COMPARATIVE MEDIA STUDIES IN THE ISOLATION

OF CANDIDA A L B I C A N S F R O M P R E G N A N T W O M E N

by

ANDREW G. SMITH, P h . D . 1), HANS D. TAUBERT, M. D.,

CHARLES M. TOWNS

(Departments o] Microbiology and Obstetrics and Gynecology,

University o/Maryland School o/Medicine, Baltimore 1, Maryland)
(27.XII. 1961)

(with 2 figs.)
A wide variety of differential media have been proposed for the
identification of Candida albicans, a yeast-like fungus commonly
accepted as having a major role in the etiology of vulvovaginitis
(24). More often than not these media have been formulated for the
purpose of stimulating chlamydospore production, for this charac-
teristic structure has been given diagnostic credence where C. albi-
cans is concerned (4, 6, 10, 11, 12, 18, 20, 21). The classic medium
used for this purpose is corn meal agar, described in 1931 by
BENHAM (2), who applied the Dalmau technique (5) of slit inocu-
lation plus the subsequent use of a coverglass to facilitate micro-
scopic observation of growth. GORDON, BRADLEY & GRANT (8)
tested four types of corn meal for their influence upon chlamydo-
spore production, and showed that the yellow unenriched variety
was superior. Covering the inoculum with a coverglass, as advo-
cated by WlCKER~AM (26), displayed no advantage in their hands.
BAKERSPIGEL (1) also found yellow corn meal to be superior.
Because of the variable results obtained with plant materials such
as potato-carrot pulp and corn meal, NICKERSON & )/IANKOWSKI
(13) formulated a medium containing soluble starch freed of re-
ducing sugars. Their "purified polysaccharide" medium 2) appeared
to them to be an improvement over corn meal agar. However, in
the experience of others this has not proven to be the case (9, 14,19).

Dr. SMITHis Associate Professor in the Department of Microbiology; Dr. TAIJBI~T

is a FeIlow in the Department of Obstetrics and Gynecology; Mr. TowNs is Labora-
tory Assistant in the Department of Microbiology.
l) Supported in part by a grant from the Lederle Medical Faculty Awards
Committee and in part by United States Public Health Service Grant E-3068.
2) Bacto-Chlamydospore Agar, Difco Laboratories, Detroit, Michigan.
 270 A.G. SMITH a . o .

t(EID, JONES & CARTER (15) devised a medium devoid of any poly-
saccharide, but containing the corn protein, zein, with which they
achieved better results than with corn meal agar.
SEELIGER (17) showed that the addition of Tween 801) to a dex-
trose-peptone-agar medium caused abundant mycelium formation
in several Candida species and also stimulated chlamydospore for-
mation in C. albicans. Working with 17 "recalcitrant" strains of
C. albicans isolated from a variety of clinical sources which failed to
form chlamydospores on rice infusion agar (22). TASCHDJIAN (23)
demonstrated that growth on a "cream of rice" infusion agar con-
taining Tween 80 resulted in abundant chlamydospore production
by all strains. ROSENTHAL (16) reported improved results also with

TABLE I.
Comparative Media Studies Regarding CMamydospore Formation by Candida albicans

Refer- Media Compared*

Investigators ence Year
No. CMA CI~AT DPAT NM RA RAT SSA ZA
13"* 19
REID, JONES & 15 1953 1-9 . . . . . . . . 1---9
CARTER 68%*** 100%
107 99 79
POLLACK & 14 1957 "1 0 9 10 9 10 9
BENHAM 98 % 91% 72 %
0 4 3 17
TASCHDJIAN 23 1957
~-G i-~ 23 I-T
0% 25% 15% 100%
22 21 29
SINA (~ REISS 19 1957 3--~ -- -- 3--6 ~-~
73% 70% 97%
19 19 15 19
I~ELLY & 9 1959 19 19 -- 1-9" 19
PUNIGIELLO 100~o 1 0 0 % 79% lOO%
17 21 21
WALKER 20 21
HUPPERT 25 1960
s5% 100% 100%

* CMA -- Corn Meal Agar.

C M A T - - C o r n M e a l A g a r - - T w e e n 80.
D P A T - - D e x t r o s e P e p t o n e A g a r - - T w e e n 80.
N17[ - - N i c k e r s o n - M a n k o w s k i M e d i u m .
RA -- Rice Infusion Agar.
R A T - - R i c e I n f u s i o n A g a r - - T w e e n 80.
SSA -- Soluble Starch Agar.
ZA -- Zein Agar.
** N u m e r a t o r - - N o . o f s t r a i n s f o r m i n g c h l a m y d o s p o r e s .
Denominator -- No. of strains tested.
*** Percentages are t o the nearest whole number.

i) Polyoxyethylene sorbitan monooleate, Atlas Powder Co., Wilmington, De].

 MEDIA FOR C. ALBICANS ISOLATION FROM PREGNANT WOMEN 271

tile addition of Tween 80 to corn meal agar.

The accumulation of a variety of media designed for such a single
purpose has led to comparative studies among the various kinds.
A summation of some of these studies is presented in Table I. It
can be seen that in two instances corn meal agar is reported to be
as good as or better than other media in the given test series; how-
ever, KEI~LY & FUNmlELI~O (9) stated that chlamydospores were
not produced in abundance on it. In all other cases it suffers b y
comparison. Nickerson-Mankowski medium is not shown to be
superlative in any case. Corn meal agar plus Tween 80 and rice agar
plus Tween 80 consistently appear to be the most efficient; however,
zein agar would seem to warrant further testing.
The purpose of the present study was to evaluate clinically
media in the isolation of C. albicans from vnlvovaginal specimens
taken from pregnant patients, and, in particular, to investigate the
reliability of chlamydospore formation as a criterion for the identi-
fication of strains of C. albicam obtained from this source.

M A T E R I A L S AND M E T H O D S

The 4 media evaluated were: corn meal agar (CMA), corn meal
agar with Tween 80 (CMAT), rice agar (RA), and rice agar with
Tween 80 (RAT). The coma meal agar was a commercial product 1!.
Rice agar was made from Cream of Rice according to TASCHDJIAN S
directions (23, 24). Tween 80 was added in a concentration of 1 9~.
The media were autoclaved, stirred well where Tween 80 was pres-
ent, and then poured into regular 90 mm Petri dishes.
Specimen material for culturing was obtained from 301 pregnant
patients in tile manner described previously (24). Sterile, dry,
cotton-tipped applicators, after having been rotated over the vagi-
nal wall and the vulva, were rolled over the surface of 1 quadrant of
a plate, and the inoculated area in part covered b y a sterile cover-
glass. Swab samplings were done in duplicate, and 1 swab served to
inoculate 2 media. Four specimens were cultured per plate. Incu-
bation was at room temperature. Using bright-field microscopy
at a magnification of 100 X, inoculated areas showing yeast-like
growth were scrutinized both through the coverglass and outside
the covered portion for chlamydospore formation. Observations
were made daily for a period of 1 week. This time limit was felt to
be a practical one for media being tested as selective and differential
for yeast-like fungi of vulvovaginal origin.
All yeast-like fungi were subcultured onto Sabouraud's dextrose
agar plates and purified b y the streaking process. Those strains
forming chlamydospores within 1 week on at least one of the media
were designated as CF (chlamydospore forming) strains. When ho-
mogeneity of colony type was ascertained, the strain was sub-

x) Baltimore Biological L a b o r a t o r y , Inc., Baltimore, Md.

272 A.G. SMITH a . o .

cultured to a slant of WICKERHAM'S malt extract-yeast extract

medium for culture maintenance (26). Strains were maintained in
stock through subculturing every 6 weeks. No loss of strains occur-
red under these conditions.
Subsequently each isolant had its fermentation pattern deter-
mined for the following carbohydrates: glucose, maltose, sucrose,
and lactose (3, 1i% 27). The composition of the fermentation me-
dium and the standardization of the inocutum were as recommended
by WICKERHAM (26). Incubation was at room temperature for 14
days as recommended by BXNI~AM (3).
The 4 media were analyzed for the incidence and the rapidity with
which they stimulated chlamydospore production, and, in view of
the fact that Candida species other than C. atbicans are capable of
forming chlamydospores (3, 7, 14, 20, 25), the isolants were analyzed
also for the degree of concurrence between their fermentation pat-
terns and chlamydospore production.

I~ESULTS
One hundred and eighteen patients (39.2 %) gave positive cultures
for yeast-like fungi. Eight (6.8 %) presented mixed cultures which
were of the following combinations: C. albicans and Candida stetla-
toidea (3 cases); C. albicans and Candida t~arapsilosis (2 cases);
C. albicans and Candida tropicalis (2 cases); C. stell~toidea and C.
tropicalis (1 case). Of the total 126 isolants, 8 died before the 6-weeks
subcutturing schedule was effected. Sixty (47.6 %) of the isolants
were identified as CF strains. The incidence with which these 60 CF
strains formed chlamydospores on the 4 media is summarized in
Table II, where it can be seen that only 27 (45.0~o) of them formed
chlamydospores on all the media. R A T appears to be the most
efficient in eliciting a chlamydospore response with 58 (97 %)
being positive on it.Only 32 (53 %) formed chlamydospores on CMA.
R A is shown to be definitelybetter than CMA, but the addition of
Tween 80 to both caused a remarkable improvement in theirability
to stimulate chlamydospore production. There were 2 strains which
formed chlamydospores on C M A T that failedto form them on RAT.
Conversely, there were 9 strains which formed eh]amydospores on
R A T that failed to form them on C M A T . No strains were found
which formed chlamydospores on IRA and failed to form them on
RAT. Three strains formed chlamydospores on C M A and failed to
form them on C M A T .
The rapidity with which chlamydospores were produced is pre-
sented graphically in Fig. I. The day of chlamydospore formation
on each medium was recorded for 50 of the CF strains. Sporulation
was earlier on RAT than on CMAT, 88 (76.0 %) strains having
formed chlamydospores on this medium within 3 days. Only 26
(52.0 °/o) had sporulated on CMAT within that time period; none
having been observed at 24 hours. Although for some strains sporu-
 MEDIA FOR C. A L B I C A N S ISOLATION FROM PREGNANT WOMEN 273

b# o

Z ~

11+o

+II
+llI
l++l ~5

I1++
t~

+1 + ~
14 P~
+l+l ~

++ll ~

~3

O
+++I Z #
++++ S

0 0 0

m
"N
~ 0 ~ 0

II iI ~
+1~

Mycopathol. et Mycol. App1. X V I t , 3. 18

274 A. G. SMITIt a,o.

50- ~:1 RICE AGAR

(.9 45- F'~ CORN MEAL AGAR

Z
40-
J
::3 55-
n"
0 50-
n
i
(../3
I1,- 2 5 -
LI.I
m 20-
:D
z 15-

,0 I
5

D A Y - ~ ILL~54567 I 25456..7J
WITH TWEEN 80 WITHOUT TWEEN 80

Fig. 1. Daily cumulative representation of time of chlamydospore formation b y

50 CF strains of Candida oi1 four media.
* % CF strains forming chlamydospores within one week.

lation occurred as early on RA and CMA as it did on RAT, the

incidences were lower with 22 (44.0 %) strains having formed
chlamydospores on RA within 3 days, and only 15 (30.0 %) having
done so on CMA. These results demonstrate in a striking manner the
stimulating influence of Tween 80 on chlamydospore formation. The
chance selection of the 50 CF strains for recording the data presented
in Fig. 1 resulted in an apparently improved overall efficiency for
all media except RAT when compared with the data presented in
Table II. Nevertheless, their relative positions, based on the final
proportion of strains forming chlamydospores, remained unchanged.
The results of the fermentation tests are summarized in Table III.
Because of the deaths referred to previously, the establishment of
the fermentation pattern was limited to 55 CF strains. Since only
41 (74.5 %) of these strains gave the fermentation pattern charac-
teristic of C. albicans, it becomes obvious that chlamydospore form-
ation per se is not sufficient for the identification of this organism.
Also, it is of interest to note the presence of C. krusei as a species
capable of forming chlamydospores. This confirms an observation
reported for this organism by FAHLBERG, DUKE & GVTHRIE (7).
The extent to which there is concurrence of the fermentation
pattern of C. albicans with chtamydospore formation is shown in
Fig. 2, where the lack of a reciprocal relationship between these
 MEDIA FOR C. ALBICANS ISOLATION FROM PREGNANT WOMEN 275

TABLE I I I .
Fermentation Reactions o/ 55 C F Strains o[ Candida

Fermentation Reactions*
No. %
Identification
G iV[ S L Strains Strains

AG** AG A 0 41 74:.5 C. albicans

AC.. AG 0 0 8 14:.5 C. stellatoidea
AG AG AG 0 4 7.3 C. t~opicalis
AG 0 0 0 2 3.6 C. krusei

G -- Glucose ** A --- A c i d
M -- Maltose G -- Gas
S -- Sucrose 0 -- No fermentatrion
L -- Lactose

two criteria is shown graphically. Of the 14 CF strains which gave

fermentation patterns of other Candida species, 12 formed chlamy-
dospores on RAT, 9 on CMAT, 7 on RA, and 8 on CMA. Statisti-
cally, this would seem to indicate that none of the media preferen-
tially stimulated chlamydospore formation in C. albicans to the
exclusion of other Candida species to a significant degree. However,

I00- A~- I ~ RICE AGAR

90- [ ] GORN MEAL AGAR
A
80" ~ x "~
A
X/>¢b

~/
1o
X),Q, ~
70-

60-

50- /-//

40- / / / =:~wr

X>(b<
30- X/'fX ....

20- X><'3
X > ( N ......
KXN~ X?43, ~ ///
10- ...... ///

~k'2'// ///

WITH TWEEN 80 WITHOUT T W E E N 80

F i g . 2. C o m p a r a t i v e a n a l y s i s o f f o u r m e d i a b a s e d u p o n c o n c u r r e n c e of t h e f e r m e n -
t a t i o n p a t t e r n of C. albieans w i t h c h l a m y d o s p o r e f o r m a t i o n .

* Percentages are to the nearest whole number.

** A = % C F s t r a i n s f o r m i n g c h l a m y d o s p o r e s .
*** B = % C F s t r a i n s i d e n t i f i e d a s C. albicans v i a f e r m e n t a t i o n r e a c t i o n s .
18"
 b.9

TABLE IV.
Summary o/ Cultural Characteristics o/ the Di//erent Candida Species

Other Than CF Strains

Fermentation Ractions* T o t a l No. No. C F A f t e r 16 D a y s on R A T * *
Identification
Strains Strains
G M S L M N

AG AG A 0 C. albica~s 69 (58.5%) 41 (59.4%) 15(21.7%) 11 (15.9%) 2 (2.8%)

AG AG 0 0 C. stellatoidea 26 (22.O%) 8 (30.8%) S(30.8%) 9 (34.6%) 1 (3.8%)
AG AG AG 0 C. tropiccdis 12 (10.2%) 4 (33.3%) 2(16.7%) 3 (25.0%) 3 (25.0%)
AG 0 0 0 C. krusei 8 (6.7%) 2 (25.0%) 0 (0.0%) I (12.5%) 5 (62.5%)
AG 0 0 0 C. parapsilosis*** 2 (1.7%) 0 (o.0%) 0 (0.0%) 2 (ioo.o%) 0 (0.0%)
AG A A 0 Candida sp. (?) i (0.9%) o (0.0%) o (0.0%) 1 (too.o%) 0 (0.0%)

Totals i l s (lOO.O%) 55 (46.6%) 25 (21.2%) 27 (~2.9%) 11 (9.3%)

* A b b r e v a t i o n s s a m e as for T a b l e I I I .
** C - - M y c e l i u m p l u s c h l a m y d o s p o r e p r o d u c t i o n .
M - h i y c e l i u m withou~ c h l a m y d o s p o r e p r o d u c t i o n .
N- No m y c e l i u m p r o d u c t i o n ; y e a s t - l i k e p h a s e only.
*** No s u r f a c e g r o w t h .
 M E D I A FOR C. ALBICANS I S O L A T I O N FROM P R E G N A N T W O M E N 277

a far greater number of CF strains formed chlamydospores on media

containing Tween 80 than on media not containing it, and, hence,
a greater number of strains of C. albicans were identified from these
media with 41 (74.5 %) from RAT giving the fermentation pattern
of C. albicans as opposed to 38 (69.1%) from CMAT.
The fermentation patterns and morphologic characteristics of
those strains which did not form chlamydospores within 1 week on
at least 1 of the 4 media at the time of primary isolation are included
in the data summarized in Table IV. C. albicans composes the great-
est number of these, and following prolonged incubation of these
strains as pure cultures on RAT an additional 15 were seen to spo-
rulate. Thus, out of a total of 118 isolants which had their fermen-
tation patterns determined, 69 (58.5 %) gave the pattern for C.
albicans. Fifty-six (81.2 %) of these eventually formed chlamydo-
spores; 11 (15.9 %) produced mycelial elements without chlamydo-
spores; and 2 (2.9 %) remained in the yeast-like phase. A total
of 26 strains (22.0 %) gave the fermentation pattern of C. stella-
toidea; of these 16 (61.5 %) formed chlamydospores, which points
up the relatively high incidence with which this sporulation process
occurs in this species. Nine strains (34.5 %) produced mycelial ele-
ments; 1 (3.8 %) remained in the yeast-like phase.
C. tropicalis was identified in 12 instances (10.2 °/o); 6 (50.0 ~o)
formed chlamydospores; 3 (25.0 %) produced mycelial elements;
and 3 (25.0 %) remained in the yeast-like phase. Eight strains
(6.8 %) were identified as C. krusei; 2 (25.0 %) formed chlamydo-
spores; 1 (12.5 %) formed mycelial elements; 5 (62.5 %) remained
in the yeast-like phase. The strains of C. parapsilosis and the nn-
identified species all formed mycelial elements, but chlamydospore
production was not observed.

DISCUSSION
Despite the inference in Table I, the results of the current study
demonstrate that not all strains of Candida giving the fermentation
pattern of C. albicans form chlamydospores on nutritionally defi-
cient media containing Tween 80. Furthermore, the divergence of
results obtained by the various investigators using the same media
must reflect the diversities of strains tested and techniques utilized.
The proclaiming of a cultural characteristic for a species as preva-
lent as C. albicans based on the results obtained with relatively few
strains can lead to precarious conclusions. The identification of C.
albicans through recognition of chlamydospore production would
appear to be a case in point, in view of the fact that, in this investi-
gation, strains of 4 of 6 species were observed to sporulate. This
may be an indication that the process is of generic importance and
we await only the development of an appropriate cultural environ-
ment to establish its universality among the Candidas.
Whether chlamydospore formation would have greater signifi-
278 .. G. SMITEa.o.

cance in identifying isolants of C. albicans from cases of leukorrhea

is open to question. TAUBERT & SMITH (24) did not establish the
fermentation patterns of isolants in their survey of pregnant and
non-pregnant patients with teukorrhea, b u t assumed that the obser-
vation of chlamydospore formation was tantamount to identifi-
cation of C. albicans. In view of the data that have been presented
here, however, such a diagnosis could be accepted only if it were to
be shown that other chlamydospore-forming species never, or rarely,
were associated with such a disorder. Furthermore, that only C.
albicans, of the yeast-like fungi, would be expected to have etiologic
significance appears unlikely since 30 (34.8 %) of the 87 strains
isolated in their series failed to produce chlamydospores on primary
isolation. In the current investigation from among those patients
giving positive cultures for yeast-like fungi, there were 2 cases of
clinical candidiasis. A sporulating strain of C. albicans was isolated
from each. There were 12 cases listed as presumptive candidiasis
from which 1 non-sporulating and 7 sporulating strains of C. albicans,
and 4 non-sporulating strains of C. stellatoidea were isolated. From
8 cases of cervicitis 1 non-sporulating and 2 sporulating strains
of C. albicans, 1 non-sporulating and 3 sporulating strains of C.
stellatoidea, and 1 sporulating strain of C. tropicalis were isolated.
The etiologic significance of such results is unclear at this time.
Each of the 4 media served satisfactorily as a directly inocu-
lable selective medium for the isolation of yeast-like fungi from
vulvovaginal specimens. Although the bacterial flora in such speci-
mens outnumbers the yeast-like flora, bacterial growth seldom was
overt, and when present was readily distinguishable. Occasionally,
only microcolony or pin point growth of yeast-like isolants was
noted on R A and RAT media in contrast to a more luxuriant growth
on CMA and CMAT media. Of 3 such strains, 2 gave the fermenta-
tion pattern of C. parapsitosis, and I that for C. krusei. In the use
of a rice medium, therefore, as the sole medium for direct culturing,
it is conceivable that cursory examinations of inoculated areas could
lead to missed isolations.
The number of strains forming chlamydospores early in the pre-
sent investigation did not approach those reported b y other investi-
gators using RAT. TASCHDJIAN(23) reported 10 of 17 strains of
C. albicans formed chalmydospores within 24 hours; WALKER &
HUePERT (25) reported all of 20 strains produced abundant chla-
mydospores on both R A T and CMAT in that time period. In con-
trast to their pure culture inocula, the present study utilized direct
swab inoculation of plates, and it is conceivable that nutrients
present in the clinical specimens m a y have had an inhibitory effect
on the sporulation process.
None of the media evaluated for the isolation and identifica-
tion of C. albicans to the exclusion of other Candida species relia-
bly accomplished this purpose for the reason that the criterion for
identification, chlamydospore formation, was confirmed as not being
 M E D I A F O R C. A L B I C A N S I S O L A T I O N F R O M P R E G N A N T ~VOMEN 279

a species characteristic, but was shown to be widely shared; further-

more, none of the media appeared to favor this process in C.
albicans over other species. In the final analysis, fermentation pat-
terns were relied upon for isolant speciation. Nevertheless, it can
be concluded that R A T and CMAT can serve with a high degree of
efficiency as directly inoculable media suitable for the selective
isolation from clinical specimens of yeast-like fungi of the Candida
genus.

Acknowledgment
We t h a n k Dr. CHARLES L. WISSEMAN JR., and Dr. MERRILL J. SNYDER for
their interest and counsel during this investigation.

Summary
Rice agar and corn meal agar, with and without Tween 80, were
evaluated clinically as directly inoculable selective and differen-
tial media for the isolation of C. albicans from vulvovaginal specimens
taken from pregnant women. Chlamydospore formation on these
media was investigated as a criterion for the identification of C.
albicans.
Of 301 patients cultured, 118 (39.2 °/o) gave positive cultures for
yeast-like fungi of the genus Casdids. Of 118 strains for which fer-
mentation patterns were determined, 69 (58.5 %) gave the pattern
for C. albicam. Of these, 56 (81.1 ~o) formed chlamydospores.
Tween 80 was found to exert a very stimulating influence on
chlamydospore production. Rice agar with Tween 80 appeared to
be the most efficient medium for elicitingchlamydospores. However,
since strains of 4 out of 6 spe~ ies of Candida isolated were found to
sporulate it was concluded that chlamydospore formation is not a
reliable criterion for the speciation of C. albicans.
Each of the 4 media served satisfactorily as a directly inocu-
lable selective medium for the isolation of yeast-like fungi of the
genus Casdida from vulvovaginal specimens. None of the media ap-
peared to preferentially stimulate chlamydospore production in
C. albicans.

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280 A.G. SMIT~ a.o.

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