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Vol. 11 No. 1 January–April 2023

Original Article

Germ Tube Induction Test Comparing Total of Six Liquid and Three
Solid Media in Candida albicans
Rivaldi Ruby1 , Erlangga Saputra Arifin1 , Sandy Vitria Kurniawan2 , Sem Samuel Surja3*
1School of Medicine and Health Sciences, Universitas Katolik Indonesia Atma Jaya, Jakarta, Indonesia
2Department of Pharmacology and Pharmacy, School of Medicine and Health Sciences, Universitas Katolik Indonesia Atma

Jaya, Jakarta, Indonesia

3
Department of Parasitology, School of Medicine and Health Sciences, Universitas Katolik Indonesia Atma Jaya,
Jakarta, Indonesia

Received: March 3rd, 2022; Revised: November 6th, 2022; Accepted: February 27th, 2023

ABSTRACT
Invasive candidiasis (IC) has a high mortality rate of 70%, thus diagnosis should be established without delay.
Given its fast result, serological test such as β-d-glucan (BDG) test is one alternative diagnosis modalities.
However, it lacks specificity. Candida albicans germ tube antibody (CAGTA) test is an alternative serological test
which has a high sensitivity of 76.2% and specificity of 80.3%. Manufacturing CAGTA serological test requires
provision of specific germ tube antigen. In this study, various culture media were tested to find the best media for
germ tube induction. This study was an experimental in vitro study. The number and length of the germ tube were
recorded in two- and three-hour incubation periods. A total of six samples containing one C. albicans ATCC 90028,
four C. albicans wild type strains, and one C. krusei wild type strain were used. Nine media were tested to induce
germ tube formation: human and sheep serum, fetal bovine serum, mueller hinton agar and broth, tryptic soy agar
and broth, brain heart infusion agar and broth. At both incubation periods, the medium with the highest number of
germ tube was human serum (p=0.001 and p=0). The longest germ tube was found in sheep serum at two-hour
incubation period (p=0.005). Mueller hinton broth (MHB) showed comparable results with human and sheep
serum (p>0.05). Human serum is a superior inducer of morphogenesis. However, the use of MHB is recommended
in this study, since provision of fresh human and sheep serum on a regular basis is impractical.
Keywords: Candida albicans; germ tube; human serum; mueller hinton broth; sheep serum

Highlights: Several media could induce not only numbers of germ tube, but also its length. Therefore, they could
benefit for easier diagnosis and also higher amounts of germ tube protein.

How to Cite: Ruby, R., Arifin., E. S., Kurniawan, S. V., Surja, S. S. Germ Tube Induction Test Comparing Total
of Six Liquid and Three Solid Media in Candida albicans. Indonesian Journal of Tropical and Infectious Disease.
11(1). 18–26. Apr. 2023.

DOI: 10.20473/ijtid.v11i1.34097

* Corresponding Author:
[email protected]

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Indonesian Journal of Tropical and Infectious Disease, Vol. 11 No. 1 January–April 2023: 8–26 19

INTRODUCTION tube and hyphae is important for

pathogenicity of C. albicans.8,10 Combination
Candidiasis is a disease with a high of BDG and CAGTA serological tests is
prevalence rate globally. This disease recommended for IC early diagnosis.11
generally affects the skin and mucosal tissue, The first step in manufacturing CAGTA
causing mild conditions such as oral and serological test is isolation of the germ tube
vulvovaginal candidiasis.1 At the systemic antigen. It is important to seek the best
level, it has high mortality and morbidity, medium for C. albicans since this antigen is
referred to as invasive candidiasis (IC). It is obtained by inducing its growth in a suitable
associated with prolonged intensive care unit environment. Various media can be used for
(ICU) admission and immunocompromised induction of germ tube, each with unique
conditions such as acquired immune compositions and function. Human serum is
deficiency syndrome (AIDS).2 Globally, the most used medium for germ tube test.12 Its
candidiasis occupies the top three incidences main limitation is the requirement of fresh
of diseases caused by fungi in the year human serum on a regular basis. For this
2017—the first is oral candidiasis with an reason, this study aims to find the best media
incidence rate of 2,000,000, followed by in inducing germ tube formation of C.
esophageal candidiasis with 1,300,000, and albicans. While previous studies mainly
then IC with 750,000 incidences.1 Invasive assessed the sensitivity of each medium for
candidiasis yields a high mortality rate of germ tube test, this study also measured the
70%.3 number and length of germ tube formed after
Invasive candidiasis is caused by Candida certain incubation period.
spp. with the most common etiology being
Candida albicans.4 In humans, this fungus is
a normal flora of the skin, oropharynx,
MATERIALS AND METHODS
digestive, and urogenital tract.5 Infection
occurs when there is hyphal growth and Study Design
biofilm formation in the tissue. These This experimental in vitro study was
mechanisms also allow resistance of C. conducted in the Parasitology Laboratory,
albicans to traditional antifungal agents.6 School of Medicine and Health Sciences,
Timely diagnosis is required in order to Atma Jaya Catholic University of Indonesia
reduce mortality. Currently, IC is diagnosed from August 2020 to October 2020. Ethical
through the findings of hyphae on microscopic clearance was obtained from the Atma Jaya
examination or through a time- consuming ethical committee with the number
culture.1 Serological tests provide relatively 01/06/KEP-FKUAJ/2020.
faster and easier way to diagnose IC. β-d-
glucan (BDG) test is one widely used Fungi Strains
Candida serological test. However, it lacks Candida albicans wild type, C. albicans
specificity due to cross reaction with other ATCC 90028, and C. krusei wild type were
fungi.7 A serological test detecting antibody used in this study. All C. albicans wild type
against germ tube could be used as an were obtained from patient’s sputum in
alternative to diagnose IC, namely C. albicans Microbiology Laboratory, School of
germ tube antibody (CAGTA) test.8 Germ tubes Medicine and Health Sciences, Atma Jaya
are formed by C. albicans in a number of Catholic University of Indonesia, while C.
conditions such as starvation, presence of albicans ATCC 90028 and C. krusei wild
serum or N-acetylglucosamine, physiological type were obtained from the collection of
temperature, and CO2.9 It has high sensitivity Department Parasitology, School of
of 76.2% and specificity of 80.3% since Medicine and Health Sciences, Atma Jaya
morphological transition from yeast to germ Catholic University of Indonesia. Each strain

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20 Rivaldi Ruby, et al. Germ Tube Induction Test

was identified and confirmed through 30, 53, and 37 g of MHA, MHB, TSA, TSB,
macroscopic and microscopic examination. BHIA, and BHIB, respectively. These
Macroscopic identification was made using suspensions were heated until completely
CHROMAgar (Oxoid, United Kingdom) to dissolved, followed by sterilization using an
analyze the color and characteristics of the autoclave at 121°C with a pressure of 15 Psi
fungal colonies. Candida albicans was for 15 minutes. Broth medium was poured
characterized by the formation of green into the 1.5 ml test tube, while agar medium
colonies, in contrast with C. krusei which was poured into a petri dish. For all media,
appeared as pink colonies.13 Microscopic pH was adjusted at 7.4 which is confirmed by
identification was made through lactophenol pH meter.
cotton blue (LPCB) staining and germ tube
test. Light microscope (Olympus CX21) is Gem Tube Induction
used to identify the morphologies. Ovoid and Candida albicans and C. krusei were
spherical yeast cell shapes are a inoculated in sabouraud dextrose agar (SDA,
characterization of C. albicans, distinguished Oxoid, United Kingdom) for 48 hours at room
from C. krusei that commonly appear as a temperature (25°C). Two hundred μL of 3
more elongated (long grain rice) shape. A McFarland fungi suspension was added into
positive germ tube test is also only found in 800 μL of each serum and broth medium. The
C. albicans.14,15 A total of six isolates mixture was incubated for 24 hours at 37°C.12
containing one C. albicans ATCC 90028, The number and length of the germ tube were
four C. albicans wild type, and one C.krusei recorded in two-, three-, and 24-hour
wild type were used. Candida albicansATCC incubation periods. Ten μL of the mixture
90028 and C. krusei were used as the positive was dripped into an improved Neubauer
and negative control, respectively. counting chamber and the germ tube was
observed under the microscope (Figure 1a).17
Medium All processes were performed in duplicate.
The media used for induction of germ tube Germ tube induction in agar media was
were serum, broth, and agar. Sera used were conducted by dripping 10 μL of 0.5
human serum, sheep serum, and fetal bovine McFarland fungi suspension into 1 x 1 cm2
serum (FBS, Biowest, France). The FBS used agar.18 Cover slip was placed to facilitate
was not diluted with 100% concentration. easier examination. The agar was then
Human serum was prepared by incubated for three hours at 37°C. The
centrifugating blood from a healthy donor. 16 number and length of germ tube induction
The broth and agar media used were mueller were recorded in two- and three-hour
hinton agar (MHA, Oxoid, United Kingdom), incubation periods using the microscope
mueller hinton broth (MHB, Conda, Spain), (Figure 1b). All processes were performed in
tryptic soy agar (TSA, Oxoid, United duplicate.
Kingdom), tryptic soy broth (TSB, Merck,
Germany), brain heart infusion agar (BHIA, Germ Tube Calculation
Oxoid, United Kingdom), and brain heart This study measured the number and
infusion broth (BHIB, Oxoid, United length of germ tubes formed. The number of
Kingdom). All media were obtained from the germ tube was calculated in five small
Department of Microbiology, Parasitology, squares of the counting chamber using
and Pharmacology, School of Medicine and standardized formula (Figure 2).19 Germ tube
Health Sciences, Atma Jaya Catholic length measurement was conducted by
University of Indonesia. comparing the length of the germ tube and the
Broth and agar media were prepared by counting chamber small squares. The longest
combining 1 L of aquadest with 38, 21, 45, germ tube in the five small squares was

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Indonesian Journal of Tropical and Infectious Disease, Vol. 11 No. 1 January–April 2023: 8–26 21

recorded. Germ tube length measurement Data Analysis

was done only in serum and broth media. Data analysis was done using Statistical
There are no difficulties in performing the Product and Service Solution (SPSS) version 22.
measurement methods. Data on serum and broth medium was analyzed
using a One-Way ANOVA statistical test or
Kruskal-Wallis test, depending on data normality.
It was then followed by a Bonferroni post hoc test
if significant results were found. In agar medium,
Fisher-Exact or Chi-Square test were used
depending on the terms and criteria of the
statistical test. A significant value was yielded if
p<0.05.

Figure 1. Germ tube appearance under the RESULTS AND DISCUSSION

microscope in different preparations. (a)
Improved Neubauer Counting Chamber – Fungi Identification
Germ tube (red circle); (b) Slide culture Macroscopic identification of all samples
was done using CHROMagar. It is depicted
in Figure 3. Moreover, the morphologies
found in microscopic identification using
LPCB confirmed the samples’ species
(Figure 4).

𝑛
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑔𝑒𝑟𝑚 𝑡𝑢𝑏𝑒 / 𝜇𝐿 =
𝑉
1
𝑉 = 𝑖𝑚𝑝𝑟𝑜𝑣𝑒𝑑 𝑁𝑒𝑢𝑏𝑎𝑢𝑒𝑟 𝑐𝑜𝑢𝑛𝑡𝑖𝑛𝑔 𝑐ℎ𝑎𝑚𝑏𝑒𝑟 𝑣𝑜𝑙𝑢𝑚𝑒 = 𝑚𝑚3
50
𝑛 = 𝑡𝑜𝑡𝑎𝑙 𝑜𝑓 𝑔𝑒𝑟𝑚 𝑡𝑢𝑏𝑒 𝑖𝑛 5 𝑠𝑚𝑎𝑙𝑙 𝑠𝑞𝑢𝑎𝑟𝑒𝑠

Figure 2. Improved Neubauer Counting

Chamber

Calculation of the number of germ tube on Figure 3. Macroscopic characterizations on

agar media was carried out by taking three CHROMagar. The green colonies are C.
albicans and the pink colony is C. krusei
representative images using the high-power
field (HPF) microscope. Then the number of
germ tubes was grouped into several
categories (Table 1).

Table 1. Germ Tubes Counts Categories Using

Agar Media Inductions
Categories Germ Tube Count/HPF
1+ 1-10 (a) (b)
2+ 11-20
3+ 21-30 Figure 4. Microscopic characterizations of
4+ 31-40
Candida species grown in SDA. (a) Ovoid
5+ 41-50
6+ >50 appearance of C. albicans (black circle) (b)
HPF – high-power field Elongated appearance of C. krusei (red circle)

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22 Rivaldi Ruby, et al. Germ Tube Induction Test

Germ Tube Induction in Serum and Broth media at two- and three-hour incubation
All Candida samples were used in germ period. At two-hour incubation period, it was
tube induction. Candida albicans ATCC found that the medium with the highest
90028 and C. krusei function serve as a number of germ tube was human serum.
positive and negative control, respectively. In Sheep serum also showed comparable results
all media, C. albicans ATCC 90028 showed in germ tube induction. Roughly, the order of
positive germ tube results, while C. krusei had media that could facilitate germ tube
negative results. Candida albicans wild type induction was as follows: human serum, sheep
was used for the evaluation of each media’s serum, FBS, MHB, TSB, and BHIB; while at
ability to induce germ tube formation. three-hour incubation period, the order was as
Several media could facilitate germ tube follows: human serum, MHB, sheep serum,
formation. Results were variable between FBS, TSB, and BHIB as shown in Table 2.

Table 2. Number of Germ Tubes Formed on Serum and Broth Media (number/μL)
Media
Time Fungi p-value
Human Serum Sheep Serum FBS MHB TSB BHIB
CA 1 38125 40625 26875 11250 5625 5000
CA 2 23125 20625 10000 22500 11250 3750
2 hours 0.001
CA 3 25000 23125 8750 15625 13750 0
CA 4 21250 15000 18125 11250 0 625
CA 1 42500 16250 13750 15625 3750 0
CA 2 26250 15000 5000 37500 15625 6875
3 hours 0
CA 3 32500 20625 7500 25000 16250 0
CA 4 31875 13125 18750 15000 3750 0
*Post hoc (2 hours) Human Serum vs. BHIB p=0.003; Human Serum vs. TSB p=0.024
**Post hoc (2 hours) Sheep Serum vs. BHIB p=0.006
***Post hoc (3 hours) MHB vs. BHIB p=0.004
****Post hoc (3 hours) Human Serum vs. BHIB p=0; Human Serum vs. Sheep Serum p=0.03; Human Serum vs. TSB p=0.001; Human Serum
vs. FBS p=0.003

No difference was shown between MHB Table 3. Number of Germ Tubes Formed on
and human serum in post hoc analysis Broth Media (number/μL)
(p>0.05). The number of germ tubes between Media
Time Fungi p-value
broth media were highest in MHB, compared MHB TSB BHIB
CA 1 11250 5625 5000
to TSB and BHIB (p=0.015 and p=0.009 at CA 2 22500 11250 3750
2 hours 0.115
two- and three-hour incubation periods, CA 3 15625 13750 0
CA 4 11250 0 625
respectively) as shown in Table 3. At the 24- CA 1 15625 3750 0
hour incubation period, the fungi experienced CA 2 37500 15625 6875
3 hours 0.009
rapid growth into hyphae. Therefore, further CA 3 25000 16250 0
CA 4 15000 3750 0
analysis was not performed in this incubation *Post hoc (2 hours) MHB vs. BHIB p=0.015
period. **Post hoc (3 hours) MHB vs. BHIB p=0.009

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Table 2. Number of Germ Tubes Formed on Serum and Broth Media (number/μL)
Media
Time Fungi p-value
Human Serum Sheep Serum FBS MHB TSB BHIB
CA 1 14 28.89 22.22 17.775 24.445 2.22
CA 2 26 38 37.78 22.22 26 17.78
2 hours 0.005
CA 3 20 33.335 26.665 23.33 31.115 0
CA 4 30 55.555 38 33.335 0 10
CA 1 26.665 53.33 26 31.115 14 0
CA 2 57.78 50 42 77.775 95.555 48.89
3 hours 0.155
CA 3 28.89 36 34 37.78 57.78 0
CA 4 77.78 44.665 22 73.335 26.665 0
*Post hoc (2 hours) BHIB vs. Sheep Serum p=0.003
*Post hoc (2 hours) BHIB vs. FBS p=0.039

In the measurement of length, the longest Hyphal morphogenesis is one of the most
germ tube was found in sheep serum at two- investigated virulence attributes of C.
hour incubation period, followed by FBS, albicans. The ability of C. albicans to
MHB, TSB, human serum, and BHIB. No undergo reversible morphological transition
difference was found at the three-hour could be triggered by variety of
incubation period as shown in Table 4. No broth environmental condition.20–22 Serum,
media was found superior to the other in terms especially human serum, contains several
of the germ tubes length as shown in Table 5. important components that promote germ
tube formation; therefore, it accounts as
Table 5. Length of the Germ Tube Formed on strong inducer for yeast-to-hyphae formation.
Broth Media (μm) Burch et al. (2018) stated that human serum
Media fraction revealed signs of bacterial
Time Fungi p-value
MHB TSB BHIB peptidoglycan (PGN)-like molecules which
CA 1 17.775 24.445 2.22 highly active for hyphae induction.23 Glucose
CA 2 22.22 26 17.78
2 hours
CA 3 23.33 31.115 0
0.998 could also act as morphogen, which in the
CA 4 33.335 0 10 certain amount could stimulate
24,25
CA 1 31.115 14 0 morphogenesis of C. albicans.
CA 2 77.775 95.555 48.89 Human serum, sheep serum, and FBS have
3 hours 0.132
CA 3 37.78 57.78 0
CA 4 73.335 26.665 0 approximately 1 mM to 10 mM of glucose.26–
28 This amount of glucose is optimal for germ

Germ Tube Induction in Agar tube formation according to previous study.24

The number of germ tubes formed was Moreover, combined with exposure of 37°C
highest in MHA. However, results were not and neutral pH environment, serum could
significant both in two- and three-hour inhibit NRG1 transcription, a potent inhibitor
incubation periods as shown in Table 6. for hyphal formation (Su et al. 2018).29 This
exposures to 37°C and neutral to alkaline pH,
Table 6. Length of the Germ Tube Formed on could induce hyphal growth through the Cek1
Agar Media (μm) mitogen-activated protein kinase pathway
(MAPK pathway) and the Rim101-pH
Media
Time Fungi
MHA TSA BHIA
p-value sensing pathway, respectively.21 Studies by
CA 1 +3 +1 0 Hilmioglu et al. (2007) found that human
2 hours
CA 2 +6 +1 +2
0.408
serum was superior with the highest number
CA 3 +4 +1 +1 of positive germ tube.30 In concordance to
CA 4 +1 0 0
CA 1 +1 0 0
previous data, present study found that
CA 2 +6 +1 +1 human and sheep serum were capable of
3 hours 1
CA 3 +2 +1 +1 inducing the highest number and longest
CA 4 0 0 0 germ tube, respectively, compared to other

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24 Rivaldi Ruby, et al. Germ Tube Induction Test

media. This study also found that extended explanation is that it contained starch
incubation period on serum led to an components with protective colloid roles
increasing number of hyphae. Long against toxic compounds in the medium.34
incubation period causes deprivation of BHIB is the most nutritious medium with
nutrient and energy which leads to more combination of brain and beef infusion (a
effective hyphal growth.31,32 total of 17.5 g/l), protease peptone (10 g/l),
Utilization of human serum for germ tube and dextrose (2 g/l) which provide carbon,
induction has few drawbacks despite its nitrogen, amino acids, and other nutrients.34
superiority to other media. Serum has to be However, C. albicans showed lowest number
fresh otherwise stored serum could decrease of germ tube in BHIB. An exact explanation
germ tube production.12 Some serum could of this phenomenon is unknown. It seems that
have biological inhibitor present in it. Wich high nutrient provision is not an inducer of
et al. (2021), found that human serum morphogenesis. According to Mba et al.
antibodies have the capability to inhibit (2020), C. albicans exhibits metabolic
adherence of C. albicans to epithelial cells.33 flexibility and filamentous growth in the
Moreover, Ding et al. (2014) stated that germ condition of nutrient starvation.34 It is
tube formation in RPMI 1640 medium was difficult to identify which media facilitate
delayed in the initial stage of the culture better morphogenesis based on previous
(within 90 min) in the presence of serum, studies, since results were mostly conflicting.
although the number of hyphae was gradually Hilmioglu et al. (2007) showed that BHIB
increased to normal after extension of surpassed TSB as morphogenesis inducer,
incubation period (from 2h to 3h).31 Presence while Yakasiri et al. (2020) concluded that
of these biological inhibitor could cause TSB exceeds MHB and BHIB.30,35 Different
inconsistent result in different batches of results could be attributed to different strain,
serum. Lastly, serum preparation poses media quality, and research conditions.
possible risk of biohazard.12 In this study, Interestingly, Atalay et al. (2017) found
several standardized media were studied to that MHA was the best media for germ tube
find comparable alternative of serum. induction compared to human serum.18
Application of commercially available media Distinct factor affects the yeast-to-hyphal
also facilitates further culture and production transition in agar media. Villa et al. (2020)
of germ tube antigen for serological test. stated that C. albicans hyphal growth in agar
Broth medium also have essential is influenced by the embedded surroundings
substances for the growth and morphogenesis or conditions. This is achievable through the
of C. albicans. MHB, TSB, and BHIB were upregulation of CZF1 transcription factor
tested in their ability to promote germ tube when the fungi are inoculated within the agar
formation. As stated above, elevated matrix.9 In present study, higher number of
temperature and neutral pH were the one germ tube was found in MHA compared to
main inducer for morphogenesis.29 other agar media although the result was not
Moreover, all media contains nutritive significant.
compounds that are necessary for fungal
growth. Amino acids are one potent inducer
present in the medium. It promotes yeast-to- STRENGTH AND LIMITATION
hyphal transition through the cAMP-PKA To our knowledge, this is the first study
pathway.21 In this study, MHB, TSB, and that compares several media by measuring
BHIB contain amino acids which further the number and length of the germ tubes.
facilitate hyphal formation.21,22 Furthermore, However, this study has several limitations.
in MHB, C. albicans showed the highest Lack of fungal strain prevents generalization
number of germ tube formation. One possible of the result to other strain of C. albicans.

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Wider variety of culture medium could be CONFLICT OF INTEREST

used, since RPMI 1640 and YEPD broth also
showed potential in previous studies.12,31 The authors declare that they have no
conflict of interest.

CONCLUSIONS
AUTHOR CONTRIBUTION
This study showed that certain media in a
specific environmental condition could Designed the study, collected and
facilitate hyphal growth that initially appears analyzed the data, and also prepared the
as germ tube formation. Human serum is a manuscript: RR and EAS. A scientific adviser
strong inducer of morphogenesis. Incubation in the field of mycology: SS and SVK. All
of C. albicans in standardized medium such authors read and approved the final
manuscript.
as MHB and TSB, coupled with 37°C
environmental temperature and neutral pH, is
also adequate to facilitate such phenomenon. REFERENCES
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