1、Designation: F838 15aStandard 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 revision
2、, 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, 2015.1. Scope1.1 This test method determines t
3、he 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 method is not intended to be used in perfor-mance o
4、f 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 should be carried out using the intendedproduct manufa
5、cturing 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 unit conversions areshown in parentheses.1.4 This st
6、andard may involve hazardous materials,operations, and equipment. This standard does not purport toaddress all of the safety concerns, if any, associated with itsuse. It is the responsibility of the user of this standard toestablish appropriate safety and health practices and deter-mine the applicab
7、ility of regulatory limitations prior to use.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 number of organisms in t
8、he 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(206 kPa) and a flo
9、w 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 developinto visible colonies
10、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 provide a high degree of
11、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 isrecognized as an industry standard for qualifying sterili
12、zingfilters. 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 fi
13、ltrationefficiency of the filter being evaluated under standard filtrationconditions. For the purpose of product sterility assurance,additional process-specific studies should be performed.1This test method is under the jurisdiction of ASTM Committee E55 onManufacture of Pharmaceutical and Biopharma
14、ceutical 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 published in 2015 as F838 15. DOI:10.1520/F0838-15A.2For referenced ASTM sta
15、ndards, 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.3Available from American Type Culture Collection (ATCC), 10801 UniversityBoulevard, M
16、anassas, VA 20110, http:/www.atcc.org.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States16. Apparatus6.1 Assemble the apparatus described below as in Fig. 1:6.1.1 Stainless Steel Pressure Vessel, 12-L capacity (orlarger), fitted witha0to
17、50-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 reading.6.1.5 Manifold, with valves (autoc
18、lavable) 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 Test Filter.7. Purity of Reagents and M
19、aterials7.1 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 indicated, referencesto water shall mean reage
20、nt 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 Wastewater.58. Reagents and Materials8.1 Saline
21、 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 lactose broth (8.1.1) to 970 mL ofsodium
22、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 gMagnesium Sulfate (MgSO43H2O) 0.35 g8.2
23、.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 Hydroxide Solution (0.1 N)Dissolve 5.61g of pot
24、assium 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.4Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society
25、, 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., Poole, Dorset, U.K., and theUnited States Pharmacopeia and National Formulary, U.S. PharmacopeialConvention, Inc. (USPC)
26、, 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-Up for Bacteria Retention TestingF838 15a28.3 Analytical Reagents and Materials:8.3.1 M-Plate Count AgarPrepare according t
27、o 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 Filters, 47-mm or 142-mmdiameter, 0.45 m pore
28、 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 presentation of these methods is not meant toexclud
29、e other equally valid methods for the preparation of B.diminuta. It is important, however, that any BP. diminutachallenge suspension used is monodisperse and meets thecriteria set forth in Section 10.9.2 Reconstitute the culture according to directions pro-vided by the American Type Culture Collecti
30、on (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 isolates of 9.2. Inoculate tryptic soy agar
31、 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 CulturesLyophilize or store inliquid nitr
32、ogen.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 broth, swirl to mix inoculum and incubate a
33、t 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 107to 108cells/mL).9.3.4 Identify the org
34、anisms 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 suspension from 9.3.1into 500 mLof Steri
35、le 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 into 500 L ofGrowth Medium A. Grow aerobic
36、ally 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 harvesting buffer.9.4.7 Centrifuge suspension an
37、d 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 dryice-acetone batch or liquid nitroge
38、n. 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 tongs to hold tube.9.5.2 Aseptically r
39、emove 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-mL aliquot of frozencell paste into 1 L
40、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 Determine the concentration of viable ce
41、lls 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 yellow-beige, slightlyconvex, complete an
42、d shiny.10.1.2 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 lightmicroscope fitted with a calibrated ocular micromete
43、r 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.F838 15a310.2.1.2 Stained preparations should reveal a Gram-negative, smal
44、l, 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 of the following biochemicalcharacteriz
45、ation 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 Aerobe +Catalase +Cytochrome (Indopheno
46、l) 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 determine the total number,viable and nonvi
47、able, 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 we
48、ll.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 through 106using 0.1 % Peptonewater.11.5 Perform viable cell assay, in duplicate, using themembrane filter assay or direct spre
49、ad plate assay underconditions that are similar to those specified for sterility testingin the current edition of the United States Pharmacopeia.711.5.1 For the membrane filter assay, use 1 mL from the104through the 106dilutions. Place 50 mL of sterile 0.9 %NaCl solution into the funnel of the filter holder prior to addingthe 1.0 mL aliquots of the decimal dilutions. Filter and washthe walls of the funnel with 50 mL of sterile 0.9 % NaClsolution. Remove assay membrane from funnel, and place onagar medium.11.5.2 For
