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The Kingdom Fungi is composed of diverse eukaryotic and According to a conservative estimate, there are 1.5 million heterotrophic organisms, including filamentous molds and species of fungi.2 Fungi exist and survive in almost every hab- itat,3 although less than 100 000 are formally described. Fungal Most fungi have cell walls composed of rigid, covalently populations may fluctuate seasonally4 and usually increase when linked polymers, including chitin and glucan. Most are aerobic, organic loading in water or soil increases. Large numbers of although some are obligate anaerobes and a few are facultative similar fungi suggest fungal amplification due to excessive or- anaerobes. Most are mesophilic (grow at temperatures between ganic loading, while a diversified mycobiota indicates stable 18 and 25°C), but some are thermophilic (grow at temperatures from Ͼ20 up to ϳ50°C) and some are psychrophilic (grow at a. Fungi in potable water: Fungi have been found in potable temperatures between 0 and 5°C, up to a maximum of about 16 water and on the inner surface of distribution-system pipes.5–10In Norway, researchers identified 94 fungal species (belonging to 30 genera) in groundwater- and surface water-derived drinking Most fungi are saprophytic, secreting extracellular enzymes water.9 The dominant mycobiota were species of Aspergillus, and absorbing nutrients from dead or decaying organic matter.
Penicillium, and Trichoderma, and some occurred throughout Some fungi are parasitic; however, relatively few species are overt pathogens and some fungi live in a symbiotic relationship These fungi either survive water treatment or enter the system with plants or other microorganisms (e.g., lichens and mycor- after treatment and remain viable for extended periods. Tuber- culate macroconidia of Histoplasma capsulatum11 can pass Fungi are ubiquitous in water environments and areas associ- through a 0.75-m rapid sand filter, but sedimentation or alum ated with water (e.g., shoreline edges and leaking pipes). Spring flocculation and settling removed 80 to 99% of the spores. If water (near the source) usually contains a minimal number of these relatively large (8- to 14-␮m), globose fungal cells pass fungal spores, although the species may be diverse. Unpolluted through treatment, it is not surprising that species with smaller stream water also may contain multiple species, including true aquatic fungi (species with flagellated zoospores and gametes), Pathogenic fungi have remained viable in distilled water aquatic fungal-like species, and soil fungi. Moderately polluted stored for relatively long periods.12 Spores of H. capsulatum, water may contain cells or spores of all types, but more of them stored in raw Ohio River water and sterile tap water, remained will be soil fungi. Heavily polluted water contains large numbers of soil fungi. Soil fungi include yeast-like fungi, many species of Tastes and odors in potable water are often associated with which have been isolated from polluted waters.
prokaryotic organisms (e.g., bacteria, actinomycetes, and cy- The association between fungal densities and organic loading anobacteria), but fungi also may be involved.9–10,14 Researchers suggests that fungi may be useful indicators of pollution, but to have found members of the genera Acremonium, Penicillium, date no single species or group of fungi has been identified for and Phialophora to be responsible for taste and odor problems in this role. There may be some exceptional cases. For example, the principal phenotypic distinction between the yeasts Candida Propagules from 19 genera of filamentous fungi have been lambica and C. krusei is the ability to use pentose sugars; there isolated from a chlorinated surface water system and an unchlo- are nucleic acid sequence differences as well. The former species rinated groundwater distribution system;5 researchers found a grows well on pentoses, so theoretically it might be used to mean of 18 colony-forming units (CFU) /100 mL in the ground-water system and 34 CFU/100 mL in the surface water system.
indicate pulp and paper mill wastes, which contain high levels of Densities of filamentous fungi and yeasts ranged from 4.0 to such sugars. Likewise, certain thermophilic species of yeasts and 25 CFU/cm2 and 0 to 9 CFU/cm2, respectively, in the fungal filamentous fungi might be useful indicators of thermal pollu- biofilms of a municipal drinking water system.16 Certain soil- inhabiting genera—Aspergillus, Penicillium, Mucor, Alternaria, Fungi can degrade or deteriorate a wide range of complex and Cladosporium—seem to be common colonizers of biofilms natural materials and hazardous compounds.1 Because they can produce diverse enzymes, they have been used to treat various In Finland,5 researchers isolated fungi from rivers, lakes, and ponds supplying nine communities with sand-filtered water,three with artificially recharged groundwater (two of which usedchemical coagulation), and three with chemically coagulated and * Approved by Standard Methods Committee, 2008.
disinfected water. They found that Aspergillus fumigatus was the Joint Task Group: Margo E. Hunt (chair), Stephen N. Bland, Hilisa B. Esteban, most common fungus. Mesophilic fungi were common in all raw Jack W. Fell, Christable L. Fernandez-Monteiro, Philip A. Geis, Harbhajan Singh,Kailash C. Srivastava water samples; however, thermotolerant fungi were more abun- dant in river than in lake water. Chemical coagulation and The kingdom Fungi seems to have evolved separately in at disinfection was found to remove fungi far more effectively than least three groups.36–38 As with bacteria, identifying fungi de- pends primarily on colonial morphology on a solid medium, In the United States, one study reported an average of about growth and reproduction morphology, and (for yeasts) physio- 5.5 CFU/100 mL per positive sample from five chlorinated logical activity in laboratory cultures.39 Molecular detection and groundwater systems.6 In France,7 researchers recovered yeasts identification techniques, such as analysis of rRNA se- from 50% and filamentous fungi from 81% of 38 samples.
quences,40,41 commercial 18S sequencing services, and real-time Except for certain Aspergillus spp.,5–7,19 the fungi isolated polymerase chain reaction (RT-PCR) are increasingly being from potable water usually are not considered medically impor- used.42,43 There are commercial identification systems that use molecular methods, especially for yeasts. Methods for extracting b. Fungi in recreational waters: Fungi may be found in both DNA from fungal conidia and yeast cells in air and water fresh and marine recreational waters. Marine fungi may have a samples have been evaluated for RT-PCR.44 Analysts have used range of salinity requirements to consider when attempting iso- RT-PCR with a molecular beacon probe to detect fungi in lation.20 Some fungi pathogenic to humans may be expected in recreational waters (e.g., pools and beaches) and in accompany- Fungal quantification is complicated by the fact that a fungal ing washing facilities (e.g., shower stalls and changing rooms).
colony may develop from one cell (spore), an aggregate of cells They may survive longer than vegetative bacteria due to spore (a cluster of spores or one multi-celled spore), or from a hyphal or pseudohyphal fragment (containing more than one viable Studies revealed dermatophytes (fungi that grow on the skin, cell). Each fungal colony that develops in laboratory culture is nails, and hair) at 42% of the beaches surveyed.23,24 The most assumed to have originated from one colony-forming unit common species were Microsporum nanum and Trichophyton (CFU), which may or may not be a single cell.
mentagrophytes, the cause of tinea pedis (athlete’s foot). Thereare also reports of finding yeasts (e.g., Candida albicans and other Candida species) at beaches in the United States, Portugal,and France.25–29 Scopulariopsis and Candida are suggested en- 1. SINGH, H. 2006. Mycoremediation: Fungal Bioremediation. John vironmental indicators of sand beach quality for the sand 2. HAWKSWORTH, D.L. 1991. The fungal dimension of diversity: mag- c. Survival after chlorination: Fungi are more resistant to nitudes, significance, and conservation. Presidential address, 1990.
chlorination than most bacteria.30,31 In one study, more chlorine was required to inactivate C. parapsilosis32,33 (a commonly 3. BRIZZIO, S., B. TURCHETTI, V. DEGARCIA, D. LIBKIND, P. BUZZINIT & isolated yeast known to cause health problems in the tropics) M. VANBROOCK. 2007. Extracellular enzymatic activities of basid- than to inactivate coliform bacteria. Inactivation mechanisms via iomycetous yeasts isolated from glacial and subglacial water of chlorine on assimilative stages of yeasts and other microorgan- northwest Patagonia. Can. J. Micro. 53:519.
isms have been suggested.34 Fungal cells, especially conidia, can 4. ESSER, K. & P.A. LEMKE, EDS. 2001. The Mycota: A comprehensive treatise on fungi as experimental systems for basic and applied survive higher doses of chlorine than coliform bacteria.35 research, Volume VII: Systematics and Evolution, Part A. Springer-Verlag, Heidelberg, Germany.
5. NAGY, L.A. & B.H. OLSON. 1982. The occurrence of filamentous fungi in water distribution systems. Can. J. Microbiol. 28:667.
There are two basic modes of fungal growth in water. True 6. NIEMI, R.M., S. KUNTH & K. LUNDSTROM. 1982. Actinomycetes and aquatic fungi produce zoospores or gametes that are motile via fungi in surface waters and potable water. Appl. Environ. Micro- flagella. Aquatic fungi typically are collected by exposing suit- able baits (solid foodstuffs, such as wood, insects, and seeds) in 7. HINZELIN, F. & J.C. BLOCK. 1985. Yeast and filamentous fungi in the habitat being examined or in a laboratory sample. This is drinking water. Environ. Technol. Lett. 6:101.
most effective if the material is kept in a moist chamber (e.g., a 8. ROSENZWEIG, W.D., H. MINNIGH & W.O. PIPES. 1986. Fungi in potable water distribution systems. J. Am. Water Works Assoc. large Petri dish with water added). Direct plating involves plac- ing the material or water sample directly onto an agar surface or 9. HAGESKAL, G., A.K. KNUTSEN, P. GAUSTAD, G. SYBREN DE HOOG & mixing it with agar and then pouring the mixture into Petri I. SKAAR. 2006. Diversity and significance of mold species in dishes. The material also can be diluted and then directly plated.
Norwegian drinking water. Appl. Environ. Microbiol. 72:7586.
Cell culture purification may require several transfers.
10. BURMAN, N.P. 1965. Symposium on consumer complaints. 4. Taste The second fungal growth mode is nonmotile in all stages of and odour due to stagnation and local warming in long lengths of the life cycle. Growth and reproduction usually are asexual piping. Proc. Soc. Water Treat. Exam. 14:125.
(anamorphic). Two growth processes have been recognized: 11. METZLER, D.F., C. RITTER & R.L. CULP. 1956. Combined effect of • filamentous growth with blastic spores or spores produced in water purification processes on the removal of Histoplasma capsu- special structures and which includes single-celled growth on latum from water. Am. J. Pub. Health 46:1571.
each parent cell (called budding), typical of such yeasts as ASTELLANI, A. 1963. The cultivation of pathogenic fungi in sterile distilled water. Commentarii 1:1.
Candida and Cryptococcus, which include human pathogens, 13. COOKE, W.B. & P.W. KABLER. 1953. The survival of Histoplasma capsulatum in water. Lloydia 16:252.
• filamentous growth in which the filaments fragment to form 14. BAYS, L.R., N.P. BURMAN & W.M. LEWIS. 1970. Taste and odour in separate spores called arthroconidia (as in Geotrichum, Tricho- water supplied in Great Britain: A survey of the present position and problems for the future. Water Treat. Exam. 19:136.
15. NYSTROM, A., A. GRIMVALL, C. KRANTZ-RULCKER, R. SAVENHED & 37. BLACKWELL, M. & J.W. SPATAFORA. 2004. Fungi and their allies. In K. AKERSTRAND. 1992. Drinking water off-flavour caused by 2,4,6- Biodiversity of Fungi, Inventory and Monitoring Methods. Elsevier trichloroanisole. Water Sci. Technol. 25:241.
Academic Press, New York, N.Y., p. 7.
16. DOGGETT, M.S. 2000. Characterization of fungal biofilms within a 38. MULLER, G.M., G.F. BILLS & M.S. FOSTER, EDS. 2004. Biodiversity municipal water distribution system. Appl. Environ. Microbiol. of Fungi, Inventory and Monitoring Methods. Elsevier Academic 17. NAGY, L.A. & B.H. OLSON. 1985. Occurrence and significance of 39. KURTZMAN, C.P. & J.W. FELL. 1998. The Yeasts, A Taxonomic bacteria, fungi, and yeasts associated with distribution pipe sur- faces. In Proc. Water Quality Technology Conference, Dec. 8 –11, 40. KURTZMAN, C.P. & C.J. ROBNETT. 1998. Identification and phylog- 1985, Houston, Tex., p. 213. American Water Works Assoc., Den- eny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie van Leewenhoek 18. GELDRICH, E.E. 1995. Microbial Quality of Water Supply in Distri- bution Systems. Lewis Publishers, New York, N.Y.
41. SCORZETTI, G., J.W. FELL, A. FONSECA & A. STATZELL-TALLMAN.
19. LATGE´, J-P. 1999. Aspergillus fumigatus and aspergillosis. Clin. 2002. Systematics of basidiomycetous yeasts: a comparison of large sub-unit D1D2 and internal transcribed spacer rDNA regions.
20. VISHNIAC, H.S. 1957. Salt requirements of marine phycomycetes.
42. GUARRO, J., J. GENE & A.M. STICHIGEL. 1999. Development in 21. WORLD HEALTH ORGANIZATION. 2003. Chapter 6. Microbial aspects Fungal taxonomy. Clin. Microbiol. Rev. 12:454.
of beach sand quality. In Guidelines for Safe Recreational Water 43. SCHENA, L., A. IPPOLITO & D. GALITELLI. 2004. Real-time quantita- Environments, Vol. 1. Coastal and Fresh Waters, WHO, Geneva, tive PCR: a new technology to detect and study phytopathogenic and antagonistic fungi. Eur. J. of Plant Pathol. 110:893.
22. AJELLO, L. & M.E. GETZ. 1954. Recovery of dermatophytes from shoes and shower stalls. J. Invest. Derm. 22:17.
AUGLAND, R.A., N. BRINKMAN & S.J. VESPER. 2002. Evaluation of rapid DNA extraction methods for the quantitative detection of HO, R. & H. HIRN. 1981. A survey of fungi and some indicator bacteria in chlorinated water of indoor public swimming pools.
fungi using real-time PCR analysis. J. Microbiol. Methods 50:319.
Zentralblatt fu¨r Bakteriologie und Hygiene B. 173:242.
45. BOWYER, P., L. HOARE & E. DENNING. 2007. Detection of fungi in hospital water supplies using molecular beacons. In Proc. 17th AMIHAMA, T., T. KIMURA, J.-I. HOSOKAWA, M. UEJI, T. TAKASE & European Congress of Clinical Microbiology and Infectious Dis- AGAMI. 1997. Tinea pedis outbreak in swimming pools in Japan.
eases (ECCMID) & 25th International Congress of Chemotherapy (ICC), March 31–April 3, 2007, Munich, Germany. European So- ISHIMOTO, R.A. & G.E. BAKER. 1969. Pathogenic and potentially pathogenic fungi isolated from beach sands and selected soils of ciety of Clinical Microbiology and Infectious Diseases, Basel, Oahu, Hawaii. Mycologia 61:539.
26. MULLER, G. 1973. Occurrence of dermatophytes in the soils of European beaches. Sci. Total Environ. 2:116.
27. MENDES, B., P. URBANO, C. ALVES, J. MORAIS, N. LAPA & J.S.
OLIVEIRA. 1998. Fungi as environmental microbiological indicators.
Water Sci. Technol. 25:241.
EMERSON, R. 1958. Mycological organization. Mycologia 50:589.
28. FIGUEIRA, D. & M. BARATA. 2007. Marine Fungi from two sandy SPARROW, F.K. 1959. Fungi (Ascomycetes, Phycomycetes); including beaches in Portugal. Mycologia 99:20.
W.W. Scott, Key to genera, Fungi Imperfecti (Aquatic Hyphomy- 29. VOGEL, C., A. ROGERSON, S. SCHATZ, H. LAUBACH, A. TALLMAN & cetes only). In W.T. Edmondson, ed. Ward & Whipple’s Fresh J.W. FELL. 2007. Prevalence of yeasts in beach sand at three bathing Water Biology, 2nd ed. John Wiley & Sons, New York, N.Y.
beaches in south Florida. Water Res. 41:1915.
COOKE, W.B. 1963. A Laboratory Guide to Fungi in Polluted Waters, 30. ENGELBRECHT, R.S., D.H. FOSTER, E.O. GREENING & S.H. LEE. 1974.
Sewage, and Sewage Treatment Systems, Their Identification and New microbial indicators of waste water efficiency. Environ. Pro- Culture, USPHS Publ. 999-WP-1. U.S. Public Health Service, tect. Technol., Series No. 670/2-73-082.
31. JONES, J. & J.A. SCHMITT. 1978. The effect of chlorination on the COOKE, W.B. & G.S. MATSUURA. 1969. Distribution of fungi in a waste survival of cells of Candida albicans. Mycologia 70:684.
stabilization pond system. Ecology 50:689.
32. ENGELBRECHT, R.S. & C.N. HAAS. 1977. Acid-fast bacteria and yeasts as disinfection indicators: Enumeration methodology. In ONES, E.B.G. 1971. Aquatic Fungi. In C. Booth, ed. Methods in Mi- Proc. Water Quality Technology Conference, Dec. 4 –7, 1977.
crobiology, Vol. 4. Academic Press, New York, N.Y., p. 335.
Kansas City, Mo. American Water Works Association, Denver, GARETH JONES, E.B., ed. 1976. Recent Advances in Aquatic Mycology.
FULLER, M.S., ed. 1978. Lower Fungi in the Laboratory. Palfrey Contrib. AAS, C.N. & R.S. ENGELBRECHT. 1980. Chlorine dynamics during inactivation of coliforms, acid-fast bacteria, and yeasts. Water Res. in Botany 1:1–212. Athens, Ga.
COOKE, W.B. 1986. The Fungi of “Our Mouldy Earth.” Beihefte zur AAS, C.N. & R.S. ENGELBRECHT. 1980. Physiological alterations of vegetative microorganisms resulting from chlorination. J. Water ALEXOPOULOS, C.J. & C.W. MIMS. 1996. Introductory Mycology, 4th ed.
Pollut. Control Fed. 52:1976.
John Wiley & Sons, New York, N.Y.
35. ROSENZWEIG, D.W., H.A. MINNIGH & W.O. PIPES. 1983. Chlorine BROCK, T.D. 1997. Biology of Microorganisms. Prentice-Hall, Engle- demand and inactivation of fungal propagules. Appl. Environ. Mi- DEACON, J., ed. 2006. Fungal Biology, 4th ed. Blackwell Publishing, 36. BLACKWELL, M., D.S. HIBBETT, J.W. TAYLOR & J.W. SPATAFORA.
2006. Research coordination networks: a phylogeny for kingdom BUCKLEY, M. 2008. The Fungal Kingdom: Diverse and Essential Roles in Earth’s Ecosystem. Amer. Acad. Microbiol., Washington, D.C.
Dipotassium hydrogen phosphate, K HPO . . . . . . . . . . . . . . . .
Magnesium sulfate, MgSO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
a. Containers: Collect samples as directed in Sections 9060A Potassium chloride, KCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
and 9610A.3. Alternatively, use sterile cylindrical plastic vials Ferrous sulfate, FeSO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
with snap-on caps. Transport them in an upright position to Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.0 g minimize the chance of leakage, and discard after use.
Reagent-grade water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
b. Storage: Hold samples no more than 24 h. If analysis is not begun promptly after sample collection, refrigerate at 2 to 8°C.
Combine these ingredients and heat to dissolve. Autoclave for 15 min at 121°C. The pH should be 7.3 Ϯ 0.2 after This medium is useful for isolating species of Aspergillus, Various media (e.g., potato dextrose agar, cornmeal agar, and Penicillium, Paecilomyces, and some other fungi with similar malt extract agar) are used to isolate, identify, and enumerate yeasts and molds.1,2 Neopeptone-glucose-rose bengal-aureomy- c. Yeast extract-malt extract-glucose agar: cin® agar is the usual medium of choice when estimating viableunits of most fungi (molds or yeasts), especially if bacterial Yeast extract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
contamination may be present. However, experience may indi- Malt extract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
cate that Czapek agar (often used for Aspergillus, Penicillium, Neopeptone (or equivalent) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
and related fungi), yeast extract-malt extract-glucose agar, or Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.0 g malt extract agar (often used for yeasts) may be preferable.
Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.0 g For consistency in media quality, use commercially prepared Reagent-grade water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
versions of the following media, whenever available. For inven-tory or culture maintenance, use neopeptone-glucose agar. Re- Combine these ingredients and heat to dissolve. Autoclave for agent-grade water (as defined in 9020B.4d) or laboratory-puri- 15 min at 121°C. No pH adjustment is required.
This medium is useful for isolating yeasts.
a. Neopeptone-glucose-rose bengal aureomycin® agar: Neopeptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Maltose, technical. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.75 g Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.0 g Dextrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Rose bengal solution (1g/100 mL reagent-grade water) . . .
Glycerol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.0 g Peptone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reagent-grade water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.0 gReagent-grade water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CAUTION: Rose bengal is irritating to eyes, respiratory
system, and skin. Combine these ingredients, bring to boil, and
Combine these ingredients and heat to dissolve. Autoclave for then sterilize via autoclaving for 15 min at 121°C. The final pH 15 min at 121°C. No pH adjustment is required. The medium will be turbid, but filtration is unnecessary.
Because this medium is used to make pour plates, prepare and This medium is useful in purifying yeast isolates and studying store basal agar either in bulk or (more conveniently) in tubes in yeast species in various specified tests. It is also useful for maintaining stock cultures. This medium is comparable to neo- Separately prepare a solution of aureomycin [1.0 g of chlor- peptone-glucose-rose bengal aureomycin agar but contains nei- tetracycline (water-soluble antibiotic) and 150 mL of distilled ordeionized water] and refrigerate. Before use, sterilize via filtra- tion through a 0.2-␮m pore size sterilizing-grade membrane. To complete the medium, add 0.05 mL sterile aureomycin solutionto 10 mL sterilized basal agar tempered at 44 to 46°C.
Neopeptone (or equivalent) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
If this medium is unavailable in dehydrated form, prepare it Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.0 g from the basic ingredients. Dehydrated Cooke’s rose bengal agar Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.0 g may be used in place of the agar base, but then store and incubate Reagent-grade water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
the resulting medium in the dark to prevent the photosensitizeddye from inhibiting fungi.
Combine these ingredients and autoclave for 15 min at This medium is useful for isolating a broad spectrum of fungal 121°C. The pH should be 6.5 Ϯ 0.2 after sterilization. (This medium is similar to Sabouraud Agar or Sabouraud Dextrose This medium is useful for maintaining stock cultures. It is Sucrose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30.0 g comparable to neopeptone-glucose-rose bengal aureomycin® Sodium nitrate (NaNO ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
agar but contains neither rose bengal nor an antibiotic.
inventory will give relative importance to, at least, the morereadily identifiable species or genera.
a. Preparation and dilution: To a sterile 250-mL erlenmeyer When preparing plates, use sample portions that will give flask, add 135 mL sterile reagent-grade water and 15 mL sample about 50 to 60 colonies on a plate. Determine this volume by trial to obtain a 1:10 sample dilution. Use a sterile measuring device and error. When first examining a new habitat, plate at least two for each sample, or (less preferably) rinse the device with sterile sample dilutions. Estimates of up to 300 colonies may be made, reagent-grade water between samples. Mix sample well before but discard more crowded plates. The medium containing rose withdrawing the 15-mL portion. Shake flask on a rotary shaker bengal tends to produce discrete colonies and limits radial at about 120 to 150 oscillations/min for about 30 min, or transfer growth (and thereby the size of mold colonies), permitting slow- flask contents to a blender jar, cover, and blend at low speed for growing organisms to develop and be observed. Counting limits 1 min or at high speed for 30 s. Use a sterile blender jar and may be raised or lowered at the analyst’s discretion.
appurtenances for each sample, or (less preferably) wash jar If five plates are used per sample, the average number of thoroughly between samples and rinse with sterile water. Further colonies on all plates (total number of colonies counted/5) times dilutions may be made by adding 45 mL sterile water to 5 mL of the reciprocal of the dilution (10/1, 100/1, 1000/1, etc.) equals the fungus colony count per milliliter of original sample. Note For stream water samples, a dilution of 1:10 usually is ade- that each colony may have resulted from one or more hyphalfragments. For solid or semisolid samples, use a correction for quate. Dilute samples containing large amounts of organic ma- the water content to report fungus colonies per gram dry weight.
terial (e.g., sediments) to 1:100 or 1:1000. Dilute stream bank or Determine water content by drying paired 15-mL portions of original sample at 100°C overnight; the difference between wet b. Plating: Prepare five plates for each dilution to be exam- and dry weights is the amount of water lost from the sample.
ined. To use neopeptone-glucose-rose bengal-aureomycin® agar, The inventory includes the direct identification of fungi based aseptically transfer 10 mL of medium at 44 to 46°C to a 9-cm on colonial morphology and the counting of colonies assignable petri dish. Add 1 mL of appropriate sample dilution and mix to various species or genera. When discrete colonies cannot be thoroughly by tilting and rotating dish (see plating procedure identified, and identification is important, use a nichrome wire under heterotrophic plate count, Section 9215B). Alternatively, (20 to 24 gauge, with its tip bent in an L-shape and flattened by add 1 mL sample, 0.05 mL antibiotic solution, and 10 mL hammer) to pick or cut a segment of growth from each selected liquefied agar medium to petri dish at 44 to 46°C. Solidify agar colony and streak on a slant of neopeptone-glucose agar as rapidly as possible. (In arid areas, use more medium to (9160B.2e). Incubate slants at growth temperature until a lawn of growth is observed. Isolation and identification can be attempted c. Incubation: Plates should be stacked no more than three again, or the slant can be stored under refrigeration for 3 to 4 weeks.
high, but do not invert. Incubate at room conditions and ambientlighting, or in the dark at 18 to 25°C. Avoid direct sunlight.
Examine plates and count colonies on each plate after 3, 5, and 7 d. Continue incubation and observe plates weekly because ILLS, G.F. & M.S. FOSTER. 2004. Formulae for selected materials used to isolate and study fungi and fungal allies. In Biodiversity of some fungi grow slowly. Avoid opening these plate cultures Fungi, Inventory and Monitoring Methods. Elsevier Academic Press, because this may increase airborne contamination.
d. Counting and inventory: The fungal plate count will provide 2. BEUCHAT, L.R. 1992. Media for detecting and enumerating yeasts and the basis for rough quantitative comparisons among samples; the moulds. Int. J. Food Microbiol. 17:145.
The spread plate technique is another procedure for obtaining a. Neopeptone-glucose-rose bengal aureomycin® agar: See quantitative data on colony-forming units.
b. Czapek (or Czapek-Dox) agar: See 9610B.2b.
c. Yeast extract-malt extract-glucose agar: See 9610B.2c.
d. Malt extract agar: See 9610B.2d.
See Sections 9060, 9610A.3, and 9610B.1.
e. Cooke’s rose bengal medium with Aureomycin®:This medium is similar to ¶ a above but is commercially Aureomycin®-rose bengal-glucose-peptone agar (¶ e below) Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
and streptomycin-terramycin®-malt extract agar (¶ f below) are Peptone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
useful in analyzing sewage and polluted waters.1 Use commer- Potassium dihydrogen phosphate, KH PO . . . . . . . . . . . . .
cially prepared media for those listed below whenever available.
Reagent-grade water (as defined in 9020B.4.d) or laboratory Rose bengal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
purified water (distilled or deionized) may be used.
Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reagent-grade water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800 b. Plating: Pre-dry plates separately with lids slightly ajar at room temperature and about 30% relative humidity for 1 to 1.5 h.
CAUTION: Rose bengal is irritating to eyes, respiratory
A sterility control plate is needed to assess possible airborne system, and skin. Heat to dissolve, bring to a boil, and then
contamination; it should be carried through the incubation pro- autoclave for 15 min at 121°C. Dissolve 70.0 mg aureomycin cess. Using a sterile pipet, transfer 0.1 mL of sample or dilution (chlortetracycline) hydrochloride in 200 mL reagent-grade wa- onto surface of a pre-dried agar plate. Spread sample over entire ter, filter–sterilize solution using a 0.2-␮m pore size sterilizing- agar surface using a sterile L-shaped glass rod or use a mechan- grade membrane, and add to the cooled (42 to 45°C) agar base.
ical device to rotate plate and ensure proper sample distribution.
No pH adjustment is necessary. Pour 25-mL portions into sterile c. Incubation: With dish covers on, let plates dry at room petri dishes (100 ϫ15 mm) and let agar harden. Poured plates temperature, invert plates, and incubate at 18 to 25°C for up to 7 d may be held up to 4 weeks at 2 to 8°C. Check for dehydration in an atmosphere of high humidity (90 to 95%). Slow-growing fungi may not produce noticeable colonies until 6 or 7 d.
f. Streptomycin-terramycin®-malt extract agar: d. Counting and recording: Using a darkfield colony counter or a binocular microscope, count all colonies on each selected plate. If Malt extract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
counting must be delayed temporarily, hold plates at 2 to 8°C for no Peptone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
longer than 24 h to avoid contamination and further spreading of Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
colonies. Depending on colony size, plates with as many as 150 Reagent-grade water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800 colonies can be counted, but the optimal maximum number is 100colonies. Counting limits may be raised or lowered at the analyst’s Heat to dissolve and autoclave the 800 mL of agar-based medium for 15 min at 121°C. Dissolve 70.0 mg of streptomycin Multiply counts by dilution factor and then record results as and 70.0 mg of terramycin (oxytetracycline) in separate 100-mL colony-forming units (CFU)/100 mL original sample. For solid or portions of reagent-grade water, sterilize each via filtration, and semisolid samples, report CFU/g wet or dry (preferably dry). If add to the cooled (42 to 45°C) agar base. (The total volume is three or more plates are used per sample, use average number of now 1000 mL.) The pH should be 5.4 Ϯ 0.2. Pour about 20-mL colonies times the reciprocal of the dilution (see 9610B.3d) to give portions into sterile petri dishes (60 ϫ 15 mm) and let agar colony count. If no plates have colonies, record count as Ͻ1 for the harden. Poured plates may be held up to 4 weeks at 4°C. Check highest dilution. If the plate colonies are too crowded to count, record as “too numerous to count” (TNTC) but indicate a count of Ͼ150 for the appropriate dilution. If colonies are crowded andoverlapping with spreaders, record as “obscured” (OBSC) and repeat analysis with higher dilution or earlier observations.
a. Preparation and dilution: See Sections 9215A.5 and 9610B.3a. Make dilutions with buffered water (Section 1. EL-SHAARAWI, A., A.A. QURESHI & B.J. DUTKA. 1977. Study of 9050C.1a) and select dilutions that yield 200 to 1500 colonies microbiological and physical parameters in Lake Ontario adjacent to the Niagara River. J. Great Lakes Res. 3:196.
For general information on the membrane filter technique and ified media described in 9610C.2e and f except that the concen- apparatus needed, see Section 9222. However, except for com- tration of each antibiotic is increased from 70 to 200 mg/L.
parisons of different manufacturers’ membranes, there are no Dispense media in portions of 5 to 7 mL in glass or plastic petri critical test reports for the fungal isolation efficiency of mem- dishes (60 ϫ15 mm); plastic dishes with tight-fitting lids are brane filters. Media components, pH levels, and antibiotics have been used in routine plating procedures. The reported proceduresseem to be satisfactory.
a. Preparation and dilution: See Sections 9215A.5 and 9610B.3a. Select dilutions to yield 20 to 100 colonies per mem- b. Filtration: Filter appropriate volumes of well-shaken sam- ple or dilution, in triplicate, through membrane filters with porediameter of 0.45 or 0.8 ␮m (see Section 9222.) Use aureomycin-rose bengal-glucose-peptone agar, Cooke’s c. Incubation: Transfer filters to dishes, invert dishes, and rose bengal agar, or modified streptomycin-terramycin-malt ex- incubate at 18 to 25°C for 3 to 5 d in a humid atmosphere to tract agar.1 These media are prepared identically to the unmod- d. Counting and recording: Using a binocular dissecting mi- croscope at a magnification of 10ϫ, count all colonies on eachselected plate. If counting must be delayed temporarily, hold 1. QURESHI, A.A. & B.J. DUTKA. 1978. Comparison of various brands of plates at 4°C for no longer than 24 h. Ideal plates have 20 to 80 membrane filter for their ability to recover fungi from water. Appl. colonies per filter (see 9610C.3d.) Yeasts are single-cell fungi with typically one nucleus per cell; c. Isolation: Remove flasks from shaker and let settle 4 to 5 h.
they do not produce filaments and reproduce via binary fission Yeast cells, if present, will settle to the bottom, bacteria and (e.g., Schizosacchromyces pombe) or budding (e.g., Cryptococ- filamentous fungi will remain in suspension, and filamentous cus spp.). Sparse to extensive hyphal growth may or may not fungi will float on the surface or attach to the glass surface at or occur. Candida albicans was found to form mycelial growth above the meniscus. With a nichrome wire loop, remove a under anaerobic conditions.1 Solid media, such as those described loopful of sediment at the sediment–supernatant interface from a above, do not permit all yeasts to grow, so an enrichment technique tilted flask and smear/streak on yeast extract-malt extract- may be useful in addition to the plate count (see also 9610H).
glucose agar. Use three plates per flask. Incubate at room tem-perature but out of direct sunlight for 2 to 3 d. It is not necessary to invert dishes. To obtain pure cultures, pick from reasonablyisolated colonies and restreak on the same medium or on malt For enrichment, use yeast nitrogen base-glucose broth; for extract agar plates. Obtain pure cultures of as many colonies as isolation, use yeast extract-malt extract-glucose agar or malt d. Counting: It is impossible to obtain a meaningful plate a. Yeast nitrogen base-glucose broth: Dissolve 13.4 g yeast count after this type of enrichment isolation because this is a nitrogen base in 1 L reagent-grade water; sterilize via filtration.
qualitative test. An estimate of density could be made if several Prepare 500 mL each of 2% and 40% aqueous glucose solutions flasks were used and they were treated as a serial dilution test.
and sterilize separately via filtration. To make final medium, An MPN-type calculation could estimate the original density.
aseptically add 25 mL yeast nitrogen base solution and 25 mL ofeither 2% or 40% glucose solutions to a sterile 250-mL erlen- meyer flask. The final glucose concentration should be 1 or 20%,respectively. Stopper flask with a gauze-wrapped cotton stopper 1. DUMITRU, R., J.M. HORNNBY & K.W. NICKERSON. 2004. Defined anaerobic growth medium for studying Candida albicans, basic b. Yeast extract-malt extract-glucose agar: See 9610B.2c.
biology and resistance to eight antifungal drugs. Antimicrob. Agents c. Malt extract agar: See 9610B.2d.
a. Sample preparation and dilution: Prepare as directed in LODDER, J., ED. 1970. The Yeasts, a Taxonomic Study, 2nd ed. North b. Enrichment: In 250-mL erlenmeyer flasks, prepare one flask BUCK, J.D. 1975. Distribution of aquatic yeasts— effect of inoculation of yeast nitrogen base medium containing 1% and one contain- temperature and chloramphenicol concentration on isolation. My- ing 20% glucose. Inoculate with 1 mL of appropriate sample dilution and incubate at room temperature on a rotary shaker KURTZMAN, C.P. & J.W. FELL. 2004. Yeasts. In Biodiversity of Fungi, operating at 120 to 150 oscillations/min for at least 64 h. Shaken Inventory and Monitoring Methods. Elsevier Academic Press, New cultures prevent the overgrowth of filamentous fungi.
habitats, and many live in parasitic relationships with algaeand other fungi.
The phylum Chytridiomycota is the only fungal group to Oomycota (commonly called water molds) have a similar include representatives with a flagellated stage. Chytrids are morphology, but are not fungi because of their glucan/cellulose zoosporic fungi that reproduce asexually via motile, uniflag- cell walls and the diploid nuclei in their non-septate vegetative ellate spores. These fungi may be found in lake and river body.1 Oomycetes produce both oospores and biflagellate zoo- spores. (Taxonomists recently placed them with algae, but they will be discussed under fungi in this revision.) Although polluted river areas have fewer species, they contain Once grown on baiting material, chytrids can be transferred to more Oomycetes than Chytridiomycetes. Species of the Oomy- a modified nutrient agar medium containing antibiotics.3 Al- cete genera, Leptomitus and Saprolegnia (notably S. ferax), seem though most filamentous Oomycetes can be cultivated on plain to be more tolerant than other forms. Saprolegnia spp. are cornmeal agar, selective media have been developed to isolate considered opportunistic facultative parasites that cause infec- tions in fish and fish eggs,2 important in aquaculture.
Obtain pure cultures by placing a small portion of material onto a depression slide with one or two drops of distilled waterand drawing spores into a micropipet as they emerge from the sporangium. Hyphal tips could be used, but are less preferablebecause one piece of bait frequently contains several genera and These fungi rarely develop in sufficient numbers to be ob- species. Transfer the spore suspension or hyphal tip to a plate of served or collected directly, so various techniques have been cornmeal agar. Once growth occurs, remove bacteria-free hyphal devised for their collection and isolation. Collect samples in tips aseptically by cutting out a small block of agar. Transfer to sterile 35-mL plastic vials, refrigerate, and (ideally) start analysis fresh medium or water. If growth is not free from contamination within 6 to 8 h. Place each sample in a sterile plate (20 ϫ 100 after one transfer, make additional transfers to ensure pure cul- mm) and dilute with 10 to 15 mL sterile reagent-grade water. As tures. Contaminants can also be cut out of the agar medium.
bait, add three to four split hemp seed halves (Cannabis sativa)† or whole mustard (Brassica) or sesame (Sesamum) seeds to each culture. (All bait material should be boiled for 3 to 5 minutes orautoclaved to reduce or remove extraneous microorganisms.2) Make serial dilutions with sterile reagent-grade water (1:1 ϫ Incubate at 18 to 25°C (the temperature closest to the environ- 105 to 1:7 ϫ 105) and spread 1 mL over the surface of a freshly mental condition where found) and examine bait daily for fungal prepared cornmeal agar plate. Remove each developing colony growth. As growth becomes evident (usually within 72 h), re- and transfer to water for identification. This method also permits move infected bait, wash it thoroughly using water from a wash numerical estimation, as well as determination of the Oomycete bottle, and then transfer the cleaned bait to a fresh plate of water community’s composition; however, at least three to five plates containing two to three halves of hemp or other seed. Alterna- per dilution are needed over several dilutions, or else count 10 tively, collect material that may include these fungi (e.g., algae, plates at one dilution for estimation purposes.
waterlogged material, or insect bodies3) and wash off. The waterwash can be placed on a depression slide for 72 h or less for microscopic observation or placed on appropriate culture media 1. SIGEE, D.S. 2005. Chapter 8. Fungi and fungal-like organisms: and incubated at the same temperature and checked daily for aquatic biota with a mycelial growth form. In Freshwater Microbi- growth and further identification or other studies.
ology: Biodiversity and Dynamic Interactions of Microorganisms in Some chytrids may develop more slowly, and incubation for the Aquatic Environment. John Wiley & Sons, Ltd., West Sussex, up to 1 week may be necessary. Other bait material can be used [e.g., purified shrimp exoskeleton (chitin) or corn straw (cellu- 2. NEISH, G.A. & G.C. HUGHES. 1980. Diseases of fishes, Book 6, Fungal Diseases of Fishes. T.W.F. Publications, Neptune, N.J.
3. SHEARER, C.A., D.M. LANGSAM & J.E. LONGCORE. 2001. Fungi in Genera may be identified via the spore arrangement in the freshwater habitats. In Biodiversity of Fungi, Inventory and Moni- sporangium and the manner in which spores are released. Spe- toring Methods. Elsevier Academic Press, New York, N.Y., p. 513.
cific determination requires microscopic examination of the sex- 4. HO, H.H. 1975. Selective media for the isolation of Saprolegnia spp.
from fresh water. Can. J. Microbiol. 21:1126.
To collect the few naturally occurring parasites or pathogens, place the host organisms in a plate containing sterile water and WILLOUGHBY, L.G. 1962. The occurrence and distribution of reproduc- tive spores of Saprolegniales in fresh water. J. Ecol. 50:733.
KAMOUN, S. 2003. Molecular genetics of pathogenic oomycetes. Eukary- † It is not legal to possess seeds of Cannabis sativa unless they are sterile/infertile.
angiosperms. Ecological investigations of freshwater hyphomy-cetes have been limited to substrate, habitat, dispersal, and their Freshwater Hyphomycetes are a specialized group of conidial role in the enhancement of leaf substrates as food for aquatic fungi that usually occur on the submerged, decaying leaves of invertebrates. The usual habitat of the fungi is well-oxygenated water (e.g., alpine brooks, mountain streams, and fast-flowing Within 1 to 2 d, mycelium and conidia develop. Conidiophores rivers), but they also have been found in slow-running, often and conidia can be observed with a dissecting microscope on any contaminated rivers, stagnant or temporary pools, melting snow, portion of a leaf surface, but they are most frequently seen on and soil. The numbers of species and individuals of aquatic petioles and veins. When released, conidia either remain sus- hyphomycetes often increase from autumn until spring and de- pended in water or settle to the bottom of the dish.
cline between late spring and early summer.
Using a dissecting microscope, pick up single conidium with The mycelium, which is branched and septate, ramifies a micropipet. Transfer each conidium in a drop of water to a through the leaf tissue, especially in petioles and veins. The microscope slide for identification. Conidia may be transferred conidiophores project into the water, and the conidia that usually with a sterile needle to a plate of 2% malt extract agar (pH 6.5) develop are liberated under water. Mature conidia also can be for colony production and then maintained on this medium at 20 found in the surface foam of most rivers, streams, and lakes.
Ϯ 2°C until recultivation or disposal is needed. Taping plates Most of these conidia are hyaline, thin-walled, and either tetra- radiately branched (four divergent arms) or sigmoid (S-shaped), Search for conidia in foam samples with a dissecting micro- with the curvature in more than one plane. The conidia do not scope and isolate single conidia as described above. Submerge germinate while suspended in water— even for long periods— mycelial plugs from stock culture isolates of aquatic Hyphomy- but on a solid surface, they will produce germ tubes within a few cetes in autoclaved pond water in deep petri dishes; conidia hours. Their spores’ size and morphology make them potentially more prominent in plankton analysis work than the spores of Conidia in all stages of development can be preserved on slides with lactophenol mounting medium in which either acidfuchsin or cotton blue (optional) is dissolved, and sealed with clear fingernail polish. To permit good adherence of the nailpolish, avoid excessive amounts of mounting medium.
For most freshwater environments, collect foam or partially decayed, submerged, angiosperm leaves in sterile bottles. Re-frigerate sample until analysis.
BARLOCHER, F. 1992. Research on aquatic hyphomycetes: historical background and overview. In F. Barlocher, ed. The Ecology of Wash leaf samples in sterile distilled water and place one to Aquatic Hyphomycetes. Springer-Verlag, Berlin, Germany.
three leaves in a sterile petri dish (about 1 cm deep) containing SATI, S.C. & S. BISHT. 2006. Utilization of various carbon sources for the sterile pond, river, or lake water. Incubate at room temperature.
growth of waterborne conidial fungi. Mycologia 98:678.
mum is the imperfect state of the fungus Pseudallescheria boydii,one of 16 species of true fungi that may cause mycetoma in Opportunistic fungi in hospital water systems are problematic humans.) Infection may be the result of a puncture wound by for hospital patients recovering from illnesses and those who are contaminated materials or of breathing contaminated air or water immunocompromised.1–4* However, routine isolations of fungi sprays. For a full discussion of pathogenic fungi, examine the from polluted streams and wastewater treatment plants usually yield relatively few species pathogenic to humans and other The presence of these fungi in stream water probably indicates higher animals. Most pathogenic fungi are ascomycetes, al- soil runoff, because soil is the natural habitat of virtually all though there are several human and animal pathogens among the zoopathogenic fungi. Other zoopathogenic fungi occasionally basidiomycetous genera (e.g., Cryptococcus, Trichosporon, and are recovered from streams (whether polluted or not).
Malassezia). Exophiala mansonii—also called Phialophora Another fungus, the yeast Candida albicans, can be recovered jeanselmei and Trichosporium heteromorphum—is isolated uni- in varying numbers from wastewater treatment plant effluents, versally and can cause one form of chromomycosis (usually in streams receiving such effluents, and recreational waters. This is the tropics). Aspergillus fumigatus, which can cause pulmonary not surprising because C. albicans is usually a commensal or- aspergillosis, is commonly isolated. Pseudallescheria (Petriel- ganism in humans, coexisting in harmony with its host and lidium, Allescheria) boydii can cause eumycotic mycetomas and resistant to several antifungal drugs.5 Up to 80% of normal, other eumycotic conditions grouped under “Pseudallescheria- healthy adults have detectable levels of C. albicans in their feces, sis”;1 it usually is recovered in its anamorphic state, Scedospo- while about 35% harbor it in their oral cavities in the absence of rium (Monosporium) apiospermum. (Scedosporium apiosper- any overt disease. Up to 50% of healthy asymptomatic femalesmay harbor C. albicans in their lower genital tract microflora.
C. albicans has been isolated on routine media heavily sup- * They are of great concern also in therapeutic pools and recreational waters, bothindoors and outdoors.
plemented with antibacterial antibiotics and cycloheximide, and it also has been isolated from estuarine and marine habitats on a arium species in a hospital water system: a new paradigm for the epidemiology of opportunistic mould infections. Clin. Infect. Dis. 2. WARRIS, A., P. GAUSTAD, J.F.G.M. MEIS, A. VOSS, P.E. VERWEIJ & T.G. ABRAHAMSEN. 2001. Recovery of filamentous fungi from waterin a paedriatic bone marrow transplantation unit. J. Hosp. Infect.
47:143.
C. albicans is a facultative anaerobe and can be detected 3. ANAISSIE, E.J., S.L. STRATTON, M.C. DIGNANI, C. LEE, R.C. SUMMER- among the white and pink yeasts growing on a 0.8-␮m black BELL, J.H. REX, T.P. MONSON & T.J. WALSH. 2003. Pathogenic molds membrane filter on maltose-yeast nitrogen base-chlorampheni- (including Aspergillus species) in hospital water distribution systems: col-cycloheximide medium. From each colony, inoculate a a 3-year prospective study and clinical implications for patients with 0.5-mL portion of calf or human blood serum, incubate at 37°C hematologic malignancies. Blood 101:2542.
for 2 to 3 h, transfer a drop or two to a slide, and examine 4. BUCK, J.D. & B.M. BUBACIS. 1978. Membrane filter procedure for microscopically for the production of germ tubes from most enumeration of Candida albicans in natural waters. Appl. Environ. cells. Inoculation must be light, or germination rate can be reduced. Of the white yeasts, only C. albicans produces these 5. DUMITRU, R., J.M. HORNBY & K.W. NICKERSON. 2004. Defined anaer- obic growth medium for studying Candida albicans basic biology short hyphae from the parent cell within 2 to 3 h of incubation.4 and resistance to eight antifungal drugs. Antimicrob. Agents Che- Germ tube test interpretation needs to address pseudohyphal formation, which can be mistaken for a germ tube (true hyphal 6. BERMAN, J. & P.E. SUDBERY. 2002. Candida albicans: a molecular revolution built on lessons from budding yeasts. Nature 3:918.
1. ANAISSIE, E.J., R.T. KUCHAR, J.H. REX, A. FRANCESCONI, M. KASAI, F.M. MU¨LLER, M. LOZANO-CHIU, R.C. SUMMERBELL, M.C. DIGNANI, RIPPON, J. 1982. Medical Mycology. W.B. Saunders Co., Philadelphia, S.J. CHANNOCK & T.J. WALSH. 2001. Fusariosis and pathogenic Fus-

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