1、Designation: E 875 00 (Reapproved 2005)Standard Test Method forEfficacy of Fungal Control Agents as Preservatives forAqueous-Based Products Used in the Paper Industry1This standard is issued under the fixed designation E 875; the number immediately following the designation indicates the year oforig
2、inal 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 laboratory test method is used to determine theefficacy o
3、f a fungal control agent to prevent spoilage ofin-process aqueous-based products used in the paper industry.1.2 For information on bacterial control agents, see TestMethod E 723.1.3 It is the responsibility of the investigator to determinewhether good laboratory practices (GLP) are required and tofo
4、llow them when appropriate (see 40 CFR, 160).1.4 A knowledge of microbiological techniques is requiredfor these procedures.1.5 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
5、-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 599 Test Method for Efficacy of Slimicides for the PaperIndustryFungal Slime3E 600 Test Method for Efficacy o
6、f Slimicides for the PaperIndustryBacterial Slime3E 723 Test Method for Efficacy of Antimicrobials as Pre-servatives for Aqueous-Based Products Used in the PaperIndustry (Bacterial Spoilage)2.2 Federal Standard:40 CFR, Part 160, Good Laboratory Practice Standards43. Terminology3.1 Definitions of Ter
7、ms 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 to prevent mi-crobial so
8、ilage 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 desired period of time, or b
9、oth. 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 control agent is one that p
10、revents 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 suspensions, dyesolutions,
11、 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 bacterial spoilage of simila
12、r products can beevaluated by Test Method E 723.NOTE 2Slimicides for control of fungal or bacterial slime can beevaluated by Test Methods E 599 and E 600.6. Apparatus6.1 Two Balances, One should be sensitive to 0.1 g at a loadof 200 g with a platform to accommodate bottles being used inthe test. The
13、 second balance (analytical) should be sensitive to0.1 mg and used for weighing test chemicals.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
14、approved Nov. 1, 2005. Published November 2005. Originallyapproved in 1982. Last previous edition approved in 2000 as E 875 00.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information
15、, refer to the standards Document Summary page onthe ASTM website.3Withdrawn.4Available from U.S. Government Printing Office Superintendent of Documents,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken,
16、PA 19428-2959, United States.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 alcohol lamp, bunsen burner,or electric device may
17、 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 borosilicateglass, sterile.6.6 pH MeasurementAny p
18、H 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 pipets should not be used for highlyviscous materi
19、als. 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 removal of fungal sporesfrom slants.6.11 Sterile F
20、unnel, 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 be understood to mean distilled water or water
21、 of equalpurity, as defined in Specification D 1193, 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, ad-hesives, dye rosin, polymer, sizing solution
22、s, 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 addition toSabouraud. Results should indicate the dat
23、a obtained with eachmedium.7.4.1 Spore Suspending Media and ContainerMilk dilu-tion bottles containing 100 mL Butterfield Buffer5with solidglass beads, for preparing sterile spore suspensions.7.4.2 Culture Media, slants of the selected agar.8. Test Organisms8.1 The test organisms selected may vary w
24、ith 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 preservationapplication, the use of spoiled produ
25、ct 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: ATCC 9644.8.2.3 Trichoderma virens: ATCC 9645
26、.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 samplecontains viable fungi. When fungal contamination i
27、s 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 temperature (basedon use conditions) until spoiled. The
28、n 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 the culture mediumselected. Grow for 7 to 14 da
29、ys 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. Cap the bottle tightly,and then shake vigoro
30、usly 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 as an inoculum for testsamples.10. Procedure10
31、.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 cultures of the test substrate beforeand after
32、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 require a preservative to prevent fungalgrowth.
33、If the test material only supports slow growth of thefungi in question, then incubation periods 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-g
34、rowth.5Butterfields Buffered Phosphate Diluent, Official Methods of Analysis of theAssociation of Official Analytical Chemists, K. Helrich, 15th ed., 1990, p. 429.E 875 00 (2005)210.2 Disperse 50-g aliquots, or any other suitable quantity,of the inoculated test material aseptically into the containe
35、rsselected. 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 other method to ensurecomplete mixing.
36、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 concentrations of thefungal control agent i
37、n 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 Incubate all samples at 28 6 2C, (or o
38、ther 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 standard streaking techniques and incub
39、ate 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 visiblegrowth with 1 mL of inoculum ob
40、tained 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, generally six weeks,unless the material
41、 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 visualgrowth of fungi, significant changes in
42、 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 other signs of degradationin the bottles,
43、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 characteristics, or other reliable criteria.12
44、. 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 patent rights asserted in connection w
45、ith 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 revision at any time by the responsible tec
46、hnical 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 comments will receive careful considera
47、tion 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 copyrighted by ASTM International, 100 Barr H
48、arbor 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).E 875 00 (2005)3
