1、Designation:E219607 Designation: E2196 12Standard Test Method forQuantification of a Quantification of Pseudomonasaeruginosa Biofilm Grown with Medium Shear andContinuous Flow Using a Rotating Disk Reactor1This standard is issued under the fixed designation E2196; the number immediately following th
2、e designation indicates the year oforiginal 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. Scope1.1This test method is used
3、for growing a repeatable1.1 This test method is used for growing a reproducible (1)2Pseudomonas aeruginosa biofilm in a continuously stirred flowreactor. In addition, the test method describes how to sample and analyze biofilm for viable cells.1.2In this test method, biofilm population density is re
4、corded as log colony forming units per surface area.1.3Basic microbiology training is required to perform this test method. This standard does not claim to address all of the safetyproblems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety
5、 practices anddetermine the applicability of regulatory limitations prior to use. biofilm in a continuously stirred tank reactor (CSTR) undermedium shear conditions. In addition, the test method describes how to sample and analyze biofilm for viable cells.1.2 Although this test method was created to
6、 mimic conditions within a toilet bowl, it can be adapted for 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 a
7、slog10colony forming units per surface area (rotating disk reactorefficacy 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 st
8、andard 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 establish appropriate safety and health practices and determine the applicability of regulatorylimitations prior to use.2. Referenced Documents2.1 O
9、ther Standards:Buffered Dilution Water PreparationMethod 9050 C.1a2.1 ASTM Standards:3Rotating Disk ReactorRepeatability and RelevanceRotating Disk ReactorEfficacy Test MethodD5465 Practice for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods2.2 Other Standards:Method 9050
10、 C.1.a Buffered Dilution Water Preparation (4)3. Terminology3.1 biofilm, n microorganisms living in a self-organized, cooperative self-organized community attached to surfaces,interfaces, or each other, embedded in a matrix of extracellular polymeric substances of microbial origin, while exhibiting
11、analtered phenotypes with respect to growth rate and gene transcription.3.1.1 DiscussionBiofilms may be comprised of bacteria, fungi, algae, protozoa, viruses, or infinite combinations of these1This test method is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Altern
12、ative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2007.2012. Published May 2007.June 2012. Originally approved in 2002. Last previous edition approved in 20022007 as E2196 027.DOI: 10.1520/E2196-07.10.1520/E2196-12.2
13、Ellison, S.L.R., M. Rosslein, A. Williams. (Eds.) 2000. Quantifying Uncertainty in Anyalytical Measurement, 2nd Edition. Eurachem.2The boldface numbers in parentheses refer to a list of references at the end of this standard.3Eaton, A.D., L.S. Clesceri, Rice, E.W., A.E. Greenberg. (Eds.) Standard Me
14、thods for the Examination of Water and Waste Water , 21st Edition. American Public HealthAssociation, American Water Works Association, Water Environment Federation. Washington D.C., 2005.3For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at servic
15、eastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.1This document is not an ASTM 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. B
16、ecauseit 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 standard as published by ASTM is to be considered the official document.Copyright ASTM International, 10
17、0 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.microorganisms. The qualitative characteristics of a biofilm, including, but not limited to, population density, taxonomic diversity,thickness, chemical gradients, chemical composition, consistency, and other materials
18、 in the matrix that are not produced by thebiofilm microorganisms;, are controlled by the physiochemical environment in which it exists.3.2 couponcoupon, nbiofilm sample surface.4. Summary of Test Method4.1 This test method is used for growing a repeatablereproducible Pseudomonas aeruginosa biofilm
19、in a rotating disk reactor.The biofilm is established by operating the reactor in batch mode (no flow) for 24 h. A steady Steady state growth (attachment isequal to detachment) is reached while the reactor operates for an additional 24 h with continuous flow of the nutrients. Theresidence time of th
20、e nutrients in the reactor is set to select for biofilm growth, and is species and reactor parameter specific. Duringthe entire 48 h, the biofilm is exposed to continuous fluid shear from the rotation of the disk. At the end of the 48 h, biofilmaccumulation is quantified by removing coupons from the
21、 disk, scrapingharvesting the biofilm from the coupon surface,disaggregating the clumps, then diluting and plating 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 th
22、e laboratory requires protocols that account 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
23、laboratory study of biofilms by clearly defining each system parameter. This method willenable a person to grow, sample, and analyze a laboratory biofilm.6. Apparatus6.1 Wooden Applicator Sticks, sterile.6.2 Inoculating Loop.6.3 Petri Dish, 100- by 15-mm, plastic, sterile and empty to hold rotor whi
24、le sampling.6.4 Culture Tubes and Culture Tube Closures, any with a volume capability of 10 mL and diameter no less than 6 cm.Recommended size is 16 by 125 mm borosilicate glass with threaded opening. , any with a volume capacity of 10 mL andminimum diameter of 16 mm. Recommended size is 16- by 125-
25、mm borosilicate glass with threaded opening.6.5 Pipetter, continuously adjustable pipetter with volume capabilitycapacity of 1 mL.6.6 Vortex, any vortex that will ensure proper agitation and mixing of culture tubes.6.7 Homogenizer, any capable of mixing at 20 500 6 5000 r/min in a 5- to 10-mL volume
26、.6.8 Homogenizer Probe, any capable of mixing at 20 500 6 5000 r/min ina5to10mLvolume andthat can withstandautoclaving or other means of sterilization.6.9 Sonicator, any noncavitatingcavitating sonicating bath that operates at 50 to 60 Hz.6.10 Syringe, sterile, 1 mL syringe used during reactor inocu
27、lation.6.10.1Needle, sterile, 22 gauge needle used with syringe to inoculate reactor.6.11Bunsen Burner, used to flame inoculating loop and other instruments.6.126.11 Stainless Steel Dissecting Tools.6.12 Stainless Steel Hemostat Clamp, with curved tip.NOTE 1Alternatively, a coupon holder4may be used
28、.6.13 Stainless Steel Hemostat Clamp with Curved Tip.6.14Environmental Shaker, capable of maintaining temperature of 3536 6 2C.6.156.14 Analytical Balance, sensitive to 0.01 g.6.16Sterilizers6.15 Sterilizer, any steam sterilizer capable of producing the conditions of sterilization is acceptable.6.17
29、6.16 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.6.186.17 Peristaltic Pump, pump head capable of holding tubing with ID 3.1 mm and OD 3.2 mm. , pump head
30、 capable of holdingtubing with inner diameter of 3.1 mm and outer diameter of 3.2 mm.4Zelver, N., M. Hamilton, B. Pitts, D. Goeres, D. Walker, P. Sturman, J. Heersink. 1999. Methods for measuring antimicrobial effects on biofilm bacteria: from laboratoryto field. In: Doyle, R.J. (Ed.), Methods in En
31、zymology-Biofilms Vol 310, Academic Press, San Diego, CA, pp. 608-628.4The sole source of supply of the apparatus (coupon holder) known to the committee at this time is BioSurface Technologies, Corp., www.biofilms.biz. If you are awareof alternative suppliers, please provide this information toASTM
32、International Headquarters. Your comments will receive careful consideration at a meeting of the responsibletechnical committee, which you may attend. The user may also build the holder.E2196 1226.18 Digital Magnetic Stir Plate, top plate 10.16 by 10.16 cm, capable of rotating at 200 6 5 r/min.6.19
33、Magnetic Stir Plate, top plate 10.16 by 10.16 cm, capable of rotating at 200 6 100 r/min.NOTE1R/min may be measured using a strobe light.6.20Silicone Tubing, two sizes of tubing: one with ID 3.1 mm and OD 3.2 mm and the other with ID 7.9 mm and OD 9.5 mm.Both sizes must withstand sterilization. , tw
34、o sizes of tubing: one with inner diameter of 3.1 mm and outer diameter of 3.2 mm,and the other with inner diameter of 7.9 mm and outer diameter of 9.5 mm. Both sizes must withstand sterilization.6.20 Norprene Tubing, inner diameter of 3.1 mm and outer diameter of 3.2 mm.6.21 Glass Flow Break, any t
35、hat will connect with tubing of ID inner diameter 3.1 mm and withstands sterilization.6.21.16.22 Clamp, used to hold flow break, extension clamp with 0.5-cm minimum grip size.6.21.26.23 Clamp Stand, height no less than 76.2 cm, used with clamp to suspend glass flow break vertically and stabilize tub
36、ingabove reactor.6.226.24 Reactor Components5:6.22.16.24.1 Berzelius Pyrex Borosilicate Glass Beaker, 1000-mL without pour spout, 9.5- 6 0.5-cm diameter. PyrexBorosili-cate barbed outlet spout added at 250 mL 250- 6 15-mL mark at 30 to 45 angle, spout should accommodate silicone tubing withan IDinne
37、r diameter of 8 to 11 mm.NOTE 2The rotor, described in 6.22.36.24.3, will displace approximately 50 mL of liquid. Therefore, an outlet spout at the 250 mL mark will resultin approximately a 200 mL operating volume. The user is encouraged to an operating volume of approximately 200 mL. Before use, th
38、e user shouldconfirm the actual liquid volume in the reactor, whenafter the rotor is in place, before use. place and the stir plate is turned on. The measured volumeis used to calculate an exact pump flow rate.6.22.26.24.2 Reactor Top, size 15 rubber or machined stopper, 3 to 4 holes bored through s
39、topper to accommodate 6 cm piecesof fire-polished glass tubing or other suitable rigid autoclavable tubing with OD 4 to 6 mm, as shown in , size 15 rubber ormachined stopper, with three holes bored through top to accommodate 6-cm pieces of stainless steel tubing or other suitable rigidautoclavable t
40、ubing with an outside diameter of 4 to 6 mm. One port accommodates 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. Another hole5Zelver, N., M. Hamilton, D. Goeres, J. Heersink. 200
41、1. Development of a Standardized Antibiofilm Test. In: Doyle, R.J. (Ed.), Methods in Enzymology-Biofilms Vol 337,Academic Press, San Diego, CA, pp. 363-376.5The sole source of supply of the apparatus (rotating disk reactor) known to the committee at this time is BioSurface Technologies, Corp., www.b
42、iofilms.biz. If you areaware of alternative suppliers, please provide this information to ASTM International Headquarter. Your comments will receive careful consideration at a meeting of theresponsible technical committee, which you may attend. The user may also build the reactor.FIG. 1 Rotating Dis
43、ck Reactor SystemE2196 123can be added to the stopper to contain an inoculum port. The inoculum port consists ofa6cmpiece of fire-polished glass tubingor other suitable rigid autoclavable tubing fitted with a septum.6.22.3.6.24.3 Rotor or Disk, nominal (see Note 3) 1.6 mm 1.6-mm thick PTFE sheet cut
44、 into a disk with a diameter of 7.0 6 0.2 cmcontaining 6six 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 fromthe center of the disk. 4.5 to 7.0 mm thick Vitonrubber sheet, or other suitable autoclavable material, cut into a disk with a diame
45、terof 7.0 6 0.2 cm containing 6six evenly spaced holes with a diameter of 1.27 6 0.15 cm (the holes in the Vitonrubber are alignedwith the holes in the PTFE) and a small hole in the center to house the disk retrieving port. PTFE washer with disk retrieving port.Four nylon screws. PTFE-coated 4.0- by
46、 1.4-cm star-head magnetic stir bar, set flush against PTFE disk and disk, with holes drilledfor assembly withusing nylon screws. The PTFE ridges on one side of the magnet may be shaved to provide a flush mountingsurface. Assemble the pieces conforming to the general details shown in Fig. 2.NOTE 3No
47、minal implies that the manufacturers tolerance is acceptable.6.22.4Cylindrical Polycarbonate Coupons6.24.4 Six Cylindrical Polycarbonate Coupons, with a diameter of 1.27 6 0.013 cm and a height of 1.5 to 4.0 mm.6.236.25 Carboys, two 15 to 20 L autoclavable carboys, to be used for waste and nutrients
48、.6.23.1, two 20-L autoclavable carboys, to be used for waste and nutrients.6.25.1 Carboy Lids, two carboy lids: two: one carboy lid with at least 2 barbed fittings to accommodate tubing ID 3.1 mm (onefor nutrient line and one for bacterial air vent). Onevent), one carboy lid with at least 2-1 cmtwo
49、1-cm holes bored in the samefashion (one for effluent waste and one for bacterial air vent (filter). vent).NOTE 4Carboy tops can be purchased with fittings.6.23.2Bacterial Air Vent (Filter), autoclavable 0.2 m pore size, to be spliced into tubing on waste carboy, nutrient carboy andreactor top, recommended diameter 37 mm.6.25.2 Bacterial Air Vent, autoclavable 0.2-m pore size, to be spliced into tubing on waste carboy, nutrient carboy, and reactortop (37-mm diameter recommended).7. Reagents and Materials7.1 Purity of WaterAll reference to water as diluent o
