Yonago Acta medica 1998;41:65–72

Germ Tube Formation of Candida albicans in Corn Meal Broth Using
the Non-Slip Slide Glass Incubation Method
Sachiko Nakamoto
Department of Medical Technology, Tottori University, Institute of Medical Care Technology,
Yonago 683-0826, Japan
Candida albicans IFO 1385 yeast cells dominantly develop germ tubes in a dilute cornmeal broth at 37˚C. For germ tube (GT) development the broth concentration is optimal
at 75% of the original, and the inoculum is optimal at 105 cells/mL. Incubation procedures were compared and the non-slip slide glass incubation (NSSI) method was foundto be more available than the tube incubation method or slid-covered slide glass incubation method. The GT formation was completed in 1 to 2 h in the NSSI method andclear-cut GTs were observed under optical microscopy. GTs were formed in all 111
test-strains of C. albicans but not at all in other species of Candida. Therefore, under
optimal conditions, the rate of identifying C. albicans is 100%, demonstrating that GT
detection with the NSSI method is a rapid and reliable identification method. The
humid NSSI method is suited for continuous observation of a GT. The initially formed
GT elongated to resemble a true hypha, attaching no chlamydoconidium. The hyphasubsequently dissociated yeast-like cells.
Key words: Candida albicans; corn meal broth; germ tube formation; non-slip slide glass incubation
methodcans is required. Thus, efforts to evaluate or
improve the medium to achieve rapid GT induction has not yet ended.
Corn meal medium is usually used for theformation of pseudohyphae or chlamydoconidium of Candida with the slide culture method,in which fungi are inoculated onto a small pieceof corn meal agar medium placed on a slide
glass for culturing. Therefore, observing GT inthis agar medium did not seem rational. However, the use of corn meal broth made it possible
to induce high rates of GT formation, which led
to the development of a rapid clinical mycological method for identifying Candida. In thisstudy, using corn meal broth as the medium for
GT formation, simple and rapid practical conditions were evaluated to obtain high rates of GT
formation by establishing an optimal concentration of inoculating fungal solution or an optimal
concentration of corn meal broth.

Since the germ tube (GT) is a characteristic
morphology observed only in C. albicans, confirmation of GT is available as a rapid method
for identifying C. albicans. GT formation was
first reported by Reynolds and Braude (1956).
They observed yeast cells transforming into
filamentation (germ) tubes in human blood,sera, plasma and cerebrospinal fluids. Since
then, many substances were reported as GT
inducers, such as serum (Taschdjian et al.,1960; Mackenzie, 1962; Andleigh, 1964; Dolan
and Ihrke, 1971), egg white (Buckley and Uden,
1963; Andleigh, 1964), N-acetylglucosamine(Simonetti et al., 1974), proline (Holmes andShepherd, 1987), ethanol (Pollack andHashimoto, 1985) and peptone (Joshi et al.,1973a, 1973b). Among them, serum is utilized
as the most potent GT inducer in laboratory
studies. However, all the present methods for
GT induction have been, so far, time-consuming when the urgent identification of C. albi-

Abbreviations: GT, germ tube; NSSI, non-slip slide glass incubation; T50, time for germ cells to reach at 50%of original; TI, tube incubation

65

S. Nakamoto

fungal solution was placed on a sterile slide
glass, and the slide glass was placed in a wet
Petri dish without a cover glass. The Petri dishwas further sealed with a vinyl bag, and thefungi were incubated at 37˚C. Meanwhile, theTI method was carried out as follows: 1 mL of
the same fungi specimen was transferred to atest tube and dipped in a 37˚C-water bath forincubation.
Cultured specimens were collected at various time points, and fungal morphology wasobserved in an optical microscope to calculate
the number of cells with extruding GT per 100
given yeast cells and the rate of GT formation.
Moreover, the GT formation rate determined in
50% calf serum solution was used as the control.

Materials and Methods

Strains of Candida
C. albicans IFO 1385 was used as the standard
strain. In addition, the following strains of
Candida isolated and identified from clinicalspecimens were also used in this study: 111
strains of C. albicans and other species of Candida including 8 strains of C. tropicalis, 9 of C.
glabrata, 4 of C. parapsilosis, 5 of C. guilliermondii, 2 of C. lusitaniae, 1 of C. kekyr, 1 of C.
krusei and 1 of C. lypolitica. Fresh fungi cultured overnight at 30˚C with Sabouraud 4% dextrose agar were inoculated into the corn meal
broth.

Results

MediumThe corn meal broth was prepared as follows:corn meal agar medium (Nissui Seiyaku Co.
Ltd., Tokyo, Japan) was modified to prepare thebroth; it was mixed with cold distilled water,
and continuously stirred overnight at 6˚C in acold room. On the next day, insoluble components were removed by filtration, and the filtrate was autoclaved for use as an undiluted
corn meal solution.

Conditions for GT formation
Figure 1 shows GT formation rates when 106
cells/mL of fungi were suspended in various
concentrations of corn meal broth and incubated at 37˚C. The maximal GT formation rate
was obtained by culturing in 75% corn meal
broth, because of undiluted or overdiluted brothresulting in unsatisfactory GT formation. Figure 2 shows GT formation rates when 104–106

Method of GT formation
Fresh fungi were inoculated into various concentrations of the corn meal broth to establish afungal suspension. That is, one loopful of fungi
was mixed with a 0.2 mL phosphate buffered
solution to create a dense fungal solution.
Then, this was mixed with 1 mL broth to create
a fungal suspension for incubation. The concentration thus prepared was approximately 106
cells/mL (Nakamoto, 1992). Moreover, various
concentrations of inoculating fungal solutions
were prepared based on this formula for preparing a fungal solution.
The 2 different incubation methods werecomparatively evaluated, that is, the non-slip
slide glass incubation (NSSI) method and thetube incubation (TI) method. The NSSI method
was carried out as follows: 50 µL of the above

GT formation rate

100

(%)

HJ
B

HJB

50

0

HBJ
0

1

2

(h)

Incubation time
Fig. 1. The mean GT formation rate in various
concentrations of corn meal broth. Seven strains of
C. albicans including the standard strain (106 cells/
mL each) were inoculated into various concentrations of corn meal broth, and incubated by the NSSI
, 25%; , 50%; , 75%; ,method at 37˚C.
100%.

66

Rapid GT formation of C. albicans in corn meal brothcells/mL fungal solutions were incubated in
75% corn meal broth or 50% calf serum. When
105 or 106 cells/mL fungi were incubated for 1 h
in 50% calf serum, GT formation rate was
100% in both concentrations of fungal solutions. When 105 cells/mL fungi were incubatedin 75% corn meal broth, GT formation rates
were 91% after a 1-h incubation and 100% after
a 2-h incubation. Meanwhile, when 104 cells/
mL fungi were incubated in 75% corn meal
broth, the number of fungi in specimens was too
small to observe, and the results could not becalculated.

the mean GT initially observed. Along with theextension of GTs, yeast cells gradually appeared after a 2-h incubation and were recognized in
specimens after a 20-h incubation period (Fig.
3c). In contrast, GTs formed during incubationin 50% calf serum grew to true hyphae with thedevelopment of a markedly small number ofyeast cells, perhaps blastoconidia; moreoverchlamydoconidium was not observed (Fig. 3d).
Figure 4 shows fungal specimens culturedby the TI method. Fungal clusters were formedby aggregating mutual GT-forming cells among
scattering yeast cells in culturing by the TI
method.
Table 1 shows the relationship between concentrations of fungal solutions and GT formation rates during GT formation in culturing by
the NSSI and TI methods in 50% calf serum.
Using my own experimental conditions, moresatisfactory GT formation was generallyachieved by the NSSI method than by the TI
method even when the optimal concentration of
fungal solution was roughly established byincreasing it 5 times.

Morphogenesis
Figures 3a and b show fungal morphology when
105 cells/mL fungi were incubated at 37˚C for 2
h by the NSSI method in 75% corn meal brothor 50% calf serum. There were no morphological differences between fungi incubated withboth media, and synchronized GT extrusions
from yeast cells were observed. Figures 3c andd show fungal morphology when the same
specimens were incubated by the NSSI methodfor 20 h. GTs formed in corn meal brothcontinuously elongated becoming similar totrue hyphae measuring several times as long as(%)

GT formation rate

100

J
B

J
B

1

2 (h)

Quality of GT formation test using cornmeal broth
GT formation in a total of 142 strains of Candida and related yeast-like fungi was tested by
the NSSI method using 75% corn meal broth.
GT formation was observed in all 111 strains of
C. albicans only. The mean GT formation rate
was 82.4% after a 1-h incubation, and GT formation with sufficient quality was confirmed
under a microscope. Moreover, there was no
GT formation observed in strains of other Candida species, although there is some concern
regarding the small number of strains tested(Table 2).

50

0

JB
0

Incubation time
Fig. 2. Relationship between concentration of
inoculating fungal solution and GT formation rate on
culturing in 75% corn meal broth or 50% calf serum.
C. albicans (10 5 cells/mL or 10 6 cells/mL) was
inoculated into each medium, and incubated by the
NSSI method at 37˚C. 50% calf serum: , 105 cells/
mL or 106 cells/mL fungal solutions. 75% corn meal
broth: , 105 cells/mL fungal solution; , 106 cells/
mL fungal solution.

Discussion
GT formation is a morphological characteristicof C. albicans, and it can be detected in a relatively short time when GT formation is accelerated by incubation at 37˚C in a solution similar to serum containing abundant nutrients67

S. NakamotoFig. 3. Morphology of C. albicans IFO 1385 inoculated into 2 kinds of inducer solutions, 75% corn meal
broth and 50% calf serum, and incubated by the NSSI method at 37˚C for 2 h and 20 h. a: C. albicans
inoculated into 75% corn meal broth at a concentration of 105 cells/mL, and incubated for 2 h. b: C. albicans
inoculated into 50% calf serum at a concentration of 106 cells/mL, and incubated for 2 h. c: C. albicans was
inoculated into 75% corn meal broth at a concentration of 105 cells/mL, and incubated for 20 h. GT extended
like relatively short true hyphae, and the small number of yeast cells were observed in the medium. d: C.
albicans was inoculated into 50% calf serum at a concentration of 106 cells/mL, and incubated for 20 h. In
contrast to culturing in corn meal broth, well developed true hyphae-like fungal morphology was observed
together with diffused blastoconidium-like yeast cells, while chlamydoconidia were not observed in themedium. Bar indicates 10 µm.

68Rapid GT formation of C. albicans in corn meal broth

Table 1. The mean GT formation rate in 10 strainsof C. albicans inoculated in 50% calf serum at
37˚C: relationship between concentration of inoculating fungal solution and incubation method
Incubation
time
(h)

Concentrationof fungal
suspension(cells/mL)

1

1.4 × 106
2.7 × 106
4.1 × 106
5.5 × 106
6.8 × 106

Mean GT formation rate (%)
TI
NSSI
methodmethod
90
84
68
54
44

95
97
96
95
97

1.4 × 10697100
2.7 × 106
91
100
79
100
4.1 × 106
5.5 × 106
66
100
6.8 × 106
53
100
GT, germ tube; NSSI, non-slip slide glass incubation; TI,
tube incubation.
2

Fig. 4. Fungal morphology of C. albicans
IFO 1385 inoculated into 50% calf serum, andincubated by the TI method at 37˚C for 2 h.
Bar indicates 10 µm.

30˚C. In this case, the corn meal medium used
was applied as agar but not a liquid for studying
chlamydoconidial formation, whereas few reports have been published describing chlamydoconidial morphogenesis even in corn mealliquid medium (Takagi and Nagata, 1962;
Vidotto et al., 1986; Nakamoto, in press).
Moreover, no report has been published regarding GT formation in corn meal broth. Since thecomposition of corn meal broth was the same asthat of corn meal agar medium, commonly used

(Taschdjian et al., 1960; Mackenzie, 1962;
Andleigh, 1964; Dolan and Ihrke, 1971;
Nakamoto, 1996). Meanwhile, chlamydoconidia, another morphological characteristicof C. albicans, are formed when C. albicans is
incubated in scanty nutrients, corn meal
medium, at a low temperature between 25 andTable 2. GT formation test in 75% corn meal broth at 37˚C by the NSSI method
Strains

Numberofstrains

Number of
GT-formed strains
1-h Incubation

C. albicans
111
111
C. tropicalis
80
C. glabrata
90
C. parapsilosis
40
C. guilliermondii
50
C. lusitaniae
20
C. kekyr
1
0
C. krusei
10
C. lypolitica
10
GT, germ tube; NSSI, non-slip slide glass incubation.

69

Mean GT formation rate(%)
1-h Incubation
2-h Incubation
82.4 [44–100]
0
0
0
0
0
0
0
0

94.1 [74–100]
0
0
0
0
0
0
0
0

S. Nakamoto

When the developmental process of GTforming cells was observed, GTs observed at
the initial stage of culturing were morphologically similar to those observed during GT
induction by serum (Fig. 3b), demonstratingthat at least morphologically similar GT could
be induced in corn meal broth. These GTs extended like true hyphae without chlamydoconidia after a long-term incubation (Fig. 3c).
Furthermore, dissociated proliferation of yeast
cells was gradually observed after a 2-h incubation. Considering the unexpected consistency
between the time that dissociated proliferationof yeast cells started and the time that the GT
formation rate reached 100%, nutrients contained in a limited medium became rapidly insufficient. This phenomenon might be related to
the findings that C. albicans grew to yeast
phases when incubated at 37˚C in common
media. From the results obtained in this study,
it was speculated that GT formation and thedevelopment of true hyphae transiently occurred before the development of the yeast phasewhen C. albicans was incubated in 75% cornmeal broth at 37˚C. However, further detailed
experimental models were necessary to analyze
this hypothesis. Meanwhile, it was recently
reported that when C. albicans was incubated incorn meal broth at 23˚C, short pseudohyphae
were aggregated and radially arranged in three
dimensions to form clusters of specific hyphaewith chlamydoconidia on their external tips
(Nakamoto, in press). Therefore, despite thesame media components, C. albicans showed atemperature-dependent induction of the morphological phase, which was considered different from the dimorphism observed on culture incommon media. In general, corn meal broth isan excellent medium for the development ofhyphae. However, it can be speculated that themorphogenesis of C. albicans using this cornmeal broth has led to GT formation and thesubsequent development of true hyphae withoutchlamydoconidia when the incubation temperature was initially established at 37˚C, while themorphogenesis of C. albicans has led to thedevelopment of pseudohyphae without GT
formation and the subsequent formation of
specific fungal clusters with chlamydoconidia

for chlamydoconidial detection, except for
agar, GT formation has been overlooked. In
this study, it was confirmed that high rates of
GT formation were achieved when yeast cells
were incubated at 37˚C in the corn meal broththough diluted to some degree. Concerning
the concentration of inoculating fungal solution, which influenced the efficiency of GT
formation, it was already reported that theextension of GT was more accelerated at alower concentration of fungal solution(Mackenzie, 1962; Nakamoto, 1996).
Gunasekaran and Hughes (1977) reported
that satisfactory GT formation was induced in
105 cells/mL fungal solution after incubation ina chitosan trypticase Tween-80 agar medium,
while Strippoli and Simonetti (1975) also
reported it to be between 105 and 106 cells/mL
after incubation in a medium containing Nacetyl-D-glucosamine. It was also reported that
GT induction was achieved by culturing yeast
cells with sera at a similar range of fungal
concentration, and further increases in the concentration of fungal solution resulted indecreased GT formation rates and inhibited GT
extension (Mackenzie, 1962; Joshi et al.,1973a; Nakamoto, 1996). In this study, theoptimal concentration of fungal solution in
the corn meal broth was established, which
was similar to those reported in previous
studies. However, compared to GT induction
by serum, it was confirmed that increased fungal concentration (106 cells/mL) resulted inslightly delayed GT formation and decreased
GT formation rate. However, regarding GT
formation during induction by serum, it wasreported (Nakamoto, 1996) that the initial
velocity of GT formation after a 20-min lag
time was 4.25% per min, and the time for germ
cells to reach 50% of the original (T50) was 33
min. Among many reports regarding T 50
(Walkeret et al., 1984; Pollack and Hashimoto,
1985; Sevilla and Odds, 1986; Holmes andShephered, 1988), Sevilla and Odds (1986)
recorded the shortest T50 at 40 min. Therefore,
a further reduction of T50 was recorded under
optimal conditions. In this study, it was considered that the rate of GT formation in 75%
corn meal broth was as good as that in serum.
70Rapid GT formation of C. albicans in corn meal brothwhen the incubation temperature was initiallyestablished at 23˚C.
The TI method and the slide glass incubation method are commonly used for morphological observation of C. albicans. The slide
glass incubation method is usually carried out
by placing a drop on the medium with a coverglass. In this study, though GT formation could
be observed by the TI method using corn meal
broth, aggregation of GT-formed fungi occurred as shown in Fig. 4. Therefore, evaluation of
the GT formation rate was markedly difficult.
Concerning the slide glass incubation method,
though satisfactory GT formation was observedin the fungal solution around the edges of thecover glass, poor GT formation was observed in
the middle of the cover glass. Therefore, it wasconsidered that contact with air was necessary
for GT formation even in a limited specimen of
fungal solution. Moreover, since the medium
easily evaporates in the slide glass incubation
method, observation of a long-term culturing isdifficult (Nakamoto, 1994). To improve theseculture conditions and to increase contact withair, I employed the NSSI method, in which acover glass was not placed over the fungal solution on the slide glass, but the slide glass wassealed inside a vinyl bag to create an environment with high humidity. As a result, a longterm incubation experiment lasting for 1 or 2days became possible. In the NSSI method,when a cover glass was first placed on the slide
glass before microscopic examination, cells
diffusing in the fungal solution were observed
across all areas under the cover glass, but there
were no fungal clusters due to aggregated GTformed cells observed. Therefore, this culture
condition provided an advantage, whichfacilitated precise evaluation of the GT formation rate. In addition, it was revealed that the
NSSI method had an advantage in maintaining
high rates of GT formation even though several
times the optimal concentration of inoculating
the fungal solution was used without strictly
establishing the optimal fungal concentration incomparison to the TI method (Table 1). Thisfinding may further facilitate clinical mycological techniques. On the basis of these findings, the NSSI method is markedly useful for

rapid identification of C. albicans by confirmation of GT.
Under optimal conditions, GT formation
was tested by the NSSI method in various
strains. As a result, the quality of identifying C.
albicans after a 1-h incubation was 100%. The
GT formation rate in each strain ranged between 44 and 100%, which allowed no doubt in
the evaluation.
These results concluded that corn meal
broth could replace serum, which was commonly used for GT formation test, and that the useof corn meal broth was far more advantageousthan serum with regard to cold storage, cost
efficiency and biohazard safety.
Because the NSSI method is technicallysimple, this method facilitates the rapid diagnosis of C. albicans infection.
Finally, the reason why GT was formed byremoving agar from corn meal medium remains
to be clarified. Further information is necessary
about culturing C. albicans by adding agar tothe optimal 75% corn meal broth.
Acknowledgments: I wish to thank the Dean andProf. Masumi Ueda, Dept. of Medical Technology,
Tottori University College of Medical Care Technology, for his appropriate advice and valuable suggestions in this study.
References
1 Andleigh HS. Rapid identification of Candida
albicans. Mycopathologia 1964;23:81–84.
2 Buckley HR, Van Uden N. The identification of
Candida albicans within two hours by the use ofan egg white slide preparation. Sabouraudia
1963;2:205–208.
3 Dolan T, Ihrke DM. Further studies of the germtube test for Candida albicans identification. Am
J Clin Pathol 1971;55:733–734.
4 Gunasekaran M, Hughes WF. A simple medium
for isolation and identification of Candida albicans directly from clinical specimens. Mycopathologia 1977;61:151–157.
5 Holmes AR, Shepherd MG. Proline-induced
germ-tube formation in Candida albicans: roleof proline uptake and nitrogen metabolism. J Gen
Microbiol 1987;133:3219–3228.
6 Holmes AR, Shepherd MG. Nutritional factors
determine germ tube formation in Candida albicans. J Med Vet Mycol 1988;26:127–131.

71

S. Nakamoto

7 Joshi KR, Bremner DA, Gavin JB, Herdson PB,
Parr DN. The formation of germ tubes by Candida albicans in sheep serum and trypticase soyabroth. Am J Clin Pathol 1973a;60:839–842.
8 Joshi KR, Gavin JB, Bremner DA. The formationof germ tubes by Candida albicans in various
peptone media. Sabouraudia 1973b;11:259–262.
9 Mackenzie DWR. Serum tube identification of
Candida albicans. J Clin Pathol 1962;15:563–
565.
10 Nakamoto S. Examination of germ tube formation’s test of Candida albicans. III. Original of
inoculation of fungus solution and the evaluation.
Igaku Kensa 1992;41:29–34 (in Japanese).
11Nakamoto S. Examination of germ tube formation’s test of Candida albicans. IV. Basic examination of slide glass’s method. Igaku Kensa
1994;43:881–886 (in Japanese).
12 Nakamoto S. Improved test of the germ-tube
induction to identify Candida albicans. Tottori
Daigaku Iryo Gijyutsu Tankidaigakubu Kiyo
1996;25:59–64.
13 Nakamoto S. Promotion of chlamydoconidium
formation in Candida albicans by corn meal
broth incubation. J Med Vet Mycol. In press.
14 Pollack JH, Hashimoto T. Ethanol-induced germtube formation in Candida albicans. J Gen
Microbiol 1985;131:3303–3310.
15 Reynolds R, Braude A. The filament-inducing
property of blood for Candida albicans: its natureand significance. Clin Res Proc 1956;4:40.
16 Sevilla MJ, Odds FC. Development of Candida
albicans hyphae in different growth media-variations in growth rates, cell dimensions and timingof morphogenetic events. J Gen Microbiol 1986;
132:3083–3088.
17 Simonetti N, Strippoli V, Cassone A. Yeastmycelial conversion induced by N-acetyl- D glucosamine in Candida albicans. Nature 1974;
250:344–346.
18 Strippoli V and Simonetti N. Specific inductionof chlamydospore formation in Candida albicans
by N-acetyl- D -glucosamine. Experientia
1975;31:130–131.
19 Takagi A, Nagata A. Studies on the fine structureof Candida albicans, with special reference tointracytoplasmic membrane system. Jpn J Microbiol 1962;6:95–111.
20 Taschdjian CL, Burchall JJ, Kozinn PJ. Rapid
identification of Candida albicans by filamentation on serum and serum substitutes. AMA J Dis
Child 1960;99:212–215.
21 Vidotto V, Caramello S, Gallo MG. A new medium for the production of chlamydoconidia by
Candida albicans. Mycopathologia 1986;95:73–
75.
22 Walker GM, Sullivan PA, Shepherd MG.
Magnesium and the regulation of germ-tube
formation in Candida albicans. J Gen Microbiol
1984;130:1941–1945.

(Received April 20, Accepted May 13, 1998)

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