STERILITY TESTINGMETHODOLOGY & INTERPRETATION
Contents   INTRODUCTION   PRINCIPLE   OBJECTIVES   CULTURE MEDIA   CONTROL TESTS   TEST METHODS   GENERAL METHOD  ...
Introduction   Sterilization – Process of removing microorganisams   Sterility testing – For detecting either viable for...
   Products which Are necessary to be sterilized:-   injections,   implants,    syringes,   bandages,   dressings, ...
Principle   If microorganisms are placed in a medium which provide nutritive    materials and water and kept at a favorab...
   Sterility, in the microbiological sense, means freedom from living    organisms and therefore it is not possible to cl...
Objectives   For validation of sterilization process   To check presence of microorganisms in preparation which are    s...
Culture media  Media must initiate and maintain the vigorous growth of small   numbers of aerobic and anaerobic bacteria ...
3. FOR DETECTION OF AEROBES AND ANAEROBES-   Fluid thioglycolate media   Thioglycolate broth media   Corn steep liquor-...
A. Fluid thioglycolate medium  INGREDIENTS                 QUANTITY                           PERPOSE     L-cystein       ...
B. Alternative thioglycolate medium     It contains no agar and indicator.      It is used with- 1.    Turbid suspensions...
C. Soyabean caesin digest medium  INGREDIENT           QUANTITY                    FUNCTIONPancreatic digest of     17g   ...
Control tests    There are two types of tests:-1.     Negative Control2.     Positive Control (Fertility Test)           ...
Test methods•   Method – A: - Membrane filtration•   Method – B: - Direct inoculation                             APMC Col...
Method – A:- Membrane filtration    They are of three types Of fluid used in This method:-1.     FLUID-A: -•      Dissolve...
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 l...
   METHOD: -   This method needs an exceptional skill and special knowledge.   It also calls for the routine use of pos...
   Advantages of METHOD-A:-a.   Wide applications for mostly all kind of products.b.   A very large volume can be tested....
This method is used for,a.   An oilb.   An ointment that can be put into the solutionc.   A non bacteriostatic solid not r...
B. Direct inocculation method:-    Here the sample is directly transferred aseptically into media tubes     to access pre...
General Methods     Liquids               APMC College Of Pharmaceutical               Education And Research
Solids     APMC College Of Pharmaceutical     Education And Research
Devices     APMC College Of Pharmaceutical     Education And Research
Parenteral Preparation             APMC College Of Pharmaceutical             Education And Research
Antibiotic solid          APMC College Of Pharmaceutical          Education And Research
Ophthalmic and other noninjectablepreparations                    APMC College Of Pharmaceutical                    Educat...
Bulk solid products            APMC College Of Pharmaceutical            Education And Research
Sterility Assurence   The achievement of sterility within any one item in a population of    items submitted to a sterili...
   The SAL(Sterility Assurance Level) for a given process is    expressed as the probability of a non-sterile item in tha...
Interpretation of results
Reference Indian Pharmacopoeia, 2007, Government  of India Ministry of Health and Family  Welfare, The Indian Pharmacopoe...
APMC College Of PharmaceuticalEducation And Research
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Sterility testing 112070804014

  1. 1. STERILITY TESTINGMETHODOLOGY & INTERPRETATION
  2. 2. 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
  3. 3. 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 Education And Research
  4. 4.  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. APMC College Of Pharmaceutical Education And Research
  5. 5. 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. APMC College Of Pharmaceutical Education And Research
  6. 6.  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. APMC College Of Pharmaceutical Education And Research
  7. 7. 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. APMC College Of Pharmaceutical Education And Research
  8. 8. 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 APMC College Of Pharmaceutical Education And Research
  9. 9. 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 APMC College Of Pharmaceutical Education And Research
  10. 10. 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 caseinResazurin / methylene 1ml Oxidation-reduction Blue indicator Distilled water Upto 1000ml Sterilize in an autoclave at 121C for 20min. APMC College Of Pharmaceutical Education And Research
  11. 11. 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. APMC College Of Pharmaceutical Education And Research
  12. 12. C. Soyabean caesin digest medium INGREDIENT QUANTITY FUNCTIONPancreatic digest of 17g ‘C’, ‘N’ & essential casein amino acidsPancreatic 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 APMC College Of Pharmaceutical Education And Research
  13. 13. Control tests There are two types of tests:-1. Negative Control2. Positive Control (Fertility Test) APMC College Of Pharmaceutical Education And Research
  14. 14. Test methods• Method – A: - Membrane filtration• Method – B: - Direct inoculation APMC College Of Pharmaceutical Education And Research
  15. 15. 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. APMC College Of Pharmaceutical Education And Research
  16. 16. 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. APMC College Of Pharmaceutical Education And Research
  17. 17.  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 APMC College Of Pharmaceutical organisms. Education And Research
  18. 18.  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 Education And Research
  19. 19. This method is used for,a. An oilb. An ointment that can be put into the solutionc. 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. APMC College Of Pharmaceutical Education And Research
  20. 20. 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 fora. 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 APMC College Of Pharmaceutical Education And Research
  21. 21. General Methods Liquids APMC College Of Pharmaceutical Education And Research
  22. 22. Solids APMC College Of Pharmaceutical Education And Research
  23. 23. Devices APMC College Of Pharmaceutical Education And Research
  24. 24. Parenteral Preparation APMC College Of Pharmaceutical Education And Research
  25. 25. Antibiotic solid APMC College Of Pharmaceutical Education And Research
  26. 26. Ophthalmic and other noninjectablepreparations APMC College Of Pharmaceutical Education And Research
  27. 27. Bulk solid products APMC College Of Pharmaceutical Education And Research
  28. 28. 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. APMC College Of Pharmaceutical Education And Research
  29. 29.  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. APMC College Of Pharmaceutical Education And Research
  30. 30. Interpretation of results
  31. 31. 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 APMC College Of Pharmaceutical Education And Research
  32. 32. APMC College Of PharmaceuticalEducation And Research

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