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 80-
oxgall-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 pro-
duction 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 collec-
tion of our department and were originally ob-
tained from the Spanish Collection of Type Cul-
tures (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 agar-
glucose a n d / o r Sabouraud agar-glucose-chloram-
phenicol (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 dis-
pensed 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 chlamydo-
spore 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.
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Table 1. Chlamydospore production by Candida albicans in different cultural media after

48 hours.

Species Number of Medium Number of % of Abundance

isolates tested chlamydospore positivity
studied producing
isolates

C. albicans 60 C1-A 42 70 +
60 CMA 33 55 ++
60 TOC 55 91.6 ++++
192 SM 180 93.7 +h-q-+
192 PM 182 94.7 ++++
60 PCB 54 82.8 q-+q-§
60 Rice 44 73..3 +q-++

C1-A = Chlamydospore agar

CMA = Corn meal agar
TOC = Tween 80, oxgall and caffeic acid
SM = Sterilized milk
PM = Pasteurized milk
PCB = Potato-Carrot-Bilis

-t- : Very scarce

++ : Scarce
q-q-+ : Abundant
+q--t-+ : 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 consider-
ation. The Z test for contrast of proportions was
applied to the results.
observed few chlamydospores ( + + ) and in chlamy-
dospore 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

Results and discussion Table 2. Statistical results of the application of test Z for contrast
of proportions to the chlamydospore agar medium compared
with other media tested.
Table 1 tabulates C. albicans chlamydospore
production in the different media. Chlamydospore Medium Values Significance
production was greatest in the sterilized and pas- (P values)
teurized milk media with 93.7% and 94.7% success, CMA 1,69 N.S.,
respectively. Following were the TOC medium with TOC 3,00 p 0,01
91.6% and the PCB medium with 82.8%. Chlamy- SM 4,91 p 0,001
dospore formation was less in the remaining media. PM 5,3 p 0,001
The production of resistent forms was abundant PCB 1,65 N.S.
RICE 0,40 N.S.
in the SM (sterilized milk), P M (pasteurized milk),
TOC and PCB media. In corn meal agar were N.S. = Not significant.
 127

Table 3. Statistical results of the application of test Z for contrast Table 6. Statistical results of the application of test Z for contrast
of proportions to corn meal agar medium compared with other of proportions to the pasterilized milk medium compared with
media tested. other media tested.

Medium Values Significance Medium Values Significance

(P values) (P values)

C1-A 1,69 N.S. C1-A 5,3 p 0,001

TOC 4,53 p 0,001 CMA 7,59 p 0,001
SM 7,24 p 0,001 TOC 0,88 N.S.
PM 7,59 p 0,001 SM 0,42 N.S.
PCB 3,29 p 0,01 PCB 2,60 p 0,01
RICE 2,09 p 0,05 RICE 4,38 p 0,001

N.S. = Not significant. N.S. = Not significant.

Table 4. Statistical results of the application of test Z for contrast Table 7. Statistical results of the application of test Z for contrast
of proportions to the tween 80, oxgall and caffeic acid medium of proportions to the potato-bilis-carrot medium compared with
compared with other media tested. media tested.

Medium Values Significance Medium Values Significance

(P values) (P values)

C1-A 3,00 p 0,01 C1-A 1,65 N.S.

CMA 4,53 p 0,01 CMA 3,29 p 0,05
SM 0,564 N.S. TOC 1,41 N.S.
PM 0,88 N.S. SM 2,60 p 0,01
PCB 1,41 N.S. PM 2,95 p 0,01
RICE 2,63 p 0,01 RICE 1,25 N.S.

N.S. = Not significant. N.S. = Not significant.

Table 5. Statistical results of the application of test Z for contrast Table 8. Statistical results of the applications of test Z for
of proportions to the sterilized milk medium compared with contrast of proportions to the rice medium compared with other
other media tested. media tested.

Medium Values Significance Medium Values Significance

(P values) (P values)
C1-A 4,91 p 0,01 C1-A 0,40 N.S.
CMA 7,24 p 0,001 CMA 2,09 p 0,05
TOC 0,564 N.S. TOC 2,63 p 0,01
PM 0,42 N.S. SM 4,38 p 0,001
PCB 2,60 p 0,01 PM 5,26 p 0,001
RICE 4,38 p 0,001 PCB 1,25 N.S.
N.S. = Not significant. N.S. = Not significant.

which have a high percentage of positivity, abun- 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

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 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 chlamydo-
spores produced.
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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