1、Designation: E723 13Standard Practice forEvaluation of Antimicrobials as Preservatives for Aqueous-Based Products Used in the Paper Industry (BacterialSpoilage)1This standard is issued under the fixed designation E723; the number immediately following the designation indicates the year oforiginal ad
2、option 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 practice is used to determine the efficacyof an antimi
3、crobial for preventing bacterial spoilage of in-process aqueous-based products used in the paper industry. Forinformation on fungal spoilage, see Test Method E875. Thispractice should be performed by persons who have had basicmicrobiological training.1.2 The values stated in SI units are to be regar
4、ded asstandard. No other units of measurement are included in thisstandard.1.3 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 determin
5、e the applica-bility of regulatory limitations prior to use. (See 40 CFR Part160.)2. Referenced Documents2.1 ASTM Standards:2D1193 Specification for Reagent WaterE640 Test Method for Preservatives in Water-ContainingCosmeticsE875 Test Method for Efficacy of Fungal Control Agents asPreservatives for
6、Aqueous-Based Products Used in thePaper IndustryE1054 Test Methods for Evaluation of Inactivators of Anti-microbial AgentsE1326 Guide for Evaluating Nonconventional Microbiologi-cal Tests Used for Enumerating BacteriaE1839 Test Method for Efficacy of Slimicides for the PaperIndustryBacterial and Fun
7、gal SlimeE2756 Terminology Relating to Antimicrobial and AntiviralAgents2.2 Other Standards:40 CFR Part 160 Good Laboratory Practice Standards33. Terminology3.1 For definitions of terms related to this practice, seeTerminology E2756.3.2 Definitions of Terms Specific to This Standard:3.2.1 antimicrob
8、ial, nchemical or physical agent that killsor inactivates microorganisms or suppresses their growth orreproduction.3.2.2 bactericide, na physical or chemical agent that killsbacteria, but not necessarily bacterial spores.3.2.3 biocide, na physical or chemical agent that killsorganisms.3.2.4 microbic
9、ide, na physical or chemical agent that killsmicroorganisms.3.2.5 preservatives, nchemical agent(s) added to a productto reduce or prevent microbial growth.4. Summary of Practice4.1 Aqueous material to be preserved is inoculated withappropriate bacterial inoculums followed by addition of abactericid
10、e that will reduce populations of bacteria and preventthe growth of survivors for a specified period of time. Bacterialnumbers in the sample are determined at various time periodsand compared to a contol without any bactericide. The properlevel of antimicrobial is one that reduces and keeps theorgan
11、isms to an acceptable level in the test material.5. Significance and Use5.1 This practice should be used to determine if an antimi-crobial preserves pigment suspensions, dye solutions, pulpslurries, starch solutions, polymers, sizing agents, latex1This practice is under the jurisdiction of ASTM Comm
12、ittee E35 on Pesticides,Antimicrobials, and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2013. Published May 2013. Originallyapproved in 1980. Last previous edition approved in 2007 as E723 07. DOI:10.1520
13、/E0723-13.2For 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 to the standards Document Summary page onthe ASTM website.3Available from U.S. Government Printing Office Sup
14、erintendent 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 States1emulsions, and other aqueous-based materials used in the paperindustry fro
15、m bacterial spoilage.NOTE 1Control of fungal spoilage of similar products can beevaluated by Test Method E875.NOTE 2Slimicides for control of fungal or bacterial slime can beevaluated by Test Method E1839.6. Apparatus6.1 BalanceOne sensitive to 0.1 g at a load of 200 g witha platform to accommodate
16、bottles being used in the test; anda second (analytical) sensitive to 0.1 mg used for weighing testchemicals.6.2 BottlesBorosilicate glass milk dilution bottles or othersuitable containers fitted either with screw caps or Escherrubber stoppers. These bottles are used for water blanks andaqueous-base
17、d samples.6.3 Colony CounterAny one of several types may beused, such as the Quebec, Buck, and Wolfhuegel. A hand tallyfor the recording of the bacterial count is recommended ifmanual counting is done. Alternatively, an automated videocolony counter may also be used.6.4 Culture Tube ClosuresAppropri
18、ate nontoxic closuresshould be selected.6.5 Culture TubesRecommended size is 15 by 125 mm or18 by 150 mm without lip, and preferably of borosilicate glass.6.6 BlenderAny blender that will ensure proper agitationand blending.6.7 Flaming EquipmentDepending upon circumstances,either an alcohol lamp or
19、bunsen burner may be used to flameinoculating needles and other equipment.6.8 Incubators, capable of maintaining temperatures of 28to 70 62C to provide proper incubation temperatures. Tem-perature should be consistent with the temperature of theproduct to be preserved.6.9 Petri Dishes, 100 by 15-mm,
20、 plastic or borosilicateglass, sterile.6.10 pH MeterAny suitable for standardizing the pH ofthe culture medium. Non-bleeding pH test strips may be usedfor samples.6.11 Pipets, 1.1 or 2.2-mL milk dilution-type, 1.0-mLgraduated in 0.01 mL, 10-mL graduated in 0.1 mL, andappropriately calibrated pipetto
21、rs may be used. Serologicalpipets and pipettors should not be used for highly viscousmaterials.6.12 Sterilizers, pressurized steam sterilizer or hot air ovencapable of 180 6 2C for 2 6 0.2 h.7. Microbicides and Materials7.1 Freshly prepared test solutions of the antimicrobial shallbe used in all tes
22、ts.7.2 Purity of WaterAll references to water as diluent orreagent shall mean distilled water or water of equal purity,unless otherwise noted (see Specification D1193, Type III).7.3 Test MaterialsFreshly prepared pigment slurries,adhesives, dye rosin, polymer, sizing solutions, and othermaterials to
23、 be preserved should be used as the substrate.7.4 Culture Medium:7.4.1 Standard dehydrated tryptone glucose extract agar orother medium that is known to recover organisms from thematerial to be tested is recommended.7.4.2 For some substrates it may be necessary to add a smallamount of nutrient mater
24、ial to ensure growth of the organismsin the material to be studied for preservation. For bacterialpreservation studies, add 5.0 mL/L of 0.2 % nutrient broth tothe test material to ensure sufficient populations.8. Test Organisms8.1 The test organisms will vary with the material to bepreserved and the
25、 purpose of the test. For specific materialsthat are contaminated, that material will serve as the inoculum.For general screening of activity or preventative evaluations,the inoculum may consist of a single or mixed culture oforganisms. It is best to use organisms that are known to causespoilage of
26、the material to be preserved.Alternatively, bacterialisted in Test Method E640 may be used. The viability of themicroorganisms in the material to be tested should be verifiedprior to initiating the test.8.2 To provide a uniformly inoculated substrate, the inocu-lum should be added to the entire quan
27、tity of the test substrateat one time, mixed thoroughly, and then dispensed into theseparate test bottles.8.3 The material under test should be inoculated withsufficient microorganisms either from pure cultures or con-tained in the spoiled material used as the inoculum to give abacterial count of 11
28、05to 1106CFU/mL.9. Procedure9.1 Dispense 50-g aliquots (or any other suitable quantity)of the inoculated test material aseptically into sterile bottles (ifnecessary, add nutrient). Treat the samples immediately withappropriate concentrations of the antimicrobial. Set up controlsin duplicate. Note ap
29、propriate physical characteristics such aspH, color, odor, viscosity etc., of all test samples at this time.9.1.1 Make the following additions aseptically to eachbottle in the order named and shake vigorously after eachaddition, using 20 complete cycles in a vertical motion.9.1.2 Add the desired vol
30、ume of the stock solution of theantimicrobial to be tested to give the desired concentration inparts per million or percent. Stock solution of the antimicrobialshould be of such strength so that the volume of antimicrobialsolution added is no more than 1 % of the total volume ofsample in each bottle
31、 Do not add an antimicrobial to thecontrol. Include in each test a minimum of five concentrationsof the antimicrobial under test. Suggested antimicrobial con-centrations depend upon the microbicide and its recommendedconcentrations. Record the pH of all samples at the beginningof the experiment.9.1
32、3 Incubate all samples at 28 to 37C (or other tempera-ture at which the test material will be stored, such as 65C forstarch solutions) with the bottle capped tightly to avoidE723 132evaporation. The organisms being tested need to be viable andstable at the test temperature for the duration of the t
33、est.9.1.4 Determine concentration of viable organisms in thecontrols at the time of biocide addition and of all samples atperiodic intervals after biocide addition (see Test MethodsE1054). This can be done with standard plate counts or otheraccepted alternative means of determining concentrations of
34、organisms (see Guide E1326). All plates or recovery mediumshould be incubated at the same temperature as the test.9.1.5 Time intervals for determining the level of organismsremaining in the sample depend on the length of time the testmaterial needs to be preserved in actual use and the acceptablelev
35、el of contamination when the material is to be used. Thus,samples can be taken at 3 h, 8 h, 24 h, 48 h, 72 h, or weekly,depending on how rapidly and to what extent the inoculumneeds to be killed. Inactivation of microbicides must beachieved with appropriate neutralizers or dilutions (Test Meth-ods E
36、1054).9.1.6 If the material can be re-inoculated while it is pre-served or to determine the number of re-inoculations a givenpreservative level will be able to control, the samples should bere-inoculated on a weekly or biweekly interval to give abacterial count of 1105to 1105CFU/mL (see 8.3).9.1.7 T
37、ypical test protocols range from biweekly inocula-tions with sampling 24 h post-inoculation to weekly inocula-tions with sampling 7 days post-inoculation. For comparativestudies, these intervals and the inoculum must be constant. Asin any lab test, it is difficult to duplicate the conditions thatmig
38、ht exist in an actual production facility.9.1.8 At each time interval (see 9.1.5) or after reinoculation(see 9.1.6), mix the sample thoroughly, inactivate the micro-bicide (Test Methods E1054), and immediately determine thelevel of microorganisms in the sample (see 9.1.4). The controlsmust maintain
39、a high count throughout the study or show visualsigns of deterioration. Either criterion can be used to indicatespoilage and the validity of the test.10. Calculation10.1 At each sampling time and at the end of each test,calculate the percent of bacteria killed at each microbicideconcentration tested
40、 as follows:% kill 5control plate count 2 test plate count!control plate count310010.1.1 The percent kill at any given time is indicative of theeffectiveness of the antimicrobial under test. The proper levelof antimicrobial to use for the material being tested is the onethat decreases the number of
41、organisms to the acceptable levelfor the test material in an acceptable amount of time. Visualdeterioration and other signs of degradation, such as changes inpH, color, odor, loss of viscosity, and so forth, should also beused to judge the degree of preservation obtained.NOTE 3Typically, the level o
42、f organisms should be reduced to lessthan 1103CFU/mL in a 7 day period of time. This is equivalent to a99.9 %kill when the inoculum provides an initial bacterial count in the testsample of 1106CFU/mL. No increase in bacterial count should bedetected during the duration of the test (if longer than 7
43、days) or length oftime the material needs to be preserved.11. Keywords11.1 bacterial; bactericide; paper based products; preserva-tives; pulp; spoilageASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Use
44、rs 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 technical committee and must be reviewed every
45、 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 consideration at a meeting of theresponsible technic
46、al 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
47、 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/).E723 133
