1、Designation: D 6530 00 (Reapproved 2006)Standard Test Method forTotal Active Biomass in Cooling Tower Waters (Kool KountAssay; KKA)1This standard is issued under the fixed designation D 6530; the number immediately following the designation indicates the year oforiginal adoption or, in the case of r
2、evision, 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 test method covers the determination of viableactive biomass in cooling tower water in the
3、range 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 do
4、es 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
5、 Section 9.2. Referenced Documents2.1 ASTM Standards:2D 1129 Terminology Relating to WaterD 1193 Specification for Reagent WaterD 1192 Guide for Equipment for Sampling Water andSteam in Closed Conduits3D 3370 Practices for Sampling Water from Closed Conduits3. Terminology3.1 DefinitionsFor definitio
6、ns of terms used in this testmethod, refer to Terminology D 1129.3.2 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.3.3 Symbo
7、l:cfu/mLcolony forming units per millilitre4. 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 shirt pocket, incubat
8、or, 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 quantities of the c
9、olor 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 viable active biomas
10、s 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 concentrations in cooling towe
11、r 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 the biomass conce
12、ntrationin 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. Timely test result
13、s 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 growth resulting in
14、 lengthy incubation periods beforea positive orange color is produced suggesting better waterquality. Addition of thiosulfate eliminates this interference and1This test method is under the jurisdiction of ASTM Committee D19 on Waterand is the direct responsibility of Subcommittee D19.24 on Water Mic
15、robiology.Current edition approved July 1, 2006. Published July 2006. Originally approvedin 2000. Last previous edition approved in 2000 as D 6530 00.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStand
16、ards volume information, refer to the standards Document Summary page onthe ASTM website.3Withdrawn.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.allows for testing of waters previously treated with halogens(not immediately prior t
17、o 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 the vialssuggests a chemical rather than a biological reaction. Waterscontaining reducing agents which react with the col
18、or 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 positivereactions.6.4 Testing must not take place within 24 h of biocideaddition.7. Apparatus7.1 The schematic arrangement of the KKAt
19、est 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 containing nutrient only(does not contain a glass rod); snapping cup; and plastic safetysleeve.8. Reagents and Materials8.
20、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-tee on Analytical Reagents of the American Chemical Societywhere such specifications are available.4Other grades may be
21、used, 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 otherwise indicated, referencesto water shall be understood to mean reagent water as definedby Type III of Specificatio
22、n D 1193.8.3 ReagentsKool Kount Test Kit: Fischer Scientific,Cole Parmer, Calgon, Sodium thiosulfate: Na2O3S29. Precautions9.1 Precautions should be taken when snapping the glass tipfrom the glass vial containing the reagents. The tip must besubmerged for the vial to completely fill as a result of t
23、hevacuum 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 accordance with SpecificationD 1192, and Practices D 3370 as applicable.11. Procedure11.1 Rinse snapping cup at least twice
24、 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 in thesnapping cup for 15 min.11.2 Submerge the tip of glass VialAcontaining reagents inthe sample to be tested (in
25、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 to 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
26、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 protective sleeve on the neck of each vial tocover the sharp edges. Carefully invert vials several times tocompletel
27、y 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 sample label on the appropriate vial andlabel the control vial. Incubate vials at approximately 95F (356 3C; heat block
28、, 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 sample Vial A for color change (yellownegative to orange positive) after 30 min of incubation bylooking through the flat b
29、ase 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 comparing with the control Vial B. Anincubation of 10.5 h, corresponding to 102cfu/mL, is the limitof this test method for
30、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 development time completed time initiated =total elapsed time; that is, 1:15(1315; end point) 10:15 (1015;initiation)=3h.1
31、2. Calculation12.1 Elapsed time for positive color development is directlycorrelated with viable active biomass concentration in thesample tested. Total elapsed time is converted to biomassconcentration in accordance with the table in Fig. 2. Thisbiomass concentration represents the viable active bi
32、omasslevel 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 active biomass concentration in accor-dance with the table in Fig. 2.14. Precision and Bias514.1 This test method was
33、 tested by three laboratories. Twooperators in each of the three laboratories analyzed each of four4Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For suggestions on the testing of reagents notlisted by the American Chemical Society, see Analar
34、 Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.5Supporting data have been filed at ASTM International Headquarters and maybe obtained by requesting Research Report R
35、R: D1168.D 6530 00 (2006)2FIG. 1 Schematic of Kool Kount Test KitD 6530 00 (2006)3samples (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 a
36、nd cooling tower 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 e
37、ither the precision 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 procedu
38、re.14.1.2 BiasComparison 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 laborator
39、ies (and a total of 7 opera-tors) participated in the round-robin study. Under the allow-ances (exception to the precision and bias statement requiredby Practice D 2777 recommended by the results advisor) madein 1.3 of Practice D 277, these precision and bias data do meetexisting requirements for in
40、terlaboratory studies of CommitteeD-19 test method.15. Keywords15.1 colorimetric bioassay; cooling tower water; triphenyltetrazolium chloride; viable active biomassASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this
41、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 technical committee and must be r
42、eviewed 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 consideration at a meeting of therespon
43、sible 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
44、 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).Time (Hours) to p
45、ositive 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 Laboratory 2 Laboratory 3 Laboratory 4Sample No. 1A
46、9h,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, 10.5 hSample No. 3 6 h, 6 h, 6 h 6 h, 6 h
47、, 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 aqueous phase did not turn a positive color until
48、 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.TABLE 2 Comparison of KKA, Dip Slide, and Standard Plate CountsSample KKA Dip Slide Plate CountNo. 1 10283 102cfu/mLA1 3 103cfu/mL 8 3 101c
49、fu/mLNo. 2 102 102cfu/mL 1 3 103cfu/mL 5 3 102cfu/mLNo. 3 2 3 10423 105cfu/mL 1 3 105cfu/mL 2 3 105cfu/mLNo. 4 2 3 106cfu/mL 1 3 107cfu/mL 1 3 107cfu/mLADoes not include the results from Laboratory No. 2 representing the presence of pink particulates, not positive color development in the aqueous phase.D 6530 00 (2006)4
