Mycopathologia 83, 17-18 (1983). �9 Dr W. Junk Publishers, The Hague. Printed in The Netherlands.

A convenient and inexpensive method for preparing fungal conidia for scanning electron microscopy

C. Ramirez Laboratory of General and Applied Mycology, lnstituto "Jaime Ferrdn' de Microbiologia, Consejo Superior de Investigaciones Cientificas, Joaquin Costa 32, Madrid-6, Spain

Abstract

An inexpensive and easy method for the preparation of fungal conidia for SEM is described. 10% formaldehyde solution is used as fixative agent and a specimen support consisting of a 2% agar layer resting on a narrow and short strip of cotton fabric, affixed by a proper adhesive to a narrow and short tin strip is employed.

Introduct ion

Samson & Stalpers (1) obtained satisfactory re- sults with fungi having broad, thin-walled struc- tures, by fixation with 6% glutaraldehyde or 1% OsO4 (separately or combined) followed by dehy- dration and critical point drying. The dehydration was performed using 2-methoxy-ethanol (= me- thylcel losolve) , a rapid chemically dehydrating agent, reducing the dehydration time to 20 minutes. Before critical point drying, the specimens were washed in 100% acetone. Martinez & Ramirez (2) described an improved technique for the prepara- tion of Penicillium conidia when a great number of samples were involved, using small blocks of 3% agar, which were pressed against well-sporulated fungal colonies, in order to get a great number of conidia adhering to the agar surface. The blocks were then introduced into perforated Beem'sl cap- sules and processed following the Samson & Stalpers' technique (1). The disadvantage of the later mentioned method was the impossibility of direct labeling ofagar blocks, leading thus to possi- ble errors in the sample identification.

Balzers Union Aktiengesellschaft Zubeh6re ftir die Elektro- nenmikroskopie, Postfach 75, FL-9496 BALZERS, Principalty of Liechtenstein.

Material and methods

The author of the present paper has obtained excellent results by fixation with 10% formalde- hyde and dehydration with ethanol.

The entire process is presented here as follows: a) A piece of cotton fabric is cut in strips 5 m m

wide. Each strip is then cut in short pieces 15 m m long.

b) A tin sheet is similarly cut in strips of identical width (5 mm) and pieces 15 mm long.

c) Each piece of cotton fabric is then affixed to each piece of tin by means of a convenient adhesive (U.H.U., from Fisher, Btihl, Germany), insolu- ble in ethanol.

d) Once the adhesive is dried, each tin piece may be properly labeled underneath by means of a hard pencil (Eberhard Faber 5H).

e) A drop of hot molten 2% agar is then deposited upon the cotton fabric surface by means of a Pasteur pipette.

f) Once the agar is solidified, its surface is gently pressed against a well-sporulated fungal colony.

g) The specimen is then air-dried in an incubator at 37 ~ C, for 60-90 minutes.

h) The dried specimens are then introduced separ- ately into perforated Beem's capsules for further processing, as follows:

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i) Fixation with 10% formaldehyde during 24 hours.

j) Dehydration by three successive washings with 96% ethanol (1:10:10 minutes), followed by three successive washings with absolute ethanol (1:10:10 minutes). The specimens may be kept in the last solvent until needed for the next step.

k) Critical point drying in CO2. This step is neces- sary to complete the dehydration process. A Samdri-Put-3 automatic apparatus, with a 15 minutes purging time was used by the author.

1) Before coating with palladium-gold (40-60) in a vacuum evaporator, the specimens are taken out from the capsules and affixed to the standard specimen stub by means of a silver electrocon- ductive adhesive 'Dotite', (JEOL Co., Tokyo, Japan). The conidia were viewed and photographed with

a JEOL JSM-50A scanning electron microscope at an accelerating voltage of 15 20 Kv. The obvious advantages of this method are the use of unexpensive fixative agents, with excel- lent results, and the easiness in the manipulation and permanent labeling of specimens.

References

1. Samson, R. A. & Stalpers, J. A., 1977. Preparation tech- niques of fungal specimens for SEM. Annual Conference of the Netherlands Soc. of Electron Microscopy. Leyden. The Netherlands.

2. Martinez, A.T. & Ramirez, C., 1981. Contribution to the preparation techniques of conidia for scanning electron mi- croscopy. Abstract of the IV International Conference on Culture Collections. BRNO, Czechoslovakia.