1、Designation: E 2196 07Standard Test Method forQuantification of a Pseudomonas aeruginosa Biofilm Grownwith Shear and Continuous Flow Using a Rotating DiskReactor1This standard is issued under the fixed designation E 2196; the number immediately following the designation indicates the year oforiginal
2、 adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method is used for growing a repeatable2Pseudomonas aeru
3、ginosa biofilm in a continuously stirred flowreactor. In addition, the test method describes how to sampleand analyze biofilm for viable cells.1.2 In this test method, biofilm population density is re-corded as log colony forming units per surface area.1.3 Basic microbiology training is required to
4、perform thistest method. This standard does not claim to address all of thesafety problems associated with its use. It is the responsibilityof the user of this standard to establish appropriate safetypractices and determine the applicability of regulatory limita-tions prior to use.2. Referenced Docu
5、ments2.1 Other Standards:Buffered Dilution Water Preparation Method 9050 C.1a3Rotating Disk Reactor Repeatability and Relevance4Rotating Disk Reactor Efficacy Test Method53. Terminology3.1 biofilm, nmicroorganisms living in a self-organized,cooperative community attached to surfaces, interfaces, or
6、eachother, embedded in a matrix of extracellular polymeric sub-stances of microbial origin, while exhibiting an altered pheno-type with respect to growth rate and gene transcription.3.1.1 DiscussionBiofilms may be comprised of bacteria,fungi, algae, protozoa, viruses, or infinite combinations ofthes
7、e microorganisms. The qualitative characteristics of abiofilm, 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 controlledb
8、y the physiochemical environment in which it exists.3.2 couponbiofilm sample surface.4. Summary of Test Method4.1 This test method is used for growing a repeatablePseudomonas aeruginosa biofilm in a rotating disk reactor.The biofilm is established by operating the reactor in batchmode (no flow) for
9、24 h. A steady state growth (attachment isequal to 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
10、 the entire 48 h, the biofilm is exposed to continuousfluid shear from the rotation of the disk. At the end of the 48 h,biofilm accumulation is quantified by removing coupons fromthe disk, scraping the biofilm from the coupon surface,disaggregating the clumps, then diluting and plating for viablecel
11、l enumeration.5. Significance and Use5.1 Bacteria that exist in a biofilm are phenotypicallydifferent from suspended cells of the same genotype. The studyof biofilm in the laboratory requires protocols that account forthis difference. Laboratory biofilms are engineered in growthreactors designed to
12、produce a specific biofilm type. Alteringsystem parameters will correspondingly result in a change inthe biofilm. The purpose 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
13、 laboratory biofilm.1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2007. Published May 2007. Originallyapproved in 2002. Las
14、t previous edition approved in 2002 as E 2196 02.2Ellison, S.L.R., M. Rosslein, A. Williams. (Eds.) 2000. Quantifying Uncer-tainty in Anyalytical Measurement, 2nd Edition. Eurachem.3Eaton, A.D., L.S. Clesceri, Rice, E.W., A.E. Greenberg. (Eds.) StandardMethods for the Examination of Water and Waste
15、Water , 21st Edition. AmericanPublic Health Association, American Water Works Association, Water EnvironmentFederation. Washington D.C., 2005.4Zelver, N., M. Hamilton, B. Pitts, D. Goeres, D. Walker, P. Sturman, J.Heersink. 1999. Methods for measuring antimicrobial effects on biofilm bacteria:from l
16、aboratory to field. In: Doyle, R.J. (Ed.), Methods in Enzymology-Biofilms Vol310, Academic Press, San Diego, CA, pp. 608-628.5Zelver, N., M. Hamilton, D. Goeres, J. Heersink. 2001. Development of aStandardized Antibiofilm Test. In: Doyle, R.J. (Ed.), Methods in Enzymology-Biofilms Vol 337, Academic
17、Press, San Diego, CA, pp. 363-376.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.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 samp
18、ling.6.4 Culture Tubes and Culture Tube Closures, any with avolume capability of 10 mL and diameter no less than 6 cm.Recommended size is 16 by 125 mm borosilicate glass withthreaded opening.6.5 Pipetter, continuously adjustable pipetter with volumecapability of 1 mL.6.6 Vortex, any vortex that will
19、 ensure proper agitation andmixing of culture tubes.6.7 Homogenizer, any capable of mixing at 20 500 6 5000r/min ina5to10mLvolume.6.8 Homogenizer Probe, any capable of mixing at 20 500 65000 r/min ina5to10mLvolume and can withstandautoclaving or other means of sterilization.6.9 Sonicator, any noncav
20、itating sonicating bath that oper-ates at 50 to 60 Hz.6.10 Syringe, sterile, 1 mL syringe used during reactorinoculation.6.10.1 Needle, sterile, 22 gauge needle used with syringe toinoculate reactor.6.11 Bunsen Burner, used to flame inoculating loop andother instruments.6.12 Stainless Steel Dissecti
21、ng Tools.6.13 Stainless Steel Hemostat Clamp with Curved Tip.6.14 Environmental Shaker, capable of maintaining tem-perature of 35 6 2C.6.15 Analytical Balance, sensitive to 0.01 g.6.16 Sterilizers, any steam sterilizer capable of producingthe conditions of sterilization is acceptable.6.17 Colony Cou
22、nter, any one of several types 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 ID 3.1 mm and OD 3.2 mm.6.19 Magnetic Stir Plate, top plate
23、10.16 by 10.16 cm,capable of rotating at 200 6 100 r/min.NOTE 1R/min may be measured using a strobe light.6.20 Silicone Tubing, two sizes of tubing: one with ID 3.1mm and OD 3.2 mm and the other with ID 7.9 mm and OD 9.5mm. Both sizes must withstand sterilization.6.21 Glass Flow Break, any that will
24、 connect with tubing ofID 3.1 mm and withstand sterilization.6.21.1 Clamp, used to hold flow break, extension clampwith 0.5 cm minimum grip size.6.21.2 Clamp Stand, height no less than 76.2 cm, used withclamp to suspend glass flow break vertically and stabilizetubing above reactor.6.22 Reactor Compo
25、nents6:6.22.1 Berzelius Pyrex Beaker, 1000 mL without pourspout, 9.5 6 0.5 cm diameter. Pyrex barbed outlet spout addedat 250 mL 6 15 mL mark at 30 to 45 angle, spout shouldaccommodate silicone tubing with an ID of 8 to 11 mm.NOTE 2The rotor, described in 6.22.3, will displace approximately 50mL of
26、liquid. Therefore, an outlet spout at the 250 mL mark will result inapproximately a 200 mL operating volume. The user is encouraged toconfirm the actual liquid volume in the reactor, when the rotor is in place,before use. The measured volume is used to calculate an exact pump flowrate.6.22.2 Reactor
27、 Top, size 15 rubber or machined stopper, 3 to4 holes bored through stopper to accommodate 6 cm pieces offire-polished glass tubing or other suitable rigid autoclavabletubing with OD 4 to 6 mm, as shown in Fig. 1. Another holecan be added to the stopper to contain an inoculum port. Theinoculum port
28、consists ofa6cmpiece of fire-polished glasstubing or other suitable rigid autoclavable tubing fitted with aseptum.6.22.3 Rotor or Disk, nominal (see Note 3) 1.6 mm thickPTFE sheet cut into a disk with a diameter of 7.0 6 0.2 cmcontaining 6 evenly spaced holes with a diameter of 1.27 6 0.1cm. The cen
29、ter of each hole is located 2.55 6 0.03 cm from thecenter of the disk. 4.5 to 7.0 mm thick Viton sheet, or othersuitable autoclavable material, cut into a disk with a diameterof 7.0 6 0.2 cm containing 6 evenly spaced holes with adiameter of 1.27 6 0.15 cm (the holes in the Viton are alignedwith the
30、 holes in the PTFE) and a small hole in the center tohouse the disk retrieving port. PTFE washer with disk retriev-ing port. Four nylon screws. PTFE coated 4.0 by 1.4 cm starhead magnetic stir bar, set flush against PTFE disk and withholes drilled for assembly with nylon screws. The PTFE ridgeson on
31、e side of the magnet may be shaved to provide a flushmounting surface. Assemble the pieces conforming to thegeneral details shown in Fig. 2.NOTE 3Nominal implies that the manufacturers tolerance is accept-able.6.22.4 Cylindrical Polycarbonate Coupons, with a diameterof 1.27 6 0.013 cm and a height o
32、f 1.5 to 4.0 mm.6.23 Carboys, two 15 to 20 L autoclavable carboys, to beused for waste and nutrients.6.23.1 Carboy Lids, two carboy lids: one carboy lid with atleast 2 barbed fittings to accommodate tubing ID 3.1 mm (onefor nutrient line and one for bacterial air vent). One carboy lidwith at least 2
33、-1 cm holes bored in the same fashion (one foreffluent waste and one for bacterial air vent (filter).NOTE 4Carboy tops can be purchased with fittings.6.23.2 Bacterial Air Vent (Filter), autoclavable 0.2 m poresize, to be spliced into tubing on waste carboy, nutrient carboyand reactor top, recommende
34、d diameter 37 mm.7. Reagents and Materials7.1 Purity of WaterAll reference to water as diluent orreagent shall mean distilled water or water of equal purity.7.2 Culture Media:7.2.1 Bacterial Liquid Growth Broth, soybean-casein digestmedium, or an equivalent general bacterial growth medium.Tryptic So
35、y Broth is recommended.6Rotating disk reactor is available commercially from BioSurface Technologies,Corp. or the user may build the reactor.E21960727.2.2 Bacterial Plating Medium, R2A Agar is recom-mended.NOTE 5Media concentration in this protocol differs from the manu-facturers recommendation. Tw
36、o different concentrations are used in theprotocol, 300 mg/L for the inoculum and batch reactor operation and 30mg/L for the continuous flow reactor operation.7.3 Buffered Water, 0.0425 g/L KH2PO4distilled water,filter sterilized and 0.405 g/L MgCl 6H2O distilled water,filter sterilized.FIG. 1 Rotat
37、ing Disc Reactor SystemFIG. 2 Rotor AssemblyE2196073NOTE 6Prepared according to the document referenced in 2.1.8. Culture Preparation8.1 Pseudomonas aeruginosaATCC 700888 is the organismused in this test. An isolated colony is aseptically removedfrom an R2A plate and placed into 100 mL of sterile ba
38、cterialliquid growth broth (300 mg/L) and incubated in an environ-mental shaker at 35 6 2C for 20 to 24 h. Viable bacterialdensity should be about 108CFU/mL, which may be checkedby serial dilution and plating.9. Reactor Preparation9.1 Preparation of Polycarbonate Coupons:NOTE 7Coupons can either be
39、used once and discarded or usedrepeatedly with proper cleaning and sterilization between each use. Checkeach coupon for scratching, chipping, other damage or accumulated debrisbefore each use by screening under a dissecting microscope at amagnification of at least 20X. Discard those with visible dam
40、age tosurface topography.9.1.1 Sonicate coupons for 30 s in a 1:100 dilution oflaboratory soap and tap water. The soapy water must com-pletely cover the coupons.9.1.2 Rinse coupons with reagent water and sonicate for 30s in reagent water.9.1.3 Repeat rinsing and sonication with reagent water until,u
41、pon visual inspection, no soap is left on the coupons.9.1.4 Place a coupon into each hole in the rotor, leaving thetop of the coupon flush with the Viton surface.9.1.5 Place the rotor inside of the beaker.9.2 Preparation of Reactor Stopper Top:9.2.1 Connect the bacterial air vent by fitting the vent
42、 to asmall section of appropriately sized tubing, which is thenattached to one of the pieces of glass tubing on the reactorstopper top.9.2.2 The glass flow break is spliced into the nutrient tubingline near the reactor stopper top. See Note 8.NOTE 8The other ports on the reactor top may be adapted f
43、oranaerobic 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
44、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 Sterilize the reactor system.10. Procedure10.1 Batch Phase:10.1.1 Prepare batch nutrient broth by dissolving bacterialliquid growth medium (300 mg/L
45、) in 250 mL reagent gradewater, sterilize.10.1.2 The top of the sterile reactor is removed and 200 mLof the batch nutrients are aseptically poured into the beaker.The reactor top is secured and the reactor is placed onto a stirplate.10.1.3 Inoculate the reactor with 1 mL of bacteria from theculture
46、prepared previously (see 8.1). Aseptically inject theinoculum into the beaker through the inoculation port using thesterile needle and syringe.10.1.4 The magnetic stir plate is turned on to allow the rotorto spin freely. The rotation speed should equal approximately200 revolutions per minute (6100 r
47、/min). The reactor systemis allowed to incubate in batch mode at room temperature (216 2C) for 24 h.NOTE 9The rotational speed of the disk dictates the fluid shear in thereactor. Fluid shear affects biofilm formation.10.2 Continuous Flow Operation (CSTR Mode):10.2.1 Aseptically connect the nutrient
48、tubing line to thecarboy containing the continuous flow nutrient broth.10.2.2 Prepare continuous flow nutrient broth by dissolvingbacterial liquid growth medium (30 mg/L) in 15 L sterilereagent grade water. Dissolve and sterilize the broth in asmaller volume to prevent caramelization. Aseptically po
49、ur theconcentrated broth into the carboy of sterile water to make atotal of 15 L.NOTE 10The concentration of nutrients in the bacterial liquid growthmedium will affect the biofilm density that accumulates on the coupons.10.2.3 A continuous flow of nutrients is pumped into thereactor through a pump set at a flow rate equal to 6.7 6 0.2mL/min. Unclamp tubing on the drain spout and place the endinto the waste carboy. The drain spout at the 250 mL mark onthe beaker allows overflow to occur, maintaining a constantbacterial liquid growth broth c
