1、Designation: D6530 00 (Reapproved 2013)Standard Test Method forTotal Active Biomass in Cooling Tower Waters (Kool KountAssay; KKA)1This standard is issued under the fixed designation D6530; the number immediately following the designation indicates the year oforiginal adoption or, in the case of rev
2、ision, 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 test method covers the determination of viableactive biomass in cooling tower water in the ran
3、ge from 102to108cfu/mL (1). It is a semiquantitative test method.1.2 This test method was used successfully with reagentwater, physiologic saline, and cooling tower waters. It is theusers responsibility to ensure the validity of this test methodfor waters of untested matrices.1.3 This standard does
4、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 limitations prior to use. For specific hazardstatements, see Se
5、ction 9.2. Referenced Documents2.1 ASTM Standards:2D1129 Terminology Relating to WaterD1192 Guide for Equipment for Sampling Water and Steamin Closed Conduits (Withdrawn 2003)3D1193 Specification for Reagent WaterD3370 Practices for Sampling Water from Closed Conduits3. Terminology3.1 DefinitionsFor
6、 definitions of terms used in this testmethod, refer to Terminology D1129.3.2 Definitions of Terms Specific to This Standard:3.2.1 snapping cupcontainer provided for holding thesample and snapping tip of the vial.3.2.2 vialsealed glass ampoule under vacuum containingreagents for the Kool Kount Test.
7、3.3 Symbols:3.3.1 cfu/mLcolony forming units per millilitre.4. Summary of Test Method4.1 This test method consists of adding a specific volume ofwater to nutrients and a color indicator contained in a glassvial. The contents of the vial are then mixed and incubated at95F (35 6 3C; that is, in a shir
8、t pocket, incubator, or heatblock). The color of the sample after addition into the vialcontaining the nutrients and color indicator is yellow. Viableactive biomass in the sample replicates using the nutrientsprovided and reduces the color indicator. At a critical biomassconcentration, sufficient qu
9、antities of the color indicator arereduced resulting in a visible change in the indicator from theoriginal yellow sample color to orange. The time required forconversion of the oxidized indicator to the reduced indicatorresulting in an orange color as directly correlated with theconcentration of via
10、ble active biomass in the water sampletested. High concentrations of active biomass in the sampleproduce the positive orange color more rapidly than lowconcentrations of viable biomass.5. Significance and Use5.1 This test method is useful for rapid determination ofviable active biomass concentration
11、s in cooling tower waters.The efficiency of cooling towers is directly affected by theconcentration of biomass in the cooling tower waters. Asbiomass concentrations increase, biofilm formation occursresulting in a decrease in the efficiency of heat exchange in thetower. Current tests for monitoring
12、the biomass concentrationin cooling towers require at least 36 h for growth of themicroorganisms on a solid agar surface for counting. Replica-tion of microorganisms over the 36-h period before results areavailable creates an aqueous environment which is no longerrepresented by the data generated. T
13、imely test results can assistin minimizing biocide addition to control biomass concentra-tions. Kool Kount provides data within hours to allow for moreprecise control of active biomass concentrations in the waters.6. Interferences6.1 Halogens interfere with this test method by inhibitingmicrobial gr
14、owth resulting in lengthy incubation periods before1This test method is under the jurisdiction of ASTM Committee D19 on Waterand is the direct responsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved June 1, 2013. Published July 2013. Originally approvedin 2000. Last prev
15、ious edition approved in 2006 as D6530 00 (2006). DOI:10.1520/D6530-00R13.2For 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
16、ASTM website.3The last approved version of this historical standard is referenced onwww.astm.org.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1a positive orange color is produced suggesting better waterquality. Addition of thiosulfa
17、te eliminates this interference andallows for testing of waters previously treated with halogens(not immediately prior to testing).6.2 Reducing agents (that is, beta mercaptoethanol) mayinterfere in this test method by reducing the color indicatorchemically. Rapid color development upon filling of t
18、he vialssuggests a chemical rather than a biological reaction. Waterscontaining reducing agents which react with the color indicatorare not suitable for testing with Kool Kount.6.3 Avoid prolonged exposure (greater than 30 min) of filledor unopened KKA vials to sunlight to avoid false positivereacti
19、ons.6.4 Testing must not take place within 24 h of biocideaddition.7. Apparatus7.1 The schematic arrangement of the KKAtest kit is shownin Fig. 1.7.2 (Parts) of the KKA Test KVial A (test vial), vial undervacuum containing nutrient and reagent on glass rod; Vial B(control vial), vial under vacuum co
20、ntaining nutrient only(does not contain a glass rod); snapping cup; and plastic safetysleeve.8. Reagents and Materials8.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents shall conform to the specifications of the Commit
21、-tee on Analytical Reagents of the American Chemical Societywhere such specifications are available.4Other grades may beused, provided it is first ascertained that the reagent is ofsufficiently high purity to permit its use without lessening theaccuracy of the determination.8.2 Purity of WaterUnless
22、 otherwise indicated, referencesto water shall be understood to mean reagent water as definedby Type III of Specification D1193.8.3 ReagentsKool Kount Test Kit: Fischer Scientific, ColeParmer, Calgon, Sodium thiosulfate: Na2O3S29. Precautions9.1 Precautions should be taken when snapping the glass ti
23、pfrom the glass vial containing the reagents. The tip must besubmerged for the vial to completely fill as a result of thevacuum in the vial. A protective sleeve is provided with the kitto cover any rough glass edges on the neck of the vial duringincubation.10. Sampling10.1 Collect the sample in acco
24、rdance with SpecificationD1192, and Practices D3370 as applicable.11. Procedure11.1 Rinse snapping cup at least twice with water to betested. Place at least 20 mL of the sample to be tested in thesnapping cup. Add two drops of the thiosulfate solutionprovided. Mix and allow sample to sit quiescently
25、 in thesnapping cup for 15 min.11.2 Submerge the tip of glass VialAcontaining reagents inthe sample to be tested (in the snapping cup). Place the tip inone of the grooves in the bottom of the snapping cup. Carefullypress the vial toward the opposite wall of the cup to snap thetip allowing the vial t
26、o fill as a result of the vacuum in the vial.11.3 Submerge the tip of control glass Vial B (no glass rod)in the same sample. Place the tip in one of the grooves in thebottom of the snapping cup. Carefully press the vial toward thewall of the cup to snap the tip allowing the vial to fill.11.4 Place a
27、 protective sleeve on the neck of each vial tocover the sharp edges. Carefully invert vials several times tocompletely mix the reagent powders with the water sample.11.5 Prepare the label with the sample designation, samplepH, sample temperature, and the time at which test wasinitiated. Place the sa
28、mple label on the appropriate vial andlabel the control vial. Incubate vials at approximately 95F (356 3C; heat block, shirt pocket, incubator).11.6 Examine Vials A and B after 10 to 15 min fordevelopment of pink to red color indicative of chemicalreaction, not biological activity.11.7 Examine sampl
29、e Vial A for color change (yellownegative to orange positive) after 30 min of incubation bylooking through the flat base of the vials comparing the testsample vial (A) with the control vial (B). Examine Vial A forcolor change at hourly intervals by looking through the flatbase of the vial and compar
30、ing with the control Vial B. Anincubation of 10.5 h, corresponding to 102cfu/mL, is the limitof this test method for estimation of viable active biomass.11.8 Note the time when the color change (yellow toorange) occurs.11.9 Determine the total elapsed time from test initiation topositive color devel
31、opment time completed time initiated =total elapsed time; that is, 1:15(1315; end point) 10:15 (1015;initiation)=3h.12. Calculation12.1 Elapsed time for positive color development is directlycorrelated with viable active biomass concentration in thesample tested. Total elapsed time is converted to b
32、iomassconcentration in accordance with the table in Fig. 2. Thisbiomass concentration represents the viable active biomasslevel present in the sample tested.13. Report13.1 Report the results including sample pH and elapsedtime for positive color development. The elapsed time isconverted into viable
33、active biomass concentration in accor-dance with the table in Fig. 2.4Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For suggestions on the testing of reagents notlisted by the American Chemical Society, see Analar Standards for LaboratoryChemi
34、cals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.D6530 00 (2013)2FIG. 1 Schematic of Kool Kount Test KitD6530 00 (2013)314. Precision and Bias514.1 This test method was tested by three laboratories
35、. Twooperators in each of the three laboratories analyzed each of foursamples (three cooling tower samples and one spiked controlsample) in triplicate. One operator at a fourth laboratory alsoparticipated in this test method. The collaborative test datawere obtained using reagent water and cooling t
36、ower waters.For other matrices these data may not apply. See Table 1.14.1.1 PrecisionAllowances are made to precision andbias statement formats for test methods yielding a nonnumeri-cal report of success or failure based on criteria specified in theprocedure. No statement is made about either the pr
37、ecision orthe bias of Method D-19.24(97-01), Kool Kount Test Methodfor measuring viable active biomass in cooling tower waterssince the result merely states whether there is conformance tothe criteria for success, that is, time to development of positivecolor, as specified in the procedure.14.1.2 Bi
38、asComparison of KKA test results with resultsof the dip slide (current test method for cooling tower waters)and standard plate count methods did not show bias for higheror lower estimates if bioconcentration in the waters tested withKool Kount. See Table 2.14.2 Four independent laboratories (and a t
39、otal of 7 opera-tors) participated in the round-robin study. Under the allow-ances (exception to the precision and bias statement requiredby Practice D2777 recommended by the results advisor) madein 1.3 of Practice D277, these precision and bias data do meetexisting requirements for interlaboratory
40、studies of CommitteeD19 test method.15. Keywords15.1 colorimetric bioassay; cooling tower water; triphenyltetrazolium chloride; viable active biomass5Supporting data have been filed at ASTM International Headquarters and maybe obtained by requesting Research Report RR:D19-1168. ContactASTM CustomerS
41、ervice at serviceastm.org.Time (Hours) to positive cfu/mL (bacterial concentration)0.5 $1082.5 1074.0 1065.5 1057.0 1049.0 10310.5 10212.5 101FIG. 2 Standard Table for Determination of Viable Active Biomassin Cooling Tower WatersTABLE 1 Round Robin Kool Kount Assay (KKA) ResultsLaboratory 1 Laborato
42、ry 2 Laboratory 3 Laboratory 4Sample No. 1A9h,9h,9h 6h,6h,6hA10.5 h, 10.5 h, 10.5 h 9 h, 9 h, 9 h9h,9h,9h 7h,7h,7hA10.5 h, 10.5 h, 10.5 hSample No. 2 10.5 h, 10.5 h, 10.5 hB10.5 h, 10.5 h, 10.5 h 10.5 h, 10.5 h, 10.5 h 10.5 h, 10.5 h, 10.5 h10.5 h, 10.5 h, 10.5 h 0.5 h, 10.5 h, 10.5 h 10.5 h, 10.5 h
43、, 10.5 hSample No. 3 6 h, 6 h, 6 h 6 h, 6 h, 6 h 6 h, 6 h, 6 h 6 h, 6 h, 6 h7h,7h,7h 7h,7h,7h 7h,7h,7hSample No. 4 4 h, 4 h, 4 h 4 h, 4 h, 4 h 4 h, 4 h, 4 h 4 h, 4 h, 4 h4h,4h,4h 4h,4h,4h 4h,4h,4hALaboratories 2, 3, and 4 reported the presence of pink particulates in Sample No. 1 at 67 h. The aqueou
44、s phase did not turn a positive color until much later. Laboratory2 did not record the time to development of positive color in the aqueous phase.B10.5h=102cfu/mL, the lower detection limit of the method.D6530 00 (2013)4ASTM International takes no position respecting the validity of any patent right
45、s 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 revision at any t
46、ime 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 comments will
47、 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
48、 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
49、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/).TABLE 2 Comparison of KKA, Dip Slide, and Standard Plate CountsSample KKA Dip Slide Plate CountNo. 1 1028102cfu/mLA1103cfu/mL 8 101cfu/mLNo. 2 102 102cfu/mL 1 103cfu/mL 5 102cfu/mLNo. 3 2 1042105cfu/mL 1 105cfu/mL 2 105cfu/mLNo. 4 2 106cfu/mL 1 107cfu/mL 1 107cfu/mLADoes not include the results from Laboratory No. 2 representing the presence of pink particulates, not positive
