility as per IP.
 
Preparation Content Minimum qty of Minimum qty of
5
product culture medium
(ml)
Liquids Less than 1ml Total container 15
   1ml or more but less than    20
20ml Half the contents
  5ml or more but less 2ml 20
than 20 ml
  20 ml or more but less 5ml 40
than 50ml
  50 mI or more but less  10ml  80
than 100 ml
Solids Less than 50 mg total content of 40
Container
50mg or more but less Half the Contents 80
than 200 mg of container 100mg

200 mg or more 100mg 82

Dilution fluids: Generally two type of dilution fluids are Used
  6
1) Fluid A: dissolve 1g of peptic digest of animal tissue such as bacteriological peptone
in one litre of water filter and adjust the PH to 7.1. Disperse fluid into flask and
sterilize at 1210 C for 20 minutes
2) Fluid B: If test Samples contains Lecithin or oil Use fluid to each Litre of which has
been added 1ml of Polylobate. Adjust pH 7.2[+/- 0.2] and Sterilize at 121 0 C for 20
minutes using autoclave

Method for Sterility test:-Sterility test can be carried out by using the following methods:
 
Method A: Membrane filtration
Method B: Direct inoculation
Method A: Membrane filtration 7
Methods to test: For aqueous solution, for oils and oils solution, for ointments and creams, For soluble
solids, For sterile devices
Quantities of Sample to be tested:

Formulation Quantity to be mixed Quantity to be used

in each culture
media
1] Ophthalmic solution, 10-100mL 5- 10mL
Other non-injectable
liquid preparation.
1-10 gm 0.5 – 1gm
2] Insoluble preparation    
to be Suspended or
emulsifier
ointment, Creams,
preparation Soluble in
water or solvent
3] Absorbent Cotton - Not less than 1 gm
Method B: Direct inoculation 8
Methods of test: For aqueous solution & suspension, For oils and oily emulsions, For ointment,
For solids, For sterile devices

Immerse the device in 100ml of culture medium. If immersion is impracticable flush the lumen
20units with the fluid thioglyclate medium and soybean casein digest medium. Incubate with
not less than 100 ml of each of the medium.
Aim: To check the sterility of following pharmaceutical preparations
(Method B- Direct inoculation method) 9
1. Absorbant cotton wool
2. Water for injection
3. Surgical needle

Requirements:
Apparatus: Conical flask, test tube, Petri plate.
Instruments: Autoclave, laminar air flow, incubator.
Media: A] Fluid Thioglycolate medium (FTM)
B] Soya bean Casein digest medium (SCDM)

Principle:
Ophthalmic Preparations, injections, syringes, and infusions are necessary to be sterilised. The test for Sterility
is based on the principle that micro-organisms are replaced in the medium providing nutrient material and are
kept at proper conditions such that the micro-organisms will grow and their presence is indicated by turbidity in
the medium.
Procedure: 10
 
1. Prepare four test tubes of fluid thioglycolate media and Soya Bean Casein digest
medium and sterilize them by autoclaving.
2. Add 5 ml of water for injection, surgical needle and absorbent cotton wool into one
test tube of each medium using sterile instruments such as forceps. One tube of
each medium is taken as control.
3. Keep the test tubes containing Soya Bean-Casein digest media for incubation for 20 -
250 C for less than 7 days and test tubes of fluid thioglycolate media 30-35 0 C for less
than 7 days.
4. After incubation observe for presence or absence of growth of micro organisms.
Observation and Results:
11
Observe the turbidity in the medium by comparing with the standard tube. If it has no
turbidity in both the media means it is free from micro-organisms and suitable for
use. If it has any type of turbidity in any tube then repeat the exp. For confirmation of
the results.
Aim: To perform the sterility test for sodium chloride injection (500ml)
(Method A- Membrane filteration method) 12

Requirements:
Apparatus: Conical flask, test tube, Petri plate.
Instruments: Autoclave, laminar air flow, incubator, vacuum pump.
Media: A] Fluid Thioglycolate medium (FTM)
B] Soya bean Casein digest medium (SCDM)

Principle:
Ophthalmic Preparations, injections, syringes, and infusions are necessary to be sterilised. The test for
Sterility is based on the principle that micro-organisms are replaced in the medium providing Nutrient
material and are Kept at proper conditions the micro-organisms will grow and their presence is indicated by
turbidity in the medium. Sodium chloride injection does not have any anti-microbial activity. It is not required
for inactivation of given preparation. Total volume of each container is 500ml. Hence, sterility of liquid
products where the volume in the container is 100ml or more, only method A is used.
Procedure:
 
1. First clean the membrane filteration unit and 13
sterilize the unit and membrane filter separately
by moist heat sterilization (autoclave)
2. Transfer the unit on laminar air flow bench and
place the membrane filter as shown in fig. Pass
all the solution through filter under vacuum.
3. After filteration the membrane is removed
aseptically and cut into two parts by using sterile
scissors.
4. One half part of the membrane is placed in 100ml of
fluid thioglycolate medium for incubation at 30-350
C for not less than 7 days. Other half part of the
membrane is placed in 100ml of Soya Bean-Casein
digest medium for incubation at 20 - 250 C for not
less than 7 days
5. After incubation observe for presence or absence of growth
of micro organisms.
Observation and Results:
Observe the turbidity in the medium by comparing with the standard tube. If it has no 14
turbidity in both the media means it is free from micro-organisms and suitable for use. If it
has any type of turbidity in any tube then repeat the exp. For confirmation of the results.