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Environmental Microbiology Laboratory, Inc., 1800 Sullivan Ave., Suite 209, Daly City, California 94015
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A modified mounting medium, Lacto-Cotton Blue, is described and evaluated for use with Indoor Air Quality spore-trap samples. The medium lacks phenol and is odorless, provides excellent rehydration, delicate staining, and is easy to apply.
Key words: cotton blue, lacto-phenol, microscopy, reagent, stain
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, Hawksworth et al 1996). Typically, the fixed cellular structures are examined under a standard bright-field microscope, using Kohler illumination. A myriad of chemicals is used to observe microscopic features of fungi and many are specific for particular anatomical structures. In general, these histological stains often contain compounds that are harmful in both short and long term applications. A common constituent of many histological stains is phenol, a compound that is harmful to the health of human analysts. In Indoor Air Quality analyses, a variety of stains and mounting agents are used to aid in the identification of spores deposited by inertial impaction sampling techniques, i.e., spore-trapping (Solomon 1984, Muilenberg and Burge 1994, Tsai et al 1999, Hess-Kosa 2001). The histological stain of choice for many mycological applications has been Lacto-Phenol Cotton Blue (L-P CB), also known as Amann's Solution, or Aniline Blue (Largent et al 1977, McGinnis et al 1982, St. Germain and Summerbell 1996, Jensen and Schafer 1998, Zefon International 2001). A principal component of L-P CB is phenol, a compound that is readily absorbed through the skin and known to produce a variety of adverse health effects from chronic contact, such as nausea, vomiting, and renal and hepatic damage. (Sigma-Aldrich 2000, Merck Index 2001). In order to reduce the possible volatile and long-term exposure effects of L-P CB, a reagent lacking phenol was developed to analyze inertial impaction samples. To date, approximately 20 000 individual spore-trap samples (Air-O-Cell cassette and Allergenco MK-5 Multi Trace Slide, Zefon International, St. Petersburg, Florida) have been analyzed using the new reagent, hereafter called Lacto-Cotton Blue (LCB). The recipe for Lacto-Cotton Blue mounting medium is: glycerol 250 mL (Sigma-Aldrich, St. Louis, Missouri), 85% lactic acid 100 mL (Sigma-Aldrich, St. Louis, Missouri), 3 mL of cotton blue stock, and de-ionized water 50 mL. A stock solution of Cotton Blue stain is made to add to the mounting medium: 85% lactic acid 99 mL, Aniline (Cotton) Blue crystals 1.0 g (Fisher Scientific, Los Angeles, California).
To make the Cotton Blue stock solution, first add cotton blue crystals to lactic acid while stirring vigorously on a stir-plate. Stir until cotton-blue crystals are dissolved. Next, vacuum filter the solution through a #50 Whatmann 90 mm filter disc (Fisher Scientific, Los Angeles, California) in a Büchner porcelain funnel. Cover the orifice of the porcelain funnel to insure airborne particulate matter cannot enter the reagent mixture. After filtration has occurred, check the clarity of the stock dye solution. Place one drop of the dye on a clean microslide with a 22 x 22 mm cover slip and examine at 400x. Particles of dye should be approximately 2 µm or less in diameter. If large clumps of dye are observed, repeat vacuum filtration of the dye solution until the appropriate clarity is obtained.
The next step is to make the LCB mounting medium. First, mix the water, lactic acid, and glycerine (in that order) for one hour on a stir-plate. Once the solution is homogenous, add three mL of Cotton Blue stock solution to the water, lactic acid, and glycerine solution. Stir the entire mixture for an additional hour. Cover the flask with laboratory film (Parafilm, Fisher Scientific, Los Angeles, California) while the mixture is stirring to insure against airborne contamination.
In all instances, samples mounted with LCB demonstrated proper rehydration of spores. The degree of staining achieved with the concentration of dye in this medium allows for subtle staining of hyaline, thin-walled spores such as many smaller basidiospores, while thicker-walled or pigmented spores (e.g., Penicillium, Aspergillus, Cladosporium, Stachybotrys) remain virtually unstained. Adequate staining of other biological material, i.e., epithelial cells, pollen, insect parts, and hyphal fragments, was also observed. Even the most hyaline and delicate structures show remarkable contrast in bright-field microscopy, eliminating the need for phase-contrast in delineating micromorphology of debris on spore-trap samples. Spore-trap samples placed in storage were found to exhibit no significant degradative alteration for up to six months. Longevity of mounts past six months is currently being established. LCB can also be used for making tease-mounts of fungal hyphae and conidiophores from cultures. Pretreatment with 95% ETOH as a wetting agent is recommended for this application.
The advantages of using LCB are (i) a reagent lacking phenol with no odor; (ii) high viscosity, allowing for rapid, easy preparation of spore-trap samples and reduced likelihood of trapped air bubbles during sample preparation; (iii) adequate rehydration of microbiological particles; and (iv) staining of very small, <5 µm diameter sized particles.
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FOOTNOTES |
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2 Current address: akaMold Lab, Sparks, Nevada
Accepted for publication March 22, 2002.
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LITERATURE CITED |
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TOPABSTRACTARTICLELITERATURE CITED |
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Hess-Kosa K., 2001 Indoor air quality sampling methodologies. New York: Lewis Publishers. 300 p
Jensen PA, Schafer MP., 1998 Sampling and characterization of bioaerosols. In: NIOSH manual of analytical methods. Method 0800, Issue 1. p 82–112
Largent DL, Johnson D, Watling R., 1977 How to identify mushrooms to genus III. Microscopic features. Eureka, California: Mad River Press. 147 p
Malloch DW., 1981 Moulds: their isolation, cultivation, and identification. Toronto, Ontario: University of Toronto Press. 97 p
McGinnis MR, D'Amato RF, Land GA., 1982 Pictorial handbook of medically important fungi and aerobic actinomycetes. New York: Praeger Publishers. 160 p
Merck Index: An encyclopedia of chemicals, drugs, & biologicals. 2001 MJ Oneil and A Smith, eds. Merck & Co., Rahway, New Jersey. 7323 p
Muilenberg ML, Burge HA., 1994 Filter cassette sampling for bacterial and fungal aerosols. In: Samson RA, Flannigan B, Flannigan ME, Verhoeff AP, Adan OCG, Hoekstra ES, eds. Health implications of fungi in indoor environments. New York: Elsevier Science. p 75–89
Sigma-Aldrich. 2000 Material safety data sheet P4161 Phenol ACS reagent. St. Louis, Missouri: Sigma Chemical Co. 6 p
Solomon WR., 1984 Sampling techniques for airborne fungi. In: Al-Doory Y, Domson JF, eds. Mould allergy. Philadelphia, Pennsylvania: Lea and Febiger. p 41–65
St. Germain G, Summerbell R., 1996 Identifying filamentous fungi: a clinical laboratory handbook. Belmont, California: Star Publishing Company. 314 p
Tsai SM, Yang CS, Moffett P, Puccetti A., 1999 Comparative studies of collection efficiency of airborne fungal matter using Andersen single stage sampler and Air-O-Cell cassettes. In: Johanning E, ed. Bioaerosols, fungi, and mycotoxins: health effects, assessment, prevention, and control. Albany, New York: Eastern New York Occupational and Environmental Health Center. p 457–464
Zefon International. 2001 User manual for Air-O-Cell Air Quality Particle Sampler. North St. Petersburg, Florida: Zefon International. 11 p
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