Sterility testing 112070804014

STERILITY TESTING
METHODOLOGY & INTERPRETATION

Contents
 INTRODUCTION

 PRINCIPLE

 OBJECTIVES

 CULTURE MEDIA

 CONTROL TESTS

 TEST METHODS

 GENERAL METHOD

 STERILITY ASSURANCE

 INTERPRETATION OF RESULTS

 REFERENCES APMC College Of Pharmaceutical
Education And Research

Introduction

 Sterilization – Process of removing microorganisams

 Sterility testing – For detecting either viable form of
microorganisms are present or not in or on pharmacopoeial
preparation.

APMC College Of Pharmaceutical

 Products which Are necessary to be sterilized:-
 injections,
 implants,
 syringes,
 bandages,
 dressings,
 needles,
 surgical instruments,
 Ophthalmic product, etc.

 The test must be carried out in an aseptic area to avoid accidental
contamination.


Principle
 If microorganisms are placed in a medium which provide nutritive

materials and water and kept at a favorable temperature the

organism will grow and their growth can be indicated by turbidity in

the originally clear medium.

 The sterility tests provide optimum conditions for the growth and

multiplication of every organism, vegetative spore, healthy or

injured, that might be a contaminant.


 Sterility, in the microbiological sense, means freedom from living

organisms and therefore it is not possible to claim that a batch of

products is sterile unless-the entire content of each and every

container in the batch has been tested.

 these conditions are not possible because The article or preparation

under test is either destroyed (example an injectable solution) or

made unstable (a syringe). Therefore only a part of the batch can be

sampled.


Objectives

 For validation of sterilization process

 To check presence of microorganisms in preparation which are
sterile.

 To prevent the issue of contaminated product in market.


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 range,
 adequate nutrients
 suitable redox potential.
 CLASSIFICATION OF MEDIA:-
1. FOR DETECTION OF AEROBES-
 Peptone broth
 Glucose peptone broth
2. FOR DETECTION OF ANAEROBES-
 Cooked meat medium
 Semi fluid meat medium
 Liver broth

3. FOR DETECTION OF AEROBES AND ANAEROBES-

 Fluid thioglycolate media

 Thioglycolate broth media

 Corn steep liquor-sodium thioglycolate medium

 Semi-fluid hydrosulphite medium.

4. FOR DETECTION OF AEROBIC AND LOWER FUNGI

 Soyabean caesin digest medium

 Sabourould’s (fluid) medium


A. Fluid thioglycolate medium
INGREDIENTS QUANTITY PERPOSE
L-cystein 0.5g -SH contg amino acid
Sodium chloride 2.5g Isotonicity
Dextrose 5.5g Energy source, reducing
agent
Agar 0.75g Viscosity enhancer,
growth promoter
Yeast extract 5g ‘C’, ‘N’ source and
vitamins
Sod. Thioglycolate 0.5g Reducing agent
Pancreatic digest of 15g Nutrient
casein
Resazurin / methylene 1ml Oxidation-reduction
Blue indicator
Distilled water Upto 1000ml
Sterilize in an autoclave at 121C for 20min.

B. Alternative thioglycolate medium

 It contains no agar and indicator.

It is used with-

1. Turbid suspensions and viscid products (creams).

2. For devices having tubes with small Lumina.


C. Soyabean caesin digest medium
INGREDIENT QUANTITY FUNCTION
Pancreatic digest of 17g ‘C’, ‘N’ & essential
casein amino acids

Pancreatic digest of 3g ‘C’, ‘N’ & essential
soyabean meal amino acids

Sodium chloride 5g Isotonicity
Dibasic potassium 2.5g Source if ions and
phosphate buffer

Dextrose 2.5g Reducing agent, ‘C’
source

Distilled water 1000ml


Control tests

 There are two types of tests:-

1. Negative Control

2. Positive Control (Fertility Test)


Test methods

• Method – A: - Membrane filtration

• Method – B: - Direct inoculation


Method – A:- Membrane filtration

They are of three types Of fluid used in This method:-

1. FLUID-A: -

• Dissolve 1g of peptic digest of animal tissue such as

bacteriological peptone or its

• equivalent in water to make 1L,

• Filter or centrifuge to clarify,

• Adjust the pH to 7.1+ 0.2.

• Dispense into flasks in 100ml quantities and sterilize at 121C for

20 min.

2. FLUID-B (as per IP) or FLUID-D (as per USP): -

• If the test sample contains lecithin or oil, use fluid A to each literf of

• which has been added 1ml of polysorbate-80,

• adjust pH 7.1+0.2.

• Dispense into flasks and sterilize it at 121C for 20 min.

3. FLUID-K (USP):-

• Dissolve 5g of peptic digest of animal tissue, 3g of beef extract, and 10g

of polysorbate-80 in water to make 1L.

• Adjust pH to obtain, after sterilization, a pH of 6.9+0.2.

• Dispense into containers, and sterilize via appropriate process.

 METHOD: -

 This method needs an exceptional skill and special knowledge.

 It also calls for the routine use of positive and negative controls. A
suitable positive control is the occasional use of a known solution
containing a fix number of microorganisms.

 The specific quantity of test specimen is taken and is made to pass
through a membrane with fix pore diameter.

 That membrane is then collected and washed using diluting fluids
and then is incubated in several Culture Medias for growth of
organisms. Education And Research

 Advantages of METHOD-A:-

a. Wide applications for mostly all kind of products.

b. A very large volume can be tested.

c. Smaller volume of broth required.

d. Applicable to substances to which no effective inactivators are
known.

e. Subculturing is eliminated.

 Disadvantages of METHOD-A:-

a. Possibility of adsorption of sufficient medicament to vitiate the test
cannot be discounted entirely.

b. High skilled staff and exceptionally good aseptic techniques are
necessary.

c. High cost. APMC College Of Pharmaceutical

This method is used for,

a. An oil

b. An ointment that can be put into the solution

c. A non bacteriostatic solid not readily soluble in the culture
medium.

d. A soluble powder or a liquid that possess inherent bacteriostatic or
fungistatic properties.

e. For liquid products where the volume in a container is 100ml or
more only method A is employed.


B. Direct inocculation method:-

 Here the sample is directly transferred aseptically into media tubes
to access presence of any viable organism.

 This method is applicable only for

a. Powders which are soluble in water.

b. Solutions which are miscible in water.

c. Substances like gauge, cotton.

 Here great care should be taken in order to avoid accidental
contamination


General Methods
Liquids


Solids


Devices


Parenteral Preparation


Antibiotic solid


Ophthalmic and other noninjectable
preparations


Bulk solid products


Sterility Assurence
 The achievement of sterility within any one item in a population of
items submitted to a sterilisation process cannot be guaranteed that
it is 100% sterile.

 there is always a finite statistical probability that a micro-organism
may survive the sterilising process.

 the probability of survival is determined by the number, types and
resistance of the microorganisms present and by the environment in
which the organisms exist during treatment.


 The SAL(Sterility Assurance Level) for a given process is
expressed as the probability of a non-sterile item in that population.

 not more than one viable micro-organism in 1x 106 sterilised items
of the final product.


Reference
 Indian Pharmacopoeia, 2007, Government
of India Ministry of Health and Family
Welfare, The Indian Pharmacopoeia
Commision, Ghaziabad,Vol.– 1, 53
 The United States Pharmacopoeia- the
National Formulary, Asian edition, 2003
 Cooper & Gunns’s Dispensing
pharmaceutical students,12th edition,CBS
publication & distributers, pp 445-446

Sterility testing 112070804014

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