- Email: [email protected]
Teaching techniques for mycology: 11. Riddell's slide cultures
•
Volume 14, Part 3, August 2000
Teaching Techniques for Mycology: 11. Riddell's Slide Cultures ROLAND W. S. WEBERl AND DENNIS PITT 2 Lehrbereich Biotechnologie, Universitiit Kaiserslautern, Paul-Ehrlich-Str. 23, 67663 Kaiserslautern, Germany. E-mail [email protected]
1
2Washington Singer Laboratories, School of Biological Sciences, University of Exeter, Perry Road, Exeter EX4 4QG, i:« E-mail [email protected]
Names of fungi Eurotium repens de Bary = Aspergillus repens de Bary; Botrytis cinerea Pers.: Fr.
Introduction: features of interest Much to the frustration of students and teachers alike, the transfer of delicate vegetative hyphae or reproductive structures of fungi from Petri dishes to a microscope slide is rarely achieved without major structural damage obliterating the features of interest. In many cases, such problems can be overcome by growing the fungi from a plug of agar directly onto a microscope coverslip (Fig 1), which can then be mounted on a microscope slide. This elegant and simple technique was devised by Roland Riddell (1950). It is essential for the professional training of mycologists, especially those working in the medical field (Beneke & Rogers, 1996; Fisher & Cook, 1998). In the present contribution, we describe a simplified method and two typical applications for mycology teaching. The Riddell technique was originally designed as a tool to obtain intact spore-producing structures for descriptive and taxonomic studies. As an example, hyphae of Aspergillus rep ens readily grow from a plug of 2% (wjv) malt extract agar (MEA) onto glass coverslips where they produce conidiophores displaying all relevant features such as the foot cell and chains of darkgreen conidia emerging from phialides on the vesicle at the conidiophore apex (Figs 2 and 3). On MEA amended with 20% (wjv) sucrose (MEA+S), conidium formation is reduced but production of the sexual Eurotium state is stimulated (Webster, 1980). In E. repens, which is homothallic, sexual reproduction is initiated when an aerial hypha coils to form an ascogonium (Fig 4) which subsequently becomes
encased in sterile investing hyphae (Fig 5). The mature cleistothecium consists of a singlelayered peridial wall enclosing numerous globose asci (Fig 5), each containing 8 ascospores (Fig 6). Thus, the sexual state (Eurotium) and asexual state (Aspergillus) of this fungus can be conveniently demonstrated side-by-side. The Riddell technique is also suitable for the study of living vegetative hyphae. By mounting the coverslip with adhering living mycelium directly in a drop of an appropriate dye, cell organelles may be stained and their distribution examined, as in the case of mitochondria using the tetrazolium stain which thus acts as a vital dye (Weber et al., 1998). Likewise, Neutral Red accumulates only in living hyphae of filamentous fungi in which it stains vacuoles bright-red (Weber & Webster, 1997). The Neutral Red molecule can diffuse across biological membranes in its neutral form, but once it encounters an environment of acid pH such as vacuoles, it becomes positively charged and is immobilized there (Lullmann-Rauch, 1979). In Botrytis cinerea, vacuoles are formed towards the base of the apical cell (Fig 7) and become more prominent in mature (intercalary) cells (Fig 8), thus demonstrating the polarity of hyphal growth in which a dense core of protoplasm moves forward, leaving vacuolated cells behind (Park & Robinson, 1967; Weber & Webster, 1998). Vacuoles are also abundant in many specialized structures such as sclerotium initials of B. cinerea (Fig 9) and are thought to be involved in generating turgor pressure for processes of differentiation such as basidiospore formation (Wells, 1965) or infection of plants from appressoria (Bourett & Howard, 1990). These and many more features can be investigated by applying the methods as outlined here.
Volume 14, Part 3, August 2000
7-
6
4
\ Fig 1. A moist chamber for slide cultures. Three days after inoculation of the centres of MEA plugs with macroconidia of Botrytis cinerea, mycelium has grown onto the coverslips. Fig 2. Conidiophore of Aspergillus repens arising from a thick-walled foot cell (arrow) and terminating in a swollen vesicle bearing phialides, each producing a chain of conidia. Bar = 50 flm. Fig 3. The vesicle with a single layer of phialides at the tip of a conidiophore of A. repens. Bar = 20 flm. Fig 4. Coiling of a hypha of A. repens to form an ascogonium of Eurotium repens on the surface of the coverslip. Bar = 20 flm. Fig 5. Formation of a cleistothecium initial of E. repens (arrow) by encasement of the ascogonium in sterile investing hyphae. Two maturing globose cleistothecia are visible nearby. Their yellowish wall (peridium) is one cell thick and encloses numerous globose asci. Clumps of detached conidia are also present. Bar = 50 flm. Fig 6. Release of asci by breakdown of the peridium in a mature cleistothecium. Each ascus contains 8 ascospores. Bar = 20 flm. Fig 7. A growing hypha of Botrytis cinerea mounted in the Neutral Red dye for 1 h. The dye has been taken up by vacuoles which become more prominent towards the base of the tip cell. Bar = 10 flm. Fig 8. Neutral Red staining of a mature hyphal cell of B. cinerea. Vacuoles have filled most of the volume of the protoplast. Bar = 10 flm. Fig 9. Neutral Red staining of a sclerotium initial of B. cinerea. Vacuoles occur prominently throughout this structure. Bar = 20 flm.
Source of material Aspergillus repens is ubiquitous and grows even in
conditions of high osmotic stress such as in jams or on dry-stored cereal grains (Raper & Fennell, 1965). It can therefore be isolated on a suitable medium amended with 20% sucrose. Our present strain was obtained some 50 years ago by J. Webster. Botrytis cinerea is the cause of grey mould on a very wide range of plants. The present strain was isolated from lettuce and is deposited as ATCC 201173 in the American Type Culture Collection. We will provide cultures of both species on request.
Maintenance of fungi Both A. rep ens and B. cinerea are easily maintained on common laboratory media such as MEA, potato dextrose agar (PDA) or corn meal agar at 4°C, subculturing every 6 months. In order to ensure that E. repens retains its sexual reproductive capacity in long-term storage, it is recommended that cleistothecia rather than conidia are used as inoculum, with MEA+S as the culture medium (see Jinks, 1954). Spores of both species can also be freeze-dried and stored in vacuum-sealed glass ampoules at
•
•
Volume 14, Part 3, August 2000 room temperature (r.t.) for many years (Smith & Onions, 1983). Preparation of material Day -21. Revive E. repens and B. cinerea by plating onto MEA or PDA. Incubate at r.t. in room light. With B. cinerea, a brief (l min) exposure to near- UV light after 4 d growth will boost the formation of macroconidia. Day -10 to -7. Autoclave glass Petri dishes containing microscope slides supported by glass rods resting on damp filter paper or tissue paper. Alternatively, because the risk of contamination is very low, unsterilized filter paper may be used with standard or large plastic Petri dishes, and the glass rods and slides can be flame-sterilized (Fig 1). Place 1 em diam. plugs of MEA and MEA+S on the microscope slides, inoculate the centres of all plugs with conidia of A. repens, and cover the plugs with flame-sterilized coverslips. Incubate at 20° in the dark. Add distilled water as required to keep the chambers moist until day O. Day -3. Inoculate Riddell slides with macroconidia of B. cinerea as described above for A. repens. Day O. Present material to the students who should remove a coverslip with mycelium of A. repens from each of the two types of agar. In order to minimize damage to the mycelium, the coverslip should be lifted vertically while holding down the agar plug with a needle or forceps. Coverslips with adhering mycelium of A. repens should be mounted in water or lactic acid. For B. cinerea, coverslips should be removed as above, but should be mounted quickly in a large drop of the Neutral Red dye solution containing 0.015% (w/v) Neutral Red (BDH 'Gurr') and 3.75% (w/v) polyvinylpyrrolidone in 50 mM Tris/maleate buffer (pH 7.5). Material should be viewed after 30 min incubation at r.t.; vacuoles will retain the dye for approx. 60 min thereafter. Useful hints Adherence of mycelium is enhanced on gelatincoated coverslips which are prepared by dipping them in a warm 1% (w/v) gelatin solution followed by air-drying. Coated coverslips can be stored dry for years at r.t. Prior to mounting, the dry gelatin coat should be removed from one side of each coverslip by gentle rubbing with an ethanol-soaked tissue paper. Each coverslip should then be flamed only briefly before being placed on an agar plug with the gelatin-coated surface facing downwards. With A. repens, trapping of air bubbles during
the mounting of coverslips can be minimized by placing the coverslip on a flat surface with the mycelium uppermost, applying a drop of mountant to the centre of the ring of mycelium, and gently lowering a microscope slide onto the coverslip. If lactic acid is used as a mountant, any residual air bubbles can be eliminated by brief heating of the mounted slide in the yellow flame of a Bunsen burner. We recommend lactic acid also because it prevents mounted slides from drying out during the course of a practical class. Permanent preparations of lactic acid mounts can be made by sealing the edges of the coverslip with nail varnish (Beneke & Rogers, 1996). We thank Mr P. M. Booth, a BMS Associate, for his support, and our anonymous referee for helpful suggestions. References Beneke, E. S. & Rogers, A. 1. (1996) Medical Mycology and Human Mycoses. Star Publishing Co.: Belmont, CA. Bourett, T. M. & Howard, R. J. (1990) In vitro development of penetration structures in the rice blast fungus Magnaporthe grisea. Canadian Journal of Botany 68: 329-342. Fisher, F. & Cook, N. B. (1998) Fundamentals of Diagnostic Mycology, WB.Saunders Co.: Philadelphia. Jinks, J. 1. (1954) Somatic selection in fungi. Nature 174: 409-410. Lilllmann-Rauch, R. (1979) Drug-induced lysosomal storage disorders. In Lysosomes in Biology and Pathology, Volume 6 (edited by Dingle, J. T., Jacques, P. J. & Shaw, H. I.), pp. 49-129. Elsevier: Amsterdam, New York. Park, D. & Robinson, P. M. (1967) A fungal hormone controlling internal water distribution normally associated with cell ageing in fungi. Symposia of the Society of Experimental Biology 21: 323-335. Raper, K. B. & Fennell, D. (1965) The Genus Aspergillus. Williams & Wilkins Co.: Baltimore. Riddell, R. W (1950) Permanent stained mycological preparations obtained by slide culture. Mycologia 42: 265-270. Smith, D. & Onions, A. H. S. (1983) The Preservation and Maintenance of Living Fungi. Commonwealth Mycological Institute: Kew, Surrey. Weber, R. W S. & Webster, J. (1997) The coprophilous fungus Sphaeronaemella [imicola, a facultative mycoparasite. Mycologist 11: 50-51. Weber, R. W S. & Webster, J. (1998)Teaching techniques for mycology: 5. Basidiobolus ranarum. Mycologist 12: 149-151. Weber, R. W. S., Wakley, G. E. & Pitt, D. (1998) Histochemical and ultrastructural characterization of fungal mitochondria. Mycologist 12: 174-180. Webster, J. (1980) Introduction to Fungi (second edition). Cambridge University Press: Cambridge. Wells, K. (1965)Ultrastructural features of developing and mature basidia and basidiospores of Schizophyllum commune. Mycologia 57: 236-261.
Volume 14, Part 3, August 2000
Teaching Techniques for Mycology: 11. Riddell's Slide Cultures ROLAND W. S. WEBERl AND DENNIS PITT 2 Lehrbereich Biotechnologie, Universitiit Kaiserslautern, Paul-Ehrlich-Str. 23, 67663 Kaiserslautern, Germany. E-mail [email protected]
1
2Washington Singer Laboratories, School of Biological Sciences, University of Exeter, Perry Road, Exeter EX4 4QG, i:« E-mail [email protected]
Names of fungi Eurotium repens de Bary = Aspergillus repens de Bary; Botrytis cinerea Pers.: Fr.
Introduction: features of interest Much to the frustration of students and teachers alike, the transfer of delicate vegetative hyphae or reproductive structures of fungi from Petri dishes to a microscope slide is rarely achieved without major structural damage obliterating the features of interest. In many cases, such problems can be overcome by growing the fungi from a plug of agar directly onto a microscope coverslip (Fig 1), which can then be mounted on a microscope slide. This elegant and simple technique was devised by Roland Riddell (1950). It is essential for the professional training of mycologists, especially those working in the medical field (Beneke & Rogers, 1996; Fisher & Cook, 1998). In the present contribution, we describe a simplified method and two typical applications for mycology teaching. The Riddell technique was originally designed as a tool to obtain intact spore-producing structures for descriptive and taxonomic studies. As an example, hyphae of Aspergillus rep ens readily grow from a plug of 2% (wjv) malt extract agar (MEA) onto glass coverslips where they produce conidiophores displaying all relevant features such as the foot cell and chains of darkgreen conidia emerging from phialides on the vesicle at the conidiophore apex (Figs 2 and 3). On MEA amended with 20% (wjv) sucrose (MEA+S), conidium formation is reduced but production of the sexual Eurotium state is stimulated (Webster, 1980). In E. repens, which is homothallic, sexual reproduction is initiated when an aerial hypha coils to form an ascogonium (Fig 4) which subsequently becomes
encased in sterile investing hyphae (Fig 5). The mature cleistothecium consists of a singlelayered peridial wall enclosing numerous globose asci (Fig 5), each containing 8 ascospores (Fig 6). Thus, the sexual state (Eurotium) and asexual state (Aspergillus) of this fungus can be conveniently demonstrated side-by-side. The Riddell technique is also suitable for the study of living vegetative hyphae. By mounting the coverslip with adhering living mycelium directly in a drop of an appropriate dye, cell organelles may be stained and their distribution examined, as in the case of mitochondria using the tetrazolium stain which thus acts as a vital dye (Weber et al., 1998). Likewise, Neutral Red accumulates only in living hyphae of filamentous fungi in which it stains vacuoles bright-red (Weber & Webster, 1997). The Neutral Red molecule can diffuse across biological membranes in its neutral form, but once it encounters an environment of acid pH such as vacuoles, it becomes positively charged and is immobilized there (Lullmann-Rauch, 1979). In Botrytis cinerea, vacuoles are formed towards the base of the apical cell (Fig 7) and become more prominent in mature (intercalary) cells (Fig 8), thus demonstrating the polarity of hyphal growth in which a dense core of protoplasm moves forward, leaving vacuolated cells behind (Park & Robinson, 1967; Weber & Webster, 1998). Vacuoles are also abundant in many specialized structures such as sclerotium initials of B. cinerea (Fig 9) and are thought to be involved in generating turgor pressure for processes of differentiation such as basidiospore formation (Wells, 1965) or infection of plants from appressoria (Bourett & Howard, 1990). These and many more features can be investigated by applying the methods as outlined here.
Volume 14, Part 3, August 2000
7-
6
4
\ Fig 1. A moist chamber for slide cultures. Three days after inoculation of the centres of MEA plugs with macroconidia of Botrytis cinerea, mycelium has grown onto the coverslips. Fig 2. Conidiophore of Aspergillus repens arising from a thick-walled foot cell (arrow) and terminating in a swollen vesicle bearing phialides, each producing a chain of conidia. Bar = 50 flm. Fig 3. The vesicle with a single layer of phialides at the tip of a conidiophore of A. repens. Bar = 20 flm. Fig 4. Coiling of a hypha of A. repens to form an ascogonium of Eurotium repens on the surface of the coverslip. Bar = 20 flm. Fig 5. Formation of a cleistothecium initial of E. repens (arrow) by encasement of the ascogonium in sterile investing hyphae. Two maturing globose cleistothecia are visible nearby. Their yellowish wall (peridium) is one cell thick and encloses numerous globose asci. Clumps of detached conidia are also present. Bar = 50 flm. Fig 6. Release of asci by breakdown of the peridium in a mature cleistothecium. Each ascus contains 8 ascospores. Bar = 20 flm. Fig 7. A growing hypha of Botrytis cinerea mounted in the Neutral Red dye for 1 h. The dye has been taken up by vacuoles which become more prominent towards the base of the tip cell. Bar = 10 flm. Fig 8. Neutral Red staining of a mature hyphal cell of B. cinerea. Vacuoles have filled most of the volume of the protoplast. Bar = 10 flm. Fig 9. Neutral Red staining of a sclerotium initial of B. cinerea. Vacuoles occur prominently throughout this structure. Bar = 20 flm.
Source of material Aspergillus repens is ubiquitous and grows even in
conditions of high osmotic stress such as in jams or on dry-stored cereal grains (Raper & Fennell, 1965). It can therefore be isolated on a suitable medium amended with 20% sucrose. Our present strain was obtained some 50 years ago by J. Webster. Botrytis cinerea is the cause of grey mould on a very wide range of plants. The present strain was isolated from lettuce and is deposited as ATCC 201173 in the American Type Culture Collection. We will provide cultures of both species on request.
Maintenance of fungi Both A. rep ens and B. cinerea are easily maintained on common laboratory media such as MEA, potato dextrose agar (PDA) or corn meal agar at 4°C, subculturing every 6 months. In order to ensure that E. repens retains its sexual reproductive capacity in long-term storage, it is recommended that cleistothecia rather than conidia are used as inoculum, with MEA+S as the culture medium (see Jinks, 1954). Spores of both species can also be freeze-dried and stored in vacuum-sealed glass ampoules at
•
•
Volume 14, Part 3, August 2000 room temperature (r.t.) for many years (Smith & Onions, 1983). Preparation of material Day -21. Revive E. repens and B. cinerea by plating onto MEA or PDA. Incubate at r.t. in room light. With B. cinerea, a brief (l min) exposure to near- UV light after 4 d growth will boost the formation of macroconidia. Day -10 to -7. Autoclave glass Petri dishes containing microscope slides supported by glass rods resting on damp filter paper or tissue paper. Alternatively, because the risk of contamination is very low, unsterilized filter paper may be used with standard or large plastic Petri dishes, and the glass rods and slides can be flame-sterilized (Fig 1). Place 1 em diam. plugs of MEA and MEA+S on the microscope slides, inoculate the centres of all plugs with conidia of A. repens, and cover the plugs with flame-sterilized coverslips. Incubate at 20° in the dark. Add distilled water as required to keep the chambers moist until day O. Day -3. Inoculate Riddell slides with macroconidia of B. cinerea as described above for A. repens. Day O. Present material to the students who should remove a coverslip with mycelium of A. repens from each of the two types of agar. In order to minimize damage to the mycelium, the coverslip should be lifted vertically while holding down the agar plug with a needle or forceps. Coverslips with adhering mycelium of A. repens should be mounted in water or lactic acid. For B. cinerea, coverslips should be removed as above, but should be mounted quickly in a large drop of the Neutral Red dye solution containing 0.015% (w/v) Neutral Red (BDH 'Gurr') and 3.75% (w/v) polyvinylpyrrolidone in 50 mM Tris/maleate buffer (pH 7.5). Material should be viewed after 30 min incubation at r.t.; vacuoles will retain the dye for approx. 60 min thereafter. Useful hints Adherence of mycelium is enhanced on gelatincoated coverslips which are prepared by dipping them in a warm 1% (w/v) gelatin solution followed by air-drying. Coated coverslips can be stored dry for years at r.t. Prior to mounting, the dry gelatin coat should be removed from one side of each coverslip by gentle rubbing with an ethanol-soaked tissue paper. Each coverslip should then be flamed only briefly before being placed on an agar plug with the gelatin-coated surface facing downwards. With A. repens, trapping of air bubbles during
the mounting of coverslips can be minimized by placing the coverslip on a flat surface with the mycelium uppermost, applying a drop of mountant to the centre of the ring of mycelium, and gently lowering a microscope slide onto the coverslip. If lactic acid is used as a mountant, any residual air bubbles can be eliminated by brief heating of the mounted slide in the yellow flame of a Bunsen burner. We recommend lactic acid also because it prevents mounted slides from drying out during the course of a practical class. Permanent preparations of lactic acid mounts can be made by sealing the edges of the coverslip with nail varnish (Beneke & Rogers, 1996). We thank Mr P. M. Booth, a BMS Associate, for his support, and our anonymous referee for helpful suggestions. References Beneke, E. S. & Rogers, A. 1. (1996) Medical Mycology and Human Mycoses. Star Publishing Co.: Belmont, CA. Bourett, T. M. & Howard, R. J. (1990) In vitro development of penetration structures in the rice blast fungus Magnaporthe grisea. Canadian Journal of Botany 68: 329-342. Fisher, F. & Cook, N. B. (1998) Fundamentals of Diagnostic Mycology, WB.Saunders Co.: Philadelphia. Jinks, J. 1. (1954) Somatic selection in fungi. Nature 174: 409-410. Lilllmann-Rauch, R. (1979) Drug-induced lysosomal storage disorders. In Lysosomes in Biology and Pathology, Volume 6 (edited by Dingle, J. T., Jacques, P. J. & Shaw, H. I.), pp. 49-129. Elsevier: Amsterdam, New York. Park, D. & Robinson, P. M. (1967) A fungal hormone controlling internal water distribution normally associated with cell ageing in fungi. Symposia of the Society of Experimental Biology 21: 323-335. Raper, K. B. & Fennell, D. (1965) The Genus Aspergillus. Williams & Wilkins Co.: Baltimore. Riddell, R. W (1950) Permanent stained mycological preparations obtained by slide culture. Mycologia 42: 265-270. Smith, D. & Onions, A. H. S. (1983) The Preservation and Maintenance of Living Fungi. Commonwealth Mycological Institute: Kew, Surrey. Weber, R. W S. & Webster, J. (1997) The coprophilous fungus Sphaeronaemella [imicola, a facultative mycoparasite. Mycologist 11: 50-51. Weber, R. W S. & Webster, J. (1998)Teaching techniques for mycology: 5. Basidiobolus ranarum. Mycologist 12: 149-151. Weber, R. W. S., Wakley, G. E. & Pitt, D. (1998) Histochemical and ultrastructural characterization of fungal mitochondria. Mycologist 12: 174-180. Webster, J. (1980) Introduction to Fungi (second edition). Cambridge University Press: Cambridge. Wells, K. (1965)Ultrastructural features of developing and mature basidia and basidiospores of Schizophyllum commune. Mycologia 57: 236-261.