1、Designation: E 1839 07Standard Test Method forEfficacy of Slimicides for the Paper IndustryBacterial andFungal Slime1This standard is issued under the fixed designation E 1839; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the ye
2、ar 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 presents a procedure to evaluate theefficacy of slimicides for the control of bacterial and fu
3、ngalslimes in paper mill systems and their counterparts.1.2 It is the responsibility of the investigator to determinewhether Good Laboratory Practices (GLP) are required and tofollow them where appropriate (40 CFR 160).1.3 This standard does not purport to address all of thesafety concerns, if any,
4、associated 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.2. Referenced Documents2.1 ASTM Standards:2D 1193 Specification for Reagent WaterE 1054 Test Met
5、hods for Evaluation of Inactivators ofAntimicrobial Agents2.2 TAPPI Standard:T 205 Forming Handsheets for Physical Tests of Pulp32.3 CFR Standard:Title 40, Code of Federal Regulations (CFR), Part 160,Good Laboratory Practice Standards43. Terminology3.1 Definitions:3.1.1 furnish, npulp slurry fed to
6、a paper machine. Thetype of pulp (sulfite, Kraft, mechanical), the source of fiber(virgin, recycled including pre- or post-consumer waste paper),and the pH are used to designate a specific type of furnish.3.1.2 pulp, nwood separated by chemical or mechanicalmeans into their fibrous components. The p
7、ulp is used to makepaper, paper board, or pulp sheets after specific treatments.Hardwood pulp is made from trees, such as maples or oaks, andsoftwood pulp is produced from trees, such as pines.3.1.3 pulp slurry, nan aqueous combination of cellulosicfibers, fillers, and other additives used for speci
8、fic grades ofpaper.3.1.4 slimicides, nchemical agent added during pulp andpaper processing to reduce the growth of slime-formingmicroorganisms.4. Summary of Test Method4.1 Bacterial cells or fungal spores are added to acid oralkaline pulp slurries, or both, treated with slimicides toachieve final co
9、ncentrations of 2 3 106to 1 3 107bacteria/mLor 105to 106fungal spores/mL, and incubated at appropriatetemperature for determined time periods. Aliquots of the testsuspension are then neutralized, plated onto bacterial or fungalmedium, and observed for growth. Results with biocide arecompared to resu
10、lts without biocide (control).4.2 As a performance standard, an effective slimicide is onethat shows a continued reduction in bacterial and fungal countsrelative to the control over the duration of the test.5. Significance and Use5.1 This test method is to be used to determine if a slimecontrol agen
11、t has application in the paper industry for controlof bacterial or fungal slime.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
12、1, 2007. Published April 2007. Originallyapproved in 1996. Last previous edition approved in 2002 as E 1839 96 (2002).2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer t
13、o the standards Document Summary page onthe ASTM website.3Available from Technical Association of the Pulp and Paper Industry (TAPPI),15 Technology Parkway South, Norcross, GA 30092, http:/www.tappi.org.4Available from U.S. Government Printing Office, Superintendent of Docu-ments, Mail Stop: SSOL, W
14、ashington, DC 20402-9328.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.5.2 This test method is run in acid, alkaline, or acid andalkaline conditions to determine the efficacy of the slimecontrol agent.5.3 The test conditions may be
15、 modified to reflect intendeduse patterns in typical paper mill systems, including use ofactual paper mill furnish.6. Apparatus6.1 Balance:6.1.1 Plant Balance, sensitive to 0.1 g and used to weightfurnish.6.1.2 Analytical Balance, sensitive to 0.1 mg and employedto weigh the candidate slime control
16、agent to be used in thepreparation of the stock solutions.6.2 Sample Containers (Sterile), 120-mL plastic specimencontainers with screw-cap lids are ideal for holding testmaterials. Other suitable containers include 150/160-mL milkdilution bottles or WHIRL-PAKS.6.3 Culture Containers, Petri plates,
17、tissue culture bottles orglass tubes (15 3 125 mm or 18 3 150 mm without lip,preferably of borosilicate glass).6.4 Closures, for tubes and containers.6.5 Disintegrators56.6 Flaming EquipmentDepending upon circumstances,either an alcohol lamp, a bunsen burner, or electric incineratormay be used to fl
18、ame inoculating needles and other equipment.6.7 Reliable incubators that control at the temperature re-quired, 6 2C. Temperatures used should be consistent withthe temperatures of the systems.6.8 pH MeterAny reliable pH meter is suitable to stan-dardize the pH of the culture.6.9 Pipets1.1-mL milk di
19、lution type, 1.0 mL graduated in0.01 mL, and 10 mL graduated in 0.1 mL. Pipetters may beused, but not for highly viscous materials.6.9.1 Pipetting AidRubber bulb or other device to accom-plish the transfer of liquid.6.10 Sterilizers, steam sterilizer (121C) or hot-air oven(180 6 2C for 2 h), or both
20、.6.11 Filter Apparatus for Filter Sterilizing, Disposable filterunits, appropriate volume, 0.22-m pore size.6.12 Sterile Funnel, with sterile glass wool or sterile cottongauze for filtration of spores.6.13 Colony counter, manual, such as the Quebec, Buck,Wolffhuegel, or equivalent; or a colony image
21、 analyzer(electronic/scanner type) are suitable for counting plates afterincubation. A hand tally for recording of bacteria count isrecommended.6.14 Swabs, sterile, for aiding in removal of fungal sporesfrom agar surface.6.15 Hemacytometer, for counting spore suspension.6.16 Microscope, that provide
22、s a magnification of 400 to10003 and is complete with a suitable light source. Phasecontrast or dark field capability is desirable.6.17 Constant Temperature ShakerA reliable constant-temperature shaker (water bath or incubator type), shall beused to provide mixing and aeration and to maintain a sele
23、ctedtemperature (6 2C) during the contact period.6.18 Mechanical StirrerMagnetic or propeller-type stir-rers or any other suitable device.7. Reagents and Materials7.1 Purity of ReagentsReagent chemicals shall be used inall tests. Unless otherwise indicated, it is intended that allreagents shall conf
24、orm to the specifications of the Committeeon Analytical Reagents of the American Chemical Society,where such specifications are available.6Other grades may beused, provided it is first ascertained that the reagent is ofsufficiently high purity to permit its use without lessening theaccuracy of the d
25、etermination.7.2 Purity of WaterUnless otherwise indicated, referencesto water shall be understood to mean distilled water or water ofequal purity (see Specification D 1193, Type III).7.3 Buffer for Suspending Spores and for Dilutions, samplecontainers having 100-mL phosphate buffer dilution water,s
26、terile, for spore suspension have solid, sterile glass beads incontainer.7.3.1 0.25 M Phosphate Buffer Stock SolutionDissolve 34g of reagent grace KH2PO4in 500 mL of distilled water andmix. Adjust to pH 7.2 with 1 N NaOH and dilute to 1 l.7.3.2 Phosphate buffer dilution water. Add 1.25 mL of 0.25M p
27、hosphate buffer stock solution to 1 L of distilled water andmix. Dispense to sample container and sterilize.7.4 Aluminum Sulfate (Alum) Al2(SO4)318H2OPreparea 0.4 % solution of the hydrated aluminum in distilled waterand sterilize in an autoclave. Any loss of water duringsterilization is made up by
28、adding sterile distilled water.Alternately, the solution may be filter sterilized.7.5 Acid and Base for pH Adjustment to Make Acid andAlkaline Furnish:7.5.1 Prepare a 2 N solution of sulfuric acid in water.Sterilize by filtration.7.5.2 Prepare a 2.0 N solution of sodium hydroxide in water.Sterilize
29、by filtration.7.6 PulpA two-third hardwood and one-third softwoodpulp, typical of current production techniques, and that hasbeen produced without slimicide is needed.7Disintegrate thesheet in distilled water until free of fiber clots and undispersedfiber bundles.5Avoid methods which involve extensi
30、ve cuttingof fibers. The concentration of the pulp in water should be 1 %.7.7 Bacterial and Fungal Culture Medium:5Forming Handsheets for Physical Tests of Pulp.AppendixA: Specifications andCare of Apparatus (Disintegrator), TAPPI Test Method T 205 on-88. 19941995.Available from Technical Associatio
31、n of the Pulp and Paper Industry (TAPPI), 15Technology Parkway South, Norcross, GA 30092, http:/www.tappi.org.6Reagent Chemicals, American Chemical Society Specifications , AmericanChemical Society, Washington, DC. For suggestions on the testing of reagents notlisted by the American Chemical Society
32、, see Analar Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.7The sole source of supply of the apparatus known to the committee at this timeis Zellerbach, 808 Rhodes A
33、ve., Columbus, OH 43205. If you are aware ofalternative suppliers, please provide this information to ASTM Headquarters. Yourcomments will receive careful consideration at a meeting of the responsibletechnical committee,1which you may attend.E18390727.7.1 BacteriaStandard dehydrated tryptone glucose
34、 ex-tract agar or equivalent is recommended. Adjust pH of culturemedium to pH of the test system.7.7.2 FungiSabouraud Dextrose Agar or Potato DextroseAgar are recommended for enumeration. Adjust pH of culturemedium to pH of the test system.7.8 Stock Slimicide SolutionWeigh appropriate amount ofslimi
35、cide and add to distilled water so that when 1 mL is addedto the furnish it gives the desired final concentration.8. Test Organisms8.1 Bacteria: Enterobacter aerogenes (ATCC 13048) andPseudomonas aeruginosa (ATCC 15442)Maintain these or-ganisms on tryptone glucose extract agar at 32 6 2C.8.2 Fungi:
36、Aspergillus niger (ATCC 6275)Maintain andgrow for sporulation on Sabouraud Dextrose Agar, grow at 256 2C. Chaetomium globosum (ATCC 6205)Maintain andgrow for sporulation on Mineral Slats Agar8with sterile filterpaper at 25 6 2C.9. Preparation of Inoculum9.1 Bacteria:9.1.1 Grow the bacterial cultures
37、 on tryptone glucose ex-tract agar at 32 6 2C for 24 h. Add 1 mL of sterile water tothe surface of the slant. Gently scrape and suspend theorganisms in the liquid.9.1.2 Transfer this volume to a tryptone glucose extract agarmilk dilution bottle flat and incubate at 32 6 2C for 24 h. Theorganisms har
38、vested from this bottle flat will serve as inoculumfor the pulp cultures.9.1.3 To obtain this inoculum, add 10 mL of sterile water tothe bottle flat from a 99 6 2-mL sterile water blank. Dispersethe growth on the flat throughout the water by gently movingit over the surface of the agar medium. Trans
39、fer 5 mL of thissuspension to the remaining 89 mL of sterile water in the waterblank and shake the contents of the water blank vigorously.Add 1 mL of this inoculum (2 3 108to 1 3 109cells/mL) toeach bottle containing the pulp substrate in the test. Thisinoculum can also be cultured on tryptone gluco
40、se agar Petriplates and harvested in a similar fashion.9.2 Fungal Spores:9.2.1 Fungi are grown on appropriate agar slants at 2562C for 7 days. The fungal spores are harvested by adding 2mL of sterile, distilled water to the surface of the slant. Gentlyscrape and suspend the spores in the liquid.9.2.
41、2 Inoculate these spores onto appropriate agar andincubate at 25 6 2C for 7 days. Spores are harvested andserve as the inoculum for the pulp cultures.9.2.3 To obtain this inoculum, add 5 to 10 mL of sterile,phosphate buffer dilution water to the surface of the agar.Disperse the fungal spores and myc
42、elia in the bottle by gentlyscraping the surface of the medium. Alternately, remove thegrowth from the surface of the medium, add it to a screw-capcontainer with buffer and glass beads, and shake vigorously.Set aside for 5 to 10 min to let the hyphae settle.9.2.4 Carefully remove the supernate with
43、a pipet and filterthrough cotton gauze or glass wool plugged sterile funnel.9.2.5 Count the conidial suspension with a hemacytometerand standardize to 107to 108/mL with phosphate dilutionbuffer.10. Pulp Cultures10.1 Weigh 50 g of pulp slurry into a sample container. Toensure a uniform pulp suspensio
44、n in each container, constantlyagitate the stock solution with a mechanical stirrer. Use onlypulp suspension prepared the same day. Stopper or cap andsterilize.After sterilization, allow the bottles to cool to approxi-mately 25 6 2C or the temperature used for the test.10.2 Make the following additi
45、ons aseptically to each bottlein the order named and shake vigorously after each addition,using 20 complete cycles in a vertical motion.10.3 Furnish:10.3.1 For acid furnish, add a sufficient amount of 2.0 Nsulfuric acid to adjust the pH to between pH 5.0 and 5.5. Add0.1 mL of 0.4 % solution of Alum.
46、10.3.2 For alkaline furnish, add a sufficient amount of 2.0 Nsodium hydroxide to adjust the pH to between pH 8.0 and 8.5.10.3.3 Other furnishes, appropriate for the specific papermill applications.10.4 Add 1 mL of slimicide stock solution of sufficientconcentration to each furnish to achieve the des
47、ired finalconcentration in parts per million. To the negative controls, add1 mL of sterile water. Include in each test, as a positive control,a reference biocide that is known to have activity in this test.Include a minimum of five concentrations of the biocide undertest and the reference biocide in
48、 each experiment. Run each testand reference biocide in duplicate.10.5 Add the amount of sterile distilled water that isnecessary to bring the total weight of the contents of the bottleto 99 6 1 g after all other additions have been made.10.6 Add 1 mL of an aqueous suspension of the testorganism to
49、each pulp slurry. Prepare this suspension justbefore it is to be used. The time interval between the prepara-tion of the suspension of the test organism for inoculating thebottles and the addition of the suspension to each bottle mayhave some effect on the resistance of the organism. Finalbacterial concentration should be between 2 3 106to 1 3 107bacterial/mL of pulp slurry. Final fungal spore concentrationshould be between 105to 106spores/mL of pulp slurry.10.7 After the final addition of the test organism, place theinoculated bottles on an inc
