Dermatologica 131: 315-324 (1965)

From the Mycology Laboratory, Division of Medicine, Montefiore Hospital,

New York

Chlamydospore Form ation of Candida Albicans

Comparative Studies on Special Media
By F. REISS and G. SZILAGYI

Numerous culture media have been form ulated for the laboratory
identification of Candida albicans. These media are designed to
dem onstrate morphological and physiological characteristics of
C. albicans. One of the most im portant characteristics of this organism
is its ability to form chlamydospores on certain media upon which is
mainly based the morphological diagnosis of this yeast. To date, how­
ever, no inform ation is available of the mechanism of chlamydo­
spore form ation on special media. O ther differential criteria include
mycelium form ation, ferm entation of certain sugars (dextrose,
maltose, sucrose and lactose) and reduction of indicators (triphenyl-
tetrazolium chloride, bismuth) [8, 9].

Review of the Methods Used Favoring Chlamydospore Formation

The following methods have been designed to induce chlamydo­
spore form ation, which as a rule does not occur on Sabouraud glucose
agar:
Benham [1] recommended corn meal agar to provide a medium poor
in nutrients which favors chlamydospore production.
Gordon et al. [3] made a com parative study of four different types
of corn meal agar and found th at none of these appeared to be the
ideal medium for eliciting chlamydospore production by C. albicans.
The reducing sugar content of corn meal agar was considered to retard
chlamydospore form ation, while the absence of sugar favored chlam y­
dospore production. Furtherm ore, the addition of cysteine or other
source of sulfhydryl groups to a natural polysaccharide medium also
prevented filam entation and chlamydospore form ation of C. albicans.
The addition of glucose to a polysaccharide m edium provided the
yeast cells w ith reducing substances which m aintained the reduced
sulfhydryl group necessary for cell division by budding [6, 7].
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316 Reiss, Szilagyi, Cklamydospore Formation of
Other media prepared from plant material such as potato pulp,
potato-carrot pulp and more recently the rice agar suggested by
Taschdjian [15, 16] are rich in polysaccharides.
Nickerson and M ankowsky [6] also described a polysaccharide
medium of known composition favoring chlamydospore formation
of C. albicans.
A medium containing no polysaccharides but only the extractives
of the corn protein, zein, was recommended by Reid, Jones and
Carter [12]. These authors dem onstrated th at zein agar yielded
chlamydospores more constantly than the previously recommended
polysaccharide media.
Liu and Newton [5] using a slide culture technique, showed th at a
medium of low nutritional value, with pH 8.2, gave good chlamydo­
spore production under anaerobic conditions.
Comparative studies of a series of culture media designed to favor
or enhance chlamydospore formation were conducted by Sina and
Reiss [14] who came to the conclusion, that rice infusion agar is the
most suitable for the form ation of chlamydospores. Nickerson’s agar
was not considered suitable for the routine procedure to identify
C. albicans, since a num ber of other species, even not belonging to the
genus Candida, were also capable of reducing bism uth.

Methods for Enhancement o f Chlamydospore Formation

In order to enhance chlamydospore formation in C. albicans various
m aterials have been used. Pavlatou and Marcelou [10, 11] were the
first to introduce bile as a surfactant for the prim ary purpose of
chlamydospore form ation.
Recently Taschdjian [15], Kelly and Funigiello [4] reported that
the addition of Tween 80 to rice agar and corn meal agar enhanced
chlamydospore form ation of C. albicans.
Seeliger [13] described a semisolid dextrose-Tween 80 medium
which enhanced mycelium and chlamydospore production. According
to him Tween 20, Tween 40 and Tween 60 are less active than Tween
80 in their ability to stim ulate chlamydospore production in C. albi­
cans.
Present Investigation
Because no explanation has as yet been offered of the mechanism
of chlamydospore form ation on special media and growth conditions,
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 Candida Albicans Comparative Studies on Special Media 317
we deemed it of interest to conduct a com parative study with the
following compounds and conditions:
(1) sorbitan, polyoxyethylene and polyoxyethylene sorbitan com­
pounds,
(2) bile salt,
(3) methylene blue,
(4) carbon dioxide,
(5) glucose.
Materials and Methods
The followin'; compounds with the exception of glucose were used in a 1% con­
centration in rice extract agar (BBL).
The substances tested were:
(1) Arlacel 80 (sorbitan monooleatc),
(2) Tween 20 (polyoxyethylene sorbitan monolaurate),
(3) Tween 21 (polyoxyethylene sorbitan monolaurate),
(4) Tween 40 (polyoxyethylene sorbitan monopalmitate),
(5) Tween 60 (polyoxyethylene sorbitan inonostearate)
(6) Tween 61 (polyoxyethylene sorbitan monostearate),
(7) Tween 6.3 (polyoxyethylene sorbitan tristearate),
(8) Tween 80 (polyoxyethylene sorbitan monooleate),
(9) Tween 81 (polyoxyethylene sorbitan monooleate),
(10) Tween 85 (polyoxyethylene sorbitan trioleate),
(11) G.3920 (polyoxyethylene oleyl ether),
(12) Na taurocholate,
(13) Methylene blue.
Although Tween 20 and 21, Tween 60 and 61, and Tween 80 and 81 respectively
have an identical chemical composition, they differ in their physical properties such
as viscosity, solidifying point and solubility tendencies.
In addition glucose, a readily metabolizable substrate, was tested for its ability
to prevent filamentation and chlamvdosporc formation. It was added to rice agar
medium in the proportions of 0.5, 1.0, 1.5, 2.0, 3.0 and 4.0 g to 100 ml of medium.
Each experiment was performed in quadruplicate.
These media, containing the different substances to be tested were inoculated
with C. albicans * according to Dalmau's [2] technique. The inoculated culture media
were incubated at room temperature and the filamentation and chlamydospore
formation were observed after 24, 48 and 72 hours. A similar series of culture media
inoculated with the same strain of C. albicans was incubated at room temperature
in a Brewer anaerobic jar in approximately 10% C02. The glucose-rice agar media
were observed only under aerobic conditions. Readings were made on plates right
side tip and lid removed through the cover glass, recording the following: (1) the
* Strain §154,63 isolated in our laboratory from a case of inframammary inter-
trigo.
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Dermatologic». Yol. 131. No. 4 (1965)

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318 Reiss, Szilagyi, Chlamydospore Formation of
growth developing under the center of the cover ¡¡lass (anaerobic zone), (2) the
growth developing under the edge of the cover glass (semi-aerobic zone) and (3) the
growth developing on the uncovered area (aerobic zone). Semi-quantitative estimates
of the amount of mycelium and chlamydosporcs were recorded on a 1 to 4 plus scale.

Results
Results of the different culture media test readings for mycelium
and chlamydospore form ation are summarized in Table I, II and III.

TABLE I
Effect o f Surface Active Agents and Methylene Blue
on Chlamydospore Formation by Candida Albicans *
Substances tested Mycelium Chlamydospore
Tween 20 +++ + ++++
Tween 40 ++ + +++
Tween 60 ++ + ++ +
Tween 80 ++ + + ++++
Arlacel 80 ++ + +++
G .3920 ++ + +++
Na taurochlorate ++ + + ++ + +
Methylene blue 0 0
* Inoculated on rice extract agar (BBL) and incubated 48 hours at 25°C.

TABLE II
Effect of CO., on Chlamydospore Formation by Candida Albicans*
Substances tested Mycelium Chlamydospore
Tween 20 j ____[_ _i_

Tween 40 +++ .L
Tween 60 + -r
Tween 80 ++ _L

Arlacel 80 + +
G..3920 +++ +
Na taurochlorate +++ +
* Inoculated on rice extract agar with addition of surface active agents and incubat­
ed 48 hours at 25f C in 10% C 02 atmosphere.
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 Candida Albicans Comparative Studies on Special Media 319
TABLE III
Lack of Prevention of Chlamydospore Formation
by Candida Albicans* on a Glucose Containing Medium
Per Cent of Glucose
0.5 1.0 1.5 2.0 3.0 4.0
Mycelium + 4- 4* + + + + + + + + + +++ +++ ++
Chlamydospore +++ + +++ + + ++ +++ ++ ++
* Inoculated on rice extract agar with addition of glucose up to 4% and incubated
48 hours at 25° C.

The most potent non-ionic surfactants in chlamydospore production

by C. albicans were: Tween 20, Tween 40, Tween 60, Tween 80,
Arlacel 80, and G.3920. These agents were all equally efficacious (Fig.
1 to 6).
Tween 21, Tween 61, Tween 65, Tween 81 and Tween 85, are
capable of inducing chlamydospore formation but they cannot be
used because of their insolubility in water. The milky medium produc­
ed was unsuitable for direct microscopic examination.
The addition of bile salt also promotes chlamydospore formation to
the same extent as the Tweens. However, the mycelium is short, curl­
ed and fragmented in contrast to th at formed by Tweens (Fig. 7).
In the methylene blue-containing medium filamentation and
chlamydospore form ation was completely inhibited, m aintaining
C. albicans in the yeast phase (Fig. 8).
Incubation of the cultures in a 10% C 02 atmosphere, decreased the
ability of all the Tweens and of bile salt to enhance chlamydospore
form ation (Fig. 9 and 10).
The addition of glucose in various concentrations ranging from
0.5 to 4.0% in rice agar medium did not prevent the filamentation
and chlamydospore formation as reported by Nickerson et al. [6],
Mycelium and chlamydospore form ation was more intense under
the edge of the cover glass than in the center. In the uncovered zone
in 24 hours no filaments or chlamydosporcs could be seen. After 48
and 72 hours of incubation very rare filaments but no chlamydo-
spores were observed.
Finally it should be stated that chlamydospore formation Mas al-
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 Heiss, Szilagji 321
ready observed in 24 hours in all media containing Tweens and bile
salt, but the optim um time was 48 hours of incubation.

Discussion
Despite the profuse literature concerning chlamydospore formation
the mechanism of the production of this cell type is still lacking an
exact understanding. The surface tension-lowering effect of the
Tweens apparently is not the only factor responsible for this morpho­
logical characteristic. A similar mechanism is probably responsible
for the chlamydospore formation on bile-containing rice agar.
No explanation has been offered for the mechanism of interference
of surface tension-lowering agents with the metabolic pathw ay of
C. albicans. The only possible and reasonable effect of this compound
could be the disruption of the cell mem brane or intracellular mem­
brane in a m anner similar to th at observed by Toschi [17] by adding
surface-active agents to brain homogenates. The disruption of cell
membranes of subcellular particles, as seen by Toschi, was not

Fig. 1. Mycelia and profuse chlamydospores on rice extract agar (BBL) plus Tween
20. (100 x )
Fig. 2. Mycelia and profuse chlamydospores on rice extract agar (BBL) plus Tween
40. (100 x )
Fig. 3. Mycelia and profuse chlamydospores on rice extract agar (BBL) plus Tween
60. (100 x )
Fig. 4. Mycelia and profuse chlamydospores on rice extract agar (BBL) plus Tween
80. (100 x )
Fig. 5. Mycelia and chlamydospores on rice extract agar (BBL) plus Arlacel 80.
(100 x)
Fig. 6. Mycelia and chlamydospores on rice extract agar (BBL) plus G. 3920. (100 x )
[‘ig. 7. Short, curled and fragmented mycelia and chlamydospores on rice extract
agar (BBL) plus sodium taurochlorate. (100 x )
Fig. 8. Non-budding yeast cells on rice extract agar (BBL) plus Methylene blue.
Note absence of mycelium and chlamydospores. (100 x )
Fig. 9. Decreased filamentation and Chlamydospore formation on rice extract agar
plus Tween 20, incubated in a 10% C02 atmosphere. (100 x )
Fig. 10. Decreased filamentation and chlamydospore formation on rice extract agar
(BBL) plus Tween 40, incubated in a 10% C02 atmosphere. (100 x )
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322 Reiss, Szilagyi, Chlamydospore Formation of
demonstrable w ith the light microscope, but it is our intention to
investigate such alterations with an electron microscope. Should this
assumption prove correct, chlamydospore form ation caused by sur­
factants could be explained by a deficient cell nutrition following
damage to the cell m embrane*.
The production of chlamydospores on polysaccharide media is
probably due to a reduced energy source on account of a diminution
or absence of amylase activity.
The redox system is not involved in chlamydospore form ation as
exemplified by the negative results in media containing methylene
blue. Similarly the excessively reduced oxygen partial pressure not
oidy does not contribute to an increased or more rapid chlam ydo­
spore formation but appreciably suppresses the production of chla­
mydospores.
The role of -SH groups in m aintaining the yeast phase of C. albicans
could not be confirmed in our experiments. It is adm itted th at glucose
provides €. albicans with reducing substances “ essential to the m ain­
tenance of reduced thiol group” which is supposed to prevent filamen­
tation and chlamydospore formation. Contrary to Nickerson et al.
[6], in our experiments neither chlamydospore form ation, nor filamen­
tation was suppressed on rice agar with the addition of glucose up to
4 %.
Summary
(1) Rice agar media containing eleven non-ionic surfactants, bile
salt and m ethylene blue were tested for ability to enhance chlamydo­
spore formation in C. albicans.
(2) Tween 20, Tween 40, Tween 60, Tween 80, G.3920, Arlaccl 80,
and bile salt are effective in enhancing chlamydospore formation.
(3) Tween 21, Tween 81 and Tween 85 are unsuitable for this
purpose because of their insolubility in water.
(4) Only non-budding yeast cells could be seen on a medium con­
taining m ethylene blue, which suppressed completely filamentation
and chlamydospore form ation.
(5) Rice agar medium inoculated with the same strain of C. albicans
and inetdtated at room tem perature in a Brewer anaerobic jar,
* Grateful acknowledgement is given to Hr. C. Smith, Anesthesiology Labora­
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tory, Montefiore Hospital, for supplying this information.

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 Candida Albicans Comparative Studies on Special Media 323
decreased the capacity of Tweens and of bile salt to enhance chlamydo-
spore form ation.
(6) The addition of glucose up to 4% to the rice agar medium did
not prevent filam entation and chlamydospore formation.
Zusammenfassung
1. Reisagar m it Zusatz von 11 nicht-ionogenen Oherfläehen-aktiven
M itteln, Gallensalzen und M ethylenblau wurde auf die Fähigkeit, die
Chlamydosporenbildung zu erhöhen, geprüft.
2. Tween 20, 40, 00, 80, G 3920, Arlacel 80 und Gallensalze erhöhen
diese Bildung.
3. Tween 21, 81 und 85 sind wegen ihrer W asserunlöslichkeit dafür
ungeeignet.
4. Auf M ethylenblau-haltigen Nährböden konnten lediglich nicht-
sprießende Hefezellen gefunden werden, auch die Fadenbildung war
aufgehoben.
5. Reisagar, der bei Zim m ertem peratur in einer Anaerobenkammer
m it dein gleichen Stamm von Candida albicans geimpft wurde, ver­
minderte die Fähigkeit von Tween und Gallensalz zur Chlamydospo­
renbildung.
6. Die Zugabe von Glykose bis zu 4% zum Reisagar konnte die
Faden- und Chlamydosporenbildung nicht verhindern.
Résumé
1 Les auteurs ont étudié la possibilité d’augm enter la formation
de chlamydospores chez le Candida albicans, au moyen de milieux
au riz et agar contenant onze éléments non ioniques, des sels biliaires
et du bleu de méthylène.
2 Le Tween 20, le Tween 40, le Tween 00, le Tween 80, le G 3920,
l’Arlacel 80 et les sels biliaires ont une action favorisante sur la for­
mation de chlamydospores.
3° Le Tween 21, le Tween 81 et le Tween 85 sont inutilisables dans
ce but, par suite de leur insolubilité dans l’eau.
4° On ne voit que des levures non bourgeonnantes dans les milieux
contenant du bleu de méthylène, lequel empêche complètement la
form ation de filaments et de chlamydospores.
5° Un milieu au riz et agar, inoculé avec la même souche de Candida
albicans et mis en incubation dans une cuve de Brewer en anaérobiose,
empêche les Tweens et les sels biliaires de favoriser la formation de
chlamydospores.
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324 Reiss, Szilagyi
6° L’addition de [dus de 4% de glucose à un milieu au riz et agar
n’a pas empêché la form ation de filaments et «le chlamydospores.
References
1. Benham, R. JF.: Certain monilias parasitic on man. Their identification by-
morphology and by agglutination. J. infect. Dis. 49: 183-215 (1931).
2. Dalmau, L. M.: Remarques sur la technique mycologiquc. Ann. Parasit. hum.
comp. 7: 536-545 (1929).
3. Gordon, M. H.; Bradley, E. G. and Grant, V. ().: The influence of different types
of corn meal agar upon chlamydospore production by Candida albicans. J. lab.
clin. mcd. 40: 316-320 (1952).
4. Kelly, J. P. and Funigiello, F .: Candida albicans: A study of media designed to
promote chlamydospore production. J. lab. clin. Med. 53: 807-809 (1959).
5. Liu, P. and Neicton, A.: Rapid chlamydospore formation by Candida albicans
in a buffered alkaline medium. Amer. J. clin. Path. 25: 93-97 (1955).
6. Nickerson, W. and Mankowsky, Z.: A polysaccharide medium of known composi­
tion favoring chlamydospore formation in Candida albicans. J. infect. Dis. 92:
20 (1953).
7. Nickerson, H . K. and van Rij, N. J. If .: The effect of sulfhydryl compounds,
penicillin and cobalt on the cell division mechanism of yeast. Biochiin. biophys.
Acta 3: 461-475 (1949).
8. Nickerson, IF.: Reduction of inorganic substances by yeast. (1) Extracellular
reduction of sulfite by species of Candida. J. infect. Dis. 93: 43-56 (1953).
9. Pagano, ./.; Letvin, J. D. and Trejo, IF.: Diagnostic medium for differentiation
of species of camlida. Antibiot. Ann. pp. 137-143 (1957/58).
10. Pavlatou, M. and Marcelou, U.: Milieu favorisant la formation «les chlamydo­
spores de Candida albicans. Ann. Inst. Pasteur 91: 410-413 (1956).
11. Pavlatou, M. and Marcelou, U.: Sur le rôle de la bile dans le milieu PCB. Autres
substances qui favorisent la production de chlamydospores par Candida albicans.
Ann. Inst. Pasteur 93: 146-149 (1957).
12. Reid, J. D.; Jones, M. M. and Carter, E. B.: A simple clear medium for demonstra­
tion of chlamydospores of Candida albicans. Amer. J. clin. Path. 23: 938-941
(1953).
13. Seeliger, H.: Ein neues Medium zur Pseudomycclbildung von Candida Albicans.
Z. Hvg. InfektKr. 141: 488-494 (1955).
14. Sina, B. and Reiss, F.: Comparative studies of special media for identifying
Candida albicans from other Candidas and molds. J. invest. Derm. 29: 263-267
(1957).
15. Taschdjian, C. L.: A simply prepared identification medium for Candida albicans.
Mycologia 45: 474-475 (1953).
16. Taschdjian, C. L.: Routine identification of Candida albicans: current methods
and a new medium. Mycologia 49: 332-338 (1957).
17. Tosclti, G.: A biochemical study of brain microsomes. Exp. Cell Res. 16: 232
(1959).
Authors' address: Frederick Reiss, M. I)., Director, Mycology Laboratory, and George Szilagyi, M. 1).,
Montcfiore Hospital, 210th St. and Rainbridge Avc., iVeic York 67, I\'.Y. (U.S.A.)
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