ASTM_D_4249_-_83_1998.pdf

Designation:D 4249 83(Reapproved 1998)An American National StandardStandard Test Method forEnumeration ofCandida albicansin Water1This standard is issued under the fixed designation D 4249;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(e)indicates an editorial change since the last revision or reapproval.1.Scope1.1 This test method covers the detection and enumerationof the yeast Candida albicans in raw sewage,waste waters,andnatural waters.1.2 It is the responsibility of the analyst to determine if thistest method yields satisfactory results in waters of othermatrices.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.For a specifichazard statement,see Section 9.2.Referenced Documents2.1 ASTM Standards:D 1129 Terminology Relating to Water2D 1193 Specification for Reagent Water2D 3370 Practices for Sampling Water from Closed Con-duits2D 3870 Practice for Establishing Performance Characteris-tics for Colony Counting Methods in Microbiology3E 200 Practice for Preparation,Standardization,and Storageof Standard and Reagent Solutions for Chemical Analysis43.Terminology3.1 Definitions For definitions of terms used in this testmethod,refer to Terminology D 1129.3.2 Definitions of Terms Specific to This Standard:3.2.1 germ tubeselongated extensions,3 to 4 m wide andup to 20 m in length,which originate from the yeast cell whenincubated for 1 to 3 h in serum.There is no constriction of thegerm tube at its point of origin;this is a critical diagnosticfeature(1).5Similar structures(elongate buds,pseudohyphae)may be produced by C.albicans and other yeasts but all havediscrete constrictions at the base where the structure is formedat the cell surface.4.Summary of Test Method4.1 This test method consists of filtering appropriate vol-umes of raw sewage,waste water,or natural water through1.2-m retentive porosity gridded membrane filters and placingthe membranes on the surface of a selective medium,hereinreferred to as mCA(2).4.2 Cultures are incubated for 2 to 4 days at 37C andtypical colonies are observed and counted using a dissectingmicroscope.4.3 At least initially,suspect colonies may be confirmed asC.albicans by picking to bovine(calf)serum,incubating for 2to 3 h,and observing cells using microscopy(preferably phase)for the presence of germ tubes that are diagnostic for C.albicans(1,3).5.Significance and Use5.1 C.albicansis a yeast that is found as a commensal inthe gastrointestinal,genitourinary,and alimentary tracts ofhealthy individuals,both human and lower animals(3,4,5).Assuch,it is a serious opportunistic pathogen of humans and maycause superficial or deep mycotic infections.Consequently,theyeast is found in raw sewage and in natural waters receivinghuman and animal wastes.C.albicans can survive in situ inseawater for at least six days(6).In vitro survival of the yeastin distilled(7)and lake water(8)has been demonstrated also.While there is at present no epidemiological evidence connect-ing human disease caused by C.albicans and use of water,theorganism may be a useful indicator of recreational waterquality(9).The test method may be applied to the monitoringof various treatment processes for efficiency in removingparticular pathogens in waste water prior to discharge inreceiving waters which in turn may be used again for a varietyof purposes.Both public health and sanitary engineeringinterests should be aware of the presence of this yeast inwastewater and the potential for disease in contiguous waters.5.2 Future studies between the incidence of C.albicans andtraditional water quality indicators(for example,total and fecalcoliforms,fecal streptococci)may reveal a correlation of value1This 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 April 29,1983.Published September 1983.2Annual Book of ASTM Standards,Vol 11.01.3Annual Book of ASTM Standards,Vol 11.02.4Annual Book of ASTM Standards,Vol 15.05.5The boldface numbers in parentheses refer to references at the end of thestandard.1Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959,United States.in the assessment of potential health risks of swimming orother recreational waters.6.Interferences6.1 In some waters,“false positive”colonies resemblingC.albicans may develop on mCAmedium.Generally,however,these can be differentiated by colony shape,color,or texture,ora combination thereof,using a high-power dissecting micro-scope.Also,they may be detected by the germ tube proceduredescribed below.6.2 Germ tube