1、Designation: E2562 12E2562 17Standard Test Method forQuantification of Pseudomonas aeruginosa Biofilm Grownwith High Shear and Continuous Flow using CDC BiofilmReactor1This standard is issued under the fixed designation E2562; the number immediately following the designation indicates the year ofori
2、ginal 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 specifies the operational parameters required
3、 to grow a reproducible (1)2 Pseudomonas aeruginosa ATCC700888 biofilm under high shear. The resulting biofilm is representative of generalized situations where biofilm exists under highshear rather than being representative of one particular environment.1.2 This test method uses the Centers for Dis
4、ease Control and Prevention (CDC) Biofilm Reactor. The CDC Biofilm Reactoris a continuously stirred tank reactor (CSTR) with high wall shear. Although it was originally designed to model a potable watersystem for the evaluation of Legionella pneumophila(2), the reactor is versatile and may also be u
5、sed for growing and/orcharacterizing biofilm of varying species (3-5).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.1.4 Basic microbiology training is required to perform this te
6、st 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 concerns, if any, associated with its use. It is the responsibilityof the user of this standard to e
7、stablish 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 recognized principles on standardizationestablished in the Decision on Principles for the Developmen
8、t 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 Determining Microbial Colony Counts from Waters Analyzed by Plating Methods2.2 Other Standards:Met
9、hod 9050 C.1.a Buffered Dilution Water Preparation according to EatonRice et al (6)3. Terminology3.1 Definitions:3.1.1 biofilm, nmicroorganisms living in a self-organized community attached to surfaces, interfaces, or each other, embeddedin a matrix of extracellular polymeric substances of microbial
10、 origin, while exhibiting altered phenotypes with respect to growthrate and gene transcription.3.1.1.1 Discussion1 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 on
11、Antimicrobial Agents.Current edition approved April 1, 2012April 1, 2017. Published June 2012May 2017. Originally approved in 2007. Last previous edition approved in 20072012 asE2562 07.E2562 12. DOI: 10.1520/E2562-12.10.1520/E2562-17.2 The boldface numbers in parentheses refer to a list of referenc
12、es 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 ASTM s
13、tandard 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 all c
14、ases 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 States1Biofilms may be comprised of bacteria, fungi, algae, protozoa, viruses, or inf
15、inite combinations of these microorganisms. Thequalitative characteristics of a biofilm, including, but not limited to, population density, taxonomic diversity, thickness, chemicalgradients, chemical composition, consistency, and other materials in the matrix that are not produced by the biofilmmicr
16、oorganisms, are controlled by the physicochemical environment in which it exists.3.1.2 coupon, nbiofilm sample surface.4. Summary of Test Method4.1 This test method is used for growing a reproducible Pseudomonas aeruginosa ATCC 700888 biofilm in a CDC BiofilmReactor. The biofilm is established by op
17、erating the reactor in batch mode (no flow of the nutrients) for 24 h. A steady statepopulation is reached while the reactor operates for an additional 24 h with a continuous flow of the nutrients. The residence timeof the nutrients in the reactor is set to select for biofilm growth, and is species
18、and reactor parameter specific. During the entire48 h, the biofilm is exposed to continuous fluid shear from the rotation of a baffled stir bar. Controlling the rate at which the baffleturns determines the intensity of the shear stress to which the coupons are exposed. At the end of the 48 h, biofil
19、m accumulationis quantified by removing coupons from suspended rods, harvesting the biofilm from the coupon surface, disaggregating the surfaceby scraping the biofilm from the coupon, homogenizing the removed biofilm to disaggregate the clumps, and diluting and platingfor viable cell enumeration.5.
20、Significance and Use5.1 Bacteria that exist in biofilms are phenotypically different from suspended cells of the same genotype. Research has shownthat biofilm bacteria are more difficult to kill than suspended bacteria (5, 7). Laboratory biofilms are engineered in growth reactorsdesigned to produce
21、a specific biofilm type.Altering system parameters will correspondingly result in a change in the biofilm. Forexample, research has shown that biofilm grown under high shear is more difficult to kill than biofilm grown under low shear (5,8). The purpose of this test method is to direct a user in the
22、 laboratory study of a Pseudomonas aeruginosa biofilm by clearlydefining each system parameter. This test method will enable an investigator to grow, sample, and analyze a Pseudomonasaeruginosa biofilm grown under high shear. The biofilm generated in the CDC Biofilm Reactor is also suitable for effi
23、cacy testing.After the 48 h growth phase is complete, the user may add the treatment in situ or harvestremove the coupons and treat themindividually.6. Apparatus6.1 Wooden Applicator Stickssterile.6.2 Inoculating Loop.6.3 Petri Dish100-100 by 15-mm, 15 mm, plastic, sterile, and empty to put beneath
24、rod while sampling.6.4 Culture Tubes and Culture Tube Closuresany with a volume capacity of 10 mL and a minimum diameter of 16 mm.Recommended size is 16-16 by 125-mm 125 mm borosilicate glass with threaded opening.6.5 PipetterPipettecontinuously adjustable pipetter with volume capacity of 1 mL.6.6 V
25、ortexany vortex that will ensure proper agitation and mixing of culture tubes.6.7 Homogenizerany that can mix at 20 500 6 5000 r/min in a 5 to 10 mL volume.6.8 Homogenizer Probeany that can mix at 20 500 6 5000 r/min in a 5-5 to 10-mL 10 mL volume and can withstandautoclaving or other means of steri
26、lization.6.9 Sonicating Water Bathany cavitating sonicating bath that operates at 50 to 60 Hz.45 to 50 kHz for cleaning coupons.6.10 Bunsen Burnerused to flame inoculating loop and other instruments.6.11 Stainless Steel Hemostat Clampwith curved tip.NOTE 1Alternatively, a coupon holdermanipulating t
27、ool4 may be used.6.12 Environmental Shakerthat can maintain a temperature of 36 6 2C.6.13 Analytical Balancesensitive to 0.01 g.6.14 Sterilizerany steam sterilizer that can produce the conditions of sterilization is acceptable.6.15 Colony Counterany one of several types may be used, such as the Queb
28、ec, Buck, and Wolfhuegel. A hand tally for therecording of the bacterial count is recommended if manual counting is done.6.16 Peristaltic Pumppump head that can hold tubing with inner diameter 3.1 mm and outer diameter 3.2 mm.4 The sole source of supply of the apparatus (coupon holder) manipulating
29、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 International Headquarters. Your comments will receive careful consideration at a meetingof the responsible technical commit
30、tee,1 which you may attend. The user may also build the holder.E2562 1726.17 Digital Magnetic Stir Platetop plate 10.16 10.16 cm, that can rotate at 125 6 5 r/min.6.18 Silicone Tubingtwo sizes of tubing: one with inner diameter 3.1 mm and outer diameter 3.2 mm, and the other with innerdiameter 7.9 m
31、m and outer diameter 9.5 mm. Both sizes must withstand sterilization.6.19 Norprene5 Tubinginner diameter 3.1 mm and outer diameter 3.2 mm.6.20 Glass Flow Breakany that will connect with tubing of inner diameter 3.1 mm and withstand sterilization.6.20.1 ClampUsed to hold flow break, extension clamp w
32、ith 0.5-cm 0.5 cm minimum grip size.6.20.2 Clamp Standheight no less than 76.2 cm, used with clamp to suspend glass flow break vertically and stabilize tubingabove reactor.6.20.3 Laboratory Screw Clampused to clamp effluent tubing during batch growth.6.21 Reactor Components.66.21.1 Berzelius Borosil
33、icate Glass Tall Beaker1000 mLwithout pour spout, 9.5-9.5 6 0.5-cm 0.5 cm diameter. Barbed outletspout added at 400-400 6 20-mL 20 mL mark. Angle the spout 30 to 45 to ensure drainage. Spout should accommodate flexibletubing with an inner diameter of 8 to 11 mm.NOTE 2The rods (see 6.21.3) and baffle
34、 (see 6.21.6) will displace approximately 50 mL of liquid when system is completely assembled. Therefore,an outlet spout at the 400-mL 400 mL mark will result in approximately a 350-mL 350 mL operating volume. The user should confirm the actual liquidvolume in the reactor, when the rods and baffle a
35、re in place and the stir plate is turned on, before use. The measured operating volume is used to calculatean exact pump flow rate.6.21.2 Reactor TopFig. 1. Ultra-high molecular weight (UHMW) polyethylene top (10.1-cm (10.1 cm diameter tapering to8.33 cm) equipped with a minimum of three holes accom
36、modating 10-cm 10 cm pieces of stainless steel or other rigid autoclavabletubing with outside diameter of 5 to 8 mm for media inlet, air exchange, and inoculation port. Center hole, 1.27-cm 1.27 cmdiameter, to accommodate the glass rod used to support the baffle assembly. Eight rod holes, 1.905-cm 1
37、.905 cm diameter, notchedto accommodate stainless steel rod alignment pin (0.236-cm (0.236 cm outside diameter).6.21.3 Polypropylene RodsFig. 2. Eight polypropylene rods, 21.08-cm 21.08 cm long, machined to hold three coupons (see6.21.4) at the immersed end. Three 316 stainless steel set screws imbe
38、dded in side to hold coupons in place. Rods fit into holesin reactor top and lock into preformed notches with alignment pin.6.21.4 Twenty-four Cylindrical Polycarbonate Couponswith a diameter of 1.27 6 0.013 cm, thickness of approximately 3.0mm.6.21.5 Small Allen Wrenchfor loosening set screws.5 Tra
39、demarked by the Saint-Gobain Performance Plastics Corporation.6 The sole source of supply of the apparatus (CDC Biofilm 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 I
40、nternational Headquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee,1 which you may attend. The user may also build the reactor.FIG. 1 Expanded Schematic of Reactor TopE2562 1736.21.6 Stir Blade Assembly (Baffled Stir Bar)Fig. 3. PTFE blade
41、(5.61-cm) (5.61 cm) fitted into cylindrical PTFE holder(8.13-cm) (8.13 cm) and held in place with a magnetic stir bar (2.54-cm). (2.54 cm). PTFE holder fits onto a glass rod (15.8-cm),(15.8 cm), fitted into the reactor top. The glass rod is held in place with a compression fitting and acts as a supp
42、ort for the movingblade assembly.6.22 Carboystwo 20-L 20 L autoclavable carboys, to be used for waste and nutrients.6.22.1 Two Carboy LidsOne carboy lid with at least two barbed fittings to accommodate tubing ID 3.1 mm (one for nutrientline and one for bacterial air vent). One carboy lid with at lea
43、st two 1-cm 1 cm holes bored in the same fashion (one for effluentwaste and one for bacterial air vent).NOTE 3Carboy tops can be purchased with fittings.6.22.2 Bacterial Air Vent (Filter)autoclavable, 0.2-m 0.2 m pore size, to be spliced into tubing on waste carboy, nutrientcarboy, and reactor top;
44、recommended diameter 37 mm.6.23 Fig. 4 illustrates a schematic of the assembled system.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 BrothTryptic Soy Broth (
45、TSB) is recommended.NOTE 4Two different TSB concentrations are used in the test method, 300 mg/L for the inoculum and batch reactor operation, and 100 mg/L for thecontinuous flow reactor operation.7.2.2 Bacterial Plating MediumR2A Agar is recommended.7.3 Buffered Water0.0425 g/LKH2PO4 distilled wate
46、r, filter sterilized, and 0.405 g/L MgCl6H2O distilled water, filtersterilized (prepared according to Method 9050 C.1.a(6).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 into 100 mL of s
47、terile TSB (300 mg/L). Incubate bacterial suspension in an environmental shaker at 36 62C for 22 6 2 h. Viable bacterial density should equal 108 CFU/mL, and may be checked by serial dilution and plating.9. Reactor Preparation9.1 Preparation of Polycarbonate Coupons:NOTE 5Coupons can be used once an
48、d 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 visible damage to sur
49、face topography.9.1.1 Sonicate coupons for 30 s in a 1+99 dilution of laboratory soap and tap water. The soapy water must completely coverthe coupons.FIG. 2 Expanded Schematic of Rod and CouponsE2562 1749.1.2 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 no soap is left on the coupons. Once the coupons are clean,care must be taken to prevent oils and other residue from contaminating the surface.NOTE 6Coupons may be ma
