The c o m p a r i s o n o f six media for c h l a m y d o s p o r e production by Candida albicans M. Casal & M. J. Linares
Departamento de Microbiologia e Higiene, Facultad de Medicina, Universidad de Cordoba, Cordoba, Espafia
Abstract Six different cultural media (corn meal agar, rice extract agar, chlamydospore agar, PCB, Tween 80oxgall-caffeic acid and diluted milk) were compared for chlamydospore production by 224 yeastlike fungi isolates. Candida albicans formed chlamydospores to a variable degree in all of the media, as did C. stellatoidea to a lesser extent. C. tropicalis, C. parasilopsis, C. guilliermondii and C. krusei did not produce chlamydospores in any of the media tested. Statistically, the most productive media were the milk and T O C media. Milk medium is particularly useful because of its simplicity and economy.
Introduction Candida albicans is an increasingly important pathogen in human clinical medicine (2). Its rapid laboratory diagnosis rests on chlamydospore production in a variety of cultural media used for that purpose. The present study compares six different media for chlamydospore production.
Material and methods Two hundred twenty-four isolates of yeastlike fungi of the following species were used: 192 Candida albicans, 16 C. stellatoidea, 11 C. tropi-
calis, 1 C. parasilopsis, 1 C. guilliermondii and 2 C. krusei. The isolates belonged to the culture collection of our department and were originally obtained from the Spanish Collection of Type Cultures (CECT) and from the Collection of Mycologic Cultures of the Wadley Institutes of Molecular Medicine, Dallas, Texas (USA), except for some wild strains isolated in the laboratory of Professor Calbo Torrecillas of M~ilaga (Spain) or in our own
laboratory. The isolates were conserved in Sabouraud agarglucose a n d / o r Sabouraud agar-glucose-chloramphenicol (Institute Pasteur) at 25-38 ~ C until used. The media tested for chlamydospore production were: - Milk medium (3) made with commercialized bovine milk, either sterilized or pasteurized. The milk was diluted to 17/100 in distilled or tap water, agitated several minutes and 3-5 ml amounts were poured into 10 X 100 tubes. After preparation, the medium was conserved in a refrigerator for up to one week. - Tween 80-oxgall-caffeic acid medium (TOC) prepared according to the formula of Fleming et al. (4). Twenty milliliters of medium was dispensed into each 90 m m Petri plate. - Others: corn meal agar (CMA), rice extract agar (rice) and chlamydospore agar (C1-A) from Difco, and PCB (Institute Pasteur) (1). The medium was distributed in Petri plates. The innoculates were taken f r o m a young colony after 24 hours growth in Sabouraud agar-glucose and planted with the usual techniques in chlamydospore agar, corn meal agar, rice agar and PCB (5,
Mycopathologia 76, 125 128 (1981). 0301 486X/81/0762-0125 $0.80. 9 Dr W. Junk Publishers, The Hague. Printed in The Netherlands.
126 Table 1. Chlamydospore production by Candida albicans in different cultural media after 48 hours. Species
Number of isolates studied
Medium tested
Number of chlamydospore producing isolates
% of positivity
Abundance
C. albicans
60 60 60 192 192 60 60
C1-A CMA TOC SM PM PCB Rice
42 33 55 180 182 54 44
70 55 91.6 93.7 94.7 82.8 73..3
+ ++ ++++ +h-q-+ ++++ q-+q-§ +q-++
C1-A CMA TOC SM PM PCB
= = = = = =
-t++ q-q-+ +q--t-+
: : : :
Chlamydospore agar Corn meal agar Tween 80, oxgall and caffeic acid Sterilized milk Pasteurized milk Potato-Carrot-Bilis Very scarce Scarce Abundant Very a b u n d a n t
6). The sterilized and pasteurized milk media was innoculated with a concentrated emulsion of yeast in sterile destilled water. In the TOC medium the surface was innoculated and no coverglass was used (1). The media were incubated at 28 ~ and read after 48 hours. C. albicans and Torulopsis glabrata isolates of k n o w n characteristics were used as controls. Each test was repeated at least two times and inconsistent results were eliminated from consideration. The Z test for contrast of proportions was applied to the results.
Results and discussion Table 1 tabulates C. albicans chlamydospore production in the different media. Chlamydospore production was greatest in the sterilized and pasteurized milk media with 93.7% and 94.7% success, respectively. Following were the TOC medium with 91.6% and the PCB medium with 82.8%. Chlamydospore formation was less in the remaining media. The production of resistent forms was abundant in the SM (sterilized milk), P M (pasteurized milk), TOC and PCB media. In corn meal agar were
observed few chlamydospores ( + + ) and in chlamydospore agar, very few (+).
C. tropicalis, C. pseudotropicalis, C. kruseL C. parasilopsis and C. guilliermondii produced no chlamydospores in any medium. The results of C. stellatoidea culture were similar to those of C. albicans although more isolates produced chlamydospores in the milk media and TOC medium than in the other media tested. Statistical results are listed in Tables 2-8. In view of the results, the most useful media for chlamydospore production are the milk media
Table 2. Statistical results of the application of test Z for contrast of proportions to the chlamydospore agar medium compared with other media tested. Medium
Values
Significance (P values)
CMA TOC SM PM PCB RICE
1,69 3,00 4,91 5,3 1,65 0,40
N.S., p 0,01 p 0,001 p 0,001 N.S. N.S.
N.S. = Not significant.
127 Table 3. Statistical results of the application of test Z for contrast of proportions to corn meal agar medium compared with other media tested.
Table 6. Statistical results of the application of test Z for contrast of proportions to the pasterilized milk medium compared with other media tested.
Medium
Values
Significance (P values)
Medium
Values
Significance (P values)
C1-A TOC SM PM PCB RICE
1,69 4,53 7,24 7,59 3,29 2,09
N.S. p 0,001 p 0,001 p 0,001 p 0,01 p 0,05
C1-A CMA TOC SM PCB RICE
5,3 7,59 0,88 0,42 2,60 4,38
p 0,001 p 0,001 N.S. N.S. p 0,01 p 0,001
N.S. = Not significant.
N.S. = Not significant. Table 4. Statistical results of the application of test Z for contrast of proportions to the tween 80, oxgall and caffeic acid medium compared with other media tested.
Table 7. Statistical results of the application of test Z for contrast of proportions to the potato-bilis-carrot medium compared with media tested.
Medium
Values
Significance (P values)
Medium
Values
Significance (P values)
C1-A CMA SM PM PCB RICE
3,00 4,53 0,564 0,88 1,41 2,63
p 0,01 p 0,01 N.S. N.S. N.S. p 0,01
C1-A CMA TOC SM PM RICE
1,65 3,29 1,41 2,60 2,95 1,25
N.S. p 0,05 N.S. p 0,01 p 0,01 N.S.
N.S. = Not significant.
N.S. = Not significant.
Table 5. Statistical results of the application of test Z for contrast of proportions to the sterilized milk medium compared with other media tested.
Table 8. Statistical results of the applications of test Z for contrast of proportions to the rice medium compared with other media tested.
Medium
Values
Significance (P values)
Medium
Values
Significance (P values)
C1-A CMA TOC PM PCB RICE
4,91 7,24 0,564 0,42 2,60 4,38
p 0,01 p 0,001 N.S. N.S. p 0,01 p 0,001
C1-A CMA TOC SM PM PCB
0,40 2,09 2,63 4,38 5,26 1,25
N.S. p 0,05 p 0,01 p 0,001 p 0,001 N.S.
N.S. = Not significant.
N.S. = Not significant.
which have a high percentage of positivity, abund a n t c h l a m y d o s p o r e f o r m a t i o n in 48 h o u r s a n d t h e a d d i t i o n a l b e n e f i t s o f e c o n o m y a n d s i m p l i c i t y in t h e i r p r e P a r a t i o n a n d use. T O C m e d i u m a l s o has a g o o d p e r c e n t a g e o f positives and abundant production. The only d r a w b a c k s to its use are its r e l a t i v e e x p e n s e a n d t h e fact t h a t t h e m e d i u m is n o t c o m m e r c i a l i z e d . C 1 - A a n d C M A did n o t give g o o d results as far as percentage of positivity or amount of chlamydospores produced.
S t a t i s t i c a l l y , the d i f f e r e n c e b e t w e e n the m e d i a with good chlamydospore formation (SM, PM, TOC) and those with poor formation (CMA, C1-A, rice) is s i g n i f i c a n t . R e s u l t s o b t a i n e d w i t h P C B w e r e inconsistent and not significantly different from T O C , C 1 - A a n d rice m e d i a . In c o n c l u s i o n , to o b t a i n r a p i d a n d a b u n d a n t c h l a m y d o s p o r e f o r m a t i o n w i t h C. a l b i c a n s , t h e p r e f e r r e d m e d i a are sterilized a n d p a s t e u r i z e d m i l k -and T O C m e d i a , f o l l o w e d by P C B a n d rice m e d i a .
128
Acknowledgements T o P r o f e s s o r F. C a l b o f r o m the M i c r o b i o l o g y L a b o r a t o r y o f t h e M e d i c a l S c h o o l o f Mfilaga. S p a i n , as w e l l as to D o c t o r W. H. F l e m i n g o f t h e Wadley Institutes of Molecular Medicine, Dallas T X 75235, U S A , f o r t h e strains m a d e a v a i l a b l e to us f o r t h e study.
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