BV Test Protocol For Bath Faucet - Nov 12, 2014
Appendix XVI B. Microbiological Examination of Non-sterile Products in BP2012
British Pharmacopoeia Volume VAppendicesAppendix XVI B. Microbiological Examination of Non-sterile Products1. Tests for Specified Micro-organisms1(Ph. Eur. method 2.6.13)1 INTRODUCTIONThe tests described hereafter will allow determination of the absence 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 used for such purposes, follow the instructions given below, including the number of samples to be taken, and interpret the results as stated below.Alternative microbiological procedures, including automated methods, may be used, provided that their equivalence to the Pharmacopoeia method has been demonstrated.2 GENERAL PROCEDURESThe 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 neutralised as described in general chapter 2.6.12.If surface-active substances are used for sample preparation, their absence of toxicity for micro-organisms and their compatibility with inactivators used 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 CONTROLSThe ability of the test to detect micro-organisms in the presence 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 strainsUse standardised stable suspensions of test strains or prepare them as stated below. Seed lot culture maintenance techniques (seed-lot systems) are used so that the viable micro-organisms used for inoculation are not more than 5 passages removed from the original master seed-lot.3-1-1 Aerobic micro-organisms Grow each of the bacterial test strains separately in casein soya bean digest broth or on casein soya bean digest agar at 30-35 °C for 18-24 h. Grow the test strain for Candida albicans separately on Sabouraud-dextrose agar or in Sabouraud-dextrose broth at 20-25 °C for 2-3 days.— Staphylococcus aureus such as ATCC 6538, NCIMB 9518, CIP 4.83 or NBRC 13276;— Pseudomonas 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 serovar Typhimurium, such as ATCC 14028 or, as an alternative,Salmonella enterica subsp. enterica serovar Abony such as NBRC 100797, NCTC 6017 or CIP 80.39;— Candida albicans such as ATCC 10231, NCPF 3179, IP 48.72 or NBRC 1594.Use buffered sodium chloride-peptone solution pH 7.0 or phosphate buffer solution pH 7.2 to make test suspensions. Use the suspensions within 2 h or within 24 h if stored at 2-8 °C.3-1-2 Clostridia Use 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 used for test inoculation. The stable spore suspension may be maintained at 2-8 °C for a validated period.3-2 Negative controlTo verify testing conditions, a negative control is performed using the chosen 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 section 4. A failed negative control requires an investigation.3-3 Growth promotion and inhibitory properties of the mediaTest 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.Test for growth promoting properties, liquid mediaInoculate 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 mediaPerform 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 mediaInoculate 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 propertiesPerform 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 those previously obtained with a previously tested and approved batch of medium.3-4 Suitability of the test methodFor each product to be tested, perform the sample preparation as described in the relevant paragraph in section 4. Add each test strain at the time of mixing, in the prescribed growth medium. Inoculate the test strains individually. Use 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 section 4 using the shortest incubation period prescribed.The specified micro-organisms must be detected with the indication reactions as described in section 4.Any antimicrobial activity of the product necessitates a modification of the test procedure (see 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 neutralised, then it is to be assumed that the inhibited micro-organism will not be present in the product.4 TESTING OF PRODUCTS4-1 Bile-tolerant gram-negative bacteria4-1-1 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 casein soya bean digest broth as the chosen 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 absence Unless otherwise prescribed, use the volume corresponding to 1 g of the product, as prepared in 4-1-1, to inoculate enterobacteria enrichment broth-Mossel. Incubate at 30-35 °C for 24-48 h. Subculture on plates of violet red bile glucose 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 test4-1-3-1 Selection and subculture Inoculate suitable quantities of enterobacteria enrichment broth-Mossel 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 glucose 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 coli4-2-1 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 use 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount (determined as described under 3-4) of casein 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 casein 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 presence of E. coli. This is confirmed by identification tests.The product complies with the test if no colonies are present or if the identification tests are negative.4-3 Salmonella4-3-1 Sample preparation and pre-incubation Prepare the product to be examined as described in general chapter 2.6.12, and use the quantity corresponding to not less than 10 g or 10 mL to inoculate a suitable amount (determined as described under 3-4) of casein 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 casein 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 xylose, lysine, deoxycholate agar. Incubate at 30-35 °C for 18-48 h.4-3-3 Interpretation The possible presence 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 present or if the confirmatory identification tests are negative.4-4 Pseudomonas aeruginosa4-4-1 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 use 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount (determined as described under 3-4) of casein 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 casein 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 presence of P. aeruginosa. This is confirmed by identification tests.The product complies with the test if colonies are not present or if the confirmatory identification tests are negative.4-5 Staphylococcus aureus4-5-1 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 use 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount (determined as described under 3-4) of casein 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 casein 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 presence 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 present or if the confirmatory identification tests are negative.4-6 Clostridia4-6-1 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 Use 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 catalase reaction indicates the presence of clostridia. This is confirmed by identification tests.The product complies with the test if colonies of the types described are not present or if the confirmatory identification tests are negative.4-7 Candida albicans4-7-1 Sample preparation and pre-incubation Prepare the product to be examined as described in general chapter 2.6.12, and use 10 mL or the quantity corresponding to not less than 1 g or 1 mL to inoculate 100 mL of Sabouraud-dextrose broth and mix. Incubate at 30-35 °C for 3-5 days.4-7-2 Selection and subculture Subculture on a plate of Sabouraud-dextrose agar and incubate at 30-35 °C for 24 -48 h.4-7-3 Interpretation Growth of white colonies may indicate the presence of C. albicans. This is confirmed by identification tests.The product complies with the test if such colonies are not present or if the confirmatory identification tests are negative.The following section is given for information.5 RECOMMENDED SOLUTIONS AND CULTURE MEDIAThe following solutions and culture media have been found to be satisfactory for the purposes for which they are prescribed in the test for microbial contamination in the Pharmacopoeia. Other media may be used 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. Dispense into containers and sterilise. 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 sterilise.Buffered sodium chloride-peptone solution pH 7.0Potassium dihydrogen phosphate 3.6 gDisodium hydrogen phosphate dihydrate7.2 g, equivalent to 0.067 M phosphateSodium chloride 4.3 gPeptone (meat or casein) 1.0 gPurified water1000 mLSterilise in an autoclave using a validated cycle.Casein soya bean digest brothPancreatic digest of casein17.0 gPapaic digest of soya bean 3.0 gSodium chloride 5.0 gDipotassium hydrogen phosphate 2.5 gGlucose monohydrate 2.5 gPurified water1000 mLAdjust the pH so that after sterilisation it is 7.3 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle. Casein soya bean digest agarPancreatic digest of casein15.0 gPapaic digest of soya bean 5.0 gSodium chloride 5.0 gAgar15.0 gPurified water1000 mLAdjust the pH so that after sterilisation it is 7.3 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle. Sabouraud-dextrose agarDextrose40.0 g Mixture of peptic digest of animal tissue and pancreatic digest of casein (1:1)10.0 g Agar15.0 g Purified water1000 mL Adjust the pH so that after sterilisation it is 5.6 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle. Potato dextrose agarInfusion from potatoes200 gDextrose20.0 gAgar15.0 gPurified water1000 mLAdjust the pH so that after sterilisation it is 5.6 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle. Sabouraud-dextrose brothDextrose20.0 g Mixture of peptic digest of animal tissue and pancreatic digest of casein (1:1)10.0 g Purified water1000 mLAdjust the pH so that after sterilisation it is 5.6 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle. Enterobacteria enrichment broth-MosselPancreatic digest of gelatin10.0 gGlucose monohydrate 5.0 g Dehydrated ox bile20.0 g Potassium dihydrogen phosphate 2.0 gDisodium hydrogen phosphate dihydrate8.0 gBrilliant green15 mgPurified water1000 mLAdjust the pH so that after heating it is 7.2 ± 0.2 at 25 °C. Heat at 100 °C for 30 min and cool immediately. Violet red bile glucose agarYeast extract 3.0 gPancreatic digest of gelatin7.0 gBile salts 1.5 gSodium chloride 5.0 gGlucose monohydrate10.0 gAgar15.0 gNeutral red30 mgCrystal violet 2 mgPurified water1000 mLAdjust the pH so that after heating it is 7.4 ± 0.2 at 25 °C. Heat to boiling; do not heat in an autoclave. MacConkey brothPancreatic digest of gelatin20.0 gLactose monohydrate10.0 gDehydrated ox bile 5.0 gBromocresol purple10 mgPurified water1000 mLAdjust the pH so that after sterilisation it is 7.3 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle. MacConkey agarPancreatic digest of gelatin17.0 gPeptones (meat and casein) 3.0 gLactose monohydrate10.0 gSodium chloride 5.0 gBile salts 1.5 gAgar13.5 gNeutral red30.0 mgCrystal violet 1 mgPurified water1000 mLAdjust the pH so that after sterilisation it is 7.1 ± 0.2 at 25 °C. Boil for 1 min with constant shaking then sterilise in an autoclave using a validated cycle.Rappaport Vassiliadis Salmonella enrichment brothSoya peptone 4.5 gMagnesium chloride hexahydrate29.0 gSodium chloride8.0 gDipotassium phosphate0.4 gPotassium dihydrogen phosphate0.6 gMalachite green0.036 gPurified water1000 mLDissolve, warming gently. Sterilise in an autoclave using a validated cycle, at a temperature not exceeding 115 °C. The pH is to be 5.2 ± 0.2 at 25 °C after heating and autoclaving.Xylose, lysine, deoxycholate agarXylose 3.5 gL-Lysine 5.0 gLactose monohydrate7.5 gSucrose7.5 gSodium chloride 5.0 gYeast extract 3.0 gPhenol red80 mgAgar13.5 gSodium deoxycholate 2.5 gSodium thiosulfate 6.8 gFerric ammonium citrate0.8 gPurified water1000 mLAdjust the pH so that after heating it is 7.4 ± 0.2 at 25 °C. Heat to boiling, cool to 50 °C and pour into Petri dishes. Do not heat in an autoclave.Cetrimide agarPancreatic digest of gelatin20.0 gMagnesium chloride 1.4 gDipotassium sulfate10.0 gCetrimide0.3 gAgar13.6 gPurified water1000 mLGlycerol10.0 mLHeat to boiling for 1 min with shaking. Adjust the pH so that after sterilisation it is 7.2 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.Mannitol salt agarPancreatic digest of casein 5.0 gPeptic digest of animal tissue 5.0 gBeef extract 1.0 gD-Mannitol10.0 gSodium chloride75.0 gAgar15.0 gPhenol red0.025 gPurified water1000 mLHeat to boiling for 1 min with shaking. Adjust the pH so that after sterilisation it is 7.4 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.Reinforced medium for clostridiaBeef extract10.0 gPeptone10.0 gYeast extract 3.0 gSoluble starch 1.0 gGlucose monohydrate 5.0 gCysteine hydrochloride0.5 gSodium chloride 5.0 gSodium acetate 3.0 gAgar0.5 gPurified water1000 mLHydrate the agar, dissolve by heating to boiling with continuous stirring. If necessary, adjust the pH so that after sterilisation it is 6.8 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.Columbia agarPancreatic digest of casein10.0 gMeat peptic digest 5.0 gHeart pancreatic digest 3.0 gYeast extract 5.0 gMaize starch 1.0 gSodium chloride 5.0 gAgar, according to gelling power10.0-15.0 gPurified water1000 mLHydrate the agar, dissolve by heating to boiling with continuous stirring. If necessary, adjust the pH so that after sterilisation it is 7.3 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle. Allow to cool to 45-50 °C; add, where necessary, gentamicin sulfate corresponding to 20 mg of gentamicin base and pour into Petri dishes.2. Microbial Enumeration Tests1(Ph. Eur. method 2.6.12)1 INTRODUCTIONThe tests described hereafter will allow quantitative enumeration of mesophilic bacteria and fungi that may grow under aerobic conditions.The tests are designed primarily to determine whether a substance or preparation complies with an established specification for microbiological quality. When used for such purposes follow the instructions given below, including the number of samples to be taken, and interpret the results as stated below.The methods are not applicable to products containing viable micro-organisms as active ingredients.Alternative microbiological procedures, including automated methods, may be used, provided that their equivalence to the Pharmacopoeia method has been demonstrated.2 GENERAL PROCEDURESCarry out the determination under conditions designed to avoid extrinsic microbial contamination of the product to be examined. The precautions taken to avoid contamination must be such that they do not affect any micro-organisms that are to be revealed in the test.If the product to be examined has antimicrobial activity, this is insofar as possible removed or neutralised. If inactivators are used for this purpose, their efficacy and their absence of toxicity for micro-organisms must be demonstrated.If surface-active substances are used for sample preparation, their absence of toxicity for micro-organisms and their compatibility with inactivators used must be demonstrated.3 ENUMERATION METHODSUse the membrane filtration method or the plate-count methods, as prescribed. The most-probable-number (MPN) method is generally the least accurate method for microbial counts, however, for certain product groups with a very low bioburden, it may be the most appropriate method.The choice of method is based on factors such as the nature of the product and the required limit of micro-organisms. The chosen method must allow testing of a sufficient sample size to judge compliance with the specification. The suitability of the method chosen must be established.4 GROWTH PROMOTION TEST, SUITABILITY OF THE COUNTING METHOD AND NEGATIVE CONTROLS4-1 General considerationsThe ability of the test to detect micro-organisms in the presence of 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.4-2 Preparation of test strainsUse standardised stable suspensions of test strains or prepare them as stated below. Seed lot culture maintenance techniques (seed-lot systems) are used so that the viable micro-organisms used for inoculation are not more than 5 passages removed from the original master seed-lot. Grow each of the bacterial and fungal test strains separately as described in Table 2.6.12.-1.Use buffered sodium chloride-peptone solution pH 7.0 or phosphate buffer solution pH 7.2 to make test suspensions; to suspend A. brasiliensis spores, 0.05 per cent of polysorbate 80 may be added to the buffer. Use the suspensions within 2 h or within 24 h if stored at 2-8 °C. As an alternative to preparing and then diluting a fresh suspension of vegetative cells of A. brasiliensis or B. subtilis, a stable spore suspension is prepared and then an appropriate volume of the spore suspension is used for test inoculation. The stable spore suspension may be maintained at 2-8 °C for a validated period of time.4-3 Negative controlTo verify testing conditions, a negative control is performed using the chosen 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 section 5. A failed negative control requires an investigation.4-4 Growth promotion of the mediaTest each batch of ready-prepared medium and each batch of medium, prepared either from dehydrated medium or from the ingredients described.Inoculate portions/plates of casein soya bean digest broth and casein soya bean digest agar with a small number (not more than 100 CFU) of the micro-organisms indicated in Table 2.6.12.-1, using a separate portion/plate of medium for each. Inoculate plates of Sabouraud-dextrose agar with a small number (not more than 100 CFU) of the micro-organisms indicated in Table 2.6.12.-1, using a separate plate of medium for each. Incubate in the conditions described in Table 2.6.12.-1.For solid media, growth obtained must not differ by a factor greater than 2 from the calculated value for a standardised inoculum. For a freshly prepared inoculum, growth of the micro-organisms comparable to that previously obtained with a previously tested and approved batch of medium occurs. Liquid media are suitable if clearly visible growth of the micro-organisms comparable to that previously obtained with a previously tested and approved batch of medium occurs.4-5 Suitability of the counting method in the presence of product4-5-1 Preparation of the sample The method for sample preparation depends upon the physical characteristics of the product to be tested. If none of the procedures described below can be demonstrated to be satisfactory, an alternative procedure must be developed.Water-soluble products Dissolve or dilute (usually a 1 in 10 dilution is prepared) the product to be examined in buffered sodium chloride-peptone solution pH 7.0, phosphate buffer solution pH 7.2 or casein soya bean digest broth. If necessary, adjust to pH 6-8. Further dilutions, where necessary, are prepared with the same diluent.Non-fatty products insoluble in water Suspend the product to be examined (usually a 1 in 10 dilution is prepared) in buffered sodium chloride-peptone solution pH 7.0, phosphate buffer solution pH 7.2 or casein soya bean digest broth. A surface-active agent such as 1 g/L of polysorbate 80 may be added to assist the suspension of poorly wettable substances. If necessary, adjust to pH 6-8. Further dilutions, where necessary, are prepared with the same diluent.Fatty products Dissolve in isopropyl myristate, sterilised by filtration or mix the product to be examined with the minimum necessary quantity of sterile polysorbate 80 or another non-inhibitory sterile surface-active agent, heated if necessary to not more than 40 °C, or in exceptional cases to not more than 45 °C. Mix carefully and if necessary maintain the temperature in a water-bath. Add sufficient of the pre-warmed chosen diluent to make a 1 in 10 dilution of the original product. Mix carefully whilst maintaining the temperature for the shortest time necessary for the formation of an emulsion. Further serial tenfold dilutions may be prepared using the chosen diluent containing a suitable concentration of sterile polysorbate 80 or another non-inhibitory sterile surface-active agent.Fluids or solids in aerosol form Aseptically transfer the product into a membrane filter apparatus or a sterile container for further sampling. Use either the total contents or a defined number of metered doses from each of the containers tested.Transdermal patches Remove the protective cover sheets ('release liners') of the transdermal patches and place them, adhesive side upwards, on sterile glass or plastic trays. Cover the adhesive surface with a sterile porous。
除菌过滤器后的完整性测试
除菌过滤器后的完整性测试除菌过滤器使用后，必需采用适当的方法赶忙对其完整性进行测试并记录。
除菌过滤器使用前，应当进行风险评估来确定是否进行完整性测试，并确定在灭菌前还是灭菌后进行。
当进行灭菌后—使用前完整性测试时，需要采取措施保证过滤器下游的无菌性。
常用的完整性测试方法有起泡点测试、扩散流/前进流试验或水侵入法测试。
进入A级和B级干净区的消毒剂，应经除菌过滤或采用其他适当方法除菌。
假如使用过滤方法除菌，应评估消毒剂与所选择滤器材质之间的适用性。
滤器使用后需进行完整性测试。
用于直接接触无菌药液或无菌设备表面的气体的过滤器，应在每批或多批次连续生产结束后对其进行完整性测试。
对于其他的应用，可以依据风险评估的结果，订立完整性测试的频率。
气体过滤器的完整性测试，可以使用低表面张力的液体润湿，进行起泡点或者扩散流/前进流的测试；也可以使用水侵入法测试。
水侵入法可作为优先选择。
对于冗余过滤，使用后应先对主过滤器进行完整性测试，假如主过滤器完整性测试通过，则冗余过滤器不需要进行完整性测试；假如主过滤器完整性测试失败，则需要对冗余过滤器进行完整性测试。
冗余过滤器完整性测试结果可作为产品放行的依据。
除菌过滤器使用前，应通过风险评估的方式确定是否进行完整性测试，以及测试哪一级过滤器或者两级过滤器都要进行检测，并确定在过滤器灭菌前还是灭菌后进行。
灭菌后的检测，应考虑确保两级过滤器之间的无菌性。
可依据工艺需要和实际条件，决议采用在线完整性测试或者离线完整性测试。
但应注意，完整性测试是检测整个过滤系统的完整性，而非仅针对过滤器自身。
在线测试能更好地保证上下游连接的完整性。
当无法满足在线测试条件时，可选择进行离线完整性测试。
此时应将过滤器保持在套筒中整体拆卸，并直接进行测试，不应将滤芯从不锈钢套筒拆卸单独测试。
考虑到完整性测试结果的客观性以及数据可靠性，应尽可能在关键使用点使用自动化完整性测试仪。
自动化完整性测试仪应在使用前，进行安装确认、运行确认和性能确认。
医用耗材说明书：BV四联检测试剂盒
BV四联检测试剂盒【导读】BV四联检测试剂盒四项指标联查；操作简单、不需特殊仪器、无需专业培训，经临床使用，灵敏度高、特异性高、准确性高；该产品适用于对妇科阴道分泌物的细菌感染的快速筛查，包括唾液酸酶、白细胞酯酶、过氧化氢、多胺的定性检测。
BV四联检测试剂盒适应症1、BV四联检测试剂盒适用于定性检测女性阴道分泌物中酸碱度(pH)、过氧化氢(H202)的浓度和唾液酸酶(SA)、白细胞酯酶(LE)的活性。
2、BV四联检测试剂盒适用于细菌性阴道病的辅助诊断。
BV四联检测试剂盒原理1.唾液酸酶检测原理：唾液酸酶能使特异性底物反应，在相应显色剂作用下呈现特异颜色变化，颜色深浅与唾液酸酶活性浓度成正比。
2.白细胞酯酶检测原理：白细胞酯酶能水解特异性底物，当样本中含有此酶时，会发生生化反应呈蓝色，蓝色深浅与白细胞酯酶活性浓度成正比。
3.过氧化氢检测原理：过氧化氢经过过氧化物酶催化下，在反应底物作用下呈蓝色，蓝色深浅与过氧化氢浓度成正比。
4.多胺检测原理：多胺在特异性物质的催化下与相应反应物发生反应生成蓝色，蓝色深浅与多胺浓度成正比。
BV四联检测试剂盒特点1.四项联合检测，可综合评估阴道酸碱环境失衡程度、阴道菌群失调程度、阴道炎症程度及诊断细菌性阴道病，综合分析阴道健康及疗效评价。
2.操作简便：卡式结构，美观且操作方便，可随时检测。
3.反应快速：15分钟出结果，检测时间短。
4.结果准确。
BV四联检测试剂盒使用方法1．取出装有检测卡的铝箔袋及样本稀释液等试剂放置室温30分钟，使其恢复室温。
2．样本采集：插入窥阴器，长棉拭子从阴道侧壁或后穹窿处尽可能多采集分泌物，立即送检；3．样本处理：取一样本管，将分泌物拭子置于该管中，滴加样本稀释液8-10滴；充分涮洗后用手指轻轻挤压试管壁，使拭子吸附的液体流回试管，弃拭子，得到样本液，立即检测或盖紧置冰箱短时间冷藏；4．从包装袋中取出检测卡，轻轻揭掉板贴，用吸管吸取样本液，在检测卡的四个孔中每孔滴加1滴样本液，之后在过氧化氢反应孔滴加1滴过氧化氢显色液，在唾液酸苷酶反应孔中滴加1滴唾液酸苷酶显色液，在多胺反应孔中滴加多胺显色液ⅰ和多胺显色液ⅱ各1滴，置37℃温育10分钟。
完整性测试方法
滤芯完整性检测
完整性检测
亲水性膜
疏水性膜
前进流 压力衰减 泡点 水侵入
© Pall Corporation 2004
滤芯的前进流和泡点检测
孔需要完全润湿 使用低表面张力的溶剂如：乙醇/水混合 物 检测完需去除溶剂 注意易燃问题
© Pall Corporation 2004
Life Sciences
冻干装瓶 无菌分装
生产过程中的产品保护
工厂间无菌容器 在生产和灌装的无菌产品储罐
© Pall Corporation 2004
在规定间歇时间内滤芯的测试
WFI 罐上的呼吸器 低生物负荷产品储罐上的呼吸器 灭菌柜上的呼吸器 发酵罐空气过滤器 发酵罐排气过滤器 仪器上空气过滤器
© Pall Corporation 2004
© Pall Corporation 2004
直接流量测量手段
恒压检测
更稳定、可靠、准确
上游流量测量 不依赖于上游体积
无需计算和确认将压力衰减 转换为流量
不需测量体积-更简单更有效
© Pall Corporation 2004
Life Sciences
产品润湿完整性测试
Filtration. Separation. Solution. SM
"实现这一点的途径就是关联过滤器的性能数 据与完整性试验数据."
U.S. Food and Drug Administration (FDA) June, 1987,
Guideline On Sterile Drug Products Produced by Aseptic Processing
© Pall Corporation 2004
美国药典微生物限度检测
61微生物限度检测（MICROBIAL LIMIT TESTS）此章提供方法来检测可能存在的好氧微生物其他制药过程中可能出现的微生物的数量，包括原材料和成品中的。
如果经过验证确认可以得到相同或更好的检测结论，也允许采用自动化的检测方法。
在样品检测过程中须进行无菌操作。
若无特别说明，则“培养（incubate）”一词指在30—35℃的培养箱内培养24至48小时；“生长（growth）”一词用于专门的判定，说明“存在和可能存在活的微生物”。
准备实验 (Preparatory Testing)本章涉及实验结果的有效性取决于：提供的被检测样品本身在实验条件下，被充分证明不会抑制可能存在的微生物的生长。
因此，在准备样品时，需要正规的实验操作和符合要求的实验条件，接种稀释样品到含有以下（微生物）培养物的培养基：金黄色（奥里斯）葡萄球菌（Staphylococcus aureus）,大肠埃希氏菌（Escherichia coli）, 铜绿假单胞菌(Pseudomonas aeruginosa), 和沙门氏菌（Salmonella）。
方法如下：将用肉汤培养基培养24小时后的（微生物）不小于10-3稀释的微生物培养物，加1 ml（微生物）培养液到磷酸(盐)缓冲液(pH 7.2)，液体大豆酪蛋白消化物培养基（Fluid Soybean-Casein Digest Medium），或者液体乳糖培养基（Fluid Lactose Medium）。
相应培养基培养失败则需要采取以下方法更改检测程序：（1）增加稀释液体积，检测样品加入量仍维持不变；或者（2）中和一定数量的干扰因子；或者（3）结合（1）、（2）得出适当条件，使接种物得以生长。
以下是一些物质的成分和浓度，该物质及浓度可用于加入培养基、阻止物质发挥抑菌作用：大豆卵磷脂（soy lecithin, 0.5%）或者聚山梨醇酯20（polysorbate 20, 4.0%）。
欧洲药典注射用水检验规程
目的使注射用水的检验操作标准化、规范化，保证注射水的质量，从而保证产品质量。
范围适用于注射用水的检验职责由化验室起草；部门经理及相关人员审核；质量总监批准；化验员执行。
内容1编制依据EP7.02定义通常用作溶剂(散装注射用水)来溶解、稀释物质或作为非肠道注射准备用水(无菌注射用水)的药物非肠道给药的准备用水3执行标准《注射用水质量标准》。
4性状本品为无色的澄明液体。
5检查5.1硝酸盐5.1.1仪器与装置：量筒、试管、移液管、水浴锅。
5.1.2试剂与试液：标准硝酸盐溶液、10%氯化钾溶液、0.1%二苯胺硫酸溶液、硫酸。
标准硝酸盐溶液：取硝酸钾0.163g，加水溶解并稀释至100ml，摇匀，再精密量取1ml，加水稀释成100ml，摇匀，再精密量取20ml，加水稀释成100ml，摇匀，即得（每1ml相当于2μgNO3-）。
5.1.3测定方法：取本品5ml置试管中，于冰浴中冷却，加10%氯化钾溶液0.4ml与0.1%二苯胺硫酸溶液0.1ml，摇匀，缓缓滴加无氮硫酸5ml，摇匀，将试管于50℃水浴中放置15分钟，溶液产生的蓝色与标准硝酸盐溶液（2ppm NO3），加无硝酸盐的水4.5ml，用同一方法处理后的颜色对比，不得更深。
5.1.4结果判定：不超过0.2ppm。
5.2电导率5.2.1电导计：最低限的分辨率为0.1μS·cm-1，准确度：±3%，温度测量：±2℃。
5.2.2步骤阶段一1.用非温度补偿或温度补偿的电导仪测定水的温度和电导率，在适当的容器中测量或在线测量。
2.在表0169- 2中找到最接近的温度的值不能大于实测温度。
以相应的温度电导率值为限值。
3.如果测量电导率不大于表0169-2中的值，则水要符合表0169-2中的电导率限值。
如果电导率高于表0169-2中的值则继续进行阶段二。
表0169-2. -温度和电导率要求（非温度补偿电导率测量）温度（℃）电导率（μS·cm-1）0 0.6 5 0.8 10 0.9 15 1.0 20 1.130 1.435 1.540 1.745 1.850 1.955 2.160 2.265 2.470 2.575 2.780 2.785 2.790 2.795 2.9100 3.1阶段二4.取至少100毫升样品到一个合适的容器中，搅拌样品。
伯杰氏细菌鉴定手册引用
伯杰氏细菌鉴定手册引用伯杰氏细菌鉴定手册引用参考内容：1. Bergey's Manual of Systematic Bacteriology- Whitman, W.B., et al. (eds.). Bergey's Manual of Systematic Bacteriology. 2nd edition. Springer, 2012.- This manual provides a comprehensive and authoritative resource for the identification and classification of bacteria. It includes information on morphological, physiological, and genetic characteristics of bacteria, along with detailed descriptions of individual taxa.2. Manual of Clinical Microbiology- Jorgensen, J.H., et al. (eds.). Manual of Clinical Microbiology. 11th edition. ASM Press, 2015.- The Manual of Clinical Microbiology is a widely used reference in clinical microbiology laboratories. It covers a broad range of topics related to bacterial identification, including specimen collection and processing, laboratory techniques, and interpretation of test results.3. Laboratory Identification of Bacteria- Elaichouni, A. (ed.). Laboratory Identification of Bacteria. CRC Press, 2014.- This book provides a practical guide to laboratory identification of bacteria, focusing on different techniques and tests used to identify and characterize bacteria. It covers topics such as microscopy, culturing, biochemical tests, and molecular techniques.4. Identification of Bacteria by Gas Chromatography-MassSpectrometry Analysis- Seng, P., et al. Identification of Bacteria by Gas Chromatography-Mass Spectrometry Analysis of Urine Samples. Clinical Microbiology and Infection, 2013, 19(4), 335-341.- This research article describes the use of gas chromatography-mass spectrometry (GC-MS) analysis for the identification of bacteria in urine samples. GC-MS analysis can provide accurate and rapid identification of bacteria based on their specific chemical profiles.5. MALDI-TOF Mass Spectrometry for Bacterial Identification- Patel, R. MALDI-TOF Mass Spectrometry for Bacterial Identification. Expert Review of Molecular Diagnostics, 2013,13(2), 167-180.- This review article discusses the applications of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for bacterial identification. It provides an overview of the technique, its advantages, and limitations, along with examples of its use in clinical microbiology.6. Molecular Methods for Bacterial Identification- Petti, C.A. Detection and Identification of Microorganisms by Gene Amplification and Sequencing. Clinical Infectious Diseases, 2007, 44(8), 1108-1114.- This review article discusses the use of molecular methods, such as gene amplification and sequencing, for the detection and identification of microorganisms. It provides an overview of different molecular techniques and their applications in clinical microbiology.Note: The above references are for informational purposes only and do not constitute an endorsement of any specific product, manufacturer, or organization. It is always important to consult multiple sources and use professional judgement in scientific research and practice.。
清洁验证-简述2024.04.11
第二部分 法规-GMP附录11
2015.05发布 第三十八条 为确认与产品直接接触设备的清洁操作规程的 有效性，应当进行清洁验证。应当根据所涉及的物料，合理 地确定活性物质残留、清洁剂和微生物污染的限度标准。 第三十九条 在清洁验证中，不能采用反复清洗至清洁的方 法。目视检查是一个很重要的标准，但通常不能作为单一可 接受标准使用。
• 第三部分
法规要求
第三部分 法规
1、药品生产质量管理规范-（2010年修订） 2、药品GMP指南- 2010 3、APIC清洁验证指南-2016 4、PDA TR29-2012 5、ICH指导原则 6、 GB/T 36030-2018《制药机械(设备)在位清洗、灭菌通 用技术要求》-2018.03.15
清洁验清证洁-验简述证 - 简 述
2024.04
• 第一部分 清洁验证的由来
第二部分 • 什么是清洁验证
• 第三部分
法规要求
• 第一部分 清洁验证的由来
第一部分 清洁验证的由来
验证是GMP发展的产物，是GMP发展史中一个非常重要的 里程碑。验证是美国FDA对污染输液所致触目惊心的药难事 件调查后采取的重要举措。
第三部分 法规-GMP附录11
第四十三条 应当在清洁验证过程中对潜在的微生物污染进 行评价，如需要，还应当评价细菌内毒素污染。应当考虑设 备使用后至清洁前的间隔时间以及设备清洁后的保存时限对 清洁验证的影响。
第三部分 法规-GMP附录11
第四十四条 当采用阶段性生产组织方式时，应当综合考虑 阶段性生产的最长时间和最大批次数量，以作为清洁验证的 评价依据。 第四十六条 清洁验证方案应当详细描述取样的位置、所选 取的取样位置的理由以及可接受标准。
第一部分 清洁验证的由来
水质标准
≤
μg/L
硝酸根
≤
μg/L
磷酸根
≤
μg/L
硫酸根
≤
μg/L
总有
机碳
≤
μg/L
EW-Ⅰ
18（95%的时间不低于17）
2பைடு நூலகம்
0.1
0.001
0.2
0.2
0.1
0.5
0.5
1
1
1
1
20
EW-Ⅱ
15（95%的时间不低于13）
10
5
0.1
1
1
1
2
2
1
1
1
1
100
蒸馏机制水与（超）纯水机制水比较表
项目
蒸馏机制水
（超）纯水机制水
0.001
0.01
-
二氧化硅（mg/L）≤
0.02
0.05
-
蒸发残渣（mg/L）≤
-
1.0
2.0
中国国家电子级超纯水标准（GB/T11446.1-1997）
项目
级别
比电阻
MΩ.cm@25℃
硅
≤
μg/L
>1μm
微粒数
<
个/ml
细菌
个数
<
个/ml
铜
≤
μg/L
锌
≤
μg/L
镍
≤
μg/L
钠
≤
μg/L
钾
≤
μg/L
≥10
硅mg/L max
-
-
-
0.01
0.01
0.01
0.05
重金属mg/L max
-
-
评估两种检测微囊藻毒素浓度的试剂盒说明书
Evaluation of Two Test Kits for Measurement of Microcystin ConcentrationsI. IntroductionThe objective of this study was to determine the suitability, feasibility and potential cost savings of using two commercially available test kits, selected by personnel of the Nova Scotia Department of Environment, which could potentially be used in the field to provide an immediate estimate of microcystins levels present within a water body. The trade names and product numbers of the test kits evaluated were the ABRAXIS Microcystin Test Strip (PN 520022) and the ENVIROLOGIX QualiTube Kit for Microcystins (PN ET 022).The trials were conducted between the 14th and 25th of August 2011 during water quality surveys carried out at ten lakes being monitored within the Carleton River area of Digby and Yarmouth counties. The results were compared to those obtained for duplicate water samples collected and sent to a commercial laboratory (ALS Environmental) located in Winnipeg, MB.II. MethodologiesBoth the ABRAXIS and ENVIRLOGIX test kits use the ELISA (Enzyme-Linked-Immunosorbent Assay) protocol for measuring microcystins. This protocol is commonly used in immunology to detect the presence of an antibody or antigen in a sample. In this procedure, an unknown amount of antigen (in this case microcystin) is affixed to a surface, and then a specific antibody is applied over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and a substance is added that the enzyme can convert to a detectable signal, most often a colour change in a chemical substrate. As a result, the more of the antigen present, the lesser is the amount of free enzyme present to react with the chemical substrate and the lesser is the degree of colour development.The ALS laboratory, to which the results of the test kits are compared, uses the ENVIROLOGIX Microcystin Plate Kit procedure to measure microcystins. This is also based on the ELISA protocol, but is a more sophisticated procedure involving color development within a series of test wells that is measured spectrophotometrically using a microtiter plate reader.Details of the procedure involved in using the ABRAXIS and ENVIROLOGIX kits are contained in APPENDICES I and II respectively.Water samples for analysis of microcystins were collected in pre-cleaned amber glass bottles, stored in a cooler and analyzed on the same day they were collected. Water samples for the ALS lab analyses were collected in clear glass sample containers provided by ALS, stored refrigerated in the dark until all lakes were sampled and then couriered to ALS in a styrofoam cooler containing ice packs.III. ResultsOf the ten lakes sampled and analyzed for microcystin concentrations using each of the two test kits, only one site had a result that was above the detection limits of the each test procedure (Table 1). This was Nowlans Lake in which the ABRAXIS test kit exhibited a microcystin concentration >10 µg/L. This result was confirmed by repeating the test three times. The ENVIROLOGIX test kit did not produce a positive result. The difference is most likely due to the cell lysing step used in the ABRAXIS procedure but absent in the ENVIROLOGIX procedure (see Section IV). The ALS laboratory analysis results also indicated a microcystin concentration above 10 µg/L for Nowlans Lake.Table 1. Comparison of microcystin analyses carried out using the field test kits with results obtained by the ALS Environmental laboratory.LAKEABRAXISTest Strip KitTotal Microcystins(µg/L)ENVIROLOGIXQualiTube KitFree Microcystins(µg/L)ALS EnvironmentalFree Microcystins(µg/L)Total Microcystins(µg/L)Hourglass < 1 < 0.5 <0.20 <0.20Placides < 1 < 0.5<0.20 <0.20 Porcupine < 1 < 0.5<0.20 <0.20 Parr < 1 < 0.5<0.20 <0.20 Ogden < 1 < 0.5<0.20 <0.20 Fanning < 1 < 0.5<0.20 <0.20 Sloans < 1 < 0.5<0.20 <0.20 Vaughan < 1 < 0.5<0.20 <0.20 Nowlans > 10 < 0.5<0.20 11.82 Provost < 1 < 0.5<0.20 <0.20 IV. DiscussionAlthough the principle is the same for both the ABRAXIS and ENVIRLOGIX test kits, there are important differences between the two. The former uses a membrane test strip and the latter an antibody coated test tube in which to visualize the degree of color development. In addition, andmost importantly, the ABRAXIS procedure involves an initial step in which the water samplebeing tested is subjected to a cell lysing agent that releases intracellular microcystins and the results represent total (i.e., free or dissolved plus intracellular) microcystin concentration. In contrast, the ENVIRLOGIX test procedure does not include a cell lysing step and the results represent only the concentration of microcystins free within the water sample. The ENVIROLOGIX Microcystin Plate Kit used by ALS Environmental, like the ENVIRLOGIX QualiTube Kit, does not routinely include an initial cell lyses step and also measures only free, as opposed to total, microcystins. However, ALS claims that freezing samples prior to analysis is a suitable means for lysing Microcystis and this was requested in addition to the usual un-lysed cell analyses.There are also important differences in the range of microcystin levels measured by the two kits. The ABRAXIS kit is designed for microcystin concentrations more suited for evaluation with reference to recreational water quality guidelines while the ENVIROLOGIX kit is designed for concentrations more suited to drinking water guidelines.There are other differences between the two test kits (Table 2), but these are mostly minor with respect to their suitability as a field test kit.Table 2. Comparison between test kits evaluated.ParameterABRAXISTest Strip Kit(520022)ENVIROLOGIXQualiTube Kit(ET 022)Protocol ELISAELISA Units Measured Total microcystins Free microcystinsAssay Range 1 - 10 µg/L 0.5 – 3 µg/LLimit of detection 1 µg/L 0.3 µg/LTime to Complete Test 30-40 min. 40-50 min.Number of water samples that can be processed per kit. 20 12to24Cost/Sample US$30 $20 to $40 Shelf Life/Recommended storage One year/Room temperature One year/RefrigeratedSuggested maximum sample storage time prior to analysis Five days if refrigerated; longerif frozen.UnspecifiedAvailability of control samples Available for $150/kit Provided with kit Optional Visualization Aids Available Test strip reader Portable photometerIt should be noted that it has been estimated that there are as many as 80 variants (congeners) in the chemical forms of microcystins which is thought to be a result of the numerous strains of Microcystis known to occur in nature. For the test kits evaluated in this study, it is thought that they will detect more than just the Microcystin-LR form, which is the form specified in the World Health Organization (WHO) and Health Canada (HC) guidelines. For both test kits the manufactures indicate that it is not known exactly how many forms each will detect. Although both WHO and HC guidelines specify Microcystin-LR levels, it appears that all microcystin variants are toxic, although to various degrees, so this difference may not be of much relevance. V. RecommendationsAs a field based test kit that can provide a rapid assessment, the ABRAXIS Test Strip Kit is the one I would recommend. It requires minimal technical skill to use, measures total microcystins which is the form most applicable to WHO and HC guidelines, and the results are available within 30 minutes. Its only real shortcoming is that without modification of the procedure, the maximum range it can detect is 10 µg/L whereas the WHO and HC guideline for recreational water use is <20 µg/L. However, this limitation can easily be overcome by dilution of a sample prior to testing. For example, if the sample were diluted 1:1 with clean water (preferably deionized) prior to analysis, and the results were still greater than 10 µg/L, this would indicate that the concentration of total microcystins is ≥20 µg/L.In comparison, although the ENVIROLOGIX QualiTube Kit is only slightly more difficult to use, it has greater shortcomings. Most importantly, because it does not contain a lysing agent the measurement of total microcystins requires that the water sample be subjected to a freeze/thaw cycle for a recommended minimum of three times. This results in a much longer time to achieve the result as well as making it impractical for use in the field.If the ABRAXIS kit is chosen, I would suggest evaluation and potential adoption of one or both of two options that are available at an additional cost. The first is the use of calibration solutions containing known amounts of microcystin that are run concurrently with the water sample being tested. In addition to providing a reference for comparing color development, use of the calibration solutions ensures that the test kit is working properly. Calibration solutions are available for US$150 if purchased with a kit, or for US$300 if purchased separately.The second option is the use of a test strip reader that would make estimating colour development of the test strip more objective than just using a visual determination. This is available from ABRAXIS at a cost of US$1800. Appendix III contains a description of this instrument.I would also recommend, as suggested by the manufacturer, that if a sample shows results >20 µg/L, this be confirmed by a more precise and accurate test such as the ENVIROLOGIX Microcystin Plate Kit procedure on lysed cells as used by ALS laboratory.APPENDIX IABRAXIS TEST STRIP PROCEDUREAPPENDIX IIENVIROLOGIX QUAITUBE KIT INSTRUCTIONSAPPENDIX III ABRASCAN DIPSTICK READER。
微晶纤维素检验标准操作规程(英文)
1. Purpose: To establish the standard operation procedure for inspection of auxiliary materialmicrocrystalline cellulose, and guarantee the smooth inspection ofmicrocrystalline cellulose.2. Applicable Scope:The procedure is applicable to inspection of auxiliary materialmicrocrystalline cellulose.3. Responsibilities: QC lab technician shall be responsible for implementation of the procedure,and lab director shall be responsible for supervising and inspecting the effectiveimplementation of the procedure.4.Contents:4.1 Inspection basis: Chinese Pharmacopoeia 2010 Part 2.4.2 Source: The product is white powder obtained from pure cotton fiber through hydrolysis.4.3 Character: The product is white or off-white powder; odorless and tasteless. If the product isinsoluble in water, ethyl alcohol, acetone or methylbenzene, it will be judged qualified.4.4 Identification4.4.1. Tools and instruments: Balance, watch glass, test tube, and dropper.4.4.2 Reagents and test solutions: Zinc chloride iodine test solution4.4.3 Operation method: Take 10mg of the product, put it on watch glass, and add 2ml zincchloride iodine test solution.4.4.4 Result and judgment: If it turns blue immediately, it can be judged that it is positivereaction.4.5 Inspection4.5.1 Fineness4.5.1.1 Operation method, result and judgment: Take 20.0g of the product, put it in the medicinalsieve, and the powder not passing No. 7 sieve cannot exceed 5.0%, and the powder passing No. 9 sieve cannot be less than 50.0%.4.5.2 PH value4.5.2.1 Tools and instruments: Balance, test tube and funnel.4.5.2.2 Reagents and test solutions: Purified water, and pH test paper4.5.2.3 Operation method: Take 2.0g of the product, add 100ml water, vibrate it for 5min, thenfilter it, and take the filter liquor, and measure its pH value in appropriate method (Appendix VI H).4.5.2.4 Result and judgment: Its pH value shall be within 5.0~7.5, and in that case, it can bejudged qualified.4.5.3 Dissolved matter in water4.5.3.1 Tools and instruments: Balance, funnel, evaporating dish, water bath, drying oven andtest tube.4.5.3.2 Reagents and test solutions: Purified water.4.5.3.3 Operation method: Take 5.0g of the product, add 80ml water, vibrate it for 10min, thenfilter it, put the filter liquor into the constant-weight evaporating dish, evaporate it on water bath, and dry it at 105℃for 1h.4.5.3.4 Result and judgment: The remaining residue shall not exceed 0.2%, and in that case, itcan be judged qualified.4.5.4 Chloride:4.5.4.1 Tools and instruments: Balance, funnel, test tube and measuring cylinder.4.5.4.2 Reagents and test solutions: Purified water and standard sodium chloride solution.4.5.4.3 Operation method: Take 0.10g of the product, add 35ml water, vibrate it, then filter it, andtake the filter liquor, inspect it in appropriate method (Appendix VIII A), and compare it with the control solution prepared with 3.0ml standard sodium chloride solution.4.5.4.4 Result and judgment: The solution will not be thicker (0.03%), and in that case, it can bejudged qualified.4.5.5 Starch4.5.5.1 Tools and instruments: Test tube, measuring cylinder and balance.4.5.5.2 Reagents and test solutions: Purified water and iodine test solution.4.5.5.3 Operation method: Table 0.10g of the product, add 5ml water, vibrate it, and then add 0.2ml iodine test solution.4.5.5.4 Result and judgment: It cannot be blue, and in that case, it can be judged qualified.4.5.6 Loss on drying4.5.6.1 Tools and instruments: Drying oven and balance.4.5.6.2 Operation method: Take the product, and dry it at 105℃until the constant weight.4.5.6.3 Result and judgment: The weight loss cannot exceed 5.0% (Appendix VIII L), and in thatcase, it can be judged qualified.4.5.7 Residue on ignition4.5.7.1 Operation method, result and judgment: Take 1.0g of the product, and inspect it inappropriate method (Appendix VIII N). The remaining residue cannot exceed 0.2%, and in that case, it can be judged qualified.4.5.8 Heavy metal4.5.8.1 Operation method, result and judgment: Take the residue under the item of residue onignition, inspect it in appropriate method (Appendix VIII H Method 2), and the heavy metal content cannot exceed ten millionths, and in that case, it can be judged qualified.4.5.9 Arsenic salt4.5.9.1 Tools and instruments: Balance, glass rod, silica gel drier, alcohol lamp, high temperaturefurnace, and measuring cylinder,4.5.9.2 Reagents and test solutions: Calcium hydroxide, purified water and hydrochloric acid. 4.5.9.3 Operation method: Take 1.0g of the product, add 1.0g calcium hydroxide, mix them, addwater to stir them evenly, and after drying, ignite them with small fire to carbonize them, and then burn them at 500～600℃until they are ashed completely, keep it cool, and then add 5ml hydrochloric acid and 23ml water to dissolve them, and then inspect it in appropriate method (Appendix VIII J Method 1)4.5.9.4 Result and judgment: It shall conform to provisions (0.0002%), and in that case, it shallbe judged qualified.4.6 Content determination4.6.1 Tools and instruments: Analytical balance, conical flask, measuring cylinder,electro-thermal furnace, measuring flask, burette, and transfer pipette.4.6.2 Reagents and test solutions: Purified water, potassium dichromate, sulfuric acid, phenanthroline indicating liquid, and ferrous sulfate.4.6.3 Operation method, result and judgment: Take 0.125g of the product, accurately weigh it,then put it into conical flask, add 25ml water, accurately add 50ml potassium dichromate solution (take 4.903g standard potassium dichromate, add an appropriate amount of water to dissolve it, and dilute it till 200ml), mix them evenly, slowly add 100ml sulfuric acid, quickly heat it till boiling, then keep it cool, and move it into 250ml measuring flask, add water to dilute it till the graduation line, then shake it evenly, accurately measure 50ml, add three drops of phenanthroline indicating liquid, titrate it with ferrous sulfate titrating solution (0.1mol/L), and correct the titration result with blank test. 1ml ferrous sulfate titrating solution (0.1mol/) is equivalent to 0.675mg cellulose.4.7 Microbial limit4.7.1 Operation method, result and judgment: Take the product, and inspect it in appropriatemethod (Appendix XI J), and among 1g test product, the bacterial count cannot exceed 1,000cfu, the mold and yeast count cannot exceed 100cfu, and no Escherichia coli can be detected, and in that case, it can be judged qualified.5. Supplementary Provisions:6. The document is drafted by QC, reviewed by QC director, and approved by QualityDepartment head.。
欧洲药典80纯化水微生物检测译
欧洲药典8.0纯化水微生物检测批量纯化水（区别于装在容器中的纯化水）微生物监测在生产和随后的贮存中，要采取适当措施以确保微生物数量得到适当控制和监测。
要设置适当的警戒限和行动限用于观察不良趋势。
正常条件下，微生物总数的行动限为100 CFU/mL，用薄膜过滤法，滤膜孔径不得过0.45μm，使用R2A琼脂培养基，于30-35℃培养不少于5天。
样品量的选择应与期望的检测结果相关联。
R2A琼脂培养基培养基灭菌后，调节pH7.2±0.2。
灭菌方式：高压灭菌锅，121℃、15分钟。
R2A琼脂培养基促生长试验- 试验菌株制备。
使用标准化的稳定的试验菌悬浮液，或按表0008-1所述配制。
试验用菌株的传代不得超过5代，从菌种保藏中心获得的原始菌株算起。
按表0008-1所述，每个菌株分开培养。
使用pH7.0氯化钠-蛋白胨缓冲液或pH7.2磷酸盐缓冲液配制试验菌悬浮液。
试验菌悬浮液配制好后，应在2小时内使用，或存于2-8℃于24小时内使用。
作为一种替代方法，可以配制然后稀释枯草芽孢杆菌（Bacillus subtilis）生长细胞的新鲜悬浮液：配制一个稳定的孢子悬液，然后使用适当体积的孢子悬液用于试验接种。
孢子悬液可在2-8℃贮藏，贮藏时间要经过验证。
- 促生长。
每批已配制好的培养基都要做促生长试验，不管是使用脱水培养基（干粉）配制，还是使用上述的成分配制。
将表0008-1所述的微生物取少量（不超过100CFU），单独接种至含R2A琼脂培养基的平皿中，按表中所述的条件培养。
培养后观察到的微生物结果不得超过理论值±2倍。
针对新配制的接种体，微生物促生长试验结果必须与之前试验过、批准过的培养基结果做对比。
表0008-1R2A琼脂促生长试验微生物试验菌株制备促生长条件。
杯碟法检测方法新
杯碟法检测青霉素酶的方法（试行）1.适用范围本方法适用于原料奶、灭菌乳、乳饮料中青霉素酶的检测。
2.实验原理青霉素酶能够破坏青霉素对藤黄微球菌的抑制作用，而舒巴坦能特异性地抑制青霉素酶的这种作用，在样品中先后加入适量的舒巴坦和青霉素，采用杯碟法通过测定抑菌圈大小差异来检测样品中的青霉素酶。
酶活单位（ Active Unit ）：在室温（ 25℃）下， 1min 分解 1μ mol 的青霉素 G 所需的酶量为一个单位，即1IU 。
3.试剂与材料除另有规定外，所有试剂为分析纯，水为GB/T6682-2008 规定的三级水。
3.1菌种：藤黄微球菌（Micrococcus Luteus）,菌种号CMCC（B）28001。
3.2青霉素酶标准品：纯度≥ 90%，酶活1200IU/mg（≥ 1000万青霉素效价单位），4℃保存。
3.3舒巴坦标准品：纯度89.2%，4℃保存。
3.4青霉素G：色谱级，纯度97%（分析纯需适当增加标准工作溶液浓度），密封避光，防潮， 4℃保存。
3.5磷酸二氢钾（KH2PO4）：纯度99.5%。
3.6磷酸氢二钾（K2HPO4）：纯度98%。
3.7氯化钠（NaCL）：纯度99.5%。
3.8磷酸盐缓冲溶液（pH6.0）：取8g磷酸二氢钾（3.5）和2g磷酸氢二钾（3.6），用 1000ml 水溶解， 121℃高压灭菌 15min。
3.9生理盐水：称取 8.5g 氯化钠（ 3.7 ），溶解于 1000ml 水中，121℃高压灭菌 15min。
3.10青霉素酶标准储备溶液：准确称取（精确至0.001g ）青霉素酶标准品（ 3.2 ）用生理盐水（ 3.9 ）溶解并稀释，配成酶活力为 1000IU/ml 的标准储备溶液， 4℃保存。
可使用 1 个月。
3.11 青霉素酶标准工作液：用生理盐水（ 3.9 ）将青霉素酶标准储备溶液（ 3.10 ）稀释配制成酶活力为1IU/ml 的标准工作溶液，需当天配制使用。