1、Designation: E3161 18Standard Practice forPreparing a Pseudomonas aeruginosa or Staphylococcusaureus Biofilm using the CDC Biofilm Reactor1This standard is issued under the fixed designation E3161; the number immediately following the designation indicates the year oforiginal adoption or, in the cas
2、e of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice specifies the parameters for growing aPseudomonas aeruginosa (ATCC 15442) or
3、Staphylococcusaureus (ATCC 6538) biofilm that can be used for disinfectantefficacy testing using the Test Method for Evaluating Disinfec-tant EfficacyAgainst Pseudomonas aeruginosa Biofilm Grownin CDC Biofilm Reactor Using Single Tube Method (E2871)orin an alternate method capable of accommodating t
4、he couponsused in the CDC Biofilm Reactor. The resulting biofilm isrepresentative of generalized situations where biofilm exist onhard, non-porous surfaces under shear rather than being repre-sentative of one particular environment. Additional bacteriamay be grown using the basic procedure outlined
5、in thisdocument, however, alternative preparation procedures forfrozen stock cultures and biofilm generation (for example,medium concentrations, baffle speed, temperature, incubationtimes, coupon types, etc.) may be necessary.1.2 This practice uses the CDC Biofilm Reactor created bythe Centers for D
6、isease Control and Prevention (1).2The CDCBiofilm Reactor is a continuously stirred tank reactor (CSTR)with high wall shear. The reactor is versatile and may also beused for growing or characterizing various species of biofilm,or both (2-4) provided appropriate adjustments are made to thegrowth medi
7、a and operational parameters of the reactor.1.3 Basic microbiology training is required to perform thispractice.1.4 UnitsThe values stated in SI units are to be regardedas standard. No other units of measurement are included in thispractice.1.5 This standard does not purport to address all of thesaf
8、ety 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 applicability of regulatory limitations prior to use.1.6 This international standard was developed in accor-dan
9、ce 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 World Trade Organization TechnicalBarriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Sta
10、ndards:3E2756 Terminology Relating to Antimicrobial and AntiviralAgentsE2871 Test Method for Evaluating Disinfectant EfficacyAgainst Pseudomonas aeruginosa Biofilm Grown inCDC Biofilm Reactor Using Single Tube Method3. Terminology3.1 Definitions:3.1.1 For definition of terms used in this method refe
11、r toTerminology E2756.3.1.2 batch phase, nestablishment of the biofilm by oper-ating the reactor without the flow of nutrients (batch phasegrowth medium), but with mixing.3.1.3 biofilm, nmicroorganisms living in a self-organizedcommunity attached to surfaces, interfaces, or each other,embedded in a
12、matrix of extracellular polymeric substances ofmicrobial origin, while exhibiting altered phenotypes withrespect to growth rate and gene transcription.3.1.4 continuously stirred tank reactor (CSTR) phase,nestablishment of a steady state biofilm population achievedwith the continuous flow of nutrient
13、s (continuous flow growthmedium) in a glass vessel.3.1.5 coupon, nbiofilm sample surface.4. Summary of Practice4.1 This practice is used for growing a P. aeruginosa or S.aureus biofilm in the CDC Biofilm Reactor. The biofilm isestablished by operating the reactor in batch phase (no flow of1This prac
14、tice is under the jurisdiction of ASTM Committee E35 on Pesticides,Antimicrobials, and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2018. Published June 2018. DOI: 10.1520/E316118.2The boldface numbers in
15、parentheses refer to the list of references at the end ofthis standard.3For 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 AST
16、M website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopmen
17、t of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.1the nutrients) for 24 h. A steady state population is reachedwhile the reactor operates for an additional 24 h with continu-ous flow of the nutrients. The resi
18、dence time of the nutrients inthe reactor is set to select for biofilm growth, and is species andreactor parameter specific. During the entire 48 h, the biofilmis exposed to continuous fluid shear from the rotation of abaffled stir bar. Controlling the rate at which the baffle turnsdetermines the in
19、tensity of the shear stress to which thecoupons are exposed. At the end of the 48 h, the biofilm-ladencoupons are used for testing.5. Significance and Use5.1 Bacteria that exist in biofilms are phenotypically differ-ent from suspended cells of the same genotype. Research hasshown that biofilm bacter
20、ia are more difficult to kill thansuspended bacteria (4, 5). Laboratory biofilms are engineeredin growth reactors designed to produce a specific biofilm type.Altering system parameters will correspondingly result in achange in the biofilm. The purpose of this practice is to directa user in the growt
21、h of a P. aeruginosa or S. aureus biofilm byclearly defining the operational parameters to grow a biofilmthat can be assessed for efficacy using the Standard TestMethod for Evaluating Disinfectant Efficacy AgainstPseudomonas aeruginosa Biofilm Grown in CDC BiofilmReactor Using Single Tube Method (E2
22、871).5.2 Operating the CDC Biofilm Reactor at the conditionsspecified in this method generates biofilm at log densities(log10CFU per coupon) ranging from 8.0 to 9.5 for P.aeruginosa and 7.5 to 9.0 for S. aureus. These levels of biofilmare anticipated on surfaces conducive to biofilm formation suchas
23、 the conditions outlined in this method.5.2.1 To achieve an S. aureus biofilm with a populationcomparable to that for P. aeruginosa using the bacterial liquidgrowth medium conditions specified here, the S. aureus biofilmmust be grown at 36 62 C rather than at room temperature(21 62 C).6. Apparatus6.
24、1 Culture Tubes and Culture Tube Closuresany glass orplastic tube with a volume capacity of at least 15 mL.6.2 Calibrated Pipettercontinuously adjustable pipetterwith volume capability of 1 mL.6.3 Vortexany vortex that will ensure proper agitation andmixing of culture tubes.6.4 Ultrasonic Water Bath
25、any cavitating sonicating baththat operates at 45 65 kHz and which has a volume largeenough to accommodate 50 mL or 250 mL conical tubes.6.5 Analytical Balancesensitive to 0.01 g.6.6 Sterilizerany steam sterilizer that can produce theconditions of sterilization is acceptable.6.7 Peristaltic Pumppump
26、 head that can hold size 16 orequivalent peristaltic pump tubing. Use a separate pump foreach reactor.6.8 Digital Magnetic Stir Platetop plate of at least 10.16by 10.16 cm that can rotate at a range of 60 to 125 65 r/min.6.9 Silicone Tubingmultiple sizes: size 16 tubing orequivalent designed for use
27、 in a peristaltic pump (used for mostconnections between CSTR growth medium carboy and thereactor), and size 18 or 25 tubing or equivalent (used forreactor effluent). All sizes must withstand sterilization (forexample, platinum cured).6.10 Norprene Tubing (or equivalent)size 16 or equiva-lent Norpre
28、ne tubing. Recommended for use in the peristalticpump.6.11 Glass Flow Breakany that will connect with size 16tubing and withstand sterilization, used to prevent microbialcontamination of the nutrient reservoir from the biofilm reac-tor.6.11.1 Clampused to hold flow break, extension clampwith 0.5 cm
29、minimum grip size.6.11.2 Clamp Standheight no less than 76.2 cm, used withclamp to suspend glass flow break vertically and stabilizetubing above reactor.6.12 Reactor Components.46.12.1 Berzelius Borosilicate Glass Tall Beaker1000 mLwithout pour spout, 9.5 60.5 cm diameter. Barbed outlet spoutadded a
30、t 400 650 mL mark. Spout angled 30 to 45 to ensuredrainage. Spout should accommodate size 18 or 25 flexiblesilicone tubing.6.12.2 Reactor TopFig. 1. Ultra-high molecular weight(UHMW) polyethylene top (10.1 cm diameter tapering to 8.33cm) equipped with a minimum of 3 holes accommodating 6 to8 cm long
31、 pieces of stainless steel or other rigid autoclavabletubing with outside diameter of 5 to 8 mm for medium inlet, airexchange and inoculation port. Center hole, 1.27 cm diameter,to accommodate the glass rod used to support the baffleassembly. Eight rod holes, 1.905 cm diameter, notched toaccommodate
32、 stainless steel rod alignment pin (0.236 cmoutside diameter). O-ring, attached to underside of reactor top.6.12.3 Polypropylene RodsFig. 2. Eight polypropylenerods, 21.08 cm long, two types: coupon holder machined tohold three coupons (see 6.12.4) at the immersed end, three 316stainless steel set s
33、crews embedded in side to hold coupons inplace; and coupon holder blanks, without coupon recesses.Rods fit into holes in reactor top and lock into preformednotches with alignment pin.6.12.4 Couponstwenty-four cylindrical coupons (forexample, borosilicate glass) with a diameter of 1.27 60.013cm, thic
34、kness of approximately 3.0 mm.6.12.5 Small Allen Wrench (1.27 mm, hex)for adjustingset screws.6.12.6 Stir Blade Assembly (Baffled Stir Bar)Fig. 3. PTFEblade (5.61 cm) fitted into cylindrical PTFE holder (8.13 cm)and held in place with a magnetic stir bar (2.54 cm).6.12.6.1 PTFE holder fits onto a gl
35、ass rod (15.8 cm), fittedinto the reactor top.4The sole source of supply of the apparatus (CDC Biofilm Reactor) andassociated coupons known to the committee at this time is BioSurface Technologies,Corp. www.biofilms.biz. If you are aware of alternative suppliers, please providethis information to AS
36、TM International Headquarters. Your comments will receivecareful consideration at a meeting of the responsible technical committee,1whichyou may attend. The user may also build the reactor.E3161 1826.12.6.2 The glass rod is held in place with a compressionfitting and acts as a support for the moving
37、 blade assembly.6.13 Carboystwo 20 L autoclavable carboys, one used forwaste and one used for growth medium.6.13.1 Carboy Lidstwo.6.13.1.1 One carboy lid with at least 2 barbed fittings toaccommodate size 16 tubing (one for growth medium and onefor bacterial air vent).6.13.1.2 One carboy lid with at
38、 least two 1 cm holes boredin the same fashion (one for effluent waste and one for bacterialair vent).NOTE 1Carboy lids can be purchased with fittings.6.13.2 Bacterial Air Vent (Filter)autoclavable 0.2 m poresize, to be spliced into tubing on waste carboy, growth mediumcarboy and reactor top, recomm
39、ended diameter 37 mm.FIG. 1 Expanded Schematic of Reactor TopFIG. 2 Expanded Schematic of Rod and CouponsE3161 1836.14 Fig. 4 illustrates a schematic of the assembled system.6.15 Detergentlaboratory detergent for cleaning couponsand reactor parts.7. Reagents and Materials7.1 Purity of WaterAll refer
40、ences to water as diluent orreagent shall mean de-ionized water or water of equal purity.FIG. 3 Expanded Schematic of Baffled Stir BarFIG. 4 Schematic of the Completely Assembled Reactor SystemE3161 1847.2 Culture Media:7.2.1 CryoprotectantTryptic Soy Broth (30 g/L) with15 % (v/v) glycerol.7.2.2 Bac
41、terial Liquid Growth MediumTryptic Soy Broth(TSB).7.2.2.1 For P. aeruginosa, use 300 mg/L TSB for theinoculum and batch phase reactor operations, and 100 mg/LTSB for the continuous flow reactor operation.7.2.2.2 For S. aureus, use 30 g/L TSB for the inoculum, 3g/L TSB for the batch phase reactor ope
42、ration, and 1 g/L TSBfor the continuous flow reactor operation.7.2.3 Growth Medium for Stock Culture GenerationTrypticase Soy Agar (TSA).8. Preparation of Apparatus8.1 Preparation of Borosilicate Glass Coupons:8.1.1 Coupons may be used repeatedly with proper cleaningand screening between each use. A
43、fter use in the reactor, placecontaminated coupons in an appropriate vessel, cover withliquid (e.g., water), and, along with the other parts of thecontaminated reactor system, autoclave at 121C for 30 min orusing other parameters that ensure sterilization.8.1.2 Check each coupon under 20 magnificati
44、on forscratching, chipping, other damage, or accumulated debrisbefore each use. Discard those with visible damage to surfacetopography.8.1.3 For initial use and re-use, sonicate coupons in indi-vidual tubes or welled plates for approximately 5 min indetergent diluted per the manufacturers instructio
45、ns. Thesoapy water must completely cover the coupons.NOTE 2Process coupons individually to minimize the risk of damageto the coupons.8.1.4 Rinse coupons with reagent grade water and sonicatefor approximately 1 min in reagent grade water.8.1.5 Repeat rinsing and sonication with reagent gradewater unt
46、il no soap is left on the coupons, as demonstrated bya lack of visible suds. Once the coupons are clean, wear glovesto prevent oils and other residue from contaminating thesurface. Store screened and cleaned coupons in a Petri dish.NOTE 3Coupons may be made out of alternative materials.Adjust thecle
47、aning procedure so that it is appropriate for the coupon material beingused.8.2 Preparation of Reactor Top:8.2.1 Invert the reactor top and place baffled stir bar ontoglass rod positioned in the center of the reactor top.8.2.2 Invert the reactor beaker and place onto the assembledtop. Turn the react
48、or over so that the reactor top is upright. Thebaffled stir bar is designed to allow it to rotate freely.8.2.3 Place a cleaned and screened coupon into each hole inthe reactor rods, leaving the top of the coupon flush with theinside rod surface. Tighten set screw. If less than 24 couponsare required
49、 for testing, substitute one coupon holder blank foreach polyproplyene rod holding three (3) coupons.8.2.4 Place rods into reactor top loosely (not yet fitted intonotches).8.2.5 Connect the bacterial air vent by fitting the vent to asmall section of appropriately sized tubing and attach to one ofthe rigid tubes on the reactor top.8.2.6 Splice the glass flow break into the growth mediumtubing line near the reactor top.9. Calibration and Standardization9.1 Confirm the operating volume of each reactor (that is,new Berzelius beaker with spou
