Sterility test

Lili Fitriani
 LEARNING OUTCOME
After studying this topic, student should be
able :
1. To explain definition and application of sterility
testing
2. To explain major factor affecting the sterility
testing
3. To describe the method for sterility testing
DEFINITION
STERILITY TEST
A test which assesses whether a sterilized
pharmaceutical or medical product is free from
contaminating microorganism

• Major component as “ microbiological quality

assurance’ for sterile pharmaceutical product
PHARMACEUTICAL PRODUCT MUST BE
DONE FOR STERILITY TEST
• Injection (solution and suspension in aqueous or
oil)
• Powder for injection & biological substance ex :
heparin, hyaluronidase and antibiotic
• Eye drops, eye ointment & eye lotion
• Water for injection
• Blood & blood product
• Immunological product (vaccine, antiserum and
diagnostic preparation)
• implant
• catgut
MAJOR FACTORS AFFECTING STERILITY
TESTING

• Environment
• The culture condition
• Test method
• Sample size
• Sampling procedure
1. Environmental conditions

• avoid accidental contamination of the

product during the test

• the test is carried out under aseptic conditions

• regular microbiological monitoring should be

carried out
2. Culture conditions

• Appropriate conditions for the growth of any

surviving organism should be provided by the
culture media selection.
• Factors affecting growth of bacteria

• Phases of bacterial growth

• Culture media for sterility testing

2.1. Factors affecting growth of
bacteria
• Nutrition
• Moisture
• Air
• Temperature
• pH
• Light
• Growth inhibitors
2.2. Phases of bacterial growth
2.3.Culture media for sterility
testing
 capable of initiating and maintaining the
vigorous growth of a small number of
organisms
 sterile
 Types of media: (refer FI IV)
▫Fluid thioglycollate medium
▫Soya-bean casein digest medium
▫other media
2.3.1.Fluid thioglycollate medium
composition described in USP 29 / Ph. Eur. 4th
Ed./ FI IV page..???
specific role of some ingredients
primarily intended for the culture of anaerobic
bacteria & aerobic bacteria
incubation of the media:
14 days at 30 -35°C
2.3.2.Soya-bean casein digest medium

primarily intended for the culture of both fungi

and aerobic bacteria
specific role of some ingredients
incubation of the media:
14 days at 20 -25°C
 Test Microorganisms
• Aerobic bacteria
▫ Staphylococcus aureus
▫ Bacillus subtilis
▫ Pseudomonas aeruginosa

• Anaerobic bacteria
▫ Clostridium sporogenes

• Fungi
▫ Candida albicans
▫ Aspergillus niger
STERILITY TEST (USP 29)
Contains two qualifying assay :
1. Suitability test (Growth Promotion Test)
2. Validation test (Bacteriostatis & Fungistatis test)

Current USP..??
Suitability test
• To confirm that each lot of growth media used in
the sterility test procedure will support the growth
of less than 100 viable microorganism

• A portion of each media lot must be incubated and

assessed for sterility according to the incubation
parameters (time, temperature) – no growth of
microorganism occurs.
Suitability test
• Growth Promotion Test
Test each lot of ready prepared medium and each
batch of medium prepared
Inoculate medium with a small number of test
microorganism (<100 cfu)
Incubate : bacteria : 3 days
fungi : 5 days
Suitability test
• STERILITY
 Confirm sterility of each sterilized batch of medium
by incubating a portion of the media at specified
incubation temperature for 14 days.

 No growth of microorganism occurs.

Validation Test
• To determine if the test sample will inhibit the
growth of microorganism in the test media.

• Must be performed on each product prior and/or

during sterility testing.
 Direct Inoculation of
The Culture medium

METHOD
FOR
STERILITY
TEST
Membrane
filtration
1. DIRECT INOCULATION OF THE
CULTURE MEDIA
This method suitable for :
• Medical devices
• Oily liquids
• Ointment & creams
• Solid
• Cotton, gauze, surgical dressing
DIRECT INOCULATION OF THE
CULTURE MEDIA

• Involve introducing test sample directly into

nutrient media.

• The volume of the product is not more than 10

% of the volume of the medium
DIRECT INOCULATION OF THE
CULTURE MEDIA

Procedure :
1. Transfer the quantity of the preparation to be examined
prescribed directly into the culture medium.
2. Incubate at :
* 30 – 35 º C for bacteria
* 20 – 25 º C for fungi
3. The growth of microorganism was observed for 14 days
2. MEMBRANE FILTRATION
This method suitable for ;
• Pharmaceutical product, not for medical devices :
▫ Filterable aqueous preparation
▫ Alcoholic preparation
▫ Oils and oily preparation
▫ Ointment and cream
▫ Preparation miscible with or soluble in aqueous or oily
(solvent must not have antimicrobial effect)
 Filter
Selection of
membrane

Membrane filtration
procedure
Membrane
Filtration Sterility test using
membrane filtration

Advantage &
Disadvantage
Selection of Filter Membrane
• Pore size : 0.45 ± 0.02 µm
• Diameter : 50 mm
• Membrane attached to proper apparatus
• Effectiveness established in the retention of
microorganism
Selection of Filter Membrane
Type of filter membrane :
• Cellulose nitrate filter
▫ Used for aqueous, oily and weakly alcoholic solutions

• Cellulose acetate filter

▫ Used for strongly alcoholic solution
Membrane filtration procedure
• Sterilization of the filtration apparatus and
membrane
• Filtration of examined solution under aseptic
condition suitable volume
• Dissolution of solid particles with suitable solvents
dilution if necessary
• The filter should be pre-wetted with diluents or
solvent before filtration to minimize the
retention of sample
Sterility test using membrane
filtration
• The sample was diluted with suitable diluents

• Filtering the solution by using proper membrane filter

• If antimicrobial agent contaminated the sample rinse the

filter with peptone water after finishing filtration process

• Transfer a half or all of membrane filter into the culture

medium

• Incubate for 14 days

• Daily observation to any changes in culture medium

Sample Dilution
 Aqueous solution – dilute with sterile
diluents --peptone water
 Oily solution – dilute with sterile diluent --
miristate isopropyl
 Solid – dilute with sterile diluents-- peptone
water
Advantages of the filtration method
• wide applications
• a large volume can be tested with one filter
• smaller volume of culture media is required
• applicable to substances for which no satisfactory
inactivators are known
• neutralization is possible on the filter
• shorter time of incubation compared with direct
inoculation
Disadvantages of the filtration
method

• Drug absorption may affect sterility test

• Personal must have a good skill in aseptic
technique
• Need sterile environment
 Observation
-During the incubation period

Observation &
Interpretation
of Result

Interpretation
of result
Interpretation of Result
1. When the sample passes the test and when
fails?

2. When the test to be considered as invalid?

When the sample passes the test
and when fails?
• Passes the test :
 if no evidence of microbial growth is found

• Fails the test :

 if evidence of microbial growth is found
What should you do when the test is
fail????
• Sterility testing is repeated on the same number of new
sample
• If microorganism was found in 2nd test, repeat the
experiment except the similar microorganism was found
on both of experiment.
• The sample is pass, If no microorganism growth at the 3rd
test
• The sample is fail, if microorganism growth at the 3rd test
• Repeat test is not encouraged because high risk
contamination and involuntary contamination
The test may be considered invalid if
only :
 The data of the microbiological monitoring of the
sterility testing facility show a fault.

 A review of the testing procedure used during the test

in question reveals a fault.

 Microbial growth is found in the negative controls

 After determination of identity of the microorganism

isolated from the test, the growth of this species may
be ascribed unequivocally to faults with respect to
material and/or the technique used in conducting the
sterility test procedure
SAMPLING
 Level of Sampling

Selection of Sample

Size of Sample
Level of Sampling
Test for End of Product
 for product sterilized by moist/dry
heat.

Test for bulk powder and end of

product
 for product prepared by aseptic
technique
SELECTION OF SAMPLE
• For aseptically prepared product
–> the sample should be taken at regular interval
during the filling operations

• For terminally sterilized product

–> the sample should be made up from units drawn
from various sites throughout the sterilizer load

• Test method is membrane filtration

–> the whole contents of the container should be
tested
 SIZE OF SAMPLE

• The minimum number of item or quantity to

be tested from each batch or each container
refer to USP 29/BP
• The pharmaceutical Codex
• Walter….