1、Designation: F838 05 (Reapproved 2013)Standard 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
2、 case of revision, 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.1. Scope1.1 This test method determines the bacterial retentioncharacteristics of membrane filters for
3、liquid filtration usingPseudomonas diminuta as the challenge organism. This testmethod may be employed to evaluate any membrane filtersystem used for liquid sterilization.1.2 This standard may involve hazardous materials,operations, and equipment. This standard does not purport toaddress all of the
4、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 applicability of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1193 Specification for Reagen
5、t 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 the filtrate.4. Summary of Test Method4.1 After sterilization, the test filter is challenged with asuspension of Pseudo
6、monas diminuta (ATCC 19146)3at aconcentration of 107organisms per cm2of effective filtrationarea (EFA) at a maximum differential pressure across the testfilter of 30 psig (206 kPa) and a flow rate of 0.5 to 1.0 GPM perft2of effective filtration area (2 to410-3LPM per cm2). Theentire filtrate is then
7、 filtered through an analytical membranefiler disc which is subsequently incubated on a solidifiedgrowth medium. Organisms that are not retained by the filterbeing tested will develop into visible colonies on the analysismembrane and can then be enumerated.5. Significance and Use5.1 Since all steril
8、izing filtration processes are performedunder positive pressure, this test method is designed to assessthe retentivity of a sterilizing filter under process conditions.5.1.1 A challenge of 107bacteria per cm2of effectivefiltration area is orders of magnitude higher than one wouldexpect to encounter
9、in a sterilizing filtration process. This levelwas selected in order to provide a high degree of assurance thatthe filter would quantitatively retain large numbers of organ-isms. This concept is important, in view of the requirement toprovide a quantitative assessment in validating a sterilizationpr
10、ocess.5.1.2 The analytical procedure utilized in this test methodprovides a method to assign a numerical value to the filtrationefficiency of the filter being evaluated. This value, coupledwith a knowledge of the number and types of organisms(bioburden) indigenous to the process, may then be utilize
11、d todetermine the probability of obtaining a sterile filtrate.Conversely, the numerical value of the filtration efficiency maybe used when one must meet a specified probability of sterilityassurance to calculate the volume of fluid that may be filteredin order to maintain that level of assurance.6.
12、Apparatus6.1 Assemble the apparatus described below as in Fig. 1:6.1.1 Stainless Steel Pressure Vessel, 12-L capacity (orlarger), fitted witha0to50-psi (0 to 350-kPa) pressure gage.6.1.2 Air Regulator.6.1.3 142-mm Disc Filter Assemblies, two or more, withhose connections.6.1.4 Diaphragm-Protected 0
13、to 50-psi Pressure Gage (0 to350-kPa), for upstream pressure reading. A second equivalentgauge for downstream pressure reading is optional.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
14、 Housing, with hose connections.1This test method is under the jurisdiction of ASTM Committee E55 onManufacture of Pharmaceutical Products and is the direct responsibility of Subcom-mittee E55.03 on General Pharmaceutical Standards.Current edition approved June 1, 2013. Published June 2013. Original
15、lyapproved in 1983. Last previous edition published in 2005 as F838 05. DOI:10.1520/F0838-05R13.2For 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 Docume
16、nt Summary page onthe ASTM website.3Available 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 States16.1.8 Hose Clamps.6.1.9 Incu
17、bator, 30 6 2C.6.1.10 Laminar Flow Bench.6.1.11 Smooth-Tip Forceps.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
18、 available.47.2 Purity of WaterUnless otherwise indicated, 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 St
19、andard Methods for theExamination of Water and Wastewater.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
20、 flask with anappropriate closure.8.1.3 Add 30 mL of lactose 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).Trypticase Peptone (or Casi
21、tone) 7.5 gYeast Extract 2.5 gSodium Chloride (NaCl) 0.5 gMagnesium Sulfate (MgSO43H2O) 0.35 g8.2.2 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 wate
22、r andsterilize at 121C for 15 min.8.2.3 Potassium Hydroxide Solution (0.1 N)Dissolve 5.61g of potassium hydroxide (KOH) in water and dilute to 1 L ina volumetric flask.8.2.4 Trypticase Soy AgarPrepare according to manufac-turers instructions.8.2.5 Trypticase Soy BrothPrepare according to manufac-tur
23、ers instructions.8.3 Analytical Reagents and Materials:8.3.1 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.
24、8.4 Pseudomonas diminuta (ATCC 19146).8.5 Analytical Membrane Filters, 142-mm diameter, 0.45m 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 prepar
25、ation of P. diminuta challenge sus-pensions. The presentation of these methods is not meant to4Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC, www.chemistry.org. For suggestions on thetesting of reagents not listed by the American Chemical Socie
26、ty, see AnalarStandards for Laboratory Chemicals, BDH Ltd., 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, Washi
27、ngton, DC 20001-3710, http:/www.apha.org.FIG. 1 Test Set-Up for Bacteria Retention TestingF838 05 (2013)2exclude other equally valid methods for the preparation of P.diminuta. It is important, however, that any P. diminutachallenge suspension used is monodisperse and meets thecriteria set forth in S
28、ection 10.9.2 Reconstitute the culture according to directions pro-vided 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 P. diminuta in accordance with Se
29、ction10.9.2.1 Stock CulturesPrepare stock cultures from singlecell isolates of 9.2. Inoculate trypticase 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, trypticase soy semisolid agar stab cul
30、turesmay be substituted for the slant cultures.9.2.2 Long Term Storage of Cultures Lyophilize or storein liquid nitrogen.9.3 Preparation of Challenge Stock Suspension in SalineLactose Broth:9.3.1 Inoculate 10-mLsterile trypticase soy broth with stockculture (9.2.1) and incubate at 30 6 2C for 24 h.9
31、.3.2 Transfer 2 mLof agitated broth culture to 1 Lof sterilesaline lactose broth, 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
32、 thechallenge suspension according to Section 11 (expected con-centration is 107to 108cells/mL).9.3.4 Identify the organisms as Pseudomonas diminuta inaccordance with Section 10.9.4 Preparation of Frozen Cell Paste of P. diminuta:9.4.1 Inoculate 10 mL of sterile growth medium A (8.2.1)with the stock
33、 culture (9.2.1) and incubate at 30 6 2C for 24h.9.4.2 Transfer 10 mL of the bacteria suspension from 9.3.1into 500 mL of sterile growth medium A and 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 sterilegrow
34、th 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 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 f
35、low centrifugation.9.4.6 Resuspend cells in two to three volumes of cold sterileharvesting buffer.9.4.7 Centrifuge suspension and resuspend cells in an equalvolume of harvesting buffer. Determine the cell concentration(expected concentration of viable cells is11012cells/mL).9.4.8 Transfer aliquots (
36、for example, 50 mL) of cell pasteinto sterile plastic centrifuge tubes, and freeze using dryice-acetone batch or liquid nitrogen. Store frozen cell paste at60C.9.5 Preparation of Challenge Stock Suspension from FrozenCell Paste:9.5.1 Disinfect the tube containing the cell paste by dippingtube in 80
37、% ethyl alcohol and flaming just long enough toburn off most of the alcohol. Use sterile 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 % Na
38、Cl which contains 0.001 to 0.002 M MgCl2at roomtemperature, (for example, transfer a 50-mL 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 uni
39、t, and mixuntil the entire contents of the tube is suspended evenly (40min).9.5.4 Determine the concentration of viable cells accordingto Section 11 (expected concentration of the cell suspension is1to21010cells/mL).9.5.5 Identify the organism as Pseudomonas diminuta inaccordance with Section 10.10.
40、 Identification of Pseudomonas diminuta10.1 Colonial Morphology:10.1.1 Colonies of Pseudomonas diminuta are yellow-beige, slightly convex, complete and 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 Microsco
41、pic Examination:10.2.1 Prepare a gram stain.10.2.1.1 Examine the preparation with a compound lightmicroscope 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). Ob
42、serve several microscopic fields fororganisms size and arrangement of cells.10.2.1.2 Stained preparations 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). Pseudomonasdi
43、minuta is characterized by a single, polar flagellum.10.3 Biochemical Characterization:10.3.1 Perform a number of the following biochemicalcharacterization tests. Pseudomonas diminuta gives the resultsindicated:F838 05 (2013)3TestP. diminuta(ATCC 19146)Spore formation OF glucose medium, open OF gluc
44、ose medium, sealed OF ethanol (3 %) medium, open +OF ethanol (3 %) medium, sealed Indole Methyl red Acetylmethylcarbionol Gelatinase Aerobe +Catalase +Cytochrome (Indophenol) oxidase +Growth on MacConkey agar +Dentrification +DNAase (BBL DNase Test agar orequivalent)Centrimide tolerance 11. Preparat
45、ion of Bacterial Challenge Suspension11.1 Determine by direct microscopic count the bacterialtitre of the suspension. This will determine the total number,viable and nonviable, cells present.11.2 Using the appropriate volume of a challenge stocksuspension, prepare an appropriate volume of a challeng
46、esuspension of Pseudomonas diminuta in a saline lactose brothor sterile saline to contain a minimum total of 107organismsper square centimetre of test filter area (1010m/ft2). Mix well.11.3 Aseptically remove a sample from the prepared chal-lenge suspension of Pseudomonas diminuta.11.4 Within a lami
47、nar flow hood, aseptically prepare dilu-tions of the suspension through 10-6using 0.1 % Peptonewater.11.5 Perform viable cell assay, in duplicate, using themembrane filter assay or direct spread plate assay underconditions that are similar to those specified for sterility testingin the current editi
48、on of the United States Pharmacopeia.611.5.1 For the membrane filter assay, use 1 mLfrom the 10-4through the 10-6dilutions. Place 50 mL of sterile 0.9 % NaClsolution into the funnel of the filter holder prior to adding the1.0 mL aliquots of the decimal dilutions. Filter and wash thewalls of the funn
49、el with 50 mL of sterile 0.9 % NaCl solution.Remove assay membrane from funnel, and place on agarmedium.11.5.2 For direct spread plate assay, use 0.1 mL from 10-3,10-4,10-5dilutions.11.6 Incubate membrane or spread assay plates to 30 6 2Cfor 48 h.11.7 Count colonies on plates showing between 30 and 300colonies (20 to 200 colonies on membrane filters) and calculatethe concentration (cell/mL) of the original suspensions.11.8 Compare the viable titre with the direct microscopiccount determined in 11.1. The viable count shoul
