Proc. Indian Acad. Sci. (Plant Sci.), Vol. 93, No. 5, October 1984, pp. 561-565 Printed in India. Mucilage interference in desmids under SEM VIDYAVATI and JOHN D DODGE* Botany Department, Kakatiya University, Warangal 506 009, India * Department of Botany, Royal Holloway College, London, UK MS received 26 August 1983; revised 26 June 1984 Almtract. Various species of desmids (Cylindrocystis brebisonii, Closterium acerosum, Closterium littorale, Cosmarium bioculatum, Cosmarium botrytis, Cosmarium contractum and Cosmarium subtumidum) were studied for their surface ornamentation. After trying various methods for removal of the mucilage, it was found that pretreatment with the glusulase preparation was usually effective in cleaning the cells and enhancing their appearance under the SEM. Keywords. Desmids; Cylindrocystis; Closterium; Cosmarium; Scanning electron microscope. 1. Introduction In Desmidiaceae, the cell wall consists of two layers a thin inner layer composed mainly of cellulose and ah outer firmer and thicker layer composed of cellulose impregnated with pectic substances which often include iron compounds. External to the outer layer is a narrow/broad diffluent mucilage layer. The mucilage is secreted through a large number of pores which traverses both the layers. Some planktonic desmids have a wide mucilage envelope. Pores were not found in the walls ofany of the Mesotaeniaceae, although there is a copious production of mucilage by these desmids (figure 1). At the ultrastructural level Pickett-Heaps (1972) showed that, irregular fibrous material usually remained attached to the external o¡ of the pore, and internally there is always a prominent hemispherical invagination of the plasmalemma containing strands of mucilagenous material. The mucilage produced from the pores of Cosmarium is composed of sulphated polysaccharides. This mucilage is often very difficult to remove completely and appears as small surface deposits under the SEM. The work of Preston (1958) and co-workers on cell-wall structure in the algae as revealed by polarization microscopy, electron microscopy, x-ray diffraction analysis and chemical analysis, indicates that these mucilages are mainly constituents of the continuous amorphous phase of the cell wall. According to Myers and Preston (1959a, b) the cell walls of numerous algae examined contain 30-70 ~o of water soluble material. Relatively few mucilages have been subjected to structural examination, those investigated exhibit great diversity in their molecular architecture, corresponding in part to the wide variety of functions which they perform. Recent SEM studies of some placoderm species showed excessive (Vidyavati 1982a-e; 1983a-c; Vidyavati et al 1983) mucilage interference. Hence, it was thought desirable to try and establish certain methods to avoid mucilage interference. 561 /'--6 562 Vidyavati and John D Dod9e Figures 1-6. 1. Cylindrocystis brebisonii covered with mucilage, 2, 3. Closterium acerosum with and without mucilagr 4, 5. Closterium littorale with and without mucilage, 6. Cosmarium subtumidum muiilage covered cells. Mucilage interference in desmids 563 2. Material and methods Cytindrocystis brebisonii Menegh, 615/la; Closterium acerosum Ehrenb, 611/4; Closterium littorale Gay, 611/6; Cosmarium bioculatum Breb, 612/17; Cosmarium botrytis Menegh, 612/5; Cosmarium contractum Kirchn, 612/16; and Cosmarium subtumidum Nordst, 612/8c (cambridge culture collection, uK) species were maintained in their unialgal clonal form. The cultures were grown in Chu's 10 inorganic medium, receiving 16 hr light and 8 hr dark period at 18-20~ All cells prepared for SEM were washed in filtered water before processing. Most algal cells characteristically secrete various quantities of mucilagenous mate¡ Such secretions can undoubtedly be considered as an integral component of the cell, but they pose problems because they condense during dehydration into a fibrous or amorphous layer, partially or totally obscuring the surface of the cell or its true cell-wall. Some mucilaginous layers are soluble in acetone-water or alcohol-water mixtures and were therefore extracted in the course of dehydration. Warm water (30 ~ with or without chelating agents (e.g., 0-02 M EDTA) solubilizes pectic fractions of higher plant cells and are sometimes successful in removing surface layers from algae. Although hot (50 ~ or cold (0 ~ solutions of sodium hydroxide (0.01 N) are more strongly extractive their use caused an increasing likelihood of damaging the cell-wall proper. Certain wall-degrading enzyme preparations (e.g., snail gut juice, pectinases) were also used for removing mucilage. Acid solutions were also tried but were mostly avoided except for organisms with siliceous walls or scales. Few cells were cleaned in dilute alkali, but a majority remained completely enshrouded in their mucilage. ~ Frequent subculturing also helped to remove mucilage. Thus only freshly grown cultures were fixed for the SEM studies. Pickett-Heaps (1973) and Marchant (1973) developed methods that have been Used successfully with many small algae (e.g., Hydrodictyon, Pediastrum, Cosmarium, Staurastrum). But incubation of specimens in a solution of the polysaccharidase preparation of glusulase (Endo Laboratories, New York) was definitely better than all other previous treatments. The glusulase was used at 1 : 50 (v/v) dilution in distilled water for 12~---2hr at room temperature, washed and allowed to stand at room temperature for 1 hr in culture medium before fixation. After pretreating with glusulase, the cells were fixed in glutaraldehyde, post-fixed in osmium tetroxide and then dehydrated with varying gradr of acetone (30, 50, 70, 90 and 100 ~o). The cells were then dried (critical point-drying apparatus), coated with carbon and gold and finally observed under SEM (JEOL-JSM-25S) 3. Observations and results While studying the various species of desmids (saccoderm and placoderm), it was observed that frequently, the cell surface was obscured by mucilage. Of all the methods employed for removing the mucilage, pretreatment with glusulase preparation was most effective in cleaning them and enhancing their appearance under the SEM (Pickett- Heaps 1973, 1974). One member of the saccoderm desmids (Cylindrocystis brebisonii) and six species of placoderm (Closterium acerosum, Cl. littorale, Cosmarium bioculatum, C. botrytis, C. contractum and C. subtumidum) desmids were studied. Figures 1-12 show the control and the treated cells for all these species. Al1 these control and treated cells show 564 Fidyavati and John D Dodge Figures 7-12. 7, 8. Cosmarium bioculatum with and without mucilage, 9, 10. Cosmarium botrytis with and without mucilage, 11, 12. Cosmarium contractum with and without mucilage. Mucilaoe interference in desmids 565 the action of glusulase in removing the mucilage from the cell surface. Thus glusulase treatment is quite efficient compared to all other methods tried. Acknowledgements Vidyavati is thankful to Prof Jafar Nizam for help. Facilities provided at the Royal Holloway College, uK is acknowledged. References Marchant H J 1973 Processing small delicate biological specimens for SEM; J. Microsc. 97 369 Myer A and Preston R D 1959a Fine structure in the red algae II. The structure of the cell-wall of Rhodymenia-palmata; Proc. R. Soc. (London) B150 447-455 Myer A and Preston R D 1959b Fine structure in the red algae III. A general survey of ceil-waU structure in the red algae; Proc. R. Soc. (London) BIS0 456-459 Pickett-Heaps J D 1972 Ceil division in Cosmarium; J. Phycol. 8 343-360 Pickett-Heaps J D 1973 Stereoscanning electron microscopy of desmids; J. Microsc. 99 109-116 Pickett-Heaps J D 1974 S~M of some cultured desmids; Trans. Aro. Microsc. Soc. 93 t-23 Preston R D 1958 Biophysical and biochemical aspects of some seaweeds; Abstr. 3rd Int. Seaweed. Sym. Galway, lreland l-I l Vidyavati 1982a Cell ornamentation in Cosmarium bioculatum Breb, under SEM ; Curr. Sci. 51 569-570 Vidyavati 1982b Cell division in Staurastrum oracile Ralf, under the SEM; Proc. Indian. Acad. Sci.. (Plant Sci) 91 443-447 Vidyavati 1982c Division in Cosmarium formosulum Hoff, under SEM; Life Sci. Adv. 1 151-155 Vidyavati 1982d Staurastrum gracile, Ralfs, under SEM; J. lndian Bot. Sci. 61 444--446 Vidyavati 1982e Cell ornamentation of Cosmariumformosulum Hoff, under SEM; Proc. Indian Natl. Sci. Acad. B48 632-634 Vidyavati 1983a Cell division in Cosmarium contractum Kirchn, under SEM; Geobios. New Rep. 2 39-41 Vidyavati 1983b Euastrum verrucosum, Ehrenb, division under SEM; Curr. Sci. 52 492-493 Vidyavati 1983c Surface ornamentation in Cosmarium praemorsum, Breb, lndian J. Bot. 6 95-97 Vidyavati, Sathaiah G, Digamber Rao B and Reddy Y R 1983 Cosmarium botrytis Menegh, under SEM; Plant & Nature 1 62--64 P-7 .