ASTM_D_4200_-_82_2011.pdf

Designation:D420082(Reapproved 2011)Standard Test Method forEvaluating Inhibitory Effects of Ink Grids on MembraneFilters1This standard is issued under the fixed designation D4200;the number immediately following the designation indicates the year oforiginal 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 test method describes a procedure whereby the userof ink-gridded membrane filters in water quality studies canascertain whether or not the grid lines are toxic and inhibitoryto bacterial growth when the membrane and its entrappedbacteria are incubated on a suitable media.1.2 The values stated in SI units are to be regarded 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 determine the applica-bility of regulatory limitations prior to use.2.Referenced Documents2.1 ASTM Standards:2D1129 Terminology Relating to WaterD1193 Specification for Reagent Water3.Terminology3.1 DefinitionsFor definitions of terms used in this testmethod,refer to Terminology D1129.4.Summary of Test Method4.1 A heavy bacterial suspension is filtered through agridded membrane filter.The bacterial concentration employedis sufficient to cover much of the membrane with bacterialcolonies.4.2 After filtration the membrane is incubated on a suitablemedium and the distribution of the colonies and shape of thecolonies noted in the area around each grid line.5.Significance and Use5.1 This test method may be applied to determine thesuitability of grid-marked membrane filters for use in bacte-riological culture techniques for the detection and enumerationof bacterial organisms.5.2 A particularly sensitive organism and growth conditionshave been selected for this test method in order to maximizesensitivity to toxic materials possibly present in the inks usedfor grid-marking membrane filters.6.Apparatus6.1 Incubator,capable of maintaining temperatures of44.56 0.2C.6.2 Membrane Filtration Units.6.3 Vacuum Source with trap vessel.6.4 Forceps,blunt-nosed.6.5 Autoclave or other sterilizing equipment.6.6 Expendables:6.6.1 Gridded membrane filters.6.6.2 1-mL and 10-mL pipets.6.6.3 Petri dishes(50-mm)containing 6 to 8 mL of agarmedium or a 100-mm dish with 20 6 2 mL of agar medium,orboth.6.6.4 Erlenmeyer flasks.7.Reagents and Materials7.1 Purity of WaterUnless otherwise indicated,referenceto water shall be understood to mean reagent water conformingto reagent water Type II of Specification D1193.7.2 M-FC Agar with Rosolic Acid or equivalent(henceforthreferred to as agar medium),formulated,prepared,and dis-pensed in accordance with the manufacturers specifications.7.3 Tryptone Soya Broth or equivalent(henceforth referredto as broth medium),formulated,prepared,and dispensed inaccordance with the manufacturers specifications.7.4 Peptone Water,0.1%,sterile.7.5 Broth Culture of E.coli ATCC 11229,18-h,prepared asfollows:Add 1 mL of an 18 6 2-h broth culture of E.coli1This test method is under the jurisdiction of ASTM Committee D19 on Waterand is the direct responsibility of Subcommittee D19.08 on Membranes and IonExchange Materials.Current edition approved May 1,2011.Published June 2011.Originallyapproved in 1982.Last previous edition approved in 2003 as D420082(2003).DOI:10.1520/D4200-82R11.2For 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.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 ATCC 11229 to 99 mL of 0.1%peptone water,mix thor-oughly,then add 0.1 mL of this suspension to another flaskcontaining 99 mL of 0.1%peptone water.This is the workingconcentration and should contain approximately 103bacteriaper millilitre to provide contiguous but discrete growth.NOTE1Hydrophobic leaching may cause growth inhibition in areasadjacent to grid lines.8.Procedure8.1 Assemble the sterile membrane filtration apparatus,andconnect to vacuum trap and vacuum source.8.2 Process five randomly selected membrane filters,using1-mL aliquots of culture suspension from 7.5,and 30 mL of0.1%peptone water added to the filter funnel with standardmembrane filtration procedures.Place the membranes in petridishes containing M-FC agar medium and incubate at 44.5Cfor at least 18 h.8.3 After incubation,examine the overgrown membrane forinhibition of growth along the grid lines.8.4 Examine the membrane filter for growth of“squarecolonies.”The growth of square colonies flowing along gridlines is not necessarily an indication of toxicity,but cou