1、Designation: E1839 13Standard Test Method forEfficacy of Slimicides for the Paper IndustryBacterial andFungal Slime1This standard is issued under the fixed designation E1839; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year
2、 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 presents a procedure to evaluate theefficacy of slimicides for the control of bacterial and funga
3、lslimes 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 The values stated in SI units are to be regarded asstandard. No other units of m
4、easurement are included in thisstandard.1.4 This standard does not purport to address all of thesafety concerns, if any, 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
5、limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1193 Specification for Reagent WaterE1054 Test Methods for Evaluation of Inactivators of Anti-microbial AgentsE2756 Terminology Relating to Antimicrobial and AntiviralAgents2.2 TAPPI Standard:T 205 Forming Handsheets for Physical T
6、ests of Pulp32.3 CFR Standard:40 CFR Part 160 Good Laboratory Practice Standards43. Terminology3.1 For definitions of terms related to this practice, seeTerminology E2756.3.2 Definitions:3.2.1 furnish, npulp slurry fed to a paper machine. Thetype of pulp (sulfite, Kraft, mechanical), the source of f
7、iber(virgin, recycled including pre- or post-consumer waste paper),and the pH are used to designate a specific type of furnish.3.2.2 microbicide, na physical or chemical agent that killsmicroorganisms.3.2.3 pulp, nwood separated by chemical or mechanicalmeans into their fibrous components. The pulp
8、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.3.2.4 pulp slurry, nan aqueous combination of cellulosicfibers, fillers, and other additives used for specific
9、grades ofpaper.3.2.5 slime, nbiofilm.3.2.6 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
10、toachieve final concentrations of 2 106to1107bacteria/mLand/or 105to 106fungal spores/mL, and incubated at appro-priate temperature for determined time periods.Aliquots of thetest suspension are then neutralized, plated onto bacterial orfungal medium, and observed for growth. Results with biocideare
11、 compared to results 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
12、slimecontrol agent has application in the paper industry for controlof bacterial or fungal slime/biofilm.1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agentsand is the direct respon-sibility of Subcommittee E35.15 on Antimicro
13、bial Agents.Current edition approved April 1, 2013. Published May 2013. Originallyapproved in 1996. Last previous edition approved in 2007 as E1839 07. DOI:10.1520/E1839-13.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For A
14、nnual Book of ASTMStandards volume information, refer to 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 O
15、ffice Superintendent of Documents,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http:/www.access.gpo.gov.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States15.2 This test method is run in acid, alkaline, or acid andalkalin
16、e conditions to determine the efficacy of the slimecontrol agent.5.3 The test conditions may be 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, one sensitive to 0.1 g at load of 200 g,with
17、platform to weigh furnish and bottles used in the test.6.1.2 Analytical Balance, one sensitive to 0.1 mg used forweighing the candidate slime control agent.6.2 Sample Containers (Sterile), 120-mL plastic specimencontainers with screw-cap lids are ideal for holding testmaterials. Other suitable conta
18、iners include 150/160-mL milkdilution bottles or WHIRL-PAKS.6.3 Culture Containers, Petri plates, tissue culture bottles,or glass tubes (15 125 mm or 18 150 mm without lip,preferably of borosilicate glass).6.4 Closures, for tubes and containers.6.5 Disintegrators, Appendix A (Specifications and Care
19、 ofApparatus (Disintegrator) of T 205.6.6 Flaming EquipmentDepending upon circumstances,either an alcohol lamp, a bunsen burner, or electric incineratormay be used to flame inoculating needles and other equipment.6.7 Incubators Capable of maintaining temperatures of 28to 70 6 2C to provide proper in
20、cubation temperatures.Temperature should be consistent with the temperature of theproduct to be preserved.6.8 pH MeterAny suitable for standardizing the pH of theculture medium.6.9 Pipets1.1-mL milk dilution type, 1.0 mL graduated in0.01 mL, and 10 mL graduated in 0.1 mL. Pipetters may beused, but n
21、ot for highly viscous materials.6.9.1 Pipetting AidRubber bulb or other device to accom-plish the transfer of liquid.6.10 Sterilizers, pressurized steam sterilizer or hot-air ovencapable of 180 6 2C for 2 6 0.2 h.6.11 Filter Apparatus for Filter Sterilizing, Disposable filterunits, appropriate volum
22、e, 0.2-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 Quebec, Buck, andWolfhuegel, or proven colony image analyzer (electronic/scanner type) are suitable for counting plates after incubation.6.14 Swabs, st
23、erile, for aiding in removal of fungal sporesfrom agar surface.6.15 Hemacytometer, for counting spore suspension.6.16 Microscope, providing a magnification range of 400 to1000 with a suitable light source. Phase contrast or dark fieldcapability may be necessary.6.17 Constant Temperature ShakerA reli
24、able constant-temperature shaker (water bath or incubator type), shall beused to provide mixing and aeration and to maintain a selectedtemperature (62C) during the contact period.6.18 Mechanical StirrerMagnetic or propeller-type stir-rers or any other suitable device.7. Reagents and Materials7.1 Pur
25、ity of ReagentsReagent chemicals shall be used inall tests. Unless otherwise indicated, it is intended that allreagents shall conform to the specifications of the Committeeon Analytical Reagents of the American Chemical Society,where such specifications are available.5Other grades may beused, provid
26、ed it is first ascertained that the reagent is ofsufficiently high purity to permit its use without lessening theaccuracy of the determination.7.2 Purity of WaterUnless otherwise indicated, referencesto water shall be understood to mean distilled water or water ofequal purity (see Specification D119
27、3, Type III).7.3 Buffer for Suspending Spores and for Dilutions, samplecontainers having 100-mL phosphate buffer dilution water,sterile, for spore suspension have solid, sterile glass beads incontainer.7.3.1 0.25 M Phosphate Buffer Stock SolutionDissolve34 g of reagent grade KH2PO4in 500 mLof distil
28、led water andmix. Adjust to pH 7.2 with 1 N NaOH and dilute to 1 l.7.3.2 Phosphate Buffer Dilution WaterAdd 1.25 mL of0.25 M phosphate buffer stock solution to 1 L of distilled waterand mix. Dispense to sample container and sterilize.7.4 Aluminum Sulfate (Alum) Al2(SO4)318H2OPreparea 0.4 % solution
29、of the hydrated aluminum in distilled waterand sterilize in an autoclave. Any loss of water duringsterilization is made up by adding sterile distilled water.Alternatively, the solution may be filter sterilized.7.5 Acid and Base for pH Adjustment to Make Acid andAlkaline Furnish:7.5.1 Prepare a 2.0 N
30、 solution of sulfuric acid in water.Sterilize by filtration.7.5.2 Prepare a 2.0 N solution of sodium hydroxide in water.Sterilize by filtration.7.6 PulpBiocide-free pulp, consisting of two-third hard-wood and one-third softwood, typical of current productiontechniques, is recommended.6Disintegrate t
31、he sheet in distilledwater until free of fiber clots and undispersed fiber bundles (T205).Avoid methods which involve extensive cutting of fibers.The concentration of the pulp in water should be 1 %.5Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, D
32、C. For suggestions on the testing of reagents notlisted by the American Chemical Society, 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.6The sole source o
33、f supply of the apparatus known to the committee at this timeis Zellerbach, 808 Rhodes Ave., 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 co
34、mmittee,1which you may attend.E1839 1327.7 Bacterial and Fungal Culture Medium:7.7.1 BacteriaStandard dehydrated tryptone glucose 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
35、for enumeration. Adjust pH of culturemedium to pH of the test system.7.8 Stock Slimicide SolutionWeigh appropriate amount ofslimicide 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 (A
36、TCC 13048) andPseudomonas aeruginosa (ATCC 15442)Maintain these or-ganisms on tryptone glucose extract agar at 32 6 2C.8.2 Fungi: Aspergillus niger (ATCC 6275)Maintain andgrow for sporulation on Sabouraud dextrose agar, grow at 25 62C. Chaetomium globosum (ATCC 6205)Maintain andgrow for sporulation
37、on Mineral Salts Agar7with sterile filterpaper at 25 6 2C. Sabouraud Dextrose agar may also be usedto grow for sporulation.9. Preparation of Inoculum9.1 Bacteria:9.1.1 Grow the bacterial cultures on tryptone glucose ex-tract agar at 32 6 2C for 24 h. Add 1 mL of sterile water tothe surface of the sl
38、ant. 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 harvested from this bottle flat will serve as inoculumfor the pulp cultures.9.1.3 To obtain this inoculum, a
39、dd 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. Transfer 5 mL of thissuspension to the remaining 89 mLof sterile water in the waterblank and shake the content
40、s of the water blank vigorously tobreak up any bacterial clumps. Let settle. Take 1 ml of fromupper half of bottle, and add this inoculum (2 108to 1 109cells/mL) to each bottle containing the pulp substrate in thetest. This inoculum can also be cultured on tryptone glucoseagar petri plates and harve
41、sted in a similar fashion.9.2 Fungal Spores:9.2.1 Fungi are grown on appropriate agar slants at25 6 2C for 7 days. The fungal spores are harvested byadding 2 mL of sterile, distilled water with a non-toxic wettingagent such as sodium dioctyl sulfosuccinate (0.05 g/Lwater) tothe surface of the slant.
42、 Gently scrape and suspend the sporesin the liquid.9.2.2 Inoculate these spores onto appropriate agar andincubate at 25 6 2C for 7 days. Spores are harvested (as in9.2.3) and serve as the inoculum for the pulp cultures.9.2.3 To obtain this inoculum, add 5 to 10 mL of sterile,phosphate buffer dilutio
43、n water having a non-toxic wettingagent such as sodium dioctyl sulfosuccinate (0.05 g/Lwater) tothe surface of the agar. Disperse the fungal spores and myceliain the bottle by gently scraping the surface of the medium.Alternatively, remove the growth from the surface of themedium, add it to a screw-
44、cap container with buffer and glassbeads, and shake vigorously. Set aside for 5 to 10 min to let thehyphae settle.9.2.4 Carefully remove the supernate with a pipet and filterthrough cotton gauze or glass wool plugged sterile funnel.9.2.5 Count the conidial suspension with a hemacytometerand standard
45、ize 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 suspension in each container, constantlyagitate the stock solution with a mechanical stirrer. Use onlypulp suspension prepared the same day. Stopper or c
46、ap 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 additions aseptically to each bottlein the order named and shake vigorously after each addition,using 20 complete cycles in a vertical motion.10.3 Fur
47、nish: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.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 Add other furnis
48、hes, appropriate for the specificpaper mill applications.10.4 Add 1 mL of slimicide stock solution of sufficientconcentration to each furnish to achieve the desired finalconcentration in parts per million. To the negative controls, add1 mLof sterile water. Include in each test, as a positive control
49、,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 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 each pulp slurry. Prepare this suspension justbefore it is to be used. The t
