ASTM D4249-83(1998)
(Test Method)Standard Test Method for Enumeration of Candida albicans in Water
Standard Test Method for Enumeration of <I>Candida albicans</I> in Water
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.
1.3 This standard does not purport to address all of the safety problems, 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 9.
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An American National Standard
Designation:D 4249–83 (Reapproved 1998)
Standard Test Method for
Enumeration of Candida albicans in Water
This standard is issued under the fixed designation D 4249; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope may be produced by C. albicans and other yeasts but all have
discrete constrictions at the base where the structure is formed
1.1 This test method covers the detection and enumeration
at the cell surface.
oftheyeast Candida albicansinrawsewage,wastewaters,and
natural waters.
4. Summary of Test Method
1.2 It is the responsibility of the analyst to determine if this
4.1 This test method consists of filtering appropriate vol-
test method yields satisfactory results in waters of other
umes of raw sewage, waste water, or natural water through
matrices.
1.2-µmretentiveporositygriddedmembranefiltersandplacing
1.3 This standard does not purport to address all of the
the membranes on the surface of a selective medium, herein
safety concerns, if any, associated with its use. It is the
referred to as mCA (2).
responsibility of the user of this standard to establish appro-
4.2 Cultures are incubated for 2 to 4 days at 37°C and
priate safety and health practices and determine the applica-
typical colonies are observed and counted using a dissecting
bility of regulatory limitations prior to use. For a specific
microscope.
hazard statement, see Section 9.
4.3 At least initially, suspect colonies may be confirmed as
2. Referenced Documents C. albicans by picking to bovine (calf) serum, incubating for 2
to3h,andobservingcellsusingmicroscopy(preferablyphase)
2.1 ASTM Standards:
for the presence of germ tubes that are diagnostic for C.
D 1129 Terminology Relating to Water
albicans (1,3).
D 1193 Specification for Reagent Water
D 3370 Practices for Sampling Water from Closed Con-
5. Significance and Use
duits
5.1 C. albicans is a yeast that is found as a commensal in
D 3870 Practice for Establishing Performance Characteris-
3 the gastrointestinal, genitourinary, and alimentary tracts of
tics for Colony Counting Methods in Microbiology
healthyindividuals,bothhumanandloweranimals(3,4,5).As
E200 PracticeforPreparation,Standardization,andStorage
4 such, it is a serious opportunistic pathogen of humans and may
of Standard and Reagent Solutions for ChemicalAnalysis
cause superficial or deep mycotic infections. Consequently, the
3. Terminology yeast is found in raw sewage and in natural waters receiving
human and animal wastes. C. albicans can survive in situ in
3.1 Definitions— For definitions of terms used in this test
seawater for at least six days (6). In vitro survival of the yeast
method, refer to Terminology D 1129.
in distilled (7) and lake water (8) has been demonstrated also.
3.2 Definitions of Terms Specific to This Standard:
While there is at present no epidemiological evidence connect-
3.2.1 germ tubes—elongatedextensions,3to4µmwideand
ing human disease caused by C. albicans and use of water, the
upto20µminlength,whichoriginatefromtheyeastcellwhen
organism may be a useful indicator of recreational water
incubated for 1 to3hin serum. There is no constriction of the
quality (9). The test method may be applied to the monitoring
germ tube at its point of origin; this is a critical diagnostic
of various treatment processes for efficiency in removing
feature (1). Similar structures (elongate buds, pseudohyphae)
particular pathogens in waste water prior to discharge in
receiving waters which in turn may be used again for a variety
This test method is under the jurisdiction of ASTM Committee D19 on Water of purposes. Both public health and sanitary engineering
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
interests should be aware of the presence of this yeast in
Current edition approved April 29, 1983. Published September 1983.
wastewater and the potential for disease in contiguous waters.
Annual Book of ASTM Standards, Vol 11.01.
5.2 Future studies between the incidence of C. albicans and
Annual Book of ASTM Standards, Vol 11.02.
Annual Book of ASTM Standards, Vol 15.05.
traditionalwaterqualityindicators(forexample,totalandfecal
The boldface numbers in parentheses refer to references at the end of the
coliforms, fecal streptococci) may reveal a correlation of value
standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D 4249–83 (1998)
in the assessment of potential health risks of swimming or 10. Procedure
other recreational waters.
10.1 Preparation of mCA Medium:
10.1.1 Combine the following ingredients:
6. Interferences
6.1 In some waters, “false positive” colonies resembling
Glycine 1.0 g
Maltose 3.0 g
C.albicansmaydeveloponmCAmedium.Generally,however,
Sodium sulfite; Na SO 0.3 g
2 3
thesecanbedifferentiatedbycolonyshape,color,ortexture,or
Bismuth ammonium citrate; Bi[(NH )C H O ] 0.5 g
4 6 5 7 3
a combination thereof, using a high-power dissecting micro-
Chloramphenicol 50 mg
Cycloheximide 150 mg
scope.Also, they may be detected by the germ tube procedure
Reagent water 90 mL
described below.
10.1.2 With stirring, warm to about 50°C (slight turbidity).
6.2 Germ tubes have been reported to occur in C. stella-
toidea, a yeast closely resembling C. albicans in all respects. 10.1.3 Whilestirring,adjustthepHto7.1with1.0 NHClor
NH OH.
However, C. stellatoidea is human-associated and apparently
rare in natural waters; its occurrence probably assumes the 10.1.4 Add 1.5 g of agar, and bring slowly to the boiling
same significance as that of C. albicans. point by swirling constantly over a flame. Continue to boil
gently for 2 min and cool to 45 to 50°C in a water bath. Do not
7. Apparatus
autoclave.
7.1 pH Meter (expanded scale preferable).
10.1.5 Add10mLofmembrane-filtered(0.45-µm)commer-
7.2 Magnetic Stirrer. cially available yeast nitrogen base prepared at a 103 concen-
7.3 Water Bath, 45 to 50°C.
tration.
7.4 Membrane Filtration Apparatus (holder, tubing, trap, 10.1.6 While stirring, adjust the pH to 6.5 with 1.0 N HCl.
flasks, vacuum pump).
This is a critical step since colony color development is
7.5 Incubator,3761°C. dependent on proper pH adjustment.
7.6 Binocular Dissecting Microscope (Stereozoom Prefer-
10.1.7 Withfrequentswirlingofthemedium,pourintopetri
able) and External Light Source (Nicholas or Spot Type). dishes to a depth of 4 to 5 mm. Allow to solidify.
7.7 Research Microscope (Phase Contrast Preferable).
10.1.8 Store plates in the dark (foil-wrapped) at about 4°C.
7.8 Culture Tubes, disposable, 10 by 75 mm. Themediumisstableforabout2weeksundertheseconditions.
7.9 Hollow Plastic Straws, approximately 13.5 by 3 mm White crystal formation indicates that the medium should be
(cocktail sippers). discarded.
7.10 Sterile Petri Dishes. 10.2 Collection of Samples:
7.11 Microscope Slides and Cover Slips. 10.2.1 Use clean, sterile containers.
10.2.2 Obtain sample so as to preclude contamination.
8. Reagents and Materials
10.2.3 Large volumes of some waters will be required (for
8.1 Purity of Reagents—Reagent grade chemicals shall be example, several litres if replicate plates of recreational waters
used in all tests. Unless otherwise indicated, it is intended that
are to be prepared).
all reagents shall conform to the specifications of the Commit-
10.2.4 If chlorinated waters are sampled, add sodium thio-
tee on Analytical Reagents of the American Chemical Soci-
sulfate to the collection bottle before sterilization, at a concen-
ety. Other grades may be used provided it is first ascertained
tration of 0.1 mL of 10 % solution for each 125-mL of sample
that the reagent is of sufficiently high purity to permit its use
volume.
without lessening the accuracy of the determination.
10.2.5 Filter sample as below as soon as possible after
8.2 Purity of Water— Unless otherwise indicated, refer-
collection.
ences to water shall be understood to mean reagent water
10.3 Filtration of Sample:
conforming to Specification D 1193, Type II.
10.3.1 Sample volumes will vary depending on the water
8.3 Hydrochloric Acid (1 + 9)—Refer to Practice E 200.
sampled; 10 to 40 mL may be appropriate for raw sewage
8.4 Ammonium Hydroxide (1 + 9).
while up to 1 L or more of relatively clean and clear
8.5 Bovine (calf) Serum.
recreational water should be examined.
8.6 Sterile Membrane Filters, white, gridded, 47-mm diam- 10.3.2 Shake sample thoroughly.
eter, 1.2-µm retentive porosity.
10.3.3 Filter sample through 1.2-µm retentive porosity
membrane filter. Rinse holder with 20 to 30 mL of sterile
9. Precautions
reagent grade water.
9.1 Candida albicans isahumanpathogen;thus,handleall
10.3.4 Aseptically remove membrane and place grid side up
culture material (plates, slides, serum tubes, and straws) using
on plates of mCA medium.
accep
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