2 a 6480 (64) / Microbiological Tests usp 43 ‘tganisms and speciied microorganisms are liste in Table Fania Table # Alternative mleroblologieal procedires inciging sutomated methods, may be used, provided that their equivalence has been demonstrated. Principles of validation of alternative microbiological methods are also described in Validation of Alternative Microbiological Methods (1.223). Methods (4, dilution, butter, or membrane fitration) may be suitably modified to overcome potential interferences such as acid production resulting from growth of probiotic organisms during the enrichment step, leading to possible false-negative Fesults, Method suitability to detect the prescnce of microorganiams in the product to be tested must be established. Method Suitability must be confirmed i changes in the testing performance occur, of changes in the product composition that may affect the outcome of the test are introduced, ‘CONTAMINANT MICROORGANISMS Unless otherwise specified inthe individual monograph the'Yecomimended tests and acceptance teria for contaminant microorganisms in probiotic ingredients o finished products for oral use are listed in Table 3. ‘Table 3. Recomaicnded Tests for Contaminant Mlcroorgahisins Ia Problotle Ingredients or Flalshed Prodiicts for OFal Use Probl ret oon ssn Test Methods aula} Revisehe sca bacteria [50 13539 009" Nurs 1a? Nevopurtamng Gees 7a yeastand mods | Miz énmiraten Tes t02) RSTO sporetorninaibeeia | Totalyeansand molds | Wivab Emerton Ter02i) RaTTO0 vesarand molds Teta aerobic iro GAN | MiG Earn Te 02) Beno: " avalaleom the emaional Oroaiation Ft Standaraton (wwii ea) » SPECIFieD MicROORGANISNS Unless otherwise specified in the iilvidual micniograph, the’ recommended tests and acceptance’ criteria for specified microorganisms in probiotics o finished products for oral use ar listed in Table 4. ‘Table 4; Recommanded Tests for Spécifled Microoraanismé In ProblOtle Inarédlents or Halshed Produets fot Oral Use i ‘expan rit sn re wa ioe a ance eit ‘aa rereatcadnt09 ee ea Seance | ance neers | senonenerg The bene of itera monoqogere Sophy oar ures o Paden aerial aon the absence othe Stophylococ specie microorgased in fbl 4 shold be ested an conmed Hs pobiouc gredent ors tishe product Containing a probiotic ingredient(s) poses a risk associated withthe contamination of the above microorganisms based {99 formal sk assessment proarams such as Hazard Analysis and Critical Control Points (HACCP). The absence of ‘Clostridium perfringens and Cronobactersokazoki in addition to the absence of the listed specified microorganisms should be tested and contirmed if a probiotic ingredient or finished product is intended for infant use. The methods for Listeria. ‘monecytagenes, Staphylococcus aureus, Pseudomonas aerugioso, Clostridium peringens, and Cronobacter sakazokil must be officially accepted methods or appropriately validated methods, ADDITIONAL REQUIREMENTS + PACKAGING AND STORAGE at ‘Shipping and storage coniticis wil necessarily be’ adapted to the particular strains befng 301d Unies othenmise specie’ inthe ncvua monograph, probiotic ingredients shoul be tored in highs baie fo aminae bags and kept ator below 4° for long-term storage. Dosage forms of probiotics should be stored in tight containersin. a coo}, dry place, LABELING For probiotic ingredients the’ stain desigriation should be listed othe label foreach stain An ingredlen tora dssage form of probiotics should be labeled with the genus. species. and strain names. in cases where there is suitable scientific _ Fationale such as scientific substantiation of health Benefits that are not strain specifi, a dosage form of probiotics be abeled with the genus and species names, A total formulated enumeration of al probiotic ingredients throughout ‘product shelf life should be included at minimum in cfg or cfu/serving. sie asain (71) STERILITY TESTS “Portions of this general chapter have been harmonized with the corresponding texts of the European Pharmacopela andior the japanese Pharmacopeia Those portions that are not harmonized are marked with symbols) to specfy ths fac, These Pharmacopeal procedures are not by themselves designed to ensue that a batch of product is stelle or has been steritzed, This accomplished primarly hy validation ofthe stelization pracest a ofthe aseptic procecing poceckires www.webofpharma.comusp 43 Microbiological Tests / (71) 6481 The testi oppled to substances, preparations, orartices which, according tothe Pharmacopei, are required to be sterile However, @ satisfactory result only indicates that no contaminating microorganism has been found in the sample examined under the conditions ofthe test. PRECAUTIONS AGAINST MICROBIAL CONTAMINATION ‘The test for sterlty is carried out under aseptic conditions. In order to achieve such conditions, the test environment has to be adapted to the way in which the sterlty tests performed. The precautions taken to avoid contamination are such that they cdo not affect any microorganisms that are to be revealed in the test. The working conditions in which the tests are performed are monitored regularly by appropriate sampling of the working area and by carrying out appropriate controls. CULTURE MEDIA AND INCUBATION TEMPERATURES Media forthe test may be prepared as descibed below or equivalent commercial media may be used provided that they ‘comply with the requirements of the Growth Promotion Test of Aerobes,Ancerobes,and Fungl ‘The following cuture media have been found tobe suitable forthe test for sterity. Fluid Thiogycollate Medium is primarily intended for the culture of anaerobic bacteria, However t wil also detect aerobic bacteria. Soybean-Casein Digest Medium i suitable for the culture of both fungi and aerobic bacter Fluid Thloglycoltate Medium opine 059 Seu Chie 259 | Dextrose Monahydrate/Anhydrous __ 35/509 ewe 0759 Yeast baract(water sabe) 309 Pancreatic Digest of Caen 1509 Sedion Toghcotate 059 er Tropica Acs osm Resazin Solum Sounion (1 n T000), ely prepared Tome Porifed Water 000m pit ater sterlizaton:7.140.2. Kiet veystine, agar, sodium chiovde, dextrose, yeast extract, and pancreatic digest of casein with te purified water, and heat unt sltion effected, Disolve the sodium thiogycolate or thioglycolc acd inthe solation and, if necessary, aad T'N sodium hyroxie So that, afer seritation, the solution willhave a pH of 7-1 0.2. fitration necessary, hea te solution Again without boting, and fiter while hot through moistened fiter paper. Add the esazurin sodium solution, mix, and place {he madium in sutable vezzls that provide svat of surface to depth of medium such that aot more than the upper ha of the relum hs undergone a or change neat of oxygen ypate at heen of he ncn plod. Seise ung 2 validated proces. the mecium Is stored, store ata temperture between 2" and 25° Ina sere, alight container i mre than the upper one-third of the medium has acquired a pink color, the medium may be restored once by heating te containers ina waterbath orinfreefiowing steam unt the pink Color dsappears and by cooling qulely, taking cae to prevent the introduction of nonstere aint the container. Bo not use the medium for 3 longer storage period then has Been validated. Theid Thoghyclete Medium i to be weubated at 30°-35" For products containtng a metcural presavatie that cannot be tested by the membrane fitration method, Fuid Thioghycollote Wedum incubated at 20°25 may be used instead of Soybean Casein Digest Medium provided tat it has been validated as described in Growth Promotion Test of robes, Ancerobes, ond ful er pressor jute and authorizes, he lowing ata hogyclate medium might be i late Medlum, but omitting the agar snd the Prepare a mixture having the same composition as that ofthe Fluid 5 resazurin sodium solution. Sterilize as directed above. The pH after sterilization is 7.1 + 0.2. Heat in a water bath prior to use ia and incubate at 30°-35" tinder anaerable conditions. g Soybean-Caseln Digest Medlum i Pancreatic Digest of Caan 1708 es Pape Digest of Soybean Meal 306 Diba Pots Phosphate 259 entre Monohyeate/aniyarous 2539 Pure Water 000m pH after sterlization: 7.320.2. www. webofpharma.com6482 (71) / Microbiological Tests use 43 Dissolve the solids in the Purified Water, heating slightly to effect a solution. Coo! the solution to room temperature, and adjust the pH with 1 N sodium hydroxide so tha, after sterilization, it wil have a pH of 7.3 + 0.2. Fite, if necessary to clarity, dispense into suitable containers, and stelize using a validated procedure. Store at a temperature between 2° and 25° ina sterile wel-closed container, unless its intended for immediate use. Do not use the medium fora longer storage period than has been validated. Soybean-Casein Digest Medium is to be incubated at 22.5 + 2.5%. ‘Media for Penicillins or Cephalosporins Were tril test mea are to be used inthe Det noulation of he Culture Medium matod under Test fy tril of the Product tobe Examined, modify the preparation of Fluid Tioglycollate Medium and the Soybean-Casein Digest Medium as fllows. Toth container of each medhum, transtoraeaptlealy a quaniy of felactamagesfcent ta nactivate the amount of anviloie inthe specimen under test Determine te quantity of lactamase required to inactivate the antibiotic by using a Bactamase preparation that has been assayed previously for Is peniclin- or cephelospori-nactvating power. (NorE—Supplemented lactamase media can also be used in the membrane filtration test] ‘Alternatively (in an area completely separate from that used for sterility testing), confirm that an appropriate amount of, fHactamase is incorporated into the medium, following either method under Method Sutabity Test, using ess than 100. Colony-forming units (fy) of Staphylococcus aureus (see Table 1)as the challenge. Typical microbial growth ofthe inoculated Culture must be observed as a confirmation thatthe Platamase concentration 1s appropriate, ‘Table 1. Strains of the Test Microorganisms Suitable for Use in the Growth Promotion Test and the Method Sultability Test Aerobic bacteria Sopher areas AE 698, G43, NCTE TOT, NEB 95, NRE oc [ATCC 6633, CP 52.62, NCW 8054, NBRC3T34 Pesdemora eso [ATG 9027, NEN 8626, CP 82.178, NBRC 1327S ‘Anaerobic bacteria Fangh Conca acon [ATCC 1025, 48.72, NOPE 3179, NBRC 1598 “Apergiag oss ‘ATE 1640, 143183, 149007, NERC 9455 (Cepbgitr Noe “an akernatvemzoorganiis Kaci mop (Moco hes) ATCC 9341 2 aerrative to lsum sorogens, when 2 nonspreforming microorganisms dese is Bote igatus (ATCC 8482) ‘The media used comply with the following tests, carried out before, orn parallel with the test onthe product to be examined. Sterility Incubate portions of the media for 14 days. No growth of microorganisms occurs. Growth Promotion Test of Aerobes, Anaerobes, and Fungi Test each lt of ready-prepared medium and each batch of medium prepare either from dehydrated medium or from aredients, Suitable strains of microorganisms are indicated in Table 7 “inoclate portions of Fuld Thiogycollte Medium with a smal umber (not more than 100 cf) ofthe fllowing imicroorganisns, sing a separate portion of medium foreach ofthe flowing speces of microorganism: Cesium Sporogenes,Preidomonas aeruginosa, and Stophylococcus aureus. “noculate potions of ternative thoglycolate medium witha Fratumber {not more then 100 ch) of Coston sporogence Inoculte portions of Soybear-Cascy Digest Med vith @ Smal number (not more than 100 cf) of the fllowing mieroorgansms, using a separate portion of medium foreach of the folowing species of microorganism: Axpergilus bella, Sac subtils and Candide acon, Incubate for not more than 5 days inte case of bacteria and not more than 5 days inthe case of fungi. Seed lot culture maintenance techniques (seedot ‘mem ar ued 0 that he able miroorgeims ted for oclaton are not more than ive pages removed fom the Sriginal master seed ot "Pre rnc are suitable ia clearly le growth of the microorganisms occurs, www.webofpharma.comusp 43 Microbiological Tests / (71) 6483 ‘DILUTING AND RINSING FLUIDS FOR MEMBRANE FILTRATION Fluid A PREPARATION Dissolve 1 g of peptic digest of animal tissue in water to make 1 L fer or centrifuge to clarify, if necessary, and adjust to a pH of 7.1 + 0.2. Dispense into containers, and sterilize using a validated process. PREPARATION FOR PENICILLINS OR CEPHALOSPORINS ‘Asaptically add to the above Preporaton, if necessary, a quantity of stelle lactamase sufficient to inactivate any reidlual antibiotic activity on the membranes after the Solution ofthe test specimen has been fitered (see Media for Penciins or Cephalosporin). Fluid D To ca of fui a1 mt of poyorbate 80, adjust toa pil of 7.1 £0. dispense into containers and stele sing @ validated process. Use this fuid for articles containing lecithin oF oil, or for devices labeled as “sterile pathway.” Fluid K Dissolve 5.0 9 of peptic digest of animal tissue, 3.0 g of beef extract, and 10.0 g of polysorbate 80 in water to make 1 L. ‘Adjust the pH to obtain, attr sterlization, a pH of 6.9 = U.2. Dispense into container, and stenlize using a validate process, METHOD SUITABILITY TEST Carry out a test as described below under Test for Sterility of the Product to be Examined using exactly the same methods, except for the following modifications. Membrane Filtration After transferring the content of the container or containers to be tested to the membrane, add an inoculum of a small ‘number of viable microorqanisms (not more than 100 cfu) to the final portion of sterile diluent used to rinse the fier. Direct Inoculation Alter transferring the contents ofthe container or containers to be tested (for catgut and other surgical sutures for veterinary use: strands) to the culture medium, add an inoculum of a small number of viable microorganisms (not more than 100 cfu) tO the medium, iin both cases use the same microorganisms as those described above under Growth Promotion Test of Aerobes, Anaerobes, and Fungi. Perform a growth promotion test as a positive control. Incubate all the containers containing medium for not more than $ days. if leary visble growth of microorganisms fs obtained after the incubation, vsualy comparable otha Inthe contol vessel ‘without product, elther the product possesses no antimicrobial activity under the conditions ofthe test or such activity has been satisfactorily eliminated, The test for stelity may then be carried out without further modification. If clearly visible growth is not obtained in the presence of the product to be tested, visually comparable to that in the control vessels without product, the product possesses antimicrobial acy that has not been satisfactory eliminated under the ‘conditions of the test. Modify the conditions in order to eliminate the antimicrobial activity, and repeat the Method Sultabity Test. ‘This method suitabiliy is performed (a) when the test for sterlty has to be carried out on anew product; and (b) whenever there isa change in the experimental conditions ofthe test. The method sultablty may be performed simultaneously with the Test for Stenlty ofthe Product to be Examined. ‘TEST FOR STERILITY OF THE PRODUCT TO BE EXAMINED ‘Number of Articles to Be Tested Unless otherwise specified elsewhere in this chapter or inthe individual monograph, test the number of articles specified in Table 3. Ifthe contents of each article are of sufficient quantity (see Table 2), they may be divided so that equal appropriate portions are added to each of the specfied media, [NoTe—Perform sterliy testing employing two or more of the specified Iedia_]f each article does not contain sufficient quantities for each medium, use twice the numberof articles indicated in Table www. webofpharma.com a 3 a fai Fs6484 (71) / Microbiological Tests ‘Table 2. Minimum Quantity to usp 43 Used for Each Medium “Moimum Quantity fo be Used Quantity per Container (nes there sted and authorized) is Les than Ym __[ The whole contents ofeach container . To mt lth contents of ech container bat not ethan T mL Greate than 40m, and nok gate than 100 mL 20m reste than 100m. 10% of he conn of the container, but ot es than Z0™mL antbore is Tt ‘nile pepo ce nd iets be pended oe ‘Use the contents each container fo provide not stan 200m ‘es ‘ess than 50mg [The whole contents of each container 301mg ormore, bates tan 3007 Ha he contents of exch container, but not les thanSO™mg 300 me59 15079) reste than Sg soomg Gigi and other sarge atures or veteran ie seaions ofa wand fach 30mg) "Surge resng/eton/gnue n package) 100 mg per pecage ‘Situs ander indiiualy packaged angle ure mater he whole device Other medic devices The whale deve, ello aces oF dansembled, “Table 2. Minimum Number of Artie to be Tasted in Relation to the Number of Articles In the Batch Number of tems inthe Batch Minimum Number of tems to be Tested for Each _Mectum (ness others sid and storie)” Peel pepartion: ‘Not ore than 100 cantare TOR ord containers, whichever she grater More tan 10 but nat mre than 500 connor More than 300 conte 2 020 containers whichever ss * far lergevoline parenteral 296 oF 10 cantar whichever es anti a Praacy bah pacayer Sw) Po containes Prarmacy Buk palages 5a) container ila ana Bend See Baksh pros, Ophea ards ranneate propor ‘Not ore than 200 contalnes ior conaines, whichever iste restr More than 200 container TWeantnes the products presntd nthe form of aigla-dom container, {sy the share shown above or reparation or parenteral se, ‘atgutand other surgi sates for veterinary se Tibor packages, whichever the geste, upto masa otal of 20 pack oo "Ret more tan 100 ale 1086 races, whichever grater ‘More han 100, but rok more than 50 arcler Taner Mare than 500 are 26 020 aries, whichever es, Bak ald prot Up to fontaine ach container More than 4 conaiers but nat more han 80 enters 209 oF 4 conaines, whichever greater More than 50 eantainers iso 10 contains, whichever greater “tf he bate ze unknown, us the masimum numba of ems prescrbed the cones fone container re enough to nocd he to mel, tis column gives the numberof contin! needed for bt the me together. www.webofpharma.comusp 43 Microbiological Tests | (71) 6485 ‘The test may be carried out using the technique uf Mernbrane Filion oF by Direct Inoculation of the Culture Meelurn with the product to be examined. Appropriate negative controls are included. The technique of membrane fitration is used whenever the nature ofthe product permis, that is for fterabe aqueous preparations for leaholic or oly preparations and for Preparations mise wit, orslube in, aqueous or oly solvers, provided these solvents do net have an antimicrobial elect In the conditions of the tet. Membrane Filtration Use membrane fiters having a nominal pore size not greater than 0.45 um, in which the effectiveness to retain ‘microorganisms has been established. Cellulose nitrate filters, for example, are used for aqueous, oly, and weakly alcoholic solutions; and cellulose acetate fiters, for example, ae used for strongly alcoholic solutions. Specially adapted filters may be needed for certain products (e.g, for antibiotics). The technique described Below assumes that membranes about 50 mm in diameter will be used. I fiters of a diferent diameter are used, the volumes of the dilutions and the washings should be adjusted accordingly. The fitration apparatus and membrane ae stetlized by appropriate means. The apparatus designed so that the olution to be examined can be introduced and itered under aseptic conditions: it permits the aseptic removal of the membrane for transfer to the medium, or itis suitable for carrying out the incubation after adding the medium to the apparatus itself. AQUEOUS SOLUTIONS lf appropriate, transfer a small quantity ofa suitable, sterile diluent such as "fluid A (see Diluting and Rinsing Fluids for Membrane filtration), onto the membrane in the apparatus and iter. The diluent may contain suitable neutralizing substances and/or approprate inactivating substances, for example, nthe as of anos, ransfer the contents of the container or containers to be tested to the membrane or membranes, ifnecessary, ater diluting to the volume used in Ue Method Sultabilty Test with the chosen sterile lluent, but using not less than the quantiies of Ure product to be examined prescribed in Tables 2 and 3. Filter immediately. If the product has antimicrobial properties, wash the membrane not less than three times by filtering through it each time the volume of the chosen stelle luent used in the Method Sultobity Tet. Do not exceed a washing cle of ve times 100 mL per ite, even uring method suitably ithas been ‘demonstrated that such a cycle does not fully eliminate the antimicrobial activity Transfer the whole membrane to the culture ‘medium or cut it aseptically into two equal parts, and transfer one half to each of two suitable media. Use the same volume of ‘each medium as in the Method Sultablty Test. Alternatively, transfer the medium onto the raembrane in the apparatus. Incubate the media for not less than 14 days, SOLUBLE SOLIDS Use foreach medium not less than the quantity prescribed in Tables 2 and 3 of the product essoled ina suitable solvent, suchas the sclvent provided withthe preparation, Stare Water for injection, stare sane, ova sltable ste solution such 25 Fai A (Ditng ond Rinsing Fuds fr Membrane Ftratior,. and proceed with te testa deserved above for Aqueous Solutions using a membrane appropriate to the chosen solvent. Ls and olLY SOLUTIONS Lice for each medium nat less than the quantity ofthe product prescribed in Tables 2 and 2. Oils and oily solutions of sufficiently Tow viscosity may be fitered without dition through a dry membrane. Viscous oils may be cluted as necessary With a suitable sterile ciuent such as isopropyl myristate shown not to have antimicrobial activity in the conditions ofthe test. ‘Allow the ol to penetrate the membrane by iis own weight, and then fier, applying the pressure or suction gradually. Wash the membrane at least three times by tering through it each time about 100 mL ofa sultable stele solution such as “Fuld A (see Dilting and Rinsing Huds for Membrane Fitton, containing a sutable emulsifying agent at a concentration shown to be 2ppropate in the Method Suitability Test, for example polysorbate 80 a a concentration of 10 9 per L (Fuld K).. Transfer the membrane or membranes tothe culture medium of media or vice vers, as described above for Aqueous Solutions, and incubate {tthe same temperatures and for the same times. OINTMENTS and CREAMS Use for each medium not less than the quantities of the product prescribed in Tables 2 and 3. Ointments in a fatty base and emulsions of the water-in-ll type may be diluted to 196 in isopropyl myristate as described above, by heating, f necessary, ‘not more than 40". In exceptional cases it may be necessary to heat to not more than 44°. Filter as rapidly as possible, and proceed as described above for Oils and Oily Solutions. “*PREFILLED SYRINGES For prefiled syringes without attached sterile needles, expel the contents of each syringe into one or two separate membrane filter funnels or into separate pooling vessels prior to transfer. If a separate sterile needle is attached, directly expel the syringe contents as indicated above, and proceed as directed for Aqueous Solutions. Test the sterlty of the needle, using Direct Inoculation under Method Suitabilty Test. www. webofpharma.com ry ra 3= a s 6486 (71) / Microbiological Tests usp 43, SOLIDS FOR INJECTION OTHER THAN ANTIBIOTICS Constitute the tes articles as directed on the label, and proceed as directed for Aqueous Solutions or Oils and Oly Solutions, aiichever apples. Nove necessary, exces clluent canbe added to ald nthe constitution and lation of the costed test artide, ANTIBIOTIC SOLIDS FOR INJECTION Pharmacy Bulk Packages, <5 g—From each of 20 containers, aseptically transfer about 300 mag of solids, nto a sterile $00-mL conical ask, dissolve in about 200 ml of Fuld A (see Ditting and Rinsing Fluids for Membrane Fitrator), and mix; oF constitute, 28 directed inthe labeling, each of 20 containers and transfer a quantity of liquid or suspension, equivalent to about 300 mg Of sally nto a tere SOiaml conical lac, cen in abet 700 rl of Fits, and mix: Peed ae directed for Aue Solutions or Ois ond Oily Solutions, whichever apples. Pharmacy Bulk Packages, 5 g-—rom each of 6 coniainers, aseptically transfer about 1 g of solids into a sterile $00-ml. conical fas, dissolve in about 200 ml of Fuid A, and mix; or constitute, as directed inthe labeling, each of 6 containers and transfer a quantity of liquid, equivalent to about 1'g of slds, int asteré 500-mL. conical last, dissolve in about 200 mL of Fuld A, and tix. Proceed as directed for Aqueous Soltis. ANTIBIOTIC SOLIDS, BULKS, and BLENDS aseptically remove a sfclent quantity of sls rom the appropriate amount of containers (see Table 2), mix to obtain a ‘composite, equivalent to about 6 g of solids, and transfer toa stelle 500-mL conical flask. Dissolve in about 200 mL. of Fluid ‘A,and mix. Proceed as directed for Aqueous Solutions. STERILE AEROSOL PRODUCTS For fluid products in pressurized aerosol form, freeze the containers in an alcohol-dty ice mixture at least at -20° for about 1 hour feasible, allo the propelant io escape before asepticaly opening the container and taster the contents toa tre pooling vessel. Add 100 mL of Fluid D to the pooling vessel, and mix gently. Proceed as directed for Aqueous Solutions or Oils {and Oily Solutions, whichever applies. DEVICES WITH PATHWAYS LABELED STERILE Acs pa not es thn 10 pata volumes ol il ough each dec tte, Cole the fuldsin an appropiate Sa at ta so In the case of sterile, empty syringes, draw sterile diluent into the barrel through the sterile needle, if attached, or through a surance stated Gr popes oe tet snd apes he cote ate pskng eae Narada Direct Inoculation of the Culture Medium ‘Transfer the quantity of the preparation to be examined prescribed in Tables 2 and 3 directly into the culture medium so that the volume of the product Is hot more than 10% of the volume of the medium, unless otherwise prescribed. Ifthe product to be examined has antimicrobial activity, carry out the test after neutralizing this witha sultable neutralizing substance or by dilution in a sufficient quantity of culture medium, When itis necessary to use a large volume of the product, it may be preferable to use a concentrated culture medium prepared in such a way that it takes into account the subsequent dilution, Where appropriate, the concentrated medium may be added directly to the product in its container. olty LIQuios: Use media to which have been added a sultable emulsifying agent at a concentration shown to be appropriate in the Method Suitability Test, for example polysorbate 80 at a concentration of 10 g per L OINTMENTS and CREAMS Prepare by dating to about 1 in 10 by emulstying with the chosen emulstying agent na suitable stele dluent such as THId he Duty ond Ring Huts or Mentors it arteries precuctioa res et conaing 20 emulsiying agent. Tneubate the Inoculated media for not les than 14 days. Observe the cultures several imes during the incubation period. Spake cures conanng ay products gan each dy ower, when ud Tigo Wad used forte detecton of anaerobic mscroorgartams, Leap shaky oF mining toa mnimiim inorder to maintain aneerable concions CATGUT and OTHER SURGICAL SUTURES FOR VETERINARIAN USE Use for each medium not less than the quantities of the product prescribed in Tables 2 and 3. Open the sealed package using aseptic precautions, and remove three sections ofthe strand for each culture medium. Cary out the test on three sections, teach 30-cm long, which have been cutoff from the beginnina, the center, and the end of the strand. Use whole strands from {freshly opened cassette packs, Transfer each section of the strand to the selected medium. Use suficient medium to cover adequately the material to be tested (20 mL to 150 mL). www.webofpharma.comusp 43 Microbiological Tests / (71) 6487 *soUuDs Transfer a quantity ofthe product in the form ofa dry solid (or prepare a suspension ofthe product by adding sterile diluent to the immediate container), corresponding to not less than the quantity indicated in Tables 2 and 3. Transfer the material so blaine to 200 mL of Fd Tlogyolote Madu and mix. Sima, transfer the same quantity to 200 mi ofSoybean-Cosin Digest Medium, and mix. Proceed as directed above. PURIFIED COTTON, GAUZE, SURGICAL DRESSINGS, and RELATED ARTICLES From each package ofoton rolled gauze bandage or large surgical dressings being ested, aseptically remove two or more partons ot $00ng each rom he neon part othe ump. rom ndidualypaclaged, ngleuse ates, Septcaly remove the entre article, Immerse the porfons or atl In each medium, and proceed as Grcted above STERILE DEVICES {cles canbe immersed intact oresasembled. To ensure that device pathways are loin contact with the mesa, immerse the appropriate number of units per medium in a volume of medium sufficient to immerse the device completely, and proceed as directed above. For extremely large devices, immerse those portions ofthe device that are to come into contact with the patient in a volume of medium Sufficient to achieve complete immersion of those portions, For catheters where the inside lumen and outside are required to be stelle, either cut them into pieces such that the medium [sin contact with the entire lumen or fil the lumen with medium, and then immerse the intact unit, OBSERVATION AND INTERPRETATION OF RESULTS At intervals during the incubation period and at its conclusion, examine the media for macroscopic evidence of microbial ‘growth. I the material being tested renders the medium turbid so that the presence or absence of microbial growth cannot be Feadily determined by visual examination, 14 days after the beginning of incubation transfer portions (each not less than 1 mL) of the medium to fresh vessels of the same medium, and then incubate the original and transfer vessels for not less than 4 days. If no evidence of microbial growth is found, the product to be examined complies with the test for sterility. If evidence of eplerobil ara sfound the product to be éxamined doesnot compl withthe test fox sterity, unless can be cety demonstrated that the test was invalid for causes unrelated to the product to be examined. The test may be considered invalid only i one or more of the following conditions are fulfilled: 1, The data of the microbiological monitoring of the sterility testing facilty show a fault. 2. Areview of the testing procedure used during the tes in question reveals a faut. 3. Microbial growth is found in the negative controls. 4. After determination of the identity of the microorganisms isolated from the test, the growth of thie species (or these speci maybe ascrived unequvocaly to aus with respect tote material andor the technique used n conducting sterlity test procedure, It the testis declared to be invalid, iis repeated with the same number of units as in the original tes, If no evidence of ‘microbial growth is found in the repeat test, the product examined complies with the test for stelity, If microbial growth is found in the repeat test, the product examined does not comply with the test for strilty. APPLICATION OF THE TEST TO PARENTERAL PREPARATIONS, OPHTHALMIC, AND OTHER NONINJECTABLE PREPARATIONS REQUIRED TO COMPLY WITH THE TEST FOR STERILITY When using the technique of membrane filtration, use, whenever possible, the whole contents ofthe container, but not ss thanthe quate: ndatad in Tame 2 cating nnreneceney to aout 160 mt mia selec Staton ch as 4 Gee Disting and Rinsing Fu or Memerone Ftation) When using the technique of direct inoculation of med se the quant shown in Table 2, unless otherwise justified and authorized the tests forbacterl and fungal sterity ae cared out oh te same some ofthe product to be examined, When thee or ne guantiy ina angle cota aint cry ou ees, he contents oo more conan ed to noclate the aferent mes, MINIMUM NUMBER OF ITEMS TO BE TESTED ‘The minimum number of items to be tested in relation to the size of the batch is given in Table 3 www.webofpharma.com ray rs z