1、Designation: E2196 12E2196 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 o
2、foriginal 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)2 P
3、seudomonas aeruginosa biofilm in a continuously stirred tankreactor (CSTR) under medium shear conditions. In addition, the test method describes how to sample and analyze biofilm forviable cells.1.2 Although this test method was created to mimic conditions within a toilet bowl, it can be adapted for
4、 the growth andcharacterization of varying species of biofilm (rotating disk reactorrepeatability and relevance (2).1.3 This test method describes how to sample and analyze biofilm for viable cells. Biofilm population density is recorded aslog10 colony forming units per surface area (rotating disk r
5、eactorefficacy test method (3).1.4 Basic microbiology training is required to perform this test method.1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.6 This standard does not purport to address all of the safety concern
6、s, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatorylimitations prior to use.1.7 This international standard was developed in accordance with internationally recogni
7、zed principles on standardizationestablished in the Decision on Principles for the Development of International Standards, Guides and Recommendations issuedby the World Trade Organization Technical Barriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:3D5465 Practices for Deter
8、mining Microbial Colony Counts from 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-organized community attached to surfaces, interfaces, or each other, embeddedin a matrix of
9、extracellular polymeric substances of microbial origin, while exhibiting altered phenotypes with respect to growthrate and gene transcription.3.1.1 DiscussionBiofilms may be comprised of bacteria, fungi, algae, protozoa, viruses, or infinite combinations of these microorganisms. Thequalitative chara
10、cteristics of a biofilm, including, but not limited to, population density, taxonomic diversity, thickness, chemical1 This test method is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15
11、on Antimicrobial Agents.Current edition approved April 1, 2012April 1, 2017. Published June 2012May 2017. Originally approved in 2002. Last previous edition approved in 20072012 asE2196 07.E2196 12. DOI: 10.1520/E2196-12.10.1520/E2196-17.2 The boldface numbers in parentheses refer to a list of refer
12、ences at the end of this standard.3 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.This document is not an AST
13、M standard and is intended only to provide the 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 al
14、l cases only the current versionof the standard 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 States1gradients, chemical composition, consistency, and other materials in the ma
15、trix that are not produced by the biofilmmicroorganisms, are controlled by the physiochemical environment in which it exists.3.2 coupon, nbiofilm sample surface.4. Summary of Test Method4.1 This test method is used for growing a reproducible Pseudomonas aeruginosa biofilm in a rotating disk reactor.
16、 The biofilmis established by operating the reactor in batch mode (no flow) for 24 h. Steady state growth (attachment is equal to detachment)is reached while the reactor operates for an additional 24 h with continuous flow of the nutrients.The residence time of the nutrientsin the reactor is set to
17、select for biofilm growth, and is species and reactor parameter specific. During the entire 48 h, the biofilmis exposed to continuous fluid shear from the rotation of the disk. At the end of the 48 h, biofilm accumulation is quantified byremoving coupons from the disk, harvesting the biofilm from th
18、e coupon surface, disaggregating the clumps, then diluting andplating for viable cell enumeration.5. Significance and Use5.1 Bacteria that exist in a biofilm are phenotypically different from suspended cells of the same genotype. The study of biofilmin the laboratory requires protocols that account
19、for this difference. Laboratory biofilms are engineered in growth reactors designedto produce a specific biofilm type. Altering system parameters will correspondingly result in a change in the biofilm. The purposeof this method is to direct a user in the laboratory study of biofilms by clearly defin
20、ing each system parameter. This method willenable a person to grow, sample, and analyze a laboratory biofilm. The method was originally developed to study toilet bowlbiofilms, but may also be utilized for research that requires a biofilm grown under moderate fluid shear.6. Apparatus6.1 Wooden Applic
21、ator Sticks, sterile.6.2 Inoculating Loop.6.3 Petri Dish, 100-100 by 15-mm, 15 mm, plastic, sterile and empty to hold rotor while sampling.6.4 Culture Tubes and Culture Tube Closures, any with a volume capacity of 10 mL and minimum diameter of 16 mm.Recommended size is 16-16 by 125-mm 125 mm borosil
22、icate glass with threaded opening.6.5 Pipetter,Pipette(s), continuously adjustable pipetterpipette(s) with volume capacity of 1 mL.6.6 Micropipette(s), continuously adjustable pipette(s) with a volume capacity of 10 250 L.6.7 Vortex, any vortex that will ensure proper agitation and mixing of culture
23、 tubes.6.8 Homogenizer, any capable of mixing at 20 500 6 5000 r/min in a 5-5 to 10-mL 10 mL volume.6.9 Homogenizer Probe, any capable of mixing at 20 500 6 5000 r/min in a 5 to 10 mL volume that can withstand autoclavingor other means of sterilization.6.10 Sonicator, Sonicating Bath, any cavitating
24、 sonicating bath that operates at 5045 to 60 Hz.kHz for cleaning the coupons.6.11 Bunsen Burner, used to flame inoculating loop and other instruments.6.12 Stainless Steel Dissecting Tools.Tools, for removing the coupons.NOTE 1Alternatively, a coupon manipulation tool4 may be used.6.13 Stainless Stee
25、l Hemostat Clamp, with curved tip.NOTE 1Alternatively, a coupon holder4 may be used.6.14 Environmental Shaker, capable of maintaining temperature of 36 6 2C.6.15 Analytical Balance, sensitive to 0.01 g.6.16 Sterilizer, any steam sterilizer capable of producing the conditions of sterilization is acce
26、ptable.6.17 Colony Counter, any one of several types may be used, such as the Quebec, Buck, and Wolfhuegel. A hand tally for therecording of the bacterial count is recommended if manual counting is done.6.18 Peristaltic Pump, pump head capable of holding tubing with inner diameter of 3.1 mm and oute
27、r diameter of 3.2 mm.6.19 Digital Magnetic Stir Plate, top plate 10.16 by 10.16 cm, capable of rotating at 200 6 5 r/min.6.20 Silicone Tubing, two sizes of tubing: one with inner diameter of 3.1 mm and outer diameter of 3.2 mm, and the other withinner diameter of 7.9 mm and outer diameter of 9.5 mm.
28、 Both sizes must withstand sterilization.4 The sole source of supply of the apparatus (coupon holder) manipulation tool) known to the committee at this time is BioSurface Technologies, Corp., www.biofilms.biz.If you are aware of alternative suppliers, please provide this information toASTM Internati
29、onal Headquarters. Your comments will receive careful consideration at a meetingof the responsible technical committee,1 which you may attend. The user may also build the holder.holder,E2196 1726.21 Norprene5 Tubing, inner diameter of 3.1 mm and outer diameter of 3.2 mm.6.22 Glass Flow Break, any th
30、at will connect with tubing of inner diameter 3.1 mm and withstands sterilization.6.23 Clamp, used to hold flow break, extension clamp with 0.5-cm 0.5 cm minimum grip size.6.24 Clamp Stand, height no less than 76.2 cm, used with clamp to suspend glass flow break vertically and stabilize tubingabove
31、reactor.6.25 Reactor Components6:6.25.1 Berzelius Borosilicate Glass Beaker, 1000-mL 1000 mL without pour spout, 9.5-9.5 6 0.5-cm 0.5 cm diameter.Borosilicate barbed outlet spout added at 250-250 6 15-mL 15 mL mark at 30 to 45 angle, spout should accommodate siliconetubing with an inner diameter of
32、8 to 11 mm.NOTE 2The rotor, described in 6.24.36.25.3, will displace approximately 50 mL of liquid. Therefore, an outlet spout at the 250 mL mark will resultin an operating volume of approximately 200 mL. Before use, the user should confirm the actual liquid volume in the reactor, after the rotor is
33、 in placeand the stir plate is turned on. The measured operating volume is used 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-cm 6 cm piecesof stainless steel tubing or other suitable rigid autoclavabl
34、e tubing with an outside diameter of 4 to 6 mm. One port accommodatestubing for media, the second port is fitted with a short piece of silicone tubing that holds a bacterial air vent, and the third is aninoculum port as shown in Fig. 1.6.25.3 Rotor or Disk, nominal 1.6-mm 1.6 mm thick PTFE sheet cut
35、 into a disk with a diameter of 7.0 6 0.2 cm containingsix evenly spaced holes with a diameter of 1.27 6 0.1 cm. The center of each hole is located 2.55 6 0.03 cm from the center ofthe disk. 4.5 to 7.0 mm thick rubber sheet, or other suitable autoclavable material, cut into a disk with a diameter of
36、 7.0 6 0.2cm containing six evenly spaced holes with a diameter of 1.27 6 0.15 cm (the holes in the rubber are aligned with the holes inthe PTFE) and a small hole in the center to house the disk retrieving port. PTFE washer with disk retrieving port. Four nylonscrews. PTFE-coated 4.0-4.0 by 1.4-cm 1
37、.4 cm star-head magnetic stir bar, set flush against PTFE disk, with holes drilled forassembly using nylon screws. The PTFE ridges on one side of the magnet may be shaved to provide a flush mounting surface.Assemble the pieces conforming to the general details shown in Fig. 2.NOTE 3Nominal implies t
38、hat the manufacturers tolerance is acceptable.6.25.4 Six Cylindrical Polycarbonate Coupons, with a diameter of 1.27 6 0.013 cm and a height of 1.5 to 4.0 mm.6.26 Carboys, two 20-L 20 L autoclavable carboys, to be used for waste and nutrients.5 Trademarked by the Saint-Gobain Performance Plastics Cor
39、poration.6 The sole source of supply of the apparatus (rotating disk reactor) known to the committee at this time is BioSurface Technologies, Corp., www.biofilms.biz. If you areaware of alternative suppliers, please provide this information to ASTM International Headquarter. Your comments will recei
40、ve careful consideration at a meeting of theresponsible technical committee,1 which you may attend. The user may also build the reactor.FIG. 1 Rotating Disk Reactor SystemE2196 1736.26.1 Carboy Lids, two: one carboy lid with at least 2 barbed fittings to accommodate tubing ID 3.1 mm (one for nutrien
41、t lineand one for bacterial air vent), one carboy lid with at least two 1-cm 1 cm holes bored in the same fashion (one for effluent wasteand one for bacterial air vent).NOTE 4Carboy tops can be purchased with fittings.6.26.2 Bacterial Air Vent, autoclavable 0.2-m 0.2 m pore size, to be spliced into
42、tubing on waste carboy, nutrient carboy, andreactor top (37-mm (37 mm and 25mm diameter recommended).7. Reagents and Materials7.1 Purity of WaterAll reference to water as diluent or reagent shall mean distilled water or water of equal purity.7.2 Culture Media:7.2.1 Bacterial Liquid Growth BrothTrypt
43、ic Soy Broth (TSB) is recommended.7.2.2 Bacterial Plating MediumR2A agar is recommended.NOTE 5Two different concentrations of TSB are used in the protocol, 300 mg/L for the inoculum and batch reactor operation and 30 mg/L for thecontinuous flow reactor operation.7.3 Buffered Water0.0425 g/L KH2PO4 d
44、istilled water, filter sterilized, and 0.405 g/L MgCl 6H2O distilled water, filtersterilized, prepared according to Method 9050 C.1.a(4).8. Culture Preparation8.1 Pseudomonas aeruginosa ATCC 700888 is the organism used in this test. Aseptically remove an isolated colony from anR2A plate and place in
45、to 100 mL of sterile TSB (300 mg/L). Incubate bacterial suspension in an environmental shaker at 36 62C for 22 6 2 h. Viable bacterial density should be about 108 CFU/mL, which may be checked by serial dilution and plating.9. Reactor Preparation9.1 Preparation of Polycarbonate Coupons:NOTE 6Coupons
46、can be used once and discarded or used repeatedly with proper cleaning and sterilization between each use. Check each coupon forscratching, chipping, other damage, or accumulated debris before each use by screening under a dissecting microscope at a magnification of at least 20.Discard those with vi
47、sible damage to surface topography.NOTE 7Coupons may be made out of alternative materials such as glass or stainless steel. The user should adjust the cleaning procedure so that itis appropriate for the coupon material being used.9.1.1 Sonicate coupons for 30 s in a 1+99 dilution of laboratory soap
48、and tap water. The soapy water must completely coverthe coupons.9.1.2 Rinse coupons with reagent grade water and sonicate for 30 s in reagent grade water.9.1.3 Repeat rinsing and sonication with reagent grade water until, upon visual inspection, no soap is left on the coupons.NOTE 8WearingWear glove
49、s to handle the cleaned coupons will help prevent oils and other residues from soiling the coupon surfaces.coupon.9.1.4 Place a coupon into each hole in the rotor, leaving the top of the coupon flush with the rubber rotor surface.9.1.5 Place the rotor with the coupons facing up inside of the beaker.FIG. 2 Rotor AssemblyE2196 1749.2 Preparation of Reactor Stopper Top:9.2.1 Use a small section of appropriately-sized tubing to connect the bacterial air vent to the stainless steel tubing on the reactorstopper top.9.2.2 The glass flow break is spl
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