1、Designation: F838 05 (Reapproved 2013)F838 15Standard 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,
2、 in the 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 retention characteristics of membrane filt
3、ers for liquid filtration usingPseudomonasBrevundimonas diminuta as the challenge organism. This test method may be employed to evaluate any membranefilter system used for liquid sterilization.1.2 This test method is not intended to be used in performance of product- and process-specific validation
4、of the bacterialretention characteristics of membrane filters to be used in pharmaceutical or biopharmaceutical sterilizing filtration, or both.Process- and product-specific bacterial retention validation should be carried out using the intended product manufacturing processparameters and the produc
5、t solution or surrogate as the carrier fluid.1.3 The values stated in SI units are to be regarded as standard.1.3.1 ExceptionThe inch-pound values given for units of pressure are to be regarded as standard; SI unit conversions areshown in parentheses.1.4 This standard may involve hazardous materials
6、, operations, and equipment. This standard does not purport to address allof the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriatesafety and health practices and determine the applicability of regulatory limitations prior
7、 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 of the ratio of the number of microorganisms in the challenge to thenumber of organisms in the filtrate.4. Summary of Test Metho
8、d4.1 After sterilization, the test filter is challenged with a suspension of PseudomonasB. diminuta (ATCC 19146)191463) at aconcentration of 107 organisms per cm2 of effective filtration area (EFA) at a maximum differential pressure across the test filterof 30 psig (206 kPa) and a flow rate of 0.5 t
9、o 1.0 GPM per ft2 to 2 of effective filtration area (2 to 4 10-33 LPM per cm2). ofeffective filtration area. The entire filtrate is then filtered through an analytical membrane filer disc, which is subsequentlyincubated on a solidified growth medium. OrganismsMicroorganisms that are not retained by
10、the filter being tested will developinto visible colonies on the analysis membrane and can then be enumerated.5. Significance and Use5.1 Since all sterilizing filtration processes are performed under positive pressure, this This test method is designed to assessthe retentivity of a sterilizing filte
11、r under process standard challenge conditions.1 This test method is under the jurisdiction ofASTM Committee E55 on Manufacture of Pharmaceutical Products and is the direct responsibility of Subcommittee E55.03on General Pharmaceutical Standards.Current edition approved June 1, 2013May 1, 2015. Publi
12、shed June 2013May 2015. Originally approved in 1983. Last previous edition published in 20052013 asF838 05.F838 05 (2013). DOI: 10.1520/F0838-05R13.10.1520/F0838-15.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Boo
13、k of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.3 Available from American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110, http:/www.atcc.org.This document is not an ASTM standard and is intended only to provide t
14、he user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current versionof the stand
15、ard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States15.1.1 A challenge of 107 bacteria per cm2 of effective filtration area is orders of magnitude higher than one would exp
16、ect toencounter in a sterilizing filtration process. This level was selected in order selected to provide a high degree of assurance that thefilter would will be challenged uniformly across the membrane surface to assure it will quantitatively retain large numbers oforganisms. This concept The model
17、 challenge organism, B. diminuta, is important, in view of the requirement to provide aquantitative assessment in validating a sterilization process.widely considered to be a small bacterium and is recognized as anindustry standard for qualifying sterilizing filters. Other species may represent a wo
18、rst-case test in terms of ability to penetrate afilter. This test does not provide assurance that filters can completely retain such bacteria.5.1.2 The analytical procedure utilized in this test method provides a method to assign a numerical value to the filtrationefficiency of the filter being eval
19、uated. This value, coupled with a knowledge of the number and types of organisms (bioburden)indigenous to the process, may then be utilized to determine the probability of obtaining a sterile filtrate. Conversely, the numericalvalue of the filtration efficiency may be used when one must meet a speci
20、fied probability of sterility assurance to calculate thevolume of fluid that may be filtered in order to maintain that level of assurance.evaluated under standard filtration conditions. Forthe purpose of product sterility assurance, additional process-specific studies should be performed.6. Apparatu
21、s6.1 Assemble the apparatus described below as in Fig. 1:6.1.1 Stainless Steel Pressure Vessel, 12-L capacity (or larger), fitted with a 0 to 50-psi (0 to 350-kPa) pressure gage.gauge.6.1.2 Air Regulator.6.1.3 142-mm 47-mm142-mm Analysis Disc Filter Assemblies, two or more, with hose connections.or
22、sanitary connections asapplicable.6.1.4 Diaphragm-Protected 0 to 50-psi Pressure Gage(0 to 350-kPa) Pressure Gauge, (0 to 350-kPa), for upstream pressurereading. A second equivalent gauge for downstream pressure reading is optional.6.1.5 Manifold, with valves (autoclavable) and hose connections.6.1.
23、6 Autoclavable Tubing, (must be able to withstand a pressure 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.FIG. 1 Test Set-Up for Bacteria Retention TestingF838 1527. P
24、urity of Reagents and Materials7.1 Purity of ReagentsReagent grade chemicals shall be used. Unless otherwise indicated, all reagents shall conform to thespecifications of the American Chemical Society, where such specifications are available.47.2 Purity of WaterUnless otherwise indicated, references
25、 to water shall mean reagent water, Type IV as defined inSpecification D1193.7.2.1 Additionally, any water used in this test method must conform to the requirements for non-bacteriostatic water specifiedin the current edition of Standard Methods for the Examination of Water and Wastewater.58. Reagen
26、ts and Materials8.1 Saline Lactose Broth Medium:8.1.1 Lactose BrothDissolve 1.3 g of dehydrated lactose broth medium in 100 mL of water.8.1.2 Sodium Chloride SolutionDissolve 7.6 g of sodium chloride (NaCl) in 970 mLof water in a 2-Lflask with an appropriateclosure.8.1.3 Add 30 mL of lactose broth (
27、8.1.1) to 970 mL of sodium 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 1 L. Autoclave at 121C for 15 min (pH 6.8 to 7.0).Trypticase Peptone (or Casitone) 7.5 gTryptic Peptone (or Casitone) 7.5 gYeast Extract 2.5 gS
28、odium Chloride (NaCl) 0.5 gMagnesium Sulfate (MgSO43H2O) 0.35 g8.2.2 Harvesting BufferDissolve 0.790 g of monobasic potassium phosphate (KH2PO4) and 1.0 g of K2HPO4 in 100 mL ofglycerol (C3H8O3). Adjust to pH 7.2 with 0.1 N potassium hydroxide solution. Dilute to 1 L with water and sterilize at 121C
29、 for15 min.8.2.3 Potassium Hydroxide Solution (0.1 N)Dissolve 5.61 g of potassium hydroxide (KOH) in water and dilute to 1 L in avolumetric flask.8.2.4 TrypticaseTryptic Soy AgarPrepare according to manufacturersmanufacturers instructions.8.2.5 TrypticaseTryptic Soy BrothPrepare according to manufac
30、turersmanufacturers instructions.8.3 Analytical Reagents and Materials:8.3.1 M-Plate Count AgarPrepare according to manufacturers instructions.8.3.2 Peptone Water (1 g/L)Dissolve the peptone in water. Dispense suitable volumes, for preparing decimal dilutions, intoscrew-cap containers. Autoclave at
31、121C for 15 min.8.4 PseudomonasB. diminuta (ATCC 19146).8.5 Analytical Membrane Filters, 47-mm or 142-mm diameter, 0.45 m pore size, 130 to 160 m thick.8.6 Petri Dishes, 150-mm diameter.9. Methods for Preparation of Bacterial Challenge Stock Suspension9.1 GeneralThe following two methods have been u
32、sed extensively for the preparation of P.B. diminuta challengesuspensions. The presentation of these methods is not meant to exclude other equally valid methods for the preparation of P.B.diminuta. It is important, however, that any P.BP. diminuta challenge suspension used is monodisperse and meets
33、the criteria setforth in Section 10.9.2 Reconstitute the culture according to directions provided by the American Type Culture Collection (ATCC). Check thepurity of the reconstituted culture by means of streak plates. Examine for uniform colony morphology, and identify single-cellisolates as P.B. di
34、minuta in accordance with Section 10.9.2.1 Stock CulturesPrepare stock cultures from single cell isolates of 9.2. Inoculate trypticasetryptic soy agar slants andincubate at 30 6 2C for 24 h. Overlay slants with sterile mineral oil and store at 4C. Check weekly for viability and purity.Alternatively,
35、 trypticasetryptic soy semisolid agar stab cultures may be substituted for the slant cultures.9.2.2 Long Term Storage of CulturesLyophilize or store in liquid nitrogen.9.3 Preparation of Challenge Stock Suspension in Saline Lactose Broth:9.3.1 Inoculate 10-mL sterile trypticasetryptic soy broth with
36、 stock culture (9.2.1) and incubate at 30 6 2C for 24 h.4 Reagent Chemicals, American Chemical Society Specifications, American Chemical Society, Washington, DC, www.chemistry.org. For suggestions on the testing ofreagents not listed by theAmerican Chemical Society, see Analar Standards for Laborato
37、ry Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville, MD, http:/www.usp.org.5 Available from the American Public Health Association (APHA), 800 I Street, NW, Washington, DC 20001-3710, http:/www.a
38、pha.org.F838 1539.3.2 Transfer 2 mL of agitated broth culture to 1 L of sterile saline lactose broth, swirl to mix inoculum and incubate at 30 62C for 24 h. Check purity of seed broth.NOTE 1Saline lactose broth suspension may be stored at 4C for up to 8 h prior to use.9.3.3 Determine the concentrati
39、on of viable cells in the challenge suspension according to Section 11 (expected concentrationis 107 to 108 cells/mL).9.3.4 Identify the organisms as PseudomonasB. diminuta in accordance with Section 10.9.4 Preparation of Frozen Cell Paste of P.B. diminuta:9.4.1 Inoculate 10 mL of sterile growth med
40、iumSterile Growth Medium A (8.2.1) with the stock culture (9.2.1) and incubateat 30 6 2C for 24 h.9.4.2 Transfer 10 mL of the bacteriabacterial suspension from 9.3.1 into 500 mL of sterile growth mediumSterile GrowthMedium A and incubate at 30 6 2C for 24 h.9.4.3 Prepare 10 L of a seed culture by tr
41、ansferring 200 mL of the bacterial suspension from 9.4.2 into 10 L of sterile growthmediumSterile Growth Medium A. Incubate at 30 6 2C for 24 h.9.4.4 Inoculate the 10 L of the seed culture into 500 L of growth mediumGrowth Medium A. Grow aerobically at 30 6 2C.Monitor growth spectrophotometrically a
42、t 500 nm, and plot growth curve.9.4.5 When the culture reaches the stationary phase, harvest the cells by continuous flow centrifugation.9.4.6 ResuspendRe-suspend cells in two to three volumes of cold sterile harvesting buffer.9.4.7 Centrifuge suspension and resuspendre-suspend cells in an equal vol
43、ume of harvesting buffer. Determine the cellconcentration (expected concentration of viable cells is 1 1012 cells/mL).9.4.8 Transfer aliquots (for example, 50 mL) of cell paste into sterile plastic centrifuge tubes, and freeze using dry ice-acetonebatch or liquid nitrogen. Store frozen cell paste at
44、 60C.70C.9.5 Preparation of Challenge Stock Suspension from Frozen Cell Paste:9.5.1 Disinfect the tube containing the cell paste by dipping tube in 80 % ethyl alcohol and flaming just long enough to burnoff most of the alcohol. Use sterile tongs to hold tube.9.5.2 Aseptically remove the cap from the
45、 tube and drop the tube into a sterile Erlenmeyer flask containing a sterile magneticstirring bar and 20 cell volumes of a sterile solution of 0.9 % NaCl which contains 0.001 to 0.002 M MgCl2 at roomtemperature,temperature (for example, transfer a 50-mL aliquot of frozen cell paste into 1 L of steri
46、le solution).NOTE 2MgCl2 must be in the solution prior to adding the frozen cell paste to prevent dumping during thaw.9.5.3 Place the flask on a magnetic stirring unit, and mix until the entire contents of the tube is suspended evenly (40 (about40 min).9.5.4 Determine the concentration of viable cel
47、ls according to Section 11 (expected concentration of the cell suspension is 1 to2 1010 cells/mL).9.5.5 Identify the organism as PseudomonasB. diminuta in accordance with Section 10.10. Identification of Pseudomonas B. diminuta10.1 Colonial Morphology:10.1.1 Colonies of PseudomonasB. diminuta are ye
48、llow-beige, slightly convex, complete and shiny.10.1.2 At 30C (optimum growth temperature) colonies are microscopic to pinpoint after 24 h and 1 to 2-mm diameter after36 to 48 h.10.2 Microscopic Examination:10.2.1 Prepare a gramGram stain.10.2.1.1 Examine the preparation with a compound light micros
49、cope fitted with a calibrated ocular micrometer and an oilimmersion objective lens with good resolving power (for example, a planachromatic objective with a numerical aperture of 1.2or greater). Observe several microscopic fields for organismsorganisms size and arrangement of cells.10.2.1.2 Stained preparations should reveal a gram-negative,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). PseudomonasB. d
