Scanning Electron Microscopy Volume 1985 Number 1 1985 Article 19 10-2-1984 Calcium Oxalate Crystal Production in Two Members of the Mucorales M. D. Powell The University of Texas at Arlington H. J. Arnott The University of Texas at Arlington Follow this and additional works at: https://digitalcommons.usu.edu/electron Part of the Biology Commons Recommended Citation Powell, M. D. and Arnott, H. J. (1984) "Calcium Oxalate Crystal Production in Two Members of the Mucorales," Scanning Electron Microscopy: Vol. 1985 : No. 1 , Article 19. Available at: https://digitalcommons.usu.edu/electron/vol1985/iss1/19 This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Scanning Electron Microscopy by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. SCANNING ELECTRON MICROSCOPY/1985/I (Pa gu 183- 189) 0586-5581/85$1 . 00+. 05 SEM In c.. , AMF O'Ha1te_ (Chic.a go ), IL 60666 USA CALCIUMOXALATE CRYSTAL PRODUCTION IN TWOMEMBERS OF THE MUCORALES M. 0. Powell* and H.J. Arnott Department of Biology The University of Texas at Arlington Arlington, TX 76019-0498 (Paper receiv ed March 12 1984, Completed manusc r ipt received October 2 1984) Abstract Introduction Calcium oxalate crystals are found in Calcium oxalate formation occurs commonly association with the sporangia of Mucor in a variety of fungi (Hamlet and Plowright, hiemalis and Rhizopus oryzae. Crystals 1877). Calcium oxalate has been found in observed in each species vary in morphology association with the sporangia of the from simple crystals consisting of single Mucorales, e.g., the spines on the sporangia of spines in M. hiemalis to complex crystals with Mucor plumbeus and Cunninghamella echinulata twin spines, sometimes three-parted, on a (Jones et al., 1976) and the zygophores and common base in R. oryzae. The early sporangia or-Mucor mucedo (Urbanus et al., development of the crystals7s similar in both 1978). The two reports confirm the presence species with a layer of the cell wall covering of calcium oxalate di hydrate (weddel lite) in in the initial crystals. The spines of M. the spines produced by these species. hiemalis rapidly emerge while the crystals of The mechanism of formation of these crys­ ~ oryzae appear to remain covered with a layer tals is unknown. Some reports of wood rotting of outer wall material. The crystals of both fungi and soil fungi (Graustei n et al., 1977; species become fully developed just prior to Cromack et al . , 1979) suggest that oxalate is spore release. Details of crystal development excreteda ndcombin ed with external calcium to are compared and possible mechanisms for form crystals. In contrast, some studies of crystal development are explored. leaf litter fungi (Arnott, 1982: Horner et al., 1983) suggest the crystals may form internaTTy. Urbanus et ~ (1978) suggest the crysta l s formed in association with the zygophores of Mucor mucedo are covered by a layer of ce ll wallmaterial. This study expands observations of calcium oxalate crystals in the Mucorales and provides further evidence for the internal development of the crystals. Materials and Methods Cultures Strains of Mucor hiemalis and Rhizopus oryzae were isolated from leaf litter and soil samples at three locations in Arlington, Texas. The fungi were grown on commercially prepared Sabouraud's Dextrose Agar (Scott Laboratories). Crysta l Isolation KEYWORDS: Mucor hiema l is, Rhizopus oryzae, Mature cultures grown in petri dishes were calcium_ oxa l ate, scanning electron microscopy, rapidly frozen in liquid nitrogen, and the sporangia, sporangiophore sporangia were scraped from the frozen agar surf ace with a seal pe l blade. The sporang i a­ crystal mixture was then transferred to a *Address for correspondence: liquid nitrogen-cooled mortar and pestle and ground in a small portion of l iquid nitrogen. The University of Texas at Arlington The resulting mixture was used for microchemi­ Department of Biology cal analysis. Arlington, TX 76019-0498 Phone No. (817) 273- 3491 183 Powell MDand Arnott HJ Microchemical analysis As the sporangia age the external layer, Analys 1s was performed on whole sporangi a along with the crysta ls, begins to mold around as we11 as the powder extract des er i bed above. the spores beneath (Figs . 5 and 6). Close Reactions were observed at 400X with bright­ examination of the sporangia reveals long, field micr 'oscopy. Solubility tests (Frey, pointed spines protruding from the outer 1925; Pohl, 1965) were performed using the fol­ sporangial wall (Fig. 7), with the wall molded lowing reagents: 70% ethanol, 2% acetic acid, to spores beneath it (Figs. 8 and 9). The 4% sodium hydroxide, 4% and 60% sulfuric acid, entire process, from appearance of the sporan­ 3% nitric acid, and 10% hydrochloric acid. gium to its complete encrustation with crys­ ScanninT Electron Microscopy (SEM) tals, occurs over a period of 3-4 days with the Cu tu res were observed by means of light first crystals appearing as early as the first microscopy to determine maturity of the cul­ day. This process of encrustation appears to tures. Mature cultures were then vapor fixed be uniform among all sporangia and no mature for 4-6 h over 4% osmium tetroxide in 0.1 M sporangia were noted without at least some cov­ phosphate buffer (pH 7 .24) at 4° C. Specimens ered crystals present. were a 11owed to air dry and portions of the The individual spines reach a length of culture were affixed to the sticky side of a about 2 µm and are composed of a basal pl ate short piece of cl ear tape. The tape was then and a terminal spine. Their shape resembles a inverted and attached to SEM stubs using double carpet tack (Fig . 9). The spine sometimes stick tape. Specimens were sputter coated in a appears to be uniformly tapered but occasion­ Polaron E5100 sputter coater. Observations and ally the spine has a central "joint" where it photographs were made on a JEOL JSM-35C quickly tapers. In the mature spines it is scanning electron microscope at 15 kV and difficult to demonstrate an overlying eel l wall 100 µA. component with SEM (Fig. 9). Changes in environmental conditions seem Results to have some bearing on the numbers of crystals produced. More crystals are produced in cu 1- Crystals found in association with sporan­ tures grown in light than in darkness and at gia of both Mucor hiemalis and Rhizopus oryzae low (4-80 C) temperatures, though the develop­ have solubility characteristics consistent with ment time is increased to 14-28 days. calcium oxalate (Table 1). The crystals of Crystal production in Rhizopus oryzae is these fungi show little or no birefringence similar to that of Mucor hi emal is. The sporan­ under polarized light microscopy, though the gi a of young culturessiiow few crystals (Fig. small size of the crystals makes observation 10) on a relatively smooth surface. During difficult. sporangial maturation, crystals appear to extrude through the outer sporang i al layer Table 1. Microchemical Analysis on (Figs. 11-13) . The appearance of the crystals Mucor hiemalis and Rhizopus oryzae is rather rapid, occurring within 1-2 days of growth. As the sporang i a mature th e crystals Calcium become fully developed and much more numerous Treatment Mucor Rhizopus Oxalate on the sporangial surface (Fig. 14). The indi­ vidual crystal morphology is different from 70% ethanol that of the spines found in Mucor (Fig. 15). The crystals have a flat rectangular base 2% acetic acid with two perpendicular spines rising from it. 4% sodium hydroxide Figures 1-9. SEM views of crystals on the 4% sulfuric acid + + + sporangia of Mucor hiemalis. Figure 1. Young sporang1a showing absence of 60% sulfuric acid + + + exposed crystals. Bar= 10 µm. Figure 2. Early sporangium with newly exposed 3% nitric acid + + + crystals. Bar= 10 µm. Figure 3. Early emergence of crystals. Bar = 10% hydrochloric acid + + + 1 µm. Figure 4. Maturing sporangium. Note increased - =insoluble,+= soluble number and length of crystals. Bar= 10 µm. Young sporangia of Mucor hiemalis have a Figure 5. Mature sporangium showing molding to smooth, sometimes bumpy, surface, and sporan­ spores beneath. Bar= 10 µm. giophores show no evidence of crystal formation Figure 6. Mature sporangium at spore release. (Fig. 1). As maturation continues, the crys­ Bar = 10 µm. tals begin to appear tip first (Figs. 2 and 3), Figure 7. Closeup of spines in mature sporan­ just protruding through the outer sporangial gium. Bar= 1 µm. wall. Crystals usually develop uniformly on Figure 8. Detail view of spines molded to the sporang i al surf ace, sometimes bas i pet ally, spore beneath. Bar= 1 µm. but always within a short time span. After Figure 9. Higher magnification of spines approximately 3-4 days, the crystals in matur­ molded to spore beneath. Bar= 1 µm. ing sporang i a become fu 11y developed and more numerous (Fig. 4). 184 Calcium Oxalate Crystals in the Mucorales 185 Powell MDand Arnott HJ Calcium Oxalate Crystals in the Mucorales Figures 10-23. SEMviews of sporangia of Rhizopus oryzae. Figure 10. Young sporangium with sparse crystals. Bar= 10 µm. Figure 11. Early crystal development. Note covering of some crystals . Bar =1 µm. Figure 12. Early sporangium with some exposed and some covered crysta l s. Bar= 1 µm. Figure 13. Early sporangium with some exposed crystals. Bar= 1 µm. Figure 14. Maturing spora ngium with many exposed crystals. Bar= 10 µm. Figure 15. Mature sporangium with fully exposed crystals. Note typical twin peaked crystal morphology. Bar= 1 µm. Figure 16. Emerging spines showing similarity to spines of Mucor. Bar= 1 µm.