What does the BP appendix test say for sterility?
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 micro-organism has been found in the sample examined in the conditions of the test.
What information is given by a sterility test? Should it be used as the sole means of controlling sterile processing?
Sterility tests can only show that organisms capable of growing in the test media under the selected conditions are absent from the fraction of the batch that has been tested.
- It should not be used as the sole means of controlling sterile processing
- It is necessary to take sufficient samples, to use sensitive culture media and, during testing, to reduce accidental contamination to a minimum.
What are the two methods for sterility testing?
- Membrane filtration method
- Direct inoculation method
For membrane filtration method (sterility test);
A) When is it used?
B) What is the nominal pore size of the membrane filter
C) What are the types of the filter?
D) What to do if the product has antimicrobial properties
A)
- Used whenever the nature of the product permits (filterable aqueous preparations)
B)
- Nominal pore size of membrane filter not greater than 0.45 micrometer (this is for testing, don’t get confused with sterilisation which is not greater than 0.22 micrometer)
C)
Established effectiveness to retain microorganism:
- Cellulose nitrate filters: aqueous, oily, weakly alcoholic solutions
- Cellulose acetate filters: strongly alcoholic solutions
- Specially adapted filters: antibiotics containing solutions
D)
- If the product has antimicrobial properties, wash the membrane not less than 3 times by filtering through it each time the volume of the chosen sterile diluent used in the method suitability test
What is the method for membrane filtration?
- Filtration system and membrane are first sterilised
- Design must allow process to be carried out under aseptic conditions
- After filtration
> Transfer the whole membrane to the culture medium; or
> Cut the membrane aseptically into 2 equal parts and transfer one half to each of 2 suitable media; or
> Transfer the medium onto the membrane in the apparatus
- Incubate the media for not less than 14 days
What is the method for direct inoculation
- Transfer the quantity of the preparation prescribed in Table 2.6.1-2 (BP) directly into the culture medium so that the volume of the product is not more than 10 per cent of the volume of the medium, unless otherwise prescribed.
- Incubate the inoculated media for not less than 14 days. Observe the cultures several times during the incubation period.
- Shake cultures containing oily products gently each day.
- Neutralisation or dilution for products with antimicrobial activity.
- Large volume of product, use concentrated culture medium.
- Where appropriate, the concentrated medium may be added directly to the product in its container.
What decisions are there to be made when performing sterility testing?
- Sample size (see lecture notes)
- Sample volume (see lecture notes)
- Culture medium
- Incubation temperature
What are the two main types of culture medium used in sterility testing? What is the key difference between them?
1.
Fluid thioglycollate medium (FTM)
- FTM contains agar, increases viscosity of medium, important when want to innoculate anaerobic bacteria, dont want the medium to have air thererfore by having a thicker medium = reduce chances of having air in the medium
2.
Soya-bean casein digest medium (SCDM)
Fluid thioglycollate medium (FTM) is suitable for the culture of
- ?
- ?
Incubation temperature
- Anaerobic
- Aerobic
Incubation temperature = 30-35oC for bacteria to grow
Soya-bean casein digest medium (SCDM) is suitable for the culture of
- ?
- ?
Incubation temperature:
- Aerobic bacteria (IT: 30-35oC)
- Fungi (IT: 20-25oC)
What are the different control tests used?
A)
Sterility (Negative control)
- incubate portions of the media for 14 days. No growth of micro-organisms occurs.
- To eliminate false positive results
- Should be performed parallel with the test for sterility of the product
B)
Growth promotion test (positive control)
- Confirm the media support growth under test conditions e.g. can aerobic bacteria grow in culture media
- done BEFORE the sterility test
> FTM = Clostridium sporogenes, Pseudomonas aeruginosa, Staphylococcus aureus
> SCDM = 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 micro-organisms occur
C)
Validation test (main positive control)
- To confirm no growth inhibitory effect from the product –> innoculate product + mo together with culture media in one tube
- Should be performed parallel with the test for sterility of the product
How does the validation test differ for membrane filtration and direct inoculation?
Membrane filtration
- After transferring the contents of the container or containers to be tested to the membrane add an inoculum of a small number of viable micro-organisms (not more than 100 CFU) to the final portion of sterile diluent used to rinse the filter.
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 micro-organisms (not more than 100 CFU) to the medium
> In both cases use the same micro-organisms as those described 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.
> Should be performed parallel with the test for sterility of the product
For validation test;
What does it mean if there is clearly visible growth of micro-organisms 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. Positive control = positive result
The test for sterility may then be carried out without further modification.
For validation test;
What does mean 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.
> product may contain an antimicrobial preservative (dilute product or use neutralising agent to remove preservative)
How to observe and interpret results if;
A) If the material being tested renders the medium turbid
B) If no microbial growth
C) If evidence of microbial growth is found
A)
- If the material being tested renders the medium turbid so that the presence or absence of microbial growth cannot be readily determined by visual examination, 14 days after the beginning of incubation –> transfer portions (each not less than 1 ml) of the medium to fresh vessels of the same medium and then incubate the original and transfer vessels for not less than 4 days.
B)
- the product to be examined complies with the test for sterility.
C)
- the product to be examined does not comply with the test for sterility, unless it can be clearly demonstrated that the test was invalid for causes unrelated to the product to be examined.
What makes a sterility test invalid?
- the data of the microbiological monitoring of the sterility testing facility show a fault (not doing test in suitable environment)
- 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 the identity of the micro-organisms isolated from the test, the growth of this species or these species may be ascribed unequivocally to faults with respect to the material and/or the technique used in conducting the sterility test procedure.
What happens if;
A) the test is declared to be invalid?
B) If no evidence of microbial growth is found in the repeat test?
C) If microbial growth is found in the repeat test?
A)
- It is repeated with the same number of units as in the original test
B)
- The product examined complies with the test for sterility.
C)
- The product examined does not comply with the test for sterility
For strains of the test micro-organisms;
A) What are examples of aerobic bacteria?
B) What are examples of anaerobic bacteria?
C) What are examples of fungi?
A)
- Staphyloccous aureus
- Bacillus subtilis
- Pseudomonas aeruginosa
B)
- Clostridium sporogenes
C)
- Candida albicans
- Aspergillus niger
SA questions
- Discuss the decisions you need to make for the test for sterility
- State the purposes of each control tests
- If you have been given a set of results from the test for sterility, do you know how to interpret the results?
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Sterilisation by membrane filtration23
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Sterility Testing19
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Colligative Properties 1+255
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Formulation of eye drops27
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Cold sterilisation26
-
Parametric Release24
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CDR and TPN36
-
DDS: Pulmonary23
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DDS: Implant18
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DDS: Nasal24
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DDS: Intraocular26
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Protein and Insulin38
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DDS: Transdermal37
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Drug targeting and nanotechnology 129
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Drug targeting and nanotechnology 2 + 336
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Recombinant Pharmaceuticals 1 and 238
