1、Designation: E875 10Standard Test Method forEfficacy of Fungal Control Agents as Preservatives forAqueous-Based Products Used in the Paper Industry1This standard is issued under the fixed designation E875; the number immediately following the designation indicates the year oforiginal adoption or, in
2、 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 laboratory test method is used to determine theefficacy of a fungal control ag
3、ent to prevent spoilage ofin-process aqueous-based products used in the paper industry.1.2 For information on bacterial control agents, see TestMethod E723.1.3 It is the responsibility of the investigator to determinewhether good laboratory practices (GLP) are required and tofollow them when appropr
4、iate (see 40 CFR 160).1.4 A knowledge of microbiological techniques is requiredfor these procedures.1.5 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.6 This standard does not purport to address all of thesafety concerns, if
5、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 limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1193 Specification for Reagent WaterE723 Test M
6、ethod for Efficacy of Antimicrobials as Preser-vatives for Aqueous-Based Products Used in the PaperIndustry (Bacterial Spoilage)E1839 Test Method for Efficacy of Slimicides for the PaperIndustryBacterial and Fungal Slime2.2 Federal Standard:40 CFR 160 Good Laboratory Practice Standards33. Terminolog
7、y3.1 Definitions of Terms Specific to This Standard:3.1.1 fungal control agent, nan agent that either kills orprevents growth of fungi and either kills or prevents thegermination of fungal spores. This term is applied to chemicalbiocidal or biostatic agents.3.1.2 preservative, nchemical agent used t
8、o prevent mi-crobial soilage of products due to microbial growth.4. Summary of Test Method4.1 Aqueous material to be preserved is inoculated with anappropriate fungal inoculum followed by addition of a concen-tration of fungal control agent that will kill the fungi or preventtheir growth for a desir
9、ed period of time, or both. In addition,the agent will also prevent fungal spore germination. Fungalgrowth is determined by visible signs of deterioration in thetest sample, and by obtaining fungal numbers and comparingthem to a sample without any fungal control agent. The properlevel of fungal cont
10、rol agent is one that prevents productdeterioration and reduces and keeps the organisms to anacceptable level in the test material, as determined by the testeror user.5. Significance and Use5.1 This test method should be used to determine if a fungalcontrol agent is effective to preserve pigment sus
11、pensions, dyesolutions, pulp slurries, starch solutions, polymers, sizingagents, latex emulsions, and other specific aqueous-basedmaterials used in the paper industry. Separate evaluationsshould be made on a representative type for each specific classof product to be preserved.NOTE 1Control of bacte
12、rial spoilage of similar products can beevaluated by Test Method E723.NOTE 2Slimicides for control of fungal or bacterial slime can beevaluated by Test Method E1839.1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and Alternative Control Agents and is the direct respons
13、ibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2010. Published May 2010. Originallyapproved in 1982. Last previous edition approved in 2005 as E875 00(2005). DOI:10.1520/E087510.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact A
14、STM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from U.S. Government Printing Office Superintendent of Documents,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http
15、:/www.access.gpo.gov.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.6. Apparatus6.1 Two Balances One should be sensitive to 0.1 g at aload of 200 g with a platform to accommodate bottles beingused in the test. The second balance (an
16、alytical) should besensitive to 0.1 mg and used for weighing test chemicals.6.2 Clean Sample Containers, (120 mL containers withscrew- cap lids are ideal for test aliquots.) Other suitablecontainers include milk dilution bottle, 4 oz glass bottles, orsterile sampling bags.6.3 Flaming EquipmentAn alc
17、ohol lamp, bunsen burner,or electric device may be used to flame inoculating needles andother equipment.6.4 IncubatorsIncubators that control the temperature ofthe test 6 2C. Temperatures for test should be temperature atwhich the product will be stored.6.5 Petri Dishes, 100 by 15-mm, plastic or bor
18、osilicateglass, sterile.6.6 pH MeasurementAny pH meter is suitable to stan-dardize the pH of the culture medium or to determine pH ofsamples. Nonbleeding test strips may be used for determiningpH of test aliquots.6.7 Pipets, 1.0-mL graduated in 0.01 mL and 10-mL gradu-ated in 0.1 mL. Serological pip
19、ets should not be used for highlyviscous materials. Automatic pipettors may be used.6.8 Pipetting Aid, rubber bulb or other device to eliminatemouth pipetting.6.9 Sterilizers, pressurized steam sterilizer or hot air ovencapable of reaching 180 6 2C for 2 6 0.2h.6.10 Swabs, sterile, for aiding in rem
20、oval of fungal sporesfrom slants.6.11 Sterile Funnel, with sterile glass wool for filtration ofspore suspension.6.12 Sterile Glass Beads, (3-5 mm).6.13 Tubes, for preparation of slanted media.6.14 Milk Dilution Bottles.7. Reagent and Materials7.1 Purity of WaterUnless otherwise indicated, watershall
21、 be understood to mean distilled water or water of equalpurity, as defined in Specification D1193, Type 3.7.2 Freshly prepared test solutions of the fungal controlagent shall be used in all tests. Some preservatives can beadded with a micropipet.7.3 Test MaterialsFreshly prepared pigment slurries, a
22、d-hesives, dye rosin, polymer, sizing solutions, and other classesof aqueous-based materials to be preserved should be used asthe substrate.7.4 Culture MediumDehydrated Sabourauds agar (mal-trose or dextrose) is recommended for fungi. A more selectivemedium may be used provided it is used in additio
23、n toSabouraud. Results should indicate the data obtained with eachmedium.7.4.1 Spore Suspending Media and ContainerMilk dilu-tion bottles containing 100 mL Butterfield Buffer4with solidglass beads, for preparing sterile spore suspensions.7.4.2 Culture Media, slants of the selected agar.8. Test Organ
24、isms8.1 The test organisms selected may vary with the purposeof the test. If evaluating the basic effectiveness of a fungalcontrol agent, the use of standard fungal cultures is recom-mended (see 8.2). If attempting to qualify a fungal controlagent for a particularly difficult, or highly specific pre
25、servationapplication, the use of spoiled product or fungal organismsisolated from the problem system, or similar systems, may beappropriate (see 8.3 and 8.4).8.2 Standard fungal cultures suitable for this procedureinclude the following:8.2.1 Aspergillus niger: ATCC 6275.8.2.2 Penicillium pinophalum:
26、 ATCC 9644.8.2.3 Trichoderma virens: ATCC 9645.8.2.4 Candida albicans: ATCC 10231.8.2.5 Saccharomyces cerevisiae: ATCC 4111.8.3 To prepare an inoculum of spoilage organisms a spoiledsample should be streaked onto plates of Sabouraud MaltoseAgar (or other media selected) to verify that the samplecont
27、ains viable fungi. When fungal contamination is verified, ifsample is large enough, it may be used directly as the inoculum(see 10.1). If sample is too small for use as inoculum, add thespoiled sample to a larger sample of unprotected material andincubate for 7 to 14 days at an appropriate temperatu
28、re (basedon use conditions) until spoiled. Then proceed with testing (see10.1).8.4 If fungi isolated from spoiled material are to be used,proceed with testing as for standard fungal organisms.9. Inoculum Preparation9.1 Standard Fungi and Fungal IsolatesOrganismsshould be grown as slant cultures of t
29、he culture mediumselected. Grow for 7 to 14 days at 25 to 30C, until culturessporulate.Add 10 mL of sterile Butterfields buffer to the slants.Gently remove the spores from the surface of the agar byrubbing with a sterile swab. Add the resulting suspension to abottle containing glass beads and buffer
30、. Cap the bottle tightly,and then shake vigorously to liberate spores from fruitingbodies and to break up spore clumps. Filter the resultingsuspension through sterile glass wool in a sterile funnel into asterile container to remove mycelial fragments. This filtrate isthe spore suspension to be used
31、as an inoculum for testsamples.10. Procedure10.1 To provide a uniform inoculated substrate, the inocu-lum of spoilage organisms or fungal spore suspension shouldbe added to the entire quantity of the test substrate at one time,mixed thoroughly, then divided into test aliquots.10.1.1 Prepare streak c
32、ultures of the test substrate beforeand after fungal inoculation and determine the fungal contami-nation. Incubate plates at 25 to 30C for seven days or until thecontrol plates show sufficient growth for rating. Some testmaterials may not support growth of fungi. These materialsmost likely do not re
33、quire a preservative to prevent fungalgrowth. If the test material only supports slow growth of the4Butterfields Buffered Phosphate Diluent, Official Methods of Analysis of theAssociation of Official Analytical Chemists, K. Helrich, 15th ed., 1990, p. 429.E875 102fungi in question, then incubation p
34、eriods must be sufficientlylong to allow those fungi to grow.10.1.2 Level of growth can either be a growth, no-growthrating, or some type of rating scale such as 0 to 4, with 0 beingno-growth and 4 being heavy-growth.10.2 Disperse 50-g aliquots, or any other suitable quantity,of the inoculated test
35、material aseptically into the containersselected. Set up controls in duplicate.10.2.1 Aseptically add the stock solution of the test fungalcontrol agent to each bottle to give the desired concentration inparts per million or percent. Shake vigorously using 20complete cycles in a vertical motion or o
36、ther method to ensurecomplete mixing. Stock solution of the fungal control agentshould be of such strength that the volume of the solutionadded is no more than 1 % of the total volume of sample ineach bottle. Do not add a fungal control agent to the untreatedcontrols. Include a minimum of five conce
37、ntrations of thefungal control agent in each test. The test concentration rangeshould include at least one concentration that is ineffective andone that is effective. Record the initial pH of all samples andother physical characteristics such as signifying change incolor, odor, and viscosity.10.2.2
38、Incubate all samples at 28 6 2C, (or other suitabletemperature related to the most optimal growth temperatureencountered in use situations), with the bottles loosely capped.At weekly or other suitable time intervals, mix each samplethoroughly and immediately streak on Sabourauds agar ac-cording to s
39、tandard streaking techniques and incubate at 28Cfor seven days. In addition, observe each sample bottle for theevidence of spoilage such as visual growth of fungi, changes incolor, odor, and pH. Record the growth of the fungi for thestreaked samples.10.2.3 Rechallenge all treated samples showing no
40、visiblegrowth with 1 mL of inoculum obtained from one of theduplicate controls.10.2.4 Base each preservation experiment upon the ex-pected time of usage of the material to be preserved. Continuethe test for at least the average length of time that the specifictype of product would need protection, g
41、enerally six weeks,unless the material under test is preserved for a shorter periodof time, for example one week. Record the nature and extent ofspoilage based upon the criteria used.10.2.5 At the completion of the incubation period, observeeach sample bottle for evidence of spoilage such as visualg
42、rowth of fungi, significant changes in color, odor, pH, andviscosity. Record the relative growth of fungi on the streakplates.11. Results11.1 At each sampling time and at the end of the test, recordall results from observations of the sample bottles and thestreak plates. Visual deterioration or othe
43、r signs of degradationin the bottles, such as changes in pH color, odor, and loss ofviscosity, should also be used to judge the degree of preserva-tion obtained. The study is not valid unless fungal spoilage isevident in the control, based on visual fungal growth, obviouschanges in physical characte
44、ristics, or other reliable criteria.12. Precision and Bias12.1 A precision and bias statement cannot be made for thistest method.13. Keywords13.1 aqueous-based products; fungi; fungal control agent;fungal spores; paper; preservativeASTM International takes no position respecting the validity of any
45、patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the riskof infringement of such rights, are entirely their own responsibility.This standard is subject to revis
46、ion at any time by the responsible technical committee and must be reviewed every five years andif not revised, either reapproved or withdrawn. Your comments are invited either for revision of this standard or for additional standardsand should be addressed to ASTM International Headquarters. Your c
47、omments will receive careful consideration at a meeting of theresponsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you shouldmake your views known to the ASTM Committee on Standards, at the address shown below.This standard is copyrig
48、hted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,United States. Individual reprints (single or multiple copies) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-832-9585 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org). Permission rights to photocopy the standard may also be secured from the ASTM website (www.astm.org/COPYRIGHT/).E875 103
