1、Designation: E875 10E875 15Standard Test Method Practice forEfficacyEvaluation of Fungal Control Agents asPreservatives for Aqueous-Based Products Used in thePaper Industry1This standard is issued under the fixed designation E875; the number immediately following the designation indicates the year o
2、foriginal adoption or, in 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 the effic
3、acy of a fungal control agent to prevent spoilage of in-processaqueous-based products used in the paper industry.1.2 For information on bacterial control agents, see Test Method E723.1.3 It is the responsibility of the investigator to determine whether good laboratory practices (GLP) are required an
4、d to followthem when appropriate (see 40 CFR 160).1.4 A knowledge of microbiological techniques is required for these procedures.1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.6 This standard does not purport to address
5、 all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatorylimitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1193 Specification
6、 for Reagent WaterE723 Practice for Evaluation of Antimicrobials as Preservatives for Aqueous-Based Products Used in the Paper Industry(Bacterial Spoilage)E1839 Test Method for Efficacy of Slimicides for the Paper IndustryBacterial and Fungal Slime2.2 Federal Standard:40 CFR 160 Good Laboratory Prac
7、tice Standards33. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 fungal control agent, nan agent that either kills or prevents growth of fungi and either kills or prevents the germinationof fungal spores. This term is applied to chemical biocidal or biostatic agents.3.1.2 preser
8、vative, nchemical agent used to prevent microbial soilage of products due to microbial growth.4. Summary of Test Method4.1 Aqueous material to be preserved is inoculated with an appropriate fungal inoculum followed by addition of a concentrationof fungal control agent that will kill the fungi or pre
9、vent their growth for a desired period of time, or both. In addition, the agentwill also prevent fungal spore germination. Fungal growth is determined by visible signs of deterioration in the test sample, and1 This test method practice is under the jurisdiction ofASTM Committee E35 on Pesticides,Ant
10、imicrobials, andAlternative ControlAgents and is the direct responsibilityof Subcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2010May 1, 2015. Published May 2010 July 2015. Originally approved in 1982. Last previous edition approved in 20052010 asE875 00E875 10.(2005). D
11、OI: 10.1520/E087510.10.1520/E087515.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.3 Available from U.S. Gov
12、ernment Printing Office Superintendent of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http:/www.access.gpo.gov.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous
13、version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM Interna
14、tional, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1by obtaining fungal numbers and comparing them to a sample without any fungal control agent. The proper level of fungal controlagent is one that prevents product deterioration and reduces and keeps the organi
15、sms to an acceptable level in the test material,as determined by the tester or user.5. Significance and Use5.1 This test method should be used to determine if a fungal control agent is effective to preserve pigment suspensions, dyesolutions, pulp slurries, starch solutions, polymers, sizing agents,
16、latex emulsions, and other specific aqueous-based materials usedin the paper industry. Separate evaluations should be made on a representative type for each specific class of product to bepreserved.NOTE 1Control of bacterial spoilage of similar products can be evaluated by Test Method E723.NOTE 2Sli
17、micides for control of fungal or bacterial slime can be evaluated by Test Method E1839.6. Apparatus6.1 Two Balances One should be sensitive to 0.1 g at a load of 200 g with a platform to accommodate bottles being used inthe test. The second balance (analytical) should be sensitive to 0.1 mg and used
18、 for weighing test chemicals.6.2 Clean Sample Containers, (120 mL containers with screw- cap lids are ideal for test aliquots.) Other suitable containersinclude milk dilution bottle, 4 oz glass bottles, or sterile sampling bags.6.3 Flaming EquipmentAn alcohol lamp, bunsen burner, or electric device
19、may be used to flame inoculating needles and otherequipment.6.4 IncubatorsIncubators that control the temperature of the test 6 2C. Temperatures for test should be temperature at whichthe product will be stored.6.5 Petri Dishes, 100 by 15-mm, plastic or borosilicate glass, sterile.6.6 pH Measurement
20、Any pH meter is suitable to standardize the pH of the culture medium or to determine pH of samples.Nonbleeding test strips may be used for determining pH of test aliquots.6.7 Pipets, 1.0-mL graduated in 0.01 mL and 10-mL graduated in 0.1 mL. Serological pipets should not be used for highlyviscous ma
21、terials. Automatic pipettors may be used.6.8 Pipetting Aid, rubber bulb or other device to eliminate mouth pipetting.6.9 Sterilizers, pressurized steam sterilizer or hot air oven capable of reaching 180 6 2C for 2 6 0.2h.6.10 Swabs, sterile, for aiding in removal of fungal spores from slants.6.11 St
22、erile Funnel, with sterile glass wool for filtration of spore 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, water shall be understood to mean distilled water
23、 or water of equal purity, asdefined in Specification D1193, Type 3.7.2 Freshly prepared test solutions of the fungal control agent shall be used in all tests. Some preservatives can be added witha micropipet.7.3 Test MaterialsFreshly prepared pigment slurries, adhesives, dye rosin, polymer, sizing
24、solutions, and other classes ofaqueous-based materials to be preserved should be used as the substrate.7.4 Culture MediumDehydrated Sabourauds agar (maltrose or dextrose) is recommended for fungi.Amore selective mediummay be used provided it is used in addition to Sabouraud. Results should indicate
25、the data obtained with each medium.7.4.1 Spore Suspending Media and ContainerMilk dilution bottles containing 100 mL Butterfield Buffer4 with solid glassbeads, for preparing sterile spore suspensions.7.4.2 Culture Media, slants of the selected agar.8. Test Organisms8.1 The test organisms selected ma
26、y vary with the purpose of the test. If evaluating the basic effectiveness of a fungal controlagent, the use of standard fungal cultures is recommended (see 8.2). If attempting to qualify a fungal control agent for a particularly4 Butterfields Buffered Phosphate Diluent, Official Methods of Analysis
27、 of the Association of Official Analytical Chemists, K. Helrich, 15th ed., 1990, p. 429.E875 152difficult, or highly specific preservation application, the use of spoiled product or fungal organisms isolated from the problemsystem, or similar systems, may be appropriate (see 8.3 and 8.4).8.2 Standar
28、d fungal cultures suitable for this procedure include the following:8.2.1 Aspergillus niger: ATCC 6275.8.2.2 Penicillium pinophalum: 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 organ
29、isms a spoiled sample should be streaked onto plates of Sabouraud Maltose Agar(or other media selected) to verify that the sample contains viable fungi. When fungal contamination is verified, if sample is largeenough, it may be used directly as the inoculum (see 10.1). If sample is too small for use
30、 as inoculum, add the spoiled sample toa larger sample of unprotected material and incubate for 7 to 14 days at an appropriate temperature (based on use conditions) untilspoiled. Then proceed with testing (see 10.1).8.4 If fungi isolated from spoiled material are to be used, proceed with testing as
31、for standard fungal organisms.9. Inoculum Preparation9.1 Standard Fungi and Fungal IsolatesOrganisms should be grown as slant cultures of the culture medium selected. Growfor 7 to 14 days at 25 to 30C, until cultures sporulate. Add 10 mL of sterile Butterfields buffer to the slants. Gently remove th
32、espores from the surface of the agar by rubbing with a sterile swab. Add the resulting suspension to a bottle containing glass beadsand buffer. Cap the bottle tightly, and then shake vigorously to liberate spores from fruiting bodies and to break up spore clumps.Filter the resulting suspension throu
33、gh sterile glass wool in a sterile funnel into a sterile container to remove mycelial fragments.This filtrate is the spore suspension to be used as an inoculum for test samples.10. Procedure10.1 To provide a uniform inoculated substrate, the inoculum of spoilage organisms or fungal spore suspension
34、should be addedto 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 before and after fungal inoculation and determine the fungal contamination.Incubate plates at 25 to 30C for seven days or un
35、til the control plates show sufficient growth for rating. Some test materials maynot support growth of fungi. These materials most likely do not require a preservative to prevent fungal growth. If the test materialonly supports slow growth of the fungi in question, then incubation periods must be su
36、fficiently long to allow those fungi to grow.10.1.2 Level of growth can either be a growth, no-growth rating, 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 material asept
37、ically into the containers selected.Set up controls in duplicate.10.2.1 Aseptically add the stock solution of the test fungal control agent to each bottle to give the desired concentration in partsper million or percent. Shake vigorously using 20 complete cycles in a vertical motion or other method
38、to ensure complete mixing.Stock solution of the fungal control agent should be of such strength that the volume of the solution added is no more than 1 %of the total volume of sample in each bottle. Do not add a fungal control agent to the untreated controls. Include a minimum offive concentrations
39、of the fungal control agent in each test. The test concentration range should include at least one concentrationthat is ineffective and one that is effective. Record the initial pH of all samples and other physical characteristics such as signifyingchange in color, odor, and viscosity.10.2.2 Incubat
40、e all samples at 28 6 2C, (or other suitable temperature 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 according to standar
41、d streaking techniques and incubate at 28C for sevendays. In addition, observe each sample bottle for the evidence of spoilage such as visual growth of fungi, changes in color, odor,and pH. Record the growth of the fungi for the streaked samples.10.2.3 Rechallenge all treated samples showing no visi
42、ble growth with 1 mL of inoculum obtained from one of the duplicatecontrols.10.2.4 Base each preservation experiment upon the expected time of usage of the material to be preserved. Continue the testfor at least the average length of time that the specific type of product would need protection, gene
43、rally six weeks, unless thematerial under test is preserved for a shorter period of time, for example one week. Record the nature and extent of spoilage basedupon the criteria used.10.2.5 At the completion of the incubation period, observe each sample bottle for evidence of spoilage such as visual g
44、rowthof fungi, significant changes in color, odor, pH, and viscosity. Record the relative growth of fungi on the streak plates.E875 15311. Results11.1 At each sampling time and at the end of the test, record all results from observations of the sample bottles and the streakplates. Visual deteriorati
45、on or other signs of degradation in the bottles, such as changes in pH color, odor, and loss of viscosity,should also be used to judge the degree of preservation obtained. The study is not valid unless fungal spoilage is evident in thecontrol, based on visual fungal growth, obvious changes in physic
46、al characteristics, or other reliable criteria.12. Precision and Bias12.1 A precision and bias statement cannot be made for this test method.13. Keywords13.1 aqueous-based products; fungi; fungal control agent; fungal spores; paper; preservativeASTM International takes no position respecting the val
47、idity of any 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 sub
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49、ters. Your comments 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 copyrighted 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-
