British Pharmacopoeia Volume V
Appendices
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Appendix XVI B. Microbiological Examination of Non-sterile Products
1. Tests for Specified Micro-organisms1
(Ph. Eur. method 2.6.13)
1 INTRODUCTION
The tests described hereafter will allow determination of the abnce or limited occurrence of specified micro-organisms that may be detected under the conditions described.
The tests are designed primarily to determine whether a substance or preparation complies with an established specification for microbiological quality. When ud for such purpos, follow the instructions given below, including the number of samples to be taken, and interpret the results as stated below.
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Alternative microbiological procedures, including automated methods, may be ud, provided that their equivalence to the Pharmacopoeia method has been demonstrated.
2 GENERAL PROCEDURES
The preparation of samples is carried out as described in general chapter 2.6.12.
If the product to be examined has antimicrobial activity, this is insofar as possible removed or neutralid as described in general chapter 2.6.12.
If surface-active substances are ud for sample preparation, their abnce of toxicity for micro-organisms and their compatibility with inactivators ud must be demonstrated as described in general chapter 2.6.12.
3 GROWTH-PROMOTING AND INHIBITORY PROPERTIES OF THE MEDIA, SUITABILITY OF THE TEST AND NEGATIVE CONTROLS
The ability of the test to detect micro-organisms in the prence of the product to be tested must be established. Suitability must be confirmed if a change in testing performance, or the product, which may affect the outcome of the test is introduced.
3-1 Preparation of test strains
U standardid stable suspensions of test strains or prepare them as stated below. Seed lot culture maintenance techniques (ed-lot systems) are ud so that the viable micro-organisms ud for inoculation are not more than 5 passages removed from the original master ed-lot.
3-1-1 Aerobic micro-organisms Grow each of the bacterial test strains parately in cain soya bean digest broth or on cain soya bean digest agar at 30-35 °C for 18-24 h. Grow the test strain for Candida albicans parately on Sabouraud-dextro agar or in Sabouraud-dextro broth at 20-25 °C for 2-3 days.
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— Staphylococcus aureus such as ATCC 6538, NCIMB 9518, CIP 4.83 or NBRC 13276;
— Pudomonas aeruginosa such as ATCC 9027, NCIMB 8626, CIP 82.118 or NBRC 13275;
— Escherichia coli such as ATCC 8739, NCIMB 8545, CIP 53.126 or NBRC 3972;
— Salmonella enterica subsp. enterica rovar Typhimurium, such as ATCC 14028 or, as an alternative,汽车多少年报废
Salmonella enterica subsp. enterica rovar Abony such as NBRC 100797, NCTC 6017 or CIP 80.39;
小时代电影— Candida albicans such as ATCC 10231, NCPF 3179, IP 48.72 or NBRC 1594.
U buffered sodium chloride-peptone solution pH 7.0 or phosphate buffer solution pH 7.2 to make test suspensions. U the suspensions within 2 h or within 24 h if stored at 2-8 °C.
3-1-2 Clostridia U Clostridium sporogenes such as ATCC 11437 (NBRC 14293, NCIMB 12343, CIP 100651) or ATCC 19404 (NCTC 532 or CIP 79.03) or NBRC 14293. Grow the clostridial test strain under anaerobic conditions in reinforced medium for clostridia at 30-35 °C for 24-48 h. As an alternative to preparing and then diluting down a fresh suspension of vegetative cells of Cl. sporogenes, a stable spore suspension is ud for test inoculation. The stable spore suspension may be maintained at 2-8 °C for a validated period.
3-2 Negative control
To verify testing conditions, a negative control is performed using the chon diluent in place of the test preparation. There must be no growth of micro-organisms. A negative control is also performed
when testing the products as described in ction 4. A failed negative control requires an investigation.
3-3 Growth promotion and inhibitory properties of the media
Test each batch of ready-prepared medium and each batch of medium prepared either from dehydrated medium or from ingredients.
Verify suitable properties of relevant media as described in Table 2.6.13.-1.
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Test for growth promoting properties, liquid media
Inoculate a portion of the appropriate medium with a small number (not more than 100 CFU) of the appropriate micro-organism. Incubate at the specified temperature for not more than the shortest period of time specified in the test. Clearly visible growth of the micro-organism comparable to that previously obtained with a previously tested and approved batch of medium occurs.
Test for growth promoting properties, solid media
Perform the surface-spread method, inoculating each plate with a small number (not more than 100 CFU) of the appropriate micro-organism. Incubate at the specified temperature for not more than the shortest period of time specified in the test. Growth of the micro-organism comparable to that previously obtained with a previously tested and approved batch of medium occurs.
Test for inhibitory properties, liquid or solid media
Inoculate the appropriate medium with at least 100 CFU of the appropriate micro-organism. Incubate at the specified temperature for not less than the longest period of time specified in the test. No growth of the test micro-organism occurs.
Test for indicative properties
Perform the surface-spread method, inoculating each plate with a small number (not more than 100 CFU) of the appropriate micro-organism. Incubate at the specified temperature for a period of time within the range specified in the test. Colonies are comparable in appearance and indication reactions to tho previously obtained with a previously tested and approved batch of medium.
3-4 Suitability of the test method
For each product to be tested, perform the sample preparation as described in the relevant paragraph in ction 4. Add each test strain at the time of mixing, in the prescribed growth medium. Inoculate the test strains individually. U a number of micro-organisms equivalent to not more than 100 CFU in the inoculated test preparation.
Perform the test as described in the relevant paragraph in ction 4 using the shortest incubation period prescribed.
The specified micro-organisms must be detected with the indication reactions as described in ction 4.
Any antimicrobial activity of the product necessitates a modification of the test procedure (e 4-5-3 of general chapter 2.6.12).
If for a given product the antimicrobial activity with respect to a micro-organism for which testing is prescribed cannot be neutralid, then it is to be assumed that the inhibited micro-organism will not be prent in the product.
4 TESTING OF PRODUCTS
4-1 Bile-tolerant gram-negative bacteria
4-1-1 Sample preparation and pre-incubation Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to be examined as described in general chapter 2.6.12, but using cain soya bean digest broth as the chon diluent, mix and incubate at 20-25 °C for a time sufficient to resuscitate the bacteria but not sufficient to encourage multiplication of the organisms (usually 2 h but not more than 5 h).
4-1-2 Test for abnce Unless otherwi prescribed, u the volume corresponding to 1 g of the product, as prepared in 4-1-1, to inoculate enterobacteria enrichment broth-Mosl. Incubate at 30-3
5 °C for 24-48 h. Subculture on plates of violet red bile gluco agar. Incubate at 30-35 °C for 18-24 h.
The product complies with the test if there is no growth of colonies.
4-1-3 Quantitative test
4-1-3-1 Selection and subculture Inoculate suitable quantities of enterobacteria enrichment broth-Mosl with the preparation as described under 4-1-1 and/or dilutions of it containing respectively 0.1 g, 0.01 g and 0.001 g (or 0.1 mL, 0.01 mL and 0.001 mL) of the product to be examined. Incubate at 30-35 °C for 24-48 h. Subculture each of the cultures on a plate of violet red bile gluco agar. Incubate at 30-35 °C for 18-24 h.
4-1-3-2 Interpretation Growth of colonies constitutes a positive result. Note the smallest quantity of the product that gives a positive result and the largest quantity that gives a negative result. Determine from Table 2.6.13.-2 the probable number of bacteria.
4-2 Escherichia coli
4-2-1 Sample preparation and pre-incubation Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to be examined as described in general chapter 2.6.12, and u 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount (determined as described under 3-4) of cain soya bean digest broth, mix and incubate at 30-35 °C for 18-24 h.
4-2-2 Selection and subculture Shake the container, transfer 1 mL of cain soya bean digest broth to 100 mL of MacConkey broth and incubate at 42-44 °C for 24-48 h. Subculture on a plate of MacConkey agar at 30-35 °C for 18-72 h.
4-2-3 Interpretation Growth of colonies indicates the possible prence of E. coli. This is confirmed by identification tests.
The product complies with the test if no colonies are prent or if the identification tests are negative.
4-3 Salmonella
4-3-1 Sample preparation and pre-incubation Prepare the product to be examined as described in general chapter 2.6.12, and u the quantity corresponding to not less than 10 g or 10 mL to inoculate a suitable amount (determined as described under 3-4) of cain soya bean digest broth, mix and incubate at 30-35 °C for 18-24 h.
4-3-2 Selection and subculture Transfer 0.1 mL of cain soya bean digest broth to 10 mL of Rappaport Vassiliadis Salmonella enrichment broth and incubate at 30-35 °C for 18-24 h. Subculture on plates of xylo, lysine, deoxycholate agar. Incubate at 30-35 °C for 18-48 h.
4-3-3 Interpretation The possible prence of Salmonella is indicated by the growth of well-developed, red colonies, with or without black centres. This is confirmed by identification tests.
The product complies with the test if colonies of the types described are not prent or if the confirm
atory identification tests are negative.
4-4 Pudomonas aeruginosa
4-4-1 Sample preparation and pre-incubation Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to be examined as described in general chapter 2.6.12, and u 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount (determined as described under 3-4) of cain soya bean digest broth and mix. When testing transdermal patches, filter the volume of sample corresponding to 1 patch of the preparation described under 4-5-1 in general chapter 2.6.12 through a sterile filter membrane and place in 100 mL of cain soya bean digest broth. Incubate at 30-35 °C for 18-24 h.
4-4-2 Selection and subculture Subculture on a plate of cetrimide agar and incubate at 30-35 °C for 18-72 h.
4-4-3 Interpretation Growth of colonies indicates the possible prence of P. aeruginosa. This is confirmed by identification tests.
The product complies with the test if colonies are not prent or if the confirmatory identification tests are negative.
4-5 Staphylococcus aureus
4-5-1 Sample preparation and pre-incubation Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to be examined as described in general chapter 2.6.12, and u 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount (determined as described under 3-4) of cain soya bean digest broth and mix. When testing transdermal patches, filter the volume of sample corresponding to 1 patch of the preparation described under 4-5-1 in general chapter 2.6.12 through a sterile filter membrane and place in 100 mL of cain soya bean digest broth. Incubate at 30-35 °C for 18-24 h.
4-5-2 Selection and subculture Subculture on a plate of mannitol salt agar and incubate at 30-35 °C for 18-72 h.
4-5-3 Interpretation The possible prence of S. aureus is indicated by the growth of yellow/white colonies surrounded by a yellow zone. This is confirmed by identification tests.
The product complies with the test if colonies of the types described are not prent or if the confirmatory identification tests are negative.
4-6 Clostridia
4-6-1 Sample preparation and heat treatment Prepare a sample using a 1 in 10 dilution (with a minimum total volume of 20 mL) of not less than 2 g or 2 mL of the product to be examined as described in general chapter 2.6.12.
Divide the sample into 2 portions of at least 10 mL. Heat 1 portion at 80 °C for 10 min and cool rapidly. Do not heat the other portion.
4-6-2 Selection and subculture U 10 mL or the quantity corresponding to 1 g or 1 mL of the product to be examined of both portions to inoculate suitable amounts (determined as described under 3-4) of reinforced medium for clostridia. Incubate under anaerobic conditions at 30-35 °C for 48 h. After incubation, make subcultures from each container on Columbia agar and incubate under anaerobic conditions at 30-35 °C for 48-72 h.
4-6-3 Interpretation The occurrence of anaerobic growth of rods (with or without endospores) giving a negative catala reaction indicates the prence of clostridia. This is confirmed by identification tests.
The product complies with the test if colonies of the types described are not prent or if the confirmatory identification tests are negative.
4-7 Candida albicans
4-7-1 Sample preparation and pre-incubation Prepare the product to be examined as described in general chapter 2.6.12, and u 10 mL or the quantity corresponding to not less than 1 g or 1 mL to inoculate 100 mL of Sabouraud-dextro broth and mix. Incubate at 30-35 °C for 3-5 days.
4-7-2 Selection and subculture Subculture on a plate of Sabouraud-dextro agar and incubate at 30-35 °C for 24 -48 h.
4-7-3 Interpretation Growth of white colonies may indicate the prence of C. albicans. This is confirmed by identification tests.
The product complies with the test if such colonies are not prent or if the confirmatory identification tests are negative.
The following ction is given for information.
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5 RECOMMENDED SOLUTIONS AND CULTURE MEDIA
The following solutions and culture media have been found to be satisfactory for the purpos for wh
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ich they are prescribed in the test for microbial contamination in the Pharmacopoeia. Other media may be ud provided that their suitability can be demonstrated.
Stock buffer solution Place 34 g of potassium dihydrogen phosphate in a 1000 mL volumetric flask, dissolve in 500 mL of purified water, adjust to pH 7.2 ± 0.2 with sodium hydroxide, dilute to 1000.0 mL with purified water and mix. Dispen into containers and sterili. Store at 2-8 °C.
Phosphate buffer solution pH 7.2 Prepare a mixture of stock buffer solution and purified water (1:800 V/V) and sterili.
Buffered sodium chloride-peptone solution pH 7.0
Potassium dihydrogen phosphate 3.6 g
Disodium hydrogen phosphate dihydrate7.2 g, equivalent to 0.067 M phosphate
Sodium chloride 4.3 g
Peptone (meat or cain) 1.0 g
Purified water1000 mL
Sterili in an autoclave using a validated cycle.
Cain soya bean digest broth
Pancreatic digest of cain17.0 g
