ASTM D4249-83(2005)
(Test Method)Standard Test Method for Enumeration of Candida albicans in Water (Withdrawn 2013)
Standard Test Method for Enumeration of <I>Candida albicans</I> in Water (Withdrawn 2013)
SIGNIFICANCE AND USE
C. albicans is a yeast that is found as a commensal in the gastrointestinal, genitourinary, and alimentary tracts of healthy individuals, both human and lower animals (3, 4, 5). As such, it is a serious opportunistic pathogen of humans and may cause superficial or deep mycotic infections. Consequently, the yeast is found in raw sewage and in natural waters receiving human and animal wastes. C. albicans can survive in situ in seawater for at least six days (6). In vitro survival of the yeast in distilled (7) and lake water (8) has been demonstrated also. While there is at present no epidemiological evidence connecting human disease caused by C. albicans and use of water, the organism may be a useful indicator of recreational water quality (9). The test method may be applied to the monitoring of various treatment processes for efficiency in removing particular pathogens in waste water prior to discharge in receiving waters which in turn may be used again for a variety of purposes. Both public health and sanitary engineering interests should be aware of the presence of this yeast in wastewater and the potential for disease in contiguous waters.
Future studies between the incidence of C. albicans and traditional water quality indicators (for example, total and fecal coliforms, fecal streptococci) may reveal a correlation of value in the assessment of potential health risks of swimming or other recreational waters.
SCOPE
1.1 This test method covers the detection and enumeration of the yeast Candida albicans in raw sewage, waste waters, and natural waters.
1.2 It is the responsibility of the analyst to determine if this test method yields satisfactory results in waters of other matrices.
This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. For a specific hazard statement, see Section .
WITHDRAWN RATIONALE
This test method covers the detection and enumeration of the yeast Candida albicans in raw sewage, waste waters, and natural waters.
Formerly under the jurisdiction of Committee D19 on Water, this test method was withdrawn in June 2013. This standard is being withdrawn without replacement due to its limited use by industry.
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Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: D4249 − 83(Reapproved 2005)
Standard Test Method for
Enumeration of Candida albicans in Water
This standard is issued under the fixed designation D4249; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope feature (1). Similar structures (elongate buds, pseudohyphae)
may be produced by C. albicans and other yeasts but all have
1.1 This test method covers the detection and enumeration
discrete constrictions at the base where the structure is formed
oftheyeastCandidaalbicansinrawsewage,wastewaters,and
at the cell surface.
natural waters.
1.2 It is the responsibility of the analyst to determine if this
4. Summary of Test Method
test method yields satisfactory results in waters of other
4.1 This test method consists of filtering appropriate vol-
matrices.
umes of raw sewage, waste water, or natural water through
1.3 This standard does not purport to address all of the
1.2-µmretentiveporositygriddedmembranefiltersandplacing
safety concerns, if any, associated with its use. It is the
the membranes on the surface of a selective medium, herein
responsibility of the user of this standard to establish appro-
referred to as mCA (2).
priate safety and health practices and determine the applica-
4.2 Cultures are incubated for 2 to 4 days at 37°C and
bility of regulatory limitations prior to use. For a specific
typical colonies are observed and counted using a dissecting
hazard statement, see Section 9.
microscope.
2. Referenced Documents 4.3 At least initially, suspect colonies may be confirmed as
C. albicans by picking to bovine (calf) serum, incubating for 2
2.1 ASTM Standards:
to3h,andobservingcellsusingmicroscopy(preferablyphase)
D1129Terminology Relating to Water
for the presence of germ tubes that are diagnostic for C.
D1193Specification for Reagent Water
albicans (1,3) .
D3870PracticeforEstablishingPerformanceCharacteristics
for Colony Counting Methods in Microbiology (With-
5. Significance and Use
drawn 2000) (Withdrawn 2000)
5.1 C. albicansisayeastthatisfoundasacommensalinthe
E200Practice for Preparation, Standardization, and Storage
gastrointestinal, genitourinary, and alimentary tracts of healthy
of Standard and Reagent Solutions for ChemicalAnalysis
individuals, both human and lower animals (3, 4, 5) .As such,
itisaseriousopportunisticpathogenofhumansandmaycause
3. Terminology
superficial or deep mycotic infections. Consequently, the yeast
3.1 Definitions—For definitions of terms used in this test
is found in raw sewage and in natural waters receiving human
method, refer to Terminology D1129.
and animal wastes. C. albicans can survive in situ in seawater
3.2 Definitions of Terms Specific to This Standard:
foratleastsixdays (6). In vitrosurvivaloftheyeastindistilled
3.2.1 germ tubes—elongatedextensions,3to4µmwideand
(7) and lake water (8) has been demonstrated also.While there
upto20µminlength,whichoriginatefromtheyeastcellwhen
is at present no epidemiological evidence connecting human
incubated for 1 to3hin serum.There is no constriction of the
disease caused by C. albicans and use of water, the organism
germ tube at its point of origin; this is a critical diagnostic
maybeausefulindicatorofrecreationalwaterquality (9).The
test method may be applied to the monitoring of various
treatmentprocessesforefficiencyinremovingparticularpatho-
This test method is under the jurisdiction ofASTM Committee D19 on Water
gens in waste water prior to discharge in receiving waters
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
whichinturnmaybeusedagainforavarietyofpurposes.Both
Current edition approved Oct. 1, 2005. Published October 2005. Originally
approved in 1983. Last previous edition approved in 1998 as D4249–83 (1998).
public health and sanitary engineering interests should be
DOI: 10.1520/D4249-83R05.
aware of the presence of this yeast in wastewater and the
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
potential for disease in contiguous waters.
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
3 4
The last approved version of this historical standard is referenced on The boldface numbers in parentheses refer to references at the end of the
www.astm.org. standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D4249 − 83 (2005)
5.2 Future studies between the incidence of C. albicans and 8.5 Bovine (calf) Serum.
traditionalwaterqualityindicators(forexample,totalandfecal
8.6 Sterile Membrane Filters, white, gridded, 47-mm diam-
coliforms,fecalstreptococci)mayrevealacorrelationofvalue
eter, 1.2-µm retentive porosity.
in the assessment of potential health risks of swimming or
other recreational waters.
9. Precautions
9.1 Candida albicans is a human pathogen; thus, handle all
6. Interferences
culture material (plates, slides, serum tubes, and straws) using
6.1 In some waters, “false positive” colonies resembling C.
accepted microbiological technique including the sterilization
albicans may develop on mCA medium. Generally, however,
of all discards.
thesecanbedifferentiatedbycolonyshape,color,ortexture,or
10. Procedure
a combination thereof, using a high-power dissecting micro-
scope.Also, they may be detected by the germ tube procedure
10.1 Preparation of mCA Medium:
described below.
10.1.1 Combine the following ingredients:
6.2 Germ tubes have been reported to occur in C. stellatoi-
Glycine 1.0 g
dea, a yeast closely resembling C. albicans in all respects.
Maltose 3.0 g
However, C. stellatoidea is human-associated and apparently
Sodium sulfite; Na SO 0.3 g
2 3
rare in natural waters; its occurrence probably assumes the
Bismuth ammonium citrate; Bi[(NH )C H O ] 0.5 g
4 6 5 7 3
Chloramphenicol 50 mg
same significance as that of C. albicans.
Cycloheximide 150 mg
Reagent water 90 mL
7. Apparatus
10.1.2 With stirring, warm to about 50°C (slight turbidity).
7.1 pH Meter (expanded scale preferable).
10.1.3 Whilestirring,adjustthepHto7.1with1.0 NHClor
NH OH.
7.2 Magnetic Stirrer.
10.1.4 Add 1.5 g of agar, and bring slowly to the boiling
7.3 Water Bath, 45 to 50°C.
point by swirling constantly over a flame. Continue to boil
7.4 Membrane Filtration Apparatus (holder, tubing, trap,
gentlyfor2minandcoolto45to50°Cinawaterbath.Donot
flasks, vacuum pump).
autoclave.
10.1.5 Add10mLofmembrane-filtered(0.45-µm)commer-
7.5 Incubator, 3761°C.
cially available yeast nitrogen base prepared at a 10× concen-
7.6 Binocular Dissecting Microscope (Stereozoom Prefer-
tration.
able) and External Light Source (Nicholas or Spot Type).
10.1.6 While stirring, adjust the pH to 6.5 with 1.0 N HCl.
7.7 Research Microscope (Phase Contrast Preferable).
This is a critical step since colony color development is
dependent on proper pH adjustment.
7.8 Culture Tubes, disposable, 10 by 75 mm.
10.1.7 Withfrequentswirlingofthemedium,pourintopetri
7.9 Hollow Plastic Straws, approximately 13.5 by 3 mm
dishes to a depth of 4 to 5 mm. Allow to solidify.
(cocktail sippers).
10.1.8 Store plates in the dark (foil-wrapped) at about 4°C.
7.10 Sterile Petri Dishes.
Themediumisstableforabout2weeksundertheseconditions.
White crystal formation indicates that the medium should be
7.11 Microscope Slides and Cover Slips.
discarded.
8. Reagents and Materials
10.2 Collection of Samples:
8.1 Purity of Reagents—Reagent grade chemicals shall be 10.2.1 Use clean, sterile containers.
10.2.2 Obtain sample so as to preclude contamination.
used in all tests. Unless otherwise indicated, it is intended that
10.2.3 Large volumes of some waters will be required (for
all reagents shall conform to the specifications of the Commit-
example, several litres if replicate plates of recreational waters
tee on Analytical Reagents of the American Chemical Soci-
are to be prepared).
ety. Other grades may be used provided it is first ascertained
10.2.4 If chlorinated waters are sampled, add sodium thio-
that the reagent is of sufficiently high purity to permit its use
sulfate to the collection bottle before sterilization, at a concen-
without lessening the accuracy of the determination.
tration of 0.1 mLof 10% solution for each 125-mLof sample
8.2 Purity of Water—Unlessotherwiseindicated,references
volume.
towatershallbeunderstoodtomeanreagentwaterconforming
10.2.5 Filter sample as below as soon as possible after
to Specification D1193, Type II.
collection.
8.3 Hydrochloric Acid(1+9)—Refer to Practice E200.
10.3 Filtration of Sample:
8.4 Ammonium Hydroxide(1+9).
10.3.1 Sample volumes will vary depending on the water
sampled; 10 to 40 mL may be appropriate for raw sewage
“ReagentChemicals,AmericanChemicalSocietySpecifications,”Am.Chemi-
cal Soc., Washington, DC. For suggestions on the testing of reagents not listed by Bismuth ammonium citrate is the most critical and least readily available
theAmerican
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