USP－微生物限度检查

1、<61>micr0bial 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 substit

2、uted for the tests presented here, provided it has bee n properly validated as giving equivale nt 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

3、 container in air that is thermostatically controlled at a temperature between 30°and 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

4、 the results of the tests 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 cond

5、ucting the tests on a regular 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 aeruginosa and salmonella. this can be done by adding 1 mlof not less than 10

6、3dilution of a 24-hour broth culture of the microorganism to the first dilution (in ph7.2phosphate buffer, fluid soybean-casein digest medium, or fluid lactose medium the test material and following the test procedure. failure of the orga nism(s)to grow in the releva nt medium invalidates that porti

7、on of the examination and necessitates a modification of the procedure by (1)an in crease in the volume of dilue nt, the quantity of test material remaining the same, or by (2)the incorporation of a sufficient quantity of suitable in activating age nt(s)in the diluents, or by (3)an appropriate combi

8、nation of modifications (1)and (2)so as to 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, rep

9、eat the test as described in the preceding paragraph, using fluid casein digest-soy lecithin-polysorbate 20 mediumto dem on strate n eutralizati on of preservatives or other antimicrobial agents in the test material. where inhibitory substances are contained in the product and the latter is soluble,

10、 a suitable, validated adaptation of a procedure set forth in the section membrane filtration under test for sterility of the product to be examinedunder sterility tests71 be usedif in spite of the incorporation of suitable inactivating agents and a substantial increase in the volume of diluent, it

11、is still not possible to recover the viable 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

12、 to indicate that the article is not likely 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 dehyd

13、rated culture media may be used provided that, 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 sol

14、ids in the water, using heat, if necessary, to effect complete solution, and add solutions of hydrochloric acid or sodium hydroxide in quantities sufficient to yield the desired ph in the medium when it is ready for use. determine the ph at 25±2°.where agar is called for in a formula, use

15、agar that has a moisture content of not more than 15%.where water is called for in a formula, use purified water.ph7.2phosphate bufferstock 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

16、 sodium hydroxide ts(about 175ml),add water to volume, and mix. dispense and sterilize. store under refrigeration.for use, dilute the stock solution with water in the ratio of 1to 800,a nd sterilize.mediaunless otherwise indicated, the media should be sterilized by heating in an autoclave (see steam

17、 sterilizationundei sterilization1211),the exposure time depending on the volume to be sterilized.i.fluid casein digest-soy lecithin-polysorbate 20mediumpancreatic digest of casein20gsoy lecithin5gpolysorbate 2040mlwater960mldissolve the pancreatic digest of casein and soy lecithin in 960mlof water,

18、 heating in a water bath at 48°to 50°for about 30minutes to effect solution. add 40mlof polysorbate 20.mix,a nd dispense as desired.ll.soybean-casein digest agar mediumpancreatic digest of casein15.ogpapaic digest of soybean meal5.0gsodium chloride5.0gagar15.ogwater1000mlphafter sterilizat

19、ion:7.3±0.2.iii.fluid soybean-casein digest mediumprepare as directed for soybean-casein digest mediumunder sterility tests71iv.mannitol-salt agar mediumpancreatic digest of casein5.0gpeptic digest of animal tissue5.0gbeef extract1.0gd-mannitol10.ogsodium chloride75.ogagar15.ogphenol red0.025gw

20、ater1000mlmix,then heat with frequent agitation, and boil for 1 minute to effect solution. phafter sterilization:7.4±0.2.heat with frequent agitation,a nd boil for 1minu te.sterilize,cool to betwee n 45° and 50°,and add 10mlof sterile potassium tellurite solution (1in 100)and 50mlof e

21、gg-yolk emulsion. mix intimately but gently, and pour into plates (prepare the egg-yolk emulsion by disinfecting the surface of whole shell eggs, aseptically cracking the eggs, and separating out intact yolks into a sterile graduated cyli nder. add sterile sali ne ts to obtai n a 3to 7 rat io of egg

22、 yolk to saline add to a sterile blen der cup, and mix at high speed for 5sec on ds.) phafter sterilization.8±0.2.vl.vogel-johnson agar mediumpancreatic digest of casein10.ogyeast extract5.0gmannitol10.ogdibasic potassium phosphate5.0glithium chloride5.0gglyci ne10.ogagar16.ogphenol red25.0mgwa

23、ter1000mlboil the solution of solids for 1 minute.sterilize5cool to between 45°and 50_,andadd 20mlof sterile potassium tellurite solution (1in 100). phafter sterilization:7.2±0.2.vll.cetrimide agar mediumpancreatic digest of gelatin20.ogmagnesium chloride1.4gpotassium sulfate10.ogagar13.6g

24、cetyl trimethylam monium bromide (cetrimide)0.3gglyceri n10.0mlwater1000mldissolve all solid comp on ents in the water, and add the glycerin. heat,with frequent agitation, and boil for 1 minute to effect solution.phafter sterilizatio n:7.2±0.2.viii. pseudomonas agar medium for detection of fluo

25、rescinpancreatic digest of casein10.ogpeptic digest of animal tissue10.oganhydrous dibasic potassium phosphate1.5gmagnesium sulfate (mgso4-7h2o)1.5gglyceri n10.0mlagar15.0gwaterlooomldissolve the solid components in the water before adding theglycerin.heat,with frequent agitation,and boil for 1 minu

26、te to effect solution. phafter sterilization:7.2±0.2.ix.pseudomonas agar medium for detection of pyocyaninpancreatic digest of gelatin20.ogan hydrous magn esium chloride1.4ganhydrous potassium sulfate10.ogagar15.0gglycerin10.0mlwater1000mldissolve the solid components in the water before adding

27、 theglycerin.heat,with frequent agitation,and boil for 1 minute to effect solution. phafter sterilization :7.2±0.2.x.fluid lactose mediumbeef extract3.0gpancreatic digest of gelatin5.0glactose5.0gwater1000mlcool as quickly as possible after sterilization. phafter sterilization.9±0.2.xi.flu

28、id selenite-cystine mediumpancreatic digest of casein5.0glactose4.0gsodium phosphate10.ogsodium acid selenite4.0gl- cystine1 o.omgwater1000mlfinal ph:7.0±0.2.mix,and heat to effect solution.heat in flowing steam for 15minutes.do notsterilize.xii.fluid tetrathionate mediumpancreatic digest of ca

29、sein2.5gpeptic digest of animal tissue2.5gbile salts1.0gcalcium carb on ate10.ogsodium thiosulfate30.ogwater1000mlheat 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 br

30、illiant green (1in 1000),and mix.do not heat the medium after adding the brilliant green solution.xiii.brilliant green agar mediumyeast extract3.0gpeptic digest of animal tissue5.0gpancreatic digest of casein5.0glactose10.ogsodium chloride5.0gsucrose10.ogphenol red80mgagar20.ogbrilliant green1 2.5mg

31、water1000mlboil the solution of solids for 1 minute.sterilize just prior to use, melt the medium, pour into petri dishes, and allow to cool.phafter sterilization.9±0.2.xiv. xylose-lysine-desoxycholate agar mediumxylose3.5gl lysi ne5.0glactose7.5gsucrose7.5gsodium chloride5.0gyeast extract3.0gph

32、enol red80mgagar13.5gsodium desoxycholate2.5gsodium thiosulfate6.8gferric ammonium citrate800mgwater1000mlfinal ph:7.4±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°?

33、and pour into plates as soon as the medium has cooled.xv.bismuth sulfite agar mediumbeef extract5.0gpancreatic digest of casein5.0gpeptic digest of animal tissue5.0gdextrose5.0gsodium phosphate4.0gferrous sulfate300mgbismuth sulfite indicator8.0gagar20.ogbrilliant green25mgwater1000mlfinal ph:7.6

34、77;0.2.heat the mixture of solids and water,with swirling,just to the boiling point.do not overheat or sterilizedransfer at once to a water bath maintained at about 50°,and pour into plates as soon as the medium has cooled.xvl.triple sugar-iron-agar mediumpancreatic digest of casein10.ogpancrea

35、tic digest of animal tissue10.oglactose10.ogsucrose10.ogdextrose1.0gferrous ammonium sulfate200mgsodium chloride5.0gsodium thiosulfate200mgagar13.ogphenol red25mgwater1000mlphafter sterilizatio n:7.3±0.2.xvii.macconkey agar mediumpancreatic digest of gelatin17.0gpancreatic digest of casein1.5gp

36、eptic digest of animal tissue1.5glactose10.ogbile salts mixture1.5gsodium chloride5.0gagar13.5gneutral red30mgcrystal violet1 .omgwater1000mlboil the mixture of solids and water for 1 minute to effect solution.phafter sterilization:7.1±0.2.xviii.levine eosin-methylene blue agar mediumpancreatic

37、 digest of gelatin10.ogdibasic potassium phosphate2.0gagar15.oglactose10.ogeosin y400mgmethylene blue65mgwater1000mldissolve the pancreatic 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 solut

38、ion, add the remaining ingredients, as solutions, in the following amounts, and mix: for each loomlof the liquefied agar solution5mlof lactose solution (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.

39、1±0.2.xix. sabouraud dextrose agar mediumdextrose40gmixture of equal parts of peptic digest of animal tissue and10gpancreatic digest of caseinagar15gwater1000mlmix,and boil to effect solution.phafter sterilization.6±0.2.xx.potato dextrose agar mediumcook 300g of peeled and diced potatoes i

40、n 500mlof water prepared by distillation, filter through cheesecloth, add water prepared by distillation to make 1 000ml,and add the following:agar15gglucose20gdissolve by heating,a nd sterilize.phafter sterilization:5.6±0.2.for use, just prior to pouring the plates, adjust the melted and coole

41、d to 45° medium 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-g specimens for each of the tests called for in the individual monograph.procedureprepare the specimen to be tested by treatment that is appropria

42、te to its physical characteristics and that does not alter the number and kind of microorganisms originally present, in order to obtain a solution or suspension 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

43、 not completely, reduce the substanee to a moderately fine powder, suspend it in the vehicle specified, and 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 a fluid spe

44、cimen 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 dissolves readily and practically completely in 90mlof ph7.2phosphate buffer or the media specified, proceed as directed under total aerobic mi

45、crobial courit.and under test for staphylococcus aureus and pseudomonas aeruginosaand test for salmonella species and escherichia coli.for water-immiscible fluids, ointments, creams, and waxes, prepare a suspension with the aid of a minimal quantity of a suitable, sterile emulsifying age nt (such as

46、 one of the polysorbates), using a mecha nical blender and warming to a temperature not exceeding 45°,if necessary, and proceed with the suspe nsion as directed under total aerobic microbial count,a nd under test for staphylococcus aureus and pseudomonas aeruginosaand test for salmonella specie

47、s and escherichia coli.for a fluid specimen in aerosol form, chill the container in an alcohol-dry ice mixture for approximately 1 hour,cut open the container, allow it to reach room temperature, permit the propellant to escape, or warm to drive off the propellant if feasible, and transfer the quant

48、ity of test material required for the procedures specified in one of the two preceding paragraphs, as appropriate. where 10.og or 10.0mlof the specimen, whichever is applicable, cannot be obtained from 10containers in aerosol form, transfer the entire contents from 10chilled containers to the cultur

49、e medium, permit the propellant to escape, and proceed with the test on the residues .if the results of the test are in conclusive or doubtful, repeat the test with a specime n from 20more con tainers total aerobic microbial countfor specimens that are sufficiently soluble or translucent to permit u

50、se of the plate method, use that method; otherwise, use the multiple-tube method. with either method, first dissolve or suspend 10.og of the specimen if it is a solid, or 10ml,accurately measured, if the specimen is a liquid, in ph7.2phosphate buffer, fluid soybean-casein digest medium,or fluid case

51、in digest-soy lecithin-polysorbate 20medium to make 10oml.for viscous specimens that cannot be pipeted at this initial 1:10dilution,dilute the specimen until a suspension is obtained,i.e.,1:50or 100,etc.,that can be pipeted. perform the test for absence of inhibitory (antimicrobial)properties as des

52、cribed under preparatory testing before the determination of total aerobic microbial count.md the specimen to the medium not more than 1 hour after preparing the appropriate dilutions for inoculation.plate methoddilute further, if necessary, the fluid so that imlwill be expected to yield between 30a

53、nd 300colonies.pipet 1 mlof the final dilution onto each of two sterile petri dishes. promptly add to each dish 15to 20mlof soybean-casein digest agar medium that previously has been melted and cooled to approximately 45°.cover the petri dishes, mix the sample with the agar by tilting or rotati

54、ng 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 average for the two plates in terms of the number of microorganisms per g o

55、r per mlof specimenf 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 methodinto each of fourteen test tubes of similar size place 9.0mlof steril

56、e fluid soybean-casein 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 (u100”)and into a fourth tube (>4)pipet 1 mlof the solution or suspension of the specimen, and mix. fr

57、om tube >4,pipet 1mlof its contents into the one remaining tube (b)not included in a set, and mix. these two tubes contain 100mg (or 100nl)and 10mg (or 10|jl)of the specimen, respective!y. into each of the second set (“10”)of three tubes pipet 1 mlfrom tube >4,and into each tube of the third set (“1”)pipet 1 mlfrom tube 8.discard the unused contents of tubes /land b.ciose well,a nd in cubate all of the tubes. following the incubation period, examine the tubes for growth: the three control tubes rem