1、Designation: E2196 17Standard Test Method forQuantification of Pseudomonas aeruginosa Biofilm Grownwith Medium Shear and Continuous Flow Using RotatingDisk Reactor1This standard is issued under the fixed designation E2196; the number immediately following the designation indicates the year oforigina
2、l adoption or, in the case 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 test method is used for growing a reproducible (1)2Pseudomona
3、s aeruginosa biofilm in a continuously stirred tankreactor (CSTR) under medium shear conditions. In addition,the test method describes how to sample and analyze biofilmfor viable cells.1.2 Although this test method was created to mimic condi-tions within a toilet bowl, it can be adapted for the grow
4、th andcharacterization of varying species of biofilm (rotating diskreactorrepeatability and relevance (2).1.3 This test method describes how to sample and analyzebiofilm for viable cells. Biofilm population density is recordedas log10colony forming units per surface area (rotating diskreactorefficac
5、y test method (3).1.4 Basic microbiology training is required to perform thistest method.1.5 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.6 This standard does not purport to address all of thesafety concerns, if any, associ
6、ated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.1.7 This international standard was developed in accor-dance with internationally recognized principle
7、s 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 Standards:3D5465 Practices for Determining Micr
8、obial Colony Countsfrom Waters Analyzed by Plating Methods2.2 Other Standards:Method 9050 C.1.a Buffered Dilution Water Preparation (4)3. Terminology3.1 biofilm, n microorganisms living in a self-organizedcommunity attached to surfaces, interfaces, or each other,embedded in a matrix of extracellular
9、 polymeric substances ofmicrobial origin, while exhibiting altered phenotypes withrespect to growth rate and gene transcription.3.1.1 DiscussionBiofilms may be comprised of bacteria,fungi, algae, protozoa, viruses, or infinite combinations ofthese microorganisms. The qualitative characteristics of a
10、biofilm, including, but not limited to, population density,taxonomic diversity, thickness, chemical gradients, chemicalcomposition, consistency, and other materials in the matrix thatare not produced by the biofilm microorganisms, are controlledby the physiochemical environment in which it exists.3.
11、2 coupon, nbiofilm sample surface.4. Summary of Test Method4.1 This test method is used for growing a reproduciblePseudomonas aeruginosa biofilm in a rotating disk reactor.The biofilm is established by operating the reactor in batchmode (no flow) for 24 h. Steady state growth (attachment isequal to
12、detachment) is reached while the reactor operates foran additional 24 h with continuous flow of the nutrients. Theresidence time of the nutrients in the reactor is set to select forbiofilm growth, and is species and reactor parameter specific.During the entire 48 h, the biofilm is exposed to continu
13、ousfluid shear from the rotation of the disk.At the end of the 48 h,biofilm accumulation is quantified by removing coupons from1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents and is the directresponsibility of Subcommitte
14、e E35.15 on Antimicrobial Agents.Current edition approved April 1, 2017. Published May 2017. Originallyapproved in 2002. Last previous edition approved in 2012 as E2196 12. DOI:10.1520/E2196-17.2The boldface numbers in parentheses refer to a list of references at the end ofthis standard.3For referen
15、ced 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.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Co
16、nshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recommendations issued by the World Trade
17、Organization Technical Barriers to Trade (TBT) Committee.1the disk, harvesting the biofilm from the coupon surface,disaggregating the clumps, then diluting and plating for viablecell enumeration.5. Significance and Use5.1 Bacteria that exist in a biofilm are phenotypicallydifferent from suspended ce
18、lls of the same genotype. The studyof biofilm in the laboratory requires protocols that account forthis difference. Laboratory biofilms are engineered in growthreactors designed to produce a specific biofilm type. Alteringsystem parameters will correspondingly result in a change inthe biofilm. The p
19、urpose of this method is to direct a user in thelaboratory study of biofilms by clearly defining each systemparameter. This method will enable a person to grow, sample,and analyze a laboratory biofilm. The method was originallydeveloped to study toilet bowl biofilms, but may also beutilized for rese
20、arch that requires a biofilm grown undermoderate fluid shear.6. Apparatus6.1 Wooden Applicator Sticks, sterile.6.2 Inoculating Loop.6.3 Petri Dish, 100 by 15 mm, plastic, sterile and empty tohold rotor while sampling.6.4 Culture Tubes and Culture Tube Closures, any with avolume capacity of 10 mL and
21、 minimum diameter of 16 mm.Recommended size is 16 by 125 mm borosilicate glass withthreaded opening.6.5 Pipette(s), continuously adjustable pipette(s) with vol-ume capacity of 1 mL.6.6 Micropipette(s), continuously adjustable pipette(s) witha volume capacity of 10 250 L.6.7 Vortex, any vortex that w
22、ill ensure proper agitation andmixing of culture tubes.6.8 Homogenizer, any capable of mixing at 20 500 6 5000r/min ina5to10mLvolume.6.9 Homogenizer Probe, any capable of mixing at 20 500 65000 r/min ina5to10mLvolume that can withstandautoclaving or other means of sterilization.6.10 Sonicating Bath,
23、 any cavitating sonicating bath thatoperates at 45 to 60 kHz for cleaning the coupons.6.11 Bunsen Burner, used to flame inoculating loop andother instruments.6.12 Stainless Steel Dissecting Tools, for removing thecoupons.NOTE 1Alternatively, a coupon manipulation tool4may be used.6.13 Stainless Stee
24、l Hemostat Clamp, with curved tip.6.14 Environmental Shaker, capable of maintaining tem-perature of 36 6 2C.6.15 Analytical Balance, sensitive to 0.01 g.6.16 Sterilizer, any steam sterilizer capable of producing theconditions of sterilization is acceptable.6.17 Colony Counter, any one of several typ
25、es may be used,such as the Quebec, Buck, and Wolfhuegel.Ahand tally for therecording of the bacterial count is recommended if manualcounting is done.6.18 Peristaltic Pump, pump head capable of holding tubingwith inner diameter of 3.1 mm and outer diameter of 3.2 mm.6.19 Digital Magnetic Stir Plate,
26、top plate 10.16 by 10.16cm, capable of rotating at 200 6 5 r/min.6.20 Silicone Tubing, two sizes of tubing: one with innerdiameter of 3.1 mm and outer diameter of 3.2 mm, and theother with inner diameter of 7.9 mm and outer diameter of 9.5mm. Both sizes must withstand sterilization.6.21 Norprene5Tub
27、ing, inner diameter of 3.1 mm and outerdiameter of 3.2 mm.6.22 Glass Flow Break, any that will connect with tubing ofinner diameter 3.1 mm and withstands sterilization.6.23 Clamp, used to hold flow break, extension clamp with0.5 cm minimum grip size.6.24 Clamp Stand, height no less than 76.2 cm, use
28、d withclamp to suspend glass flow break vertically and stabilizetubing above reactor.6.25 Reactor Components6:6.25.1 Berzelius Borosilicate Glass Beaker, 1000 mL with-out pour spout, 9.5 6 0.5 cm diameter. Borosilicate barbedoutlet spout added at 250 6 15 mL mark at 30 to 45 angle,spout should accom
29、modate silicone tubing with an innerdiameter of 8 to 11 mm.NOTE 2The rotor, described in 6.25.3, will displace approximately 50mL of liquid. Therefore, an outlet spout at the 250 mL mark will result inan operating volume of approximately 200 mL. Before use, the usershould confirm the actual liquid v
30、olume in the reactor, after the rotor is inplace and the stir plate is turned on. The measured operating volume isused to calculate an exact pump flow rate.6.25.2 Reactor Top, size 15 rubber or machined stopper,with three holes bored through top to accommodate 6 cmpieces of stainless steel tubing or
31、 other suitable rigid autocla-vable tubing with an outside diameter of 4 to 6 mm. One portaccommodates tubing for media, the second port is fitted witha short piece of silicone tubing that holds a bacterial air vent,and the third is an inoculum port as shown in Fig. 1.6.25.3 Rotor or Disk, nominal 1
32、.6 mm thick PTFE sheet cutinto a disk with a diameter of 7.0 6 0.2 cm containing sixevenly spaced holes with a diameter of 1.27 6 0.1 cm. The4The sole source of supply of the apparatus (coupon manipulation tool) knownto the committee at this time is BioSurface Technologies, Corp., www.biofilms.biz.I
33、f you are aware of alternative suppliers, please provide this information to ASTMInternational Headquarters. Your comments will receive careful consideration at ameeting of the responsible technical committee,1which you may attend. The usermay also build the holder,5Trademarked by the Saint-Gobain P
34、erformance Plastics Corporation.6The sole source of supply of the apparatus (rotating disk reactor) known to thecommittee at this time is BioSurface Technologies, Corp., www.biofilms.biz. If youare aware of alternative suppliers, please provide this information to ASTMInternational Headquarter. Your
35、 comments will receive careful consideration at ameeting of the responsible technical committee,1which you may attend. The usermay also build the reactor.E2196 172center of each hole is located 2.55 6 0.03 cm from the centerof the disk. 4.5 to 7.0 mm thick rubber sheet, or other suitableautoclavable
36、 material, cut into a disk with a diameter of 7.0 60.2 cm containing six evenly spaced holes with a diameter of1.27 6 0.15 cm (the holes in the rubber are aligned with theholes in the PTFE) and a small hole in the center to house thedisk retrieving port. PTFE washer with disk retrieving port.Four ny
37、lon screws. PTFE-coated 4.0 by 1.4 cm star-headmagnetic stir bar, set flush against PTFE disk, with holesdrilled for assembly using nylon screws. The PTFE ridges onone side of the magnet may be shaved to provide a flushmounting surface. Assemble the pieces conforming to thegeneral details shown in F
38、ig. 2.NOTE 3Nominal implies that the manufacturers tolerance is accept-able.6.25.4 Six Cylindrical Polycarbonate Coupons, with a di-ameter of 1.27 6 0.013 cm and a height of 1.5 to 4.0 mm.6.26 Carboys, two 20 Lautoclavable carboys, to be used forwaste and nutrients.6.26.1 Carboy Lids, two: one carbo
39、y lid with at least 2barbed fittings to accommodate tubing ID 3.1 mm (one fornutrient line and one for bacterial air vent), one carboy lid withat least two 1 cm holes bored in the same fashion (one foreffluent waste and one for bacterial air vent).NOTE 4Carboy tops can be purchased with fittings.FIG
40、. 1 Rotating Disk Reactor SystemFIG. 2 Rotor AssemblyE2196 1736.26.2 Bacterial Air Vent, autoclavable 0.2 m pore size, tobe spliced into tubing on waste carboy, nutrient carboy, andreactor top (37 mm and 25mm diameter recommended).7. Reagents and Materials7.1 Purity of WaterAll reference to water as
41、 diluent orreagent shall mean distilled water or water of equal purity.7.2 Culture Media:7.2.1 Bacterial Liquid Growth BrothTryptic Soy Broth(TSB) is recommended.7.2.2 Bacterial Plating MediumR2A agar is recom-mended.NOTE 5Two different concentrations of TSB are used in the protocol,300 mg/L for the
42、 inoculum and batch reactor operation and 30 mg/L forthe continuous flow reactor operation.7.3 Buffered Water0.0425 g/L KH2PO4distilled water,filter sterilized, and 0.405 g/L MgCl 6H2O distilled water,filter sterilized, prepared according to Method 9050 C.1.a(4).8. Culture Preparation8.1 Pseudomonas
43、 aeruginosaATCC 700888 is the organismused in this test.Aseptically remove an isolated colony from anR2A plate and place into 100 mL of sterile TSB (300 mg/L).Incubate bacterial suspension in an environmental shaker at 366 2C for 22 6 2 h. Viable bacterial density should be about108CFU/mL, which may
44、 be checked by serial dilution andplating.9. Reactor Preparation9.1 Preparation of Polycarbonate Coupons:NOTE 6Coupons can be used once and discarded or used repeatedlywith proper cleaning and sterilization between each use. Check eachcoupon for scratching, chipping, other damage, or accumulated deb
45、risbefore each use by screening under a dissecting microscope at amagnification of at least 20. Discard those with visible damage to surfacetopography.NOTE 7Coupons may be made out of alternative materials such asglass or stainless steel. The user should adjust the cleaning procedure sothat it is ap
46、propriate for the coupon material being used.9.1.1 Sonicate coupons for 30 s in a 1+99 dilution oflaboratory soap and tap water. The soapy water must com-pletely cover the coupons.9.1.2 Rinse coupons with reagent grade water and sonicatefor 30 s in reagent grade water.9.1.3 Repeat rinsing and sonica
47、tion with reagent gradewater until, upon visual inspection, no soap is left on thecoupons.NOTE 8Wear gloves to prevent oils and other residues from soilingthe coupon.9.1.4 Place a coupon into each hole in the rotor, leaving thetop of the coupon flush with the rubber rotor surface.9.1.5 Place the rot
48、or with the coupons facing up inside ofthe beaker.9.2 Preparation of Reactor Stopper Top:9.2.1 Use a small section of appropriately-sized tubing toconnect the bacterial air vent to the stainless steel tubing on thereactor stopper top.9.2.2 The glass flow break is spliced into the nutrient tubingline
49、 near the reactor stopper top.NOTE 9The other ports on the reactor top may be adapted foranaerobic use, dilution water, or treatment solutions as necessary.9.3 Sterilization of Reactor System:9.3.1 Assemble the reactor, with the overflow (waste) lineclamped and the reactor top securely fastened to the beakerbefore sterilization.9.3.2 Cover the end of the nutrient tubing that connects tothe nutrient carboy and the end of the overflow (waste) tubingwith aluminum foil. Cover any extra openings on the reactortop with aluminum foil.9.3.3 S
