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Fungal melanin detection by the use of copper sulfide-silver

     Department of Biology, University of Western Ontario, London, Ontario, N6A 5B7 Canada

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Silver-staining procedures were investigated for their effectiveness in identifying cell wall-based fungal melanins in live and fixed plastic embedded samples, particularly 1,8-dihydroxynaphthalene (DHN) based polyketide melanins. We developed a simple and reliable melanin-staining technique based on a silver accumulation method originally published for histological demonstration of heavy metal sulfides in mammalian tissues. Copper is bound to fungal melanin followed by formation of the copper sulfide at melanin sites in fungal cell walls, which then are amplified into vivid black stains using a silver enhancement step. The method demonstrates patterns of melanization in a range of fungal hyphae and is suitable for light and electron microscopy. Albino mutant fungi and normally nonmelanized fungi do not stain with the sulfide-silver technique. Mammalian melanocytes also were labeled by the technique, indicating its universality as a melanin probe.

Key words: cell wall, copper, DOPA, Melanin, microscopy, silver

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Most life forms produce melanins, which are dark (usually black), complex, poorly characterized pigments, synthesized enzymatically or auto-oxidatively from a variety of cyclic, heterocyclic, phenolic or other resonance stabilized precursor molecules. These dark pigments have been shown to be highly resistant to decay and biodegradation and to confer protection against uv irradiation, lytic enzyme attack and a number of other environmental or biological insults depending on the producing organism and its environment. Several excellent general reviews exist on the chemical and physical properties of melanins in a comprehensive work on the human pigment cell (Prota et al 1998).

Many fungi produce melanins, which are deposited in or on the cell wall and some of which function prominently in a number of serious plant and animal infections-blast disease of rice caused by Magnaporthe grisea and the often fatal brain infections of AIDS sufferers caused by Cryptococcus neoformans. The most widely studied fungal melanin is produced by a pentaketide pathway that forms the monomer 1,8-dihydroxynaphthalene (DHN), polymerized to form so-called DHN melanin. DHN melanins function critically in rice blast disease and might play a role in a number of other fungal plant diseases. DHN melanin is produced by the fungus Wangiella dermatitidis, which like C. neoformans can cause serious infections in people with AIDS or other immunosuppressive diseases. A number of comprehensive reviews are available on fungal melanins (Bell and Wheeler 1986, Butler and Day 1998a, Henson et al 1999, Langfelder et al 2003, Nosanchuk and Casadevall 2003) and pathogenesis-related aspects of fungal melanins (Butler et al 2001, Perfect et al 1998, Horre and de Hoog 1999).

Some dyes are used to stain mammalian melanins, standard mammalian histology and histochemistry texts (Presnell and Schreibman 1997, Lillie and Fullmer 1976) prescribe Azure A and Nile Blue most commonly as melanin stains, although iron-based methods sometimes are used. Our experience was that the dyes do not stain fungal melanin intensely and iron-based methods failed with DHN melanin. Dye-based methods do not always work because some cell-wall components interfere with azure A staining (Butler and Lachance 1986). The Warthin-Starry technique used to stain melanins is silver based. The technique long has been used to stain for spirochetes and involves impregnation of sectioned material or smears with acidic silver nitrate solutions followed by development in silver nitrate, hydroquinone, and there are numerous variants of the method (Hood and Learn 1996). While we found that the Warthin-Starry method worked well with DOPA melanins, it was time consuming and gave inconsistent results when used with DHN fungal melanins.

Little has been published about histological methods for demonstration of fungal melanins by light microscopy, as opposed to mammalian melanins, for which numerous methods are available. We sought a simple and reproducible melanin stain which could be used rapidly to track the dynamics of melanin deposition in the black yeast Phaeococcomyces by light microscopy. This yeast is used as our model system for investigation of the enzymology and dynamics of DHN melanin formation (Butler and Lachance 1986, Butler et al 1989, Butler et al 2004).

	MATERIALS AND METHODS

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Cultures.— – The black yeast Phaeococcomyces sp., its albino (ALB), dehydratase negative cross feeding (CF) mutant (Butler 1987), and the sporidial cells of Microbotryum violaceum came from our laboratory culture collection. Yeasts were grown and maintained on 2% glucose–0.5% yeast-extract agar (YEG) at room temperature, and grown for experimental purposes in 200 mL YEG broth cultures in 500 mL flasks with rotary shaking 110 rpm. Broth culture cells (72 h) were centrifuged to recover cell pellets, which were washed once in 200 mL distilled water and recovered by centrifugation.

Myceliate fungi were maintained on 2% malt-extract agar at 4 C. Gaeumannomyces graminis var graminis was provided by Dr Joan Henson, Department of Microbiology, University of Montana at Bozeman; all other mycelial cultures were provided by Dr George Lazarovits of Agriculture Canada (London Ontario).

Formation of DOPA melanin by albino mutants.— – The albino mutant of Phaeococcomyces was induced to form DOPA melanin to assess the ability of the sulfide-silver copper linked stain to label DOPA based melanin deposited in fungal cell walls (Butler and Lachance 1986). Albino cells (0.25 mL of packed cells) were gathered from 50 mL of a 72 h YEG broth culture by centrifugation (3000x g, 15 min at room temperature) and washed twice by centrifugation in the same volume of distilled water. The cells (0.25 mL packed volume) were suspended in 10 mL of 50 mM Tris/HCl buffer, pH 7.5 containing 2 mg/mL of L-DOPA (Sigma Aldrich) and agitated gently at 37 C for 8 h. The black melanized cells were gathered and washed by centrifugation and stored frozen in water at –10 C.

The albino mutant of Phaeococcomyces was also induced to form DHN melanin by growing it on the same medium with the CF mutant (Butler et al 1989). A line of the albino mutant was streaked onto YEG medium, and 4 d later a line of the CF mutant culture was streaked about 1 cm from and perpendicular to the line of albino cells. The CF mutant releases scytalone, which is converted to DHN melanin by the albino cells; the albino culture will blacken over a period of several days as DHN melanin is formed and deposited in the cell walls, and samples can be removed for analysis using the silver-sulfide stain.

Sample preparations for light microscopy.— – None of the fungi used in this study were chemically fixed before staining. In some trials yeasts and mycelial fungi were stained in "free form" (unembedded). In this case staining reactions were carried out in 1.5 mL volumes of all solutions in Eppendorf-type tubes. After centrifugation the supernatant was removed by pipette (2 min in EC minifuge at room temperature); mycelial samples did not need to be centrifuged and solutions were removed with a pipette. Pellets were washed between steps in two 1.5 mL spins in distilled water; the only exception being the last step of silver stains where pelleted cells and mycelia from the silver-HQ treatment (described below) were directly and immediately treated with 1 mL of photographic fixer solution (Ilford 2000RT) without washing.

Yeasts also were incorporated in thin agarose sheets (ca. 0.2 mm) for staining, as follows: Yeast cells were removed from broth culture by centrifugation and a packed cell volume of about 20 µL was washed twice in 1.5 mL distilled water and then resuspended in about 100 µL of distilled water. Molten 2% agarose (electrophoresis grade, Sigma Type II) was prepared in distilled water and cooled to 40 C. A glass Petri dish was placed without a lid on a slide warmer to keep its temperature at approximately 40 C and 3 mL of the molten agar was added into the dish, followed by about 50 µL of the cell suspension and gently mixed. Approximately 300 µL of this preparation was pipetted along the central length of a prewarmed glass microscope slide; another warmed slide immediately was placed on that slide to spread the preparation (slides were not pressed together) and the two slides then were slid apart gently, longitudinally, to obtain a thin layer of preparation on both slides, which was allowed to solidify. The slides were stored in large Petri dishes under distilled water. A new, clean single-edge razorblade was scraped along a slide a distance of ca. 1 cm, which caused the agarose sheet to curl up onto the edge of the blade. The sheet is delicate and must be handled gently. A small spatula was used to pick the agarose sheet from the blade and drop it into 2 mL water in a 5 mL well in a plastic-culture well dish (Falcon, VWR, Mississauga, Ontario), and the agarose can be manipulated gently to cause it to fan out into a sheet form.

Mycelial fungi were grown on agar-based medium in thin layers in Petri dishes. ME agar medium was prepared and sterilized and poured into Petri dishes at 80 C and poured immediately into a glass waste receptacle. This leaves a thin layer of medium (ideally ca. 0.5 mm) onto which small cubes of fungal inoculum are placed. The plates are sealed to prevent drying and incubated 3–6 d at room temperature. The thin layer of nutrient-poor ME medium encourages most myceliate fungi to form a spreading, less crowded growth pattern, and the thinner agar layer is optimal for light microscopy. Small agar squares (ca. 1 cm²) can be cut out and manipulated into wells in disposable tissue-culture plates for silver treatment.

Mycelial samples processed through the staining procedures described above were dropped into 40 µL drops of glycerol on a slide and a cover slip was applied. Agarose embedded samples were handled in the same manner with gentle pressure on the cover slip to flatten out the sheets. Free stained yeasts were viewed by placing small drops (10–15 µL) onto thin sheets (2 mm and 2 cm²) of 1.5% water agar, letting the drop dry into the agar and then adding a cover slip. Slides were viewed and photographed through a Kodak Wratten gelatin No. 15 filter and photographed on Kodak Tri-X film.

Sulfide-silver staining procedure.— – Danscher’s method (1982) is intended for use with animal tissues in 1–5 µm sections embedded in Epon. In the original method fixed and sectioned samples are pretreated with a 1.0% sodium sulfide solution in distilled water at 45 or 60 C for an hour in the dark and after washing are dipped in a gelatine-coating solution, dried and developed in a 20 mL solution of 22 mg silver lactate and 170 mg hydroquinone in a citrate buffer (0.1 M, pH 3.7) solution (Ag–HQ solution) containing gum arabic 30 min to 1 h at 26 C.

Various modifications of this method were attempted. These included pre-incubation of samples in 10 mM copper sulphate in distilled water at room temperature for various times (1, 8 and 12 h); a range of pHs for silver development step (pH 2.5–8.0 in half pH unit increments); a range of times (1–8 min) and incubation temperatures (25, 30, 37 C) in the sulfide and silver solutions. Controls lacked either the sulfide or the silver solutions. Gelatin and gum arabic treatments described in the original method were not used in our experiments. In all cases the copper and sulfide treatments were followed by repeated washings (6 x 2 mL distilled water) before the next step.

Electron microscopy, prestain method.— – Mycelial fungi were harvested and treated. After the final step a drop of 1% agarose was placed on the top of the agar plug to secure the sample for further processing. Samples were fixed in paraformaldehyde (4%)/glutaraldehyde (0.5%) in 0.04 M potassium phosphate buffer (0.8 M Sorbitol), 1 mM magnesium chloride, 1 mM EGTA, pH 6.7) 1 h at room temperature, transferred to fresh buffer and stored overnight at 4 C. The samples were washed in buffer, followed by double distilled water, stained with 0.5% uranyl acetate 30 min, dehydrated in a graded ethanol series (10, 30, 50, 70, 90, 100%) and embedded in LR white resin. Sections were cut with a diamond knife, mounted on nickel grids and post-stained with uranyl acetate before viewing in a Philips CM 10 TEM.

Post-stain.— – Fungi were fixed and embedded as above but not prestained with uranyl acetate or copper-sulfide silver. The blocks were microtomed and the sections collected on nickel grids. Grids were floated on a drop of 10 mM copper chloride overnight at room temperature, washed with distilled water, incubated on a drop of 1% sodium sulfide 1 h. Grids were washed with distilled water and silver intensified by the method of Danscher (1981), poststained with uranyl acetate and examined in the EM.

Animal tissues.— – C3H mice (white and dark brown) were euthanized and skin biopsies taken from the back. Samples were fixed immediately in paraformaldehyde (4%)/glutaraldehydre (0.5%) in Millonig’s buffer (pH 7.3) 2 h at 4 C. Samples were dehydrated, infiltrated and treated with copper-sulfide-silver as above.

	RESULTS

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Optimum conditions for copper-sulfide-silver staining.— – Best results were obtained when using a close approximation to the original procedure. It was convenient to pre-incubate samples in copper sulphate overnight at room temperature, but optimum results also are obtained by as little as one hour of incubation with copper at room temperature. After washing, a one hour sulfide treatment follows at room temperature. Conditions were critical and varied somewhat with the sample type, DOPA melanized albino mutant cells being much more densely stained than DHN melanized cells and requiring less exposure to the Ag-HQ to obtain stains of similar intensity as those for DHN melanin. Lower pH than the optimum 3.7 resulted in poor and spotty intensity of staining, in which large and sparsely distributed grains of silver-sulfide were distributed on melanized yeast cell walls and the intensity of the stain often was barely higher than found on albino non-melanized yeasts. Higher pH resulted in more rapid and intense staining which was difficult to control. Results generally were similar for free yeast or mycelia, and for yeasts or mycelia embedded in agarose. The most efficient protocol was yeast samples embedded in agarose sheets and with mycelia grown on thin agar medium because those samples were easier to manipulate during washing and treatment. Optimized staining was obtained with a silver-HQ development step of 4 min, lesser times resulted in less intense and spottier staining, and longer times resulted in dense staining and formation of large silver sulfide crystals which obscured cell-wall structure and also caused high background staining. We found that 2.5 to 3 min gave the same result for DOPA melanins.

Dansche’s sulfide-silver amplification method used with a copper pretreatment gave excellent discriminatory melanin staining results (FIGS. 1, 2). The copper-sulfide-silver procedure gives solid staining over the entire cell surface which is purple-black when viewed using a red filter. Reasonable staining could be obtained in a number of cases when the initial copper pretreatment was omitted, although it was less intense. Melanized yeasts showed good results (FIGS. 1–4), whereas normally nonmelanized yeasts such as the basidiomycetous yeast form of Microbotryum violaceum did not stain. Albino cells did not produce melanin and did not stain (cells appear identical to nonstained black yeast cells) (FIG. 1). By contrast, albino black yeast was induced to form DHN melanin from a supply of the missing precursor during growth in proximity to the cross feeding CF mutant. As a result, the albino black yeast formed melanin and became stained using the procedure (FIG. 4). Albino yeast cells melanized by treatment with DOPA became densely stained (FIG. 3) Melanized mycelia stained well and it was possible to see various patterns of melanization, such as an unstained mycelial section immediately adjacent to or continuous with a stained hyphal segment (FIGS. 5–7). When the silver treatment was omitted cells did not stain.

View larger version (57K): [in this window] [in a new window]   FIGS. 1–4. Bright field microscopy of Phaeococcomyces (black yeast) cells. Bar = 5 µm. FIG. 1. Black yeast cells that have not been treated with the copper-silver stain. No coloration of the cell wall is evident. FIG. 2. Black yeast cells treated with copper-silver stain appear purple-black. Note the formation of a bud (arrow) that has not yet deposited melanin in its cell wall. FIG. 3. Albino cells treated with DOPA exhibit intense staining of the cell wall due to the formation of melanin. FIG. 4. Albino cells grown with the cross-feeding CF mutant are supplied with a required precursor and produce melanin as indicated by the dark staining of the cell. The degree of staining exhibited among normal cells, DOPA treated albinos or albinos cross-fed with a precursor in the melanin pathway is similar.

View larger version (62K): [in this window] [in a new window]   FIG. 5. Bright field micrograph of Cladosporium mycelium that have not been stained for melanin. FIGS. 6, 7. Light micrographs (bright field) of Cladosporium hyphae stained for melanin. Note that not all the hyphal cells have stained (arrows) indicating a difference in the degree of melanization in the mycelium. Bar = 5 µm.

When examined in the electron microscope, labeling was extensive on the cell walls of fungi which expressed melanin. Mycelial fungi showed strong labeling of the cell walls as seen in preparations of Aspergillus nidulans and C. cucumerinum (FIGS. 8, 9). The results with black yeast indicated where the staining seen in the light microscope was taking place (FIG. 10). The cell walls were labeled heavily with some cytoplasmic labeling present. Omitting the copper pretreatment produced no staining. M. violaceum, which does not have melanin in its cell wall, did not show any label when examined in the EM (FIG. 11).

View larger version (172K): [in this window] [in a new window]   FIG. 8. Electron micrograph of a cross section of an Aspergillus hyphae showing labeling of the cell wall. FIG. 9. Electron micrograph of a cross section of two Cladosporium hyphae showing labeling of cell walls. Little if any internal labeling is present. FIG. 10. Electron micrograph of black yeast showing intense staining of the cell wall and some staining in cytoplasm. FIG. 11. Microbotryum violaceum sporidial cell that has been stained with the copper-silver procedure and shows no labeling of the cell wall. FIGS. 8–11 bar = 2 µm.

View larger version (72K): [in this window] [in a new window]   FIG. 12. Light micrograph (bright field) of mouse brown skin stained with the copper-silver technique showing the characteristic melanocytes present. Note the dendritic processes (arrow) of the cells which transfer melanin to other skin cells. Bars = 50 microns. FIG. 13. Electron micrograph of mouse skin cells showing labeling of melanin in melanosomes (arrow). Note some labeling of melanin in the vicinity of collagen fibers (c). Bar = 1 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Danscher’s silver method (1983), developed as a counterstain for mammalian tissues. In his method sections are pretreated with silver lactate alone. This causes silver ions to deposit at various sites in the section such as melanins. These bound and miniscule traces of silver deposition, invisible in light microscopy, form the template site for subsequent accumulative binding of silver ions in the presence of hydroquinone, which reduces the accumulated silver at the template site to its metallic and black state. The sulfide-silver method (Danscher 1982) as a staining system for heavy metals, omits the pretreatment step with silver lactate, and instead substitutes a pretreatment with sulfide solution, which causes many metals (copper, lead, cadmium, etc) already present to form their insoluble sulfides. Subsequent treatment with silver lactate-hydroquinone causes accumulation of silver around the insoluble metal sulfides to levels visible in light microscopy.

Copper and other metals bind with melanins (Fogarty and Tobin 1996), and addition of copper ions to suspensions of pure melanin granules from the black yeast causes rapid aggregation of the granules. This property of melanins was used to remove suspended melanin from experimental treatments during melanin-degradation studies (Butler and Day 1998b). Our observation of this strong affinity of copper for melanins lead to our pretreatment of fungal melanins with copper, to cause formation of the copper sulfide, which then could be detected by Danscher’s autographic silver-staining procedure. Identification of melanin in this way is analogous to the common use of metal ligands in mordant-dye stains in histological studies.

Electron micrographs indicated that the dense labeling seen in light micrographs was restricted to the outer fungal cell. This fits with the location of melanin deposition in fungi. Melanin serves as a protective barrier against many potential damaging agents such as ultraviolet light, lytic attack, environmental stress and even might serve a structural role in the cell wall. Melanin has not been shown to have a metabolic role within the cell as is confirmed by the lack of labeling of cytoplasmic components.

Animal tissue treated in the same way also gave a positive result for melanin localization, as would be expected because DOPA melanin had been identified with Azure A, Nile Blue and the Warthin-Starry silver-stain technique. These procedures however, provided only variable results with fungal melanins. The Warthin-Starry procedure was developed for staining of spirochete bacteria (Warthin and Starry 1920) but also is used to stain for mammalian melanins. We found that the Warthin-Starry procedure had variable results when used to stain for fungal melanins, possibly because of the presence of reducing materials in fungal cell walls that cause nonspecific staining so that some fungal cell walls may stain black even where melanin is not present. This did not occur with the sulfide-silver procedure, S. cerevisiae and M. violaceum, neither of which produce melanin; they were not stained by this procedure and showed the same negative results as the albino mutant of the black yeast, and this was the case with nonmelanized mycelial fungi such as Phanerochaete chrysosporium.

No staining occurred if the sulfide pretreatment was omitted. Sulfide occasionally interacts in some manner with melanized fungal cell walls to allow reasonable staining without pretreatment with copper. The amount of copper normally found in standard fungal-culture media seem to be sufficient to bind with cell-wall melanin where subsequently they can form insoluble sulfides, detected by our staining procedure, although the staining is less intense. A study of copper deposition patterns in the melanin of Gaeumannomyces graminis var graminis used a sulfide-silver technique to disclose copper binding sites in cell-wall melanin (Caesar-Tonthat et al 1995).

The utility of a stain for any target molecule requires affinity and specificity. The first requirement generally is satisfied as long as the stain in question is vivid enough to be useful in the method used, such as light microscopy. The second point, specificity, is more problematic. There are almost no staining methods that show absolute specificity; even monoclonal antibodies are subject to nonspecific binding that can confuse interpretation if appropriate controls are not made. It is context and proper use of controls that provides much of the specificity of histological stains. Wild-type melanized black yeasts stain with the sulfide-silver method, albino (nonmelanized) mutants and natively nonmelanized control yeasts, such as M. violaceum, do not stain. This indicates the specificity of the sulfide-silver stain methods for melanin under the conditions used. We sought a method that could be used rapidly in light microscopy but also found this method to work well for electron microscopy purposes.

	ACKNOWLEDGMENTS

 
This work was financially supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada. We thank Mrs Jackie Hill for her technical assistance providing cultures and Mr Ron Smith for assistance with the electron microscope.

	FOOTNOTES

 
Accepted for publication October 13, 2004.

¹ Corresponding author. Email: mjbutler{at}uwo.ca

	LITERATURE CITED

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———. 1987. Melanin production by the black yeast Phaeococcomyces sp. [Doctoral dissertation]. The University of Western Ontario, London, Ontario, Canada.

———, Lazarovits G, Higgins VJ, Lachance MA. 1989. Identification of a black yeast isolated from oak bark as belonging to genus Phaeococcomyces sp.: Analysis of melanin produced by the yeast. Can J Microbiol 35:728–734.

———, Day AW. 1998a. Fungal melanins: a review. Can Microbiol 44:1115–1136.[CrossRef]

———, ———. 1998b. Destruction of fungal melanins by ligninases of Phanerochaete chrysosporium and other white rot fungi. Int J Plant Sci 159:989–995.[CrossRef]

———, ———, Henson JM, Money NP. 2001. Pathogenic properties of fungal melanins. Mycologia 93:1–8.[CrossRef]

———, Gardiner RB, Day AW. 2004. Use of the black yeast Phaeococcomyces fungal melanin model system for preparation of 1,3,6,8-tetrahydroxynaphthalene and the other components of the DHN melanin pathway. Int J Plant Sci 165:787–793.[CrossRef]

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Danscher, G. 1982. Exogenous selenium in the brain. A histo-chemical technique for light and electron microscopical localization of catalytic selenium bonds. Histochemistry 76:281–293.[CrossRef][Medline]

Danscher, G. 1983. A silver method for counterstaining plastic embedded tissue. Stain Technology 58:365–372.[Medline]

Fogarty RV, Tobin JM. 1996. Fungal melanins and their interaction with metals. Enz Microb Technol 19:311–317.

Henson JM, Butler MJ, Day AW. 1999. The dark side of the mycelium: melanins of phytopathogenic fungi. Annu Rev Phytopathol 37:447–471.[CrossRef][Medline]

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Langfelder K, Streibel M, Jahn B, Haase G, Brakhage AA. 2003. Biosynthesis of fungal melanins and their importance for human pathogenic fungi. Fungal Genetics and Biology 38:143–158.[CrossRef][Medline]

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Perfect JR, Wong B, Chang YC, Kwon-Chung KJ, Williamson PR. 1998. Cryptococcus neoformans: Virulence and host defences. Medical Mycology 36:79–86.

Presnell JK, Schreibman P. 1997. Humason’s Animal Tissue Techniques. 5th ed. Baltimore: The Johns Hopkins University Press.

Prota G, D’Ischia M, Napolitano A. 1998. The Chemistry of Melanins and Related Metabolites. In: Nordlund JJ, Boissy RE, Hearing VJ, King RA, Ortonne JP, eds. The Pigmentary System. Oxford: Oxford University Press. p 307–332.

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