微生物限度检查

1、á61ñMICROBIAL LIMIT TESTSThis chapter provides tests for the estimation of the number of viable aerobic microorganisms present and for freedom from designated microbial species in pharmaceutical articles of all kinds,from raw materials to the finished forms.An automated method may be subst

2、ituted for the tests presented here,provided it has been properly validated as giving equivalent or better results.In preparing for and in applying the tests,observe aseptic precautions in handling the specimens.Unless otherwise directed,where the procedure specifies simply “incubate,”hold the conta

3、iner in air that is thermostatically controlled at a temperature between 30and 35,for a period of 24to 48hours.The term “growth”is used in a special sense herein,i.e.,to designate the presence and presumed proliferation of viable microorganisms.PREPARATORY TESTINGThe validity of the results of the t

4、ests set forth in this chapter rests largely upon the adequacy of a demonstration that the test specimens to which they are applied do not,of themselves,inhibit the multiplication,under the test conditions,of microorganisms that may be present.Therefore,preparatory to conducting the tests on a regul

5、ar basis and as circumstances require subsequently,inoculate diluted specimens of the material to be tested with separate viable cultures of Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosaand Salmonella.This can be done by adding 1mLof not less than 10-3dilution of a 24-hour broth cult

6、ure of the microorganism to the first dilution (in pH7.2Phosphate Buffer,Fluid SoybeanCasein Digest Medium,or Fluid Lactose Medium)of the test material and following the test procedure.Failure of the organism(s)to grow in the relevant medium invalidates that portion of the examination and necessitat

7、es a modification of the procedure by (1)an increase in the volume of diluent,the quantity of test material remaining the same,or by (2)the incorporation of a sufficient quantity of suitable inactivating agent(s)in the diluents,or by (3)an appropriate combination of modifications (1)and (2)so as to

8、permit growth of the inocula.The following are examples of ingredients and their concentrations that may be added to the culture medium to neutralize inhibitory substances present in the sample:soy lecithin,0.5%;and polysorbate 20,4.0%.Alternatively,repeat the test as described in the preceding para

9、graph,using Fluid Casein DigestSoy LecithinPolysorbate 20Mediumto demonstrate neutralization of preservatives or other antimicrobial agents in the test material.Where inhibitory substances are contained in the product and the latter is soluble,a suitable,validated adaptation of a procedure set forth

10、 in the section Membrane Filtrationunder Test for Sterility of the Product to be Examinedunder Sterility Tests á71ñ,may be used.If in spite of the incorporation of suitable inactivating agents and a substantial increase in the volume of diluent,it is still not possible to recover the viabl

11、e cultures described above and where the article is not suitable for employment of membrane filtration,it can be assumed that the failure to isolate the inoculated organism is attributable to the bactericidal activity of the product.This information serves to indicate that the article is not likely

12、to be contaminated with the given species of microorganism.Monitoring should be continued in order to establish the spectrum of inhibition and bactericidal activity of the article.BUFFER SOLUTION AND MEDIACulture media may be prepared as follows,or dehydrated culture media may be used provided that,

13、when reconstituted as directed by the manufacturer or distributor,they have similar ingredients and/or yield media comparable to those obtained from the formulas given herein.In preparing media by the formulas set forth herein,dissolve the soluble solids in the water,using heat,if necessary,to effec

14、t complete solution,and add solutions of hydrochloric acid or sodium hydroxide in quantities sufficient to yield the desired pHin the medium when it is ready for use.Determine the pHat 25±2.Where agar is called for in a formula,use agar that has a moisture content of not more than 15%.Where wat

15、er is called for in a formula,use Purified Water.PH7.2Phosphate Buffer Stock Solution Dissolve 34g of monobasic potassium phosphate in about 500mLof water contained in a 1000-mLvolumetric flask.Adjust to pH7.2±0.1by the addition of sodium hydroxide TS(about 175mL),add water to volume,and mix.Di

16、spense and sterilize.Store under refrigeration. For use,dilute the Stock Solutionwith water in the ratio of 1to 800,and sterilize.Media Unless otherwise indicated,the media should be sterilized by heating in an autoclave (see Steam Sterilizationunder Sterilization á1211ñ),the exposure time

17、 depending on the volume to be sterilized.I.Fluid Casein DigestSoy LecithinPolysorbate 20Medium Pancreatic Digest of Casein20gSoy Lecithin5gPolysorbate 2040mLWater960mLDissolve the pancreatic digest of casein and soy lecithin in 960mLof water,heating in a water bath at 48to 50for about 30minutes to

18、effect solution.Add 40mLof polysorbate 20.Mix,and dispense as desired.II.SoybeanCasein Digest Agar Medium Pancreatic Digest of Casein15.0gPapaic Digest of Soybean Meal5.0gSodium Chloride5.0gAgar15.0gWater1000mLpHafter sterilization:7.3±0.2.III.Fluid SoybeanCasein Digest Medium Prepare as direct

19、ed for SoybeanCasein Digest Mediumunder Sterility Tests á71ñ.IV.MannitolSalt Agar Medium Pancreatic Digest of Casein5.0gPeptic Digest of Animal Tissue5.0gBeef Extract1.0gD-Mannitol10.0gSodium Chloride75.0gAgar15.0gPhenol Red0.025gWater1000mLMix,then heat with frequent agitation,and boil fo

20、r 1minute to effect solution.pHafter sterilization:7.4±0.2.Heat with frequent agitation,and boil for 1minute.Sterilize,cool to between 45and 50,and add 10mLof sterile potassium tellurite solution (1in 100)and 50mLof egg-yolk emulsion.Mix intimately but gently,and pour into plates.(Prepare the e

21、gg-yolk emulsion by disinfecting the surface of whole shell eggs,aseptically cracking the eggs,and separating out intact yolks into a sterile graduated cylinder.Add sterile saline TSto obtain a 3to 7ratio of egg yolk to saline.Add to a sterile blender cup,and mix at high speed for 5seconds.)pHafter

22、sterilization:6.8±0.2.VI.VogelJohnson Agar Medium Pancreatic Digest of Casein10.0gYeast Extract5.0gMannitol10.0gDibasic Potassium Phosphate5.0gLithium Chloride5.0gGlycine10.0gAgar16.0gPhenol Red25.0mgWater1000mLBoil the solution of solids for 1minute.Sterilize,cool to between 45and 50,and add 2

23、0mLof sterile potassium tellurite solution (1in 100).pHafter sterilization:7.2±0.2.VII.Cetrimide Agar Medium Pancreatic Digest of Gelatin20.0gMagnesium Chloride1.4gPotassium Sulfate10.0gAgar13.6gCetyl Trimethylammonium Bromide (Cetrimide)0.3gGlycerin10.0mLWater1000mLDissolve all solid component

24、s in the water,and add the glycerin.Heat,with frequent agitation,and boil for 1minute to effect solution.pHafter sterilization:7.2±0.2.VIII.Pseudomonas Agar Medium for Detection of Fluorescin Pancreatic Digest of Casein10.0gPeptic Digest of Animal Tissue10.0gAnhydrous Dibasic Potassium Phosphat

25、e1.5gMagnesium Sulfate (MgSO4·7H2O)1.5gGlycerin10.0mLAgar15.0gWater1000mLDissolve the solid components in the water before adding the glycerin.Heat,with frequent agitation,and boil for 1minute to effect solution.pHafter sterilization:7.2±0.2.IX.Pseudomonas Agar Medium for Detection of Pyoc

26、yanin Pancreatic Digest of Gelatin20.0gAnhydrous Magnesium Chloride1.4gAnhydrous Potassium Sulfate10.0gAgar15.0gGlycerin10.0mLWater1000mLDissolve the solid components in the water before adding the glycerin.Heat,with frequent agitation,and boil for 1minute to effect solution.pHafter sterilization:7.

27、2±0.2.X.Fluid Lactose Medium Beef Extract3.0gPancreatic Digest of Gelatin5.0gLactose5.0gWater1000mLCool as quickly as possible after sterilization.pHafter sterilization:6.9±0.2.XI.Fluid SeleniteCystine Medium Pancreatic Digest of Casein5.0gLactose4.0gSodium Phosphate10.0gSodium Acid Seleni

28、te4.0gL-Cystine10.0mgWater1000mLFinal pH:7.0±0.2.Mix,and heat to effect solution.Heat in flowing steam for 15minutes.Do not sterilize.XII.Fluid Tetrathionate Medium Pancreatic Digest of Casein2.5gPeptic Digest of Animal Tissue2.5gBile Salts1.0gCalcium Carbonate10.0gSodium Thiosulfate30.0gWater1

29、000mLHeat the solution of solids to boiling.On the day of use,add a solution prepared by dissolving 5g of potassium iodide and 6g of iodine in 20mLof water.Then add 10mLof a solution of brilliant green (1in 1000),and mix.Do not heat the medium after adding the brilliant green solution.XIII.Brilliant

30、 Green Agar Medium Yeast Extract3.0gPeptic Digest of Animal Tissue5.0gPancreatic Digest of Casein5.0gLactose10.0gSodium Chloride5.0gSucrose10.0gPhenol Red80mgAgar20.0gBrilliant Green12.5mgWater1000mLBoil the solution of solids for 1minute.Sterilize just prior to use,melt the medium,pour into petri d

31、ishes,and allow to cool.pHafter sterilization:6.9±0.2.XIV.XyloseLysineDesoxycholate Agar Medium Xylose3.5gL-Lysine5.0gLactose7.5gSucrose7.5gSodium Chloride5.0gYeast Extract3.0gPhenol Red80mgAgar13.5gSodium Desoxycholate2.5gSodium Thiosulfate6.8gFerric Ammonium Citrate800mgWater1000mLFinal pH:7.

32、4±0.2.Heat the mixture of solids and water,with swirling,just to the boiling point.Do not overheat or sterilize.Transfer at once to a water bath maintained at about 50,and pour into plates as soon as the medium has cooled.XV.Bismuth Sulfite Agar Medium Beef Extract5.0gPancreatic Digest of Casei

33、n5.0gPeptic Digest of Animal Tissue5.0gDextrose5.0gSodium Phosphate4.0gFerrous Sulfate300mgBismuth Sulfite Indicator8.0gAgar20.0gBrilliant Green25mgWater1000mLFinal pH:7.6±0.2.Heat the mixture of solids and water,with swirling,just to the boiling point.Do not overheat or sterilize.Transfer at o

34、nce to a water bath maintained at about 50,and pour into plates as soon as the medium has cooled.XVI.Triple SugarIronAgar Medium Pancreatic Digest of Casein10.0gPancreatic Digest of Animal Tissue10.0gLactose10.0gSucrose10.0gDextrose1.0gFerrous Ammonium Sulfate200mgSodium Chloride5.0gSodium Thiosulfa

35、te200mgAgar13.0gPhenol Red25mgWater1000mLpHafter sterilization:7.3±0.2.XVII.MacConkey Agar Medium Pancreatic Digest of Gelatin17.0gPancreatic Digest of Casein1.5gPeptic Digest of Animal Tissue1.5gLactose10.0gBile Salts Mixture1.5gSodium Chloride5.0gAgar13.5gNeutral Red30mgCrystal Violet1.0mgWat

36、er1000mLBoil the mixture of solids and water for 1minute to effect solution.pHafter sterilization:7.1±0.2.XVIII.Levine EosinMethylene Blue Agar Medium Pancreatic Digest of Gelatin10.0gDibasic Potassium Phosphate2.0gAgar15.0gLactose10.0gEosin Y400mgMethylene Blue65mgWater1000mLDissolve the pancr

37、eatic digest of gelatin,the dibasic potassium phosphate,and the agar in the water,with warming,and allow to cool.Just prior to use,liquefy the gelled agar solution,add the remaining ingredients,as solutions,in the following amounts,and mix:for each 100mLof the liquefied agar solution5mLof lactose so

38、lution (1in 5),2mLof the eosin Ysolution (1in 50),and 2mLof methylene blue solution (1in 300).The finished medium may not be clear.pHafter sterilization:7.1±0.2.XIX.Sabouraud Dextrose Agar Medium Dextrose40gMixture of equal parts of Peptic Digest of Animal Tissue and Pancreatic Digest of Casein

39、10gAgar15gWater1000mLMix,and boil to effect solution.pHafter sterilization:5.6±0.2.XX.Potato Dextrose Agar Medium Cook 300g of peeled and diced potatoes in 500mLof water prepared by distillation,filter through cheesecloth,add water prepared by distillation to make 1000mL,and add the following:A

40、gar15gGlucose20gDissolve by heating,and sterilize.pHafter sterilization:5.6±0.2.For use,just prior to pouring the plates,adjust the melted and cooled to 45medium with sterile tartaric acid solution (1in 10)to a pHof 3.5±0.1.Do not reheat the pH3.5medium.SAMPLINGProvide separate 10-mLor 10-

41、g specimens for each of the tests called for in the individual monograph.PROCEDUREPrepare the specimen to be tested by treatment that is appropriate to its physical characteristics and that does not alter the number and kind of microorganisms originally present,in order to obtain a solution or suspe

42、nsion of all or part of it in a form suitable for the test procedure(s)to be carried out.For a solid that dissolves to an appreciable extent but not completely,reduce the substance to a moderately fine powder,suspend it in the vehicle specified,and proceed as directed under Total Aerobic Microbial C

43、ount,and under Test for Staphylococcus aureus and Pseudomonas aeruginosaand Test for Salmonella species and Escherichia coli.For a fluid specimen that consists of a true solution,or a suspension in water or a hydroalcoholic vehicle containing less than 30percent of alcohol,and for a solid that disso

44、lves readily and practically completely in 90mLof pH7.2Phosphate Bufferor the media specified,proceed as directed under Total Aerobic Microbial Count,and under Test for Staphylococcus aureus and Pseudomonas aeruginosaand Test for Salmonella species and Escherichia coli.For water-immiscible fluids,oi

45、ntments,creams,and waxes,prepare a suspension with the aid of a minimal quantity of a suitable,sterile emulsifying agent (such as one of the polysorbates),using a mechanical blender and warming to a temperature not exceeding 45,if necessary,and proceed with the suspension as directed under Total Aer

46、obic Microbial Count,and under Test for Staphylococcus aureus and Pseudomonas aeruginosaand Test for Salmonella species and Escherichia coli.For a fluid specimen in aerosol form,chill the container in an alcohol-dry ice mixture for approximately 1hour,cut open the container,allow it to reach room te

47、mperature,permit the propellant to escape,or warm to drive off the propellant if feasible,and transfer the quantity of test material required for the procedures specified in one of the two preceding paragraphs,as appropriate.Where 10.0g or 10.0mLof the specimen,whichever is applicable,cannot be obta

48、ined from 10containers in aerosol form,transfer the entire contents from 10chilled containers to the culture medium,permit the propellant to escape,and proceed with the test on the residues.If the results of the test are inconclusive or doubtful,repeat the test with a specimen from 20more containers

49、.Total Aerobic Microbial Count For specimens that are sufficiently soluble or translucent to permit use of the Plate Method,use that method;otherwise,use the Multiple-Tube Method.With either method,first dissolve or suspend 10.0g of the specimen if it is a solid,or 10mL,accurately measured,if the sp

50、ecimen is a liquid,in pH7.2Phosphate Buffer,Fluid SoybeanCasein Digest Medium,or Fluid Casein DigestSoy Lecithin-Polysorbate 20Mediumto make 100mL.For viscous specimens that cannot be pipeted at this initial 1:10dilution,dilute the specimen until a suspension is obtained,i.e.,1:50or 1:100,etc.,that

51、can be pipeted.Perform the test for absence of inhibitory (antimicrobial)properties as described under Preparatory Testingbefore the determination of Total Aerobic Microbial Count.Add the specimen to the medium not more than 1hour after preparing the appropriate dilutions for inoculation.PLATE METHO

52、DDilute further,if necessary,the fluid so that 1mLwill be expected to yield between 30and 300colonies.Pipet 1mLof the final dilution onto each of two sterile petri dishes.Promptly add to each dish 15to 20mLof SoybeanCasein Digest Agar Mediumthat previously has been melted and cooled to approximately

53、 45.Cover the petri dishes,mix the sample with the agar by tilting or rotating the dishes,and allow the contents to solidify at room temperature.Invert the petri dishes,and incubate for 48to 72hours.Following incubation,examine the plates for growth,count the number of colonies,and express the avera

54、ge for the two plates in terms of the number of microorganisms per g or per mLof specimen.If no microbial colonies are recovered from the dishes representing the initial 1:10dilution of the specimen,express the results as “less than 10microorganisms per g or per mLof specimen.”MULTIPLE-TUBE METHODIn

55、to each of fourteen test tubes of similar size place 9.0mLof sterile Fluid SoybeanCasein Digest Medium.Arrange twelve of the tubes in four sets of three tubes each.Put aside one set of three tubes to serve as the controls.Into each of three tubes of one set (“100”)and into a fourth tube (A)pipet 1mL

56、of the solution or suspension of the specimen,and mix.From tube A,pipet 1mLof its contents into the one remaining tube (B)not included in a set,and mix.These two tubes contain 100mg (or 100µL)and 10mg (or 10µL)of the specimen,respectively.Into each of the second set (“10”)of three tubes pi

57、pet 1mLfrom tube A,and into each tube of the third set (“1”)pipet 1mLfrom tube B.Discard the unused contents of tubes Aand B.Close well,and incubate all of the tubes.Following the incubation period,examine the tubes for growth:the three control tubes remain clear and the observations in the tubes containing the specimen,when interpreted by reference to Table 1,indicate the most probable number of microorganisms per g or per mLof specimen.Table 1.Most Probable Total Count by Multiple-T