1、Designation: F838 15a1Standard Test Method forDetermining Bacterial Retention of Membrane FiltersUtilized for Liquid Filtration1This standard is issued under the fixed designation F838; the number immediately following the designation indicates the year of originaladoption or, in the case of revisio
2、n, the year of last revision.Anumber in parentheses indicates the year of last reapproval.Asuperscriptepsilon () indicates an editorial change since the last revision or reapproval.NOTEFig. 1 was editorially updated and the year date changed on Sept. 30, 2015.1NOTE9.1 was editorially corrected in Au
3、gust 2018.1. Scope1.1 This test method determines the bacterial retentioncharacteristics of membrane filters for liquid filtration usingBrevundimonas diminuta as the challenge organism. This testmethod may be employed to evaluate any membrane filtersystem used for liquid sterilization.1.2 This test
4、method is not intended to be used in perfor-mance of product- and process-specific validation of thebacterial retention characteristics of membrane filters to beused in pharmaceutical or biopharmaceutical sterilizingfiltration, or both. Process- and product-specific bacterialretention validation sho
5、uld be carried out using the intendedproduct manufacturing process parameters and the productsolution or surrogate as the carrier fluid.1.3 The values stated in SI units are to be regarded asstandard.1.3.1 ExceptionThe inch-pound values given for units ofpressure are to be regarded as standard; SI u
6、nit conversions areshown in parentheses.1.4 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety, health, and environmental practices and deter-mine the applicabilit
7、y of regulatory limitations prior to use.1.5 This international standard was developed in accor-dance with internationally recognized principles on standard-ization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recom-mendations issued by the Worl
8、d Trade Organization TechnicalBarriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2D1193 Specification for Reagent Water3. Terminology3.1 Definitions:3.1.1 log reduction valuethe logarithm to the base 10 ofthe ratio of the number of microorganisms in the challenge tothe numbe
9、r of organisms in the filtrate.4. Summary of Test Method4.1 After sterilization, the test filter is challenged with asuspension of B. diminuta (ATCC 191463) at a concentration of107organisms per cm2of effective filtration area (EFA) at amaximum differential pressure across the test filter of 30 psig
10、(206 kPa) and a flow rate of 2 to4103LPM per cm2ofeffective filtration area. The entire filtrate is then filteredthrough an analytical membrane filer disc, which is subse-quently incubated on a solidified growth medium. Microorgan-isms that are not retained by the filter being tested will developint
11、o visible colonies on the analysis membrane and can then beenumerated.5. Significance and Use5.1 This test method is designed to assess the retentivity ofa sterilizing filter under standard challenge conditions.5.1.1 A challenge of 107bacteria per cm2of effectivefiltration area is selected to provid
12、e a high degree of assurancethat the filter will be challenged uniformly across the mem-brane surface to assure it will quantitatively retain largenumbers of organisms. The model challenge organism, B.diminuta, is widely considered to be a small bacterium and is1This test method is under the jurisdi
13、ction of ASTM Committee E55 onManufacture of Pharmaceutical and Biopharmaceutical Products and is the directresponsibility of Subcommittee E55.03 on General Pharmaceutical Standards.Current edition approved Sept. 30, 2015. Published October 2015. Originallyapproved in 1983. Last previous edition pub
14、lished in 2015 as F838 15. DOI:10.1520/F0838-15AE01.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Availabl
15、e from American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, VA 20110, http:/www.atcc.org.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationa
16、lly recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.1recognized as an industry standard for qualifying steri
17、lizingfilters. Other species may represent a worst-case test in termsof ability to penetrate a filter. This test does not provideassurance that filters can completely retain such bacteria.5.1.2 The analytical procedure utilized in this test methodprovides a method to assign a numerical value to the
18、filtrationefficiency of the filter being evaluated under standard filtrationconditions. For the purpose of product sterility assurance,additional process-specific studies should be performed.6. Apparatus6.1 Assemble the apparatus described below as in Fig. 1:6.1.1 Stainless Steel Pressure Vessel, 12
19、-L capacity (orlarger), fitted witha0to50-psi (0 to 350-kPa) pressure gauge.6.1.2 Air Regulator.6.1.3 47-mm142-mm Analysis Disc Filter Assemblies, twoor more, with hose or sanitary connections as applicable.6.1.4 Diaphragm-Protected 0 to 50-psi (0 to 350-kPa)Pressure Gauge, for upstream pressure rea
20、ding.6.1.5 Manifold, with valves (autoclavable) and hose connec-tions.6.1.6 Autoclavable Tubing, (must be able to withstand apressure of 50 psi (350 kPa).6.1.7 Filter Housing, with hose connections.6.1.8 Hose Clamps.6.1.9 Incubator, 30 6 2C.6.1.10 Laminar Flow Bench.6.1.11 Smooth-Tip Forceps.6.1.12
21、Test Filter.7. Purity of Reagents and Materials7.1 Purity of ReagentsReagent grade chemicals shall beused. Unless otherwise indicated, all reagents shall conform tothe specifications of the American Chemical Society, wheresuch specifications are available.47.2 Purity of WaterUnless otherwise indicat
22、ed, referencesto water shall mean reagent water, Type IV as defined inSpecification D1193.7.2.1 Additionally, any water used in this test method mustconform to the requirements for non-bacteriostatic water speci-fied in the current edition of Standard Methods for theExamination of Water and Wastewat
23、er.58. Reagents and Materials8.1 Saline Lactose Broth Medium:8.1.1 Lactose BrothDissolve 1.3 g of dehydrated lactosebroth medium in 100 mL of water.8.1.2 Sodium Chloride SolutionDissolve 7.6 g of sodiumchloride (NaCl) in 970 mL of water in a 2-L flask with anappropriate closure.8.1.3 Add 30 mL of la
24、ctose broth (8.1.1) to 970 mL ofsodium chloride solution. Autoclave at 121C for 15 min.8.2 Frozen Cell Paste Method:8.2.1 Growth Medium ADissolve in water and dilute to 1L. Autoclave at 121C for 15 min (pH 6.8 to 7.0).Tryptic Peptone (or Casitone) 7.5 gYeast Extract 2.5 gSodium Chloride (NaCl) 0.5 g
25、Magnesium Sulfate (MgSO43H2O) 0.35 g4Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC, www.chemistry.org. For suggestions on thetesting of reagents not listed by the American Chemical Society, see AnalarStandards for Laboratory Chemicals, BDH Ltd.
26、, Poole, Dorset, U.K., and theUnited States Pharmacopeia and National Formulary, U.S. PharmacopeialConvention, Inc. (USPC), Rockville, MD, http:/www.usp.org.5Available from the American Public Health Association (APHA), 800 I Street,NW, Washington, DC 20001-3710, http:/www.apha.org.FIG. 1 Test Set-U
27、p for Bacteria Retention TestingF838 15a128.2.2 Harvesting BufferDissolve 0.790 g of monobasicpotassium phosphate (KH2PO4)and1.0gofK2HPO4in 100mL of glycerol (C3H8O3). Adjust to pH 7.2 with 0.1 Npotassium hydroxide solution. Dilute to 1 L with water andsterilize at 121C for 15 min.8.2.3 Potassium Hy
28、droxide Solution (0.1 N)Dissolve 5.61g of potassium hydroxide (KOH) in water and dilute to 1 L ina volumetric flask.8.2.4 Tryptic Soy AgarPrepare according to manufactur-ers instructions.8.2.5 Tryptic Soy BrothPrepare according to manufactur-ers instructions.8.3 Analytical Reagents and Materials:8.3
29、.1 M-Plate Count AgarPrepare according to manufac-turers instructions.8.3.2 Peptone Water (1 g/L)Dissolve the peptone in water.Dispense suitable volumes, for preparing decimal dilutions,into screw-cap containers. Autoclave at 121C for 15 min.8.4 B. diminuta (ATCC 19146).8.5 Analytical Membrane Filte
30、rs, 47-mm or 142-mmdiameter, 0.45 m pore size, 130 to 160 m thick.8.6 Petri Dishes, 150-mm diameter.9. Methods for Preparation of Bacterial Challenge StockSuspension9.1 GeneralThe following two methods have been usedextensively for the preparation of B. diminuta challengesuspensions. The presentatio
31、n of these methods is not meant toexclude other equally valid methods for the preparation of B.diminuta. It is important, however, that any B. diminutachallenge suspension used is monodisperse and meets thecriteria set forth in Section 10.9.2 Reconstitute the culture according to directions pro-vide
32、d by the American Type Culture Collection (ATCC).Check the purity of the reconstituted culture by means of streakplates. Examine for uniform colony morphology, and identifysingle-cell isolates as B. diminuta in accordance with Section10.9.2.1 Stock CulturesPrepare stock cultures from singlecell isol
33、ates of 9.2. Inoculate tryptic soy agar slants andincubate at 30 6 2C for 24 h. Overlay slants with sterilemineral oil and store at 4C. Check weekly for viability andpurity. Alternatively, tryptic soy semisolid agar stab culturesmay be substituted for the slant cultures.9.2.2 Long Term Storage of Cu
34、lturesLyophilize or store inliquid nitrogen.9.3 Preparation of Challenge Stock Suspension in SalineLactose Broth:9.3.1 Inoculate 10-mL sterile tryptic soy broth with stockculture (9.2.1) and incubate at 30 6 2C for 24 h.9.3.2 Transfer 2 mLof agitated broth culture to 1 Lof sterilesaline lactose brot
35、h, swirl to mix inoculum and incubate at 306 2C for 24 h. Check purity of seed broth.NOTE 1Saline lactose broth suspension may be stored at 4C for upto 8 h prior to use.9.3.3 Determine the concentration of viable cells in thechallenge suspension according to Section 11 (expected con-centration is 10
36、7to 108cells/mL).9.3.4 Identify the organisms as B. diminuta in accordancewith Section 10.9.4 Preparation of Frozen Cell Paste of B. diminuta:9.4.1 Inoculate 10 mL of Sterile Growth Medium A (8.2.1)with the stock culture (9.2.1) and incubate at 30 6 2C for 24h.9.4.2 Transfer 10 mL of the bacterial s
37、uspension from 9.3.1into 500 mLof Sterile Growth MediumAand incubate at 30 62C for 24 h.9.4.3 Prepare 10 L of a seed culture by transferring 200 mLof the bacterial suspension from 9.4.2 into 10 L of SterileGrowth Medium A. Incubate at 30 6 2C for 24 h.9.4.4 Inoculate the 10 L of the seed culture int
38、o 500 L ofGrowth Medium A. Grow aerobically at 30 6 2C. Monitorgrowth spectrophotometrically at 500 nm, and plot growthcurve.9.4.5 When the culture reaches the stationary phase, harvestthe cells by continuous flow centrifugation.9.4.6 Re-suspend cells in two to three volumes of coldsterile harvestin
39、g buffer.9.4.7 Centrifuge suspension and re-suspend cells in an equalvolume of harvesting buffer. Determine the cell concentration(expected concentration of viable cells is11012cells/mL).9.4.8 Transfer aliquots (for example, 50 mL) of cell pasteinto sterile plastic centrifuge tubes, and freeze using
40、 dryice-acetone batch or liquid nitrogen. Store frozen cell paste at70C.9.5 Preparation of Challenge Stock Suspension from FrozenCell Paste:9.5.1 Disinfect the tube containing the cell paste by dippingtube in 80 % ethyl alcohol and flaming just long enough toburn off most of the alcohol. Use sterile
41、 tongs to hold tube.9.5.2 Aseptically remove the cap from the tube and drop thetube into a sterile Erlenmeyer flask containing a sterile mag-netic stirring bar and 20 cell volumes of a sterile solution of0.9 % NaCl which contains 0.001 to 0.002 M MgCl2at roomtemperature (for example, transfer a 50-m
42、L aliquot of frozencell paste into 1 L of sterile solution).NOTE 2MgCl2must be in the solution prior to adding the frozen cellpaste to prevent dumping during thaw.9.5.3 Place the flask on a magnetic stirring unit, and mixuntil the entire contents of the tube is suspended evenly (about40 min).9.5.4 D
43、etermine the concentration of viable cells accordingto Section 11 (expected concentration of the cell suspension is1to21010cells/mL).9.5.5 Identify the organism as B. diminuta in accordancewith Section 10.10. Identification of B. diminuta10.1 Colonial Morphology:10.1.1 Colonies of B. diminuta are ye
44、llow-beige, slightlyconvex, complete and shiny.F838 15a1310.1.2 At 30C (optimum growth temperature) colonies aremicroscopic to pinpoint after 24 h and 1 to 2-mm diameter after36 to 48 h.10.2 Microscopic Examination:10.2.1 Prepare a Gram stain.10.2.1.1 Examine the preparation with a compound lightmic
45、roscope fitted with a calibrated ocular micrometer and anoil immersion objective lens with good resolving power (forexample, a planachromatic objective with a numerical apertureof 1.2 or greater). Observe several microscopic fields fororganisms size and arrangement of cells.10.2.1.2 Stained preparat
46、ions should reveal a Gram-negative, small, rod-shaped organism about 0.3 to 0.4 m by0.6 to 1.0 m in size, occurring primarily as single cells.10.2.2 Prepare a flagella stain (optional). B. diminuta ischaracterized by a single, polar flagellum.10.3 Biochemical Characterization:10.3.1 Perform a number
47、 of the following biochemicalcharacterization tests. B. diminuta gives the results indicated:6TestB. diminuta(ATCC 19146)Spore formation OF glucose medium, open OF glucose medium, sealed OF ethanol (3 %) medium, open +OF ethanol (3 %) medium, sealed Indole Methyl red Acetylmethylcarbionol Gelatinase
48、 Aerobe +Catalase +Cytochrome (Indophenol) oxidase +Growth on MacConkey agar +Denitrification +DNAase (BBL DNase Test agar orequivalent)Centrimide tolerance 11. Preparation of Bacterial Challenge Suspension11.1 Determine by direct microscopic count the bacterialtitre of the suspension. This will det
49、ermine the total number,viable and nonviable, cells present.11.2 Using the appropriate volume of a challenge stocksuspension, prepare an appropriate volume of a challengesuspension of B. diminuta in a saline lactose broth or sterilesaline to contain a minimum total of 107organisms per squarecentimetre of test filter area 1010m/ft2. Mix well.11.3 Aseptically remove a sample from the prepared chal-lenge suspension of B. diminuta.11.4 Within a laminar flow hood, aseptically prepare dilu-tions of the suspension throu
