Mycopathologia 95:73-75 (1986) 9 Martinus Nijhoff/Dr W. Junk Publishers, Dordrecht - Printed in the Netherlands

73

A new m e d i u m for the production of c h l a m y d o c o n i d i a by Candida albicans V. Vidotto*, S. Caramello** & M. G. Gallo***

*Infectious Disease Department, University of Turin, Corso Svizzera 164, 10149 Turin, Italy; **Hygiene Institute, University of Turin, Via Santena 5 bis, 10126 Turin, Italy; ***Department of Animal Pathology, Veterinary Faculty, University of Turin, Via Nizza 52, 10126 Turin, Italy

Abstract Several isolates of Candida albicans were tested for production of chlamydoconidia and metabolic changes when grown on several different solid and liquid media. A liquid medium, consisting solely of sterilized skimmed milk and a solid medium containing processed cheese stimulated more rapid and greater production of chlamydoconidia than the corn meal agar and the other media tested.

Introduction Candida albicans is an important yeast in human clinical medicine (2). It produces both yeast and mycelial cells in vivo and in vitro (1). Chlamydoconidia production is associated with its mycelial form. The production of chlamydoconidia is very useful in the identification of Candida albicans in routine laboratory tests. The purpose of this paper is to compare different media for their effect in chlamydoconidia production and to investigate the metabolic changes that may occur in the isolates grown on these media.

Materials and methods Five different isolates were tested on solid and liquid media. Three were poor chlamydoconidia producers (C.B.S. 562, B 311-10 and 0-402), whereas the other two A12 and C 1 were very good producers. We used the latter in our tests. These isolates were maintained on Malt Agar (Difco) with penicillin G 2500 U/ml and streptomycin 1 000/xg/ml to inhibit the growth of bacteria sensitive to these antibiotics (7). Each strain was successively tested six times on the different media.

(A). The following liquid media were used: 1. Rice extract was prepared by adding 10 g of cream of rice to 1000 ml of cold distilled water. Then it was brought to a boil for 30 sec. This preparation was filtered through gauze then centrifuged at 2500 r.p.m, for 20 min. The supernatant was made up to a volume of 1000 ml with 8.96 mt of distilled water (3), glucose 200 mg, peptone 200 mg, Tween 80 0.10 ml. 2. Distilled water 8.96 ml, glucose 20 mg, peptone 20 mg, Tween 80 0.10 ml. 3. Sterilized skimmed commercialized milk 1.8% fat, was diluted to 17/t00 in distilled sterile water and agitated several minutes. After this preparation the medium was conserved in a refrigerator for one week (2) 9.06 ml. 4. Corn meal liquid was prepared by adding 12.5 g yellow corn meal stirred in 300 ml of water and heated in a water bath at 60~ for 1 h and then filtered through paper. The volume of the filtrate was made up to 300 ml distilled water and sterilized for 15 rain at 120 ~ The hot liquid was refiltered through absorbent cotton wool (5) 9.06 ml, Tween 80 0.10 ml. Each medium was sterilized at 120~ for 15 min. They were inoculated with 1 ml of Candida albicans cells, suspended in sterile water 1•

74 cells/ml and after incubation at 28 ~ for 7 days. (B). The following solid media were used: 5. Corn meal agar (Difco). 6. Rice extract (3) 9.7 ml, Tween 80 0.10 ml, Agar 20 mg. 7. Distilled water 9.7 ml, processed cheese 'Crema Bel Paese Galbani' with these ingredients: cheese, water, whey powder and condensed whey, cream, butter emulsifying salts, 400 mg homogenized in 9.7 ml of distilled water, Tween 80 0.10 ml, Agar 20 mg. 8. Distilled water 9.7 ml, processed cheese 'Crem a Bel Paese Galbani' 40 mg, homogenized in 9.7 ml of distilled water, Tween 80 0.10 ml, Agar 20 mg. 9. Distilled water 9.7 ml, Tween 80 0.10 ml, Agar 20 mg. 10. Distilled water 9.7 ml, Soya flour 400 mg, Tween 80 0.10 mg, Agar 20 mg. 11. Distilled water 9.7 ml, Soya flour 4 0 m g , Tween 80 0.10 ml, Agar 20 mg. Each medium was sterilized at 120 ~ for 15 min. For the solid media we used Petri dishes with a filter paper liner, moistened with distilled water and each containing four slides laid on glass rods, sterilized at 120~ for 15 min. The slides were covered with sterile solid medium. One loopful of inoculum was cut twice into the agar. The plates were incubated at 28 ~ for seven days.

Whereas using peptone at 200 ng in 10.06 ml ( = 19.880 g/l) or 20 mg in 10.06 ml (= 1.998 g/l) we did not obtain chlamydoconidia. On solid media (Table 2) we obtained good results only with Corn meal agar (Difco) and the 400 mg cheese medium both for chlamydoconidia and mycelium production. Particularly cheese (400 mg) showed during the first days greater chlamydoconidia production than Corn meal agar (Difco). We suggest that the processed cheese medium should be used in laboratories for obtaining rapid production and high numbers of chlamydoconidia. It seems probable that there is a great affinity of chemical elements between cheese and milk for chlamydoconidia production. We obtained no results using soya flour.

Conclusions The results of this study lead us to conclude that the processed cheese medium stimulated, in the strains of C a n d i d a a l b i c a n s tested, the production of mycelium and chlamydoconidia. In addition we found that processed cheese is more practical and speedy than milk medium for the production of chlamydoconidia.

Acknowledgements Results and discussion On the liquid media (Table 1) we tried the two strains A12 and C 1 o n media 1, 2, 3, 4. We cut into the agar three times with one loopful of each C a n d i d a a l b i c a n s strain on two separate Petri dishes with Corn meal agar (Difco) used as a control. We obtained no chlamydoconidia on media 1, 2, 4, but we had abundant and speedy mycelial production on all of the media. Whereas we obtained very high chlamydoconidia production using sterilized milk medium (2). In Table 1, it appears that glucose at 200 mg/10.06 ml (=19.88 g/l) or 20 mg/10.06 ml (=1.988 g/l), inhibited chlamydoconidia formation. This agrees with the results of Jansons and Nickerson (4), (inhibition with 5 mg/ml) and Dujardin, Walbaum and Biguet (3) (inhibition with 0.5 g/l).

The authors thank Prof. Arturo Ceruti and Prof. Paolo Gioannini for their advice.

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