Mycopathologia et Mycologia applicata, vol. 41, 3-4 pag. 299-305, 1970

E X A M I N A T I O N OF SEVERAL SELECTED F U N G I BY S C A N N I N G ELECTRON

MICROSCOPE

by

Y U K I ITO*, YOSHINORI NOZAWA* & TAKAO SETOGUTI**

ABSTRACT

Scanning electron microscopy with high resolution and great depth of focus has been known to be able to Show the fine features of surface ornamenta t ion of fungi, and with this technique we could obtain the interesting results about the surface structures of the selected fungi, PeniciUium chrysogenum, Aspergillus niger, Asper- gillus oryzae and Candida utiIis.

INTRODUCTION

The surface features of fungi have until recently been obtained by the replica and shadowing techniques with transmission electron microscope. However, these techniques have only a limited practical value for examination of the surface ornamentations of cell walls, because of small depth of focus and complex preparation of speci- mens in conventional electron microscope.

Recently a new technique of scanning electron microscope, char- acterized by greater depth of focus and higher resolving power than those of the transmission type microscope, has been introduced for examination of intact solid specimens in microorganisms (1, 2, 4, 5, 6), and provides available information which has not been obtained with other techniques. Using this efficient instrument, we also could reveal some interesting features on the surface orna- mentation and the development progress of hyphae and/or spores of human pathogenic fungi, dermatophytes (8).

The present paper reports the preliminary results of examination of Aspergillus, Penicillium and Candida.

MATERIALS AND METHODS

Strains of Fungi and Cultivation All strains (Aspergillus niger 460, A. oryzae 216, Penicillium

chrysoge~mm 473 and Candida utilis 553) were kindly supplied by

*) Depar tment of Biochemistry, Gifu University School of Medicine, Gifu, Japan. **) Depar tment of Anatomy, Gifu University School of Medicine, Tsukasa-machi

40, Gifu, Japan. Accepted for publication: 13.IX.1969.

300 Y. ITO, Y. NOZAWA ~¢ T. SETOGUTI

Dr. HORITSU, Department of Applied Microbiology, Faculty of Agriculture, Gifu University.

The organisms were grown on test tube slants of Sabouraud's dextrose agar containing 0.5 dried yeast extract, for 10 days at 28 ° C.

Scanning Electron Microscopy

AII samples weie scraped carefully without deformation of their surface features from an agar slope and mounted on specimen holder. The mounts were allowed to dry under a high vacuum and coated with gold to a thickness of about 150 •. Observation of these speci- mens was made by a JSM-2 Scanning Electron Microscope at an accelerating voltage of 10 kV.

R E S U L T S A N D DISCUSSION

The results of observation by scanning electron microscope are revealed in the following figures.

F i g . 1. N u m e r o u s t y p i c a l c o n i d i a I c h a i n s of P . chrysogenum 473. × 800.

SCANNING ELECTRON MICROSCOPY 30t

Penicillium chrysogenum 473

Fig. 1 is a general view which is composed of irregularly branched, filamentous hyphae and typical conidial chains. At a high magnifi- cation (Fig. 2), the chain is shown to be consisted of numerous, relatively smooth surfaced conidia, connected to one another by a bridge which may be discernible or ahnost invisible. Some of the conidia have depressions on their surfaces presumably due to dryness under high vacuum.

Fig. 2. Magnif ied view o5 conidial cha ins in Fig. 1. × 8,000.

Aspergillus niger 460

Fig. 3, at a low magnification, represents a feature like a group of large chrysanthemum-flowers which correspond to typical mature heads with long conidiophores. Some of the heads show a character- istic splitting of conidial mass into divergent columns.

In a magnified photograph, a mature head is composed of many chains of conidia which are arranged compactly or relatively loosely (Fig. 4, 5). Each conidium is globose to somewhat elliptical in shape, having many conspicuous echinulates (Fig. 5).

3 0 2 Y. ITO, Y. NOZAWA ~ T. SETOGUTI

Fig. 3. A fea ture l ike a group of ch rysan themum-f lower s of A. niger 460. × 48.

Fig. 4 A t yp i ca l m a t u r e head composed of numerous conidia l chains. × 240,

S C A N N I N G N L E C T R O N M I C R O S C O P Y 303

Fig. 5. Conspicuous echinulate conidia of the same strain. × 800.

Aspergillus oryzae 216

Typical conidial chains seem to be like glass beads and are com- posed of numerous, large and roughed conidia arranged in a lon- gitudinal pattern, which are more strongly echinulate than those of A. niger 460 (Fig. 6).

Candida utilis 553

It shows, as a whole, uneven surface consisting of numerous large or small tubercles. The surface of each large tubercle is not ,always smooth, frequently ragged. Individual cells cannot be discernible (Fig. 7).

We have described the surface features of the several selected strains of fungi by scanning electron microscope. The results obtained suggest that the scanning type microscope is an extremely efficient technique for observation of the surface views and the development of many fungi. This method also may give valuable information to a morphological classification of fungi.

3 0 4 ¥. ITO, Y. NOZA'vVA (~ T. SETOGUTI

Fig. 6. Glass beads- l ike s t ruc tu r e of conid ia l cha ins of A, oryzae 216. x 800.

Fig. 7. Large or smal l tuberc les of Candida ulilis 553. × 8,000.

SCANNING ELECTRON MICROSCOPY 305

Summary

Several selected strains of Aspergiltus (Aspergillus niger 460, Aspergillus oryzae 216), Penicillium chrysogenum 473 and Candida utilis 553 were examined using a scanning electron microscope. The characteristic features of these strains suggest a possibility of application of this technique to classification of fungi.

Acknowledgement

We wish to express our gratitude to the Japan :Electron Opticals Laboratory Co., Ltd., Tokyo, for permitting the use of JSM-2 Scanning Electron Microscope.

References

I. ]3ARLETa:, G. A. (1967) Scanning electron microscopy: Potentials in the mor- phology of microorganisms. Science, 158: 1318--13,59.

2. HILmSRT, F. A. (1967) The use of scanning electron microscopy in the study of Carboni/erus micospores. New Phytologist, 66: 825--826,

3. ITO, Y., NOZAWA, Y., SUZLTKI, H, & SE'roGIJTI, T. (1970)Surface structures of the hyphae and spores of dermatophytes by scanning electron microscope. Sa- bouraudia, 7: 270--272.

4. Joz~Es, D. (1967) Examination of mycological specimens in the scanning electron microscope. Trans. Brit. mycoL Sot., 50: 690--691.

5, WlLLETS, S. T. & DAVIES, F. L. (I967) Use of scanning electron microscopy for the examination of Actinomyces. J. gen. Microbiol., 48: 171--176.