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Year : 2017  |  Volume : 20  |  Issue : 1  |  Page : 61-63

Can serums be replaced by Mueller-Hinton agar in germ tube test?

1 Department of Clinical Microbiology Faculty of Medicine, Erciyes University, Kayseri, Turkey
2 Department of Biostatistics, Faculty of Medicine, Erciyes University, Kayseri, Turkey
3 Department of Clinical Microbiology, Faculty of Medicine, Istanbul University, Istanbul, Turkey

Date of Acceptance03-Mar-2016
Date of Web Publication12-Dec-2016

Correspondence Address:
Dr. M A Atalay
Department of Medical Microbiology, Erciyes University, Faculty of Medicine, 38039 Melikgazi, Kayseri
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1119-3077.180046

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Background: The germ tube test (GTT) is inexpensive, easy, and well-defined test that differentiates Candida albicans (excluding Candida dubliniensis and Candida africana) from other species. The aim of this study was to evaluate various serums (i.e., human, rabbit, horse, and fetal bovine serum) used in the GTT and Mueller-Hinton agar (MHA).
Materials and Methods: Fifty species isolated from various clinical samples that were defined as C. albicans by both conventional and DNA sequence analysis methods were included in the study. One to two colonies of C. albicans were mixed into 0.5–1 ml of fetal bovine serum, horse serum, rabbit serum, and human serum. Serums and MHA were incubated at 37°C for GTT. They were removed from the incubator and evaluated after 30 min, 1 h, 2 h, and 3 h of incubation. The GTT was accepted to be positive only if germ tube was 1/2 the width and 3 times the length of the parent yeast cell and with no constriction at the point of origin.
Results: When the use of serums and MHA for GTT was statistically evaluated, according to the positive scoring, the best results were obtained with MHA and with rabbit, horse, and fetal bovine serum, respectively. The best definition over time statistically was the third hour.
Conclusion: It is suggested that inexpensive MHA is a fast, appropriate, and reliable medium for the probable diagnosis of GTT and C. albicans; however, additional studies are still needed to define other Candida species.

Keywords: Candida albicans, germ tube test, Mueller-Hinton agar, sequencing

How to cite this article:
Atalay M A, Koc A N, Parkan O M, Aydemir G, Elmali F, Sav H. Can serums be replaced by Mueller-Hinton agar in germ tube test?. Niger J Clin Pract 2017;20:61-3

How to cite this URL:
Atalay M A, Koc A N, Parkan O M, Aydemir G, Elmali F, Sav H. Can serums be replaced by Mueller-Hinton agar in germ tube test?. Niger J Clin Pract [serial online] 2017 [cited 2022 Jan 24];20:61-3. Available from:

   Introduction Top

The increased survival of cancer patients and Intensive Care Unit patients in recent years, widespread use of bone marrow and organ transplants, use of broad-spectrum antibiotics, corticosteroids, antineoplastic and immune suppressive agents, and indwelling catheters have led to an increase in the prevalence of fungal infections and mainly the infections caused by Candida species.[1],[2],[3]Candida albicans is defined as the most common causative agent, although there is a trend of increasing prevalence of species other than C. albicans.[4] Many rapid assays have been developed to identify yeasts, and most of these methods are extremely expensive and labor-intense and may not be available routinely in all laboratories.[5] The germ tube test (GTT) is a well-established, inexpensive, and easy-to-administer test used to identify C. albicans from other species (except Candida dubliniensis and Candida africana). Reynold and Braude described the germ tube formation for the 1st time in 1956, and it labeled the “Reynold Braude Phenomenon.”[5] The GTT is performed to induce the formation of hyphae at 37°C and neutral pH in the presence of serum. These conditions mimic the host environment.[6] In this test, one or two colonies of yeast are mixed with test substrate (fetal bovine serum) and incubated at 37°C for 3 h. The incubation period should not exceed 4 h, because other hyphae-producing yeasts start germinating beyond this time frame.[7]

The aim of this study was to evaluate various serums (human, rabbit, horse, and fetal bovine) used in GTT in the laboratories and Mueller-Hinton agar (MHA) used in antibiotic susceptibility tests.

   Materials and Methods Top

Ethical approval was granted for this study by the Erciyes University Ethical Committee. A total of fifty strains identified as C. albicans (using both conventional methods and DNA-sequencing) from various clinical samples (52% from blood, 32% from bronchoalveolar lavage fluid, 16% from other samples) of 50 inpatients were included in the study. DNA sequencing was performed using an automated sequencer (3130 Genetic Analyzer; Applied Biosystems, USA). For GTTs; one or two colonies of C. albicans were mixed into 0.5–1 mL fetal bovine serum (Argene-Parc Technologique, France), horse serum (E and O Laboratories, Scotland), rabbit serum (Serotec, UK), and human serum. MHA (Merck, Germany) was cut in 1 cm × 1 cm dimensions and prepared as a slide culture. An inoculum from C. albicans colony was streaked onto the plate. For GTT, sera and MHA were kept at 37°C. The plates were evaluated for growth at 30 min, 1 h, 2 h, and 3 h. One drop of the serum was placed on the slides with a cover glass, and MHA prepared as slide culture was directly observed under light microscope (×20). The test was considered positive if a short hyphal extension was seen arising laterally from a parent yeast cell with no constriction at the point of origin and if it was half the width and 3 times the length of the parent yeast cell with no presence of nucleus. The evaluation was performed as follows: 1–9/10 field 1+, 1–9/1 field 2+, 10–90/1 field 3+, >90/1 field 4+. C. albicans ATCC 64546 was used as the standard strain. The data were analyzed using the two-way repeated measures analysis of variance.

   Results Top

When the use of sera and MHA in GTT was evaluated according to positive scoring, the best results were obtained using MHA followed by rabbit, human, horse, and fetal bovine serum. The evaluation at 3 h offers the best detection time followed by 2 h, 1 h, and 30 min [Figure 1]. There were also significant differences between rabbit-fetal bovine and human fetal bovine serum at 2 h, and rabbit-fetal bovine, human-fetal bovine, and horse-fetal bovine serum at 3 h (P < 0.05).
Figure 1: Mean positivity scores of serums and Mueller-Hinton agar according to time in germ tube test

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   Discussion Top

C. albicans, a component of the normal flora of the gastrointestinal tract, vagina, and oral cavity is the leading cause of opportunistic fungal infections reported in epidemiological studies.[5],[8] Although various morphological, biochemical, and molecular methods are available for the identification of C. albicans, GTT is a simple, rapid, and highly reliable test that has been used for many years.[9],[10]

Studies conducted with human serum reported a sensitivity of 91–100% and a specificity of 95–100% for GTT, and the studies using fetal bovine serum, rabbit serum, and horse serum reported a sensitivity of 92.3%, 90%, and 35%, respectively.[11],[12],[13],[14] In the study by Hilmioglu et al.,[14] comparing 12 fluids, the best results in GTT were obtained with human serum, followed by rabbit and heart-brain infusion agar with the worst results obtained from horse serum. For GTT, human serum was found to be superior in the study by Arora et al.,[15] and horse serum was found to be superior in the study by Makwana et al.,[16] In studies that evaluated various broths, rice cream agar, 2% oxgall broth, and rice infusion-oxgall-Tween 80 agar yielded a sensitivity ranging from 98% to 100%.[17],[18] Kim et al.,[19] reported that incubation in serum-free YEPD (1% yeast extract, 2% peptone, 2% dextrose) at 39°C for 1 h provided a rapid and reliable test protocol for germ tube formation in the detection of C. albicans. On the other hand, Rimek et al.,[11] reported that such broths (like serum-free YEPD) are not available commercially and are produced specifically for GTT, and they reported 91.5% and 60.0% sensitivity rate using commercially available MHA in the identification of C. albicans and C. dubliniensis, respectively. In our study, C. albicans statistically turned out to be the best to produce germ tube in MHA followed by rabbit, human, horse, and fetal bovine serum.

Many laboratories use human serum for GTT. However, the use of human serum has some disadvantages. Serum sample must be fresh or stored frozen. The yeast inoculum must contain <107 cells/mL. Otherwise, germ tube formation is inhibited, and the use of pooled human serum poses some risks such as transmission of HIV or hepatitis virus infections.[5],[11] Mackenzie,[20] reported 50% reduction in germ tube formation when human serum is kept at +4°C for more than 15 days. According to our calculations, the cost of fetal bovine serum, horse serum and rabbit serum for GTT were approximately 130 USD, 20 USD, and 42 USD for 100 tests, respectively. However, when MHA is used (1 × 1 dimensions) for GTT, the cost was approximately 7 USD for 100 test. Hence, MHA is an inexpensive medium widely used in the microbiology laboratories and offering a long shelf life, avoids such risks.

   Conclusion Top

MHA is an appropriate and reliable medium for the GTT and the presumptive identification of C. albicans, and additional studies are required to determine the identification of other Candida species.

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Conflicts of interest

There are no conflicts of interest.

   References Top

Sardi JC, Scorzoni L, Bernardi T, Fusco-Almeida AM, Mendes Giannini MJ. Candida species: Current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options. J Med Microbiol 2013;62(Pt 1):10-24.  Back to cited text no. 1
Hachem R, Hanna H, Kontoyiannis D, Jiang Y, Raad I. The changing epidemiology of invasive candidiasis: Candida glabrata and Candida krusei as the leading causes of candidemia in hematologic malignancy. Cancer 2008;112:2493-9.  Back to cited text no. 2
Gayibova Ü, Dalyan Cilo B, Agca H, Ener B. Comparison of Phoenix™ Yeast ID Panel and API ® ID 32C commercial systems for the identification of Candida species isolated from clinical samples. Mikrobiyol Bul 2014;48:438-48.  Back to cited text no. 3
Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: A persistent public health problem. Clin Microbiol Rev 2007;20:133-63.  Back to cited text no. 4
Deorukhkar SC, Saini S, Jadhav PA. Evaluation of different media for germ tube production of Candida albicans and Candida dubliniensis. Int J Biomed Adv Res 2012;3:704-7.  Back to cited text no. 5
Kadosh D, Johnson AD. Induction of the Candida albicans filamentous growth program by relief of transcriptional repression: A genome-wide analysis. Mol Biol Cell 2005;16:2903-12.  Back to cited text no. 6
Hazen KC, Howell SA. Candida, Cryptococcus, and other yeasts of medical importance. In: Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH, editors. Manual of Clinical Microbiology. 8th ed. Washington, DC: ASM Press; 2003. p. 1693-711.  Back to cited text no. 7
Çerikcioglu N. Candida species. In: Topcu AW, Soyletir G, Doganay M, editors. Infection Diseases and Microbiology. 3rd ed. Istanbul: Nobel Medicine Press; 2008. p. 2411-26.  Back to cited text no. 8
Brown S, Traczewski M. Quality control limits for posaconazole disk susceptibility tests on Mueller-Hinton agar with glucose and methylene blue. J Clin Microbiol 2007;45:222-3.  Back to cited text no. 9
Raghunath P, Seshu Kumari K, Subbannayya K. SST broth, a new serum free germ tube induction medium for identification of Candida albicans. World J Microbiol Biotechnol 2014;30:1955-8.  Back to cited text no. 10
Rimek D, Fehse B, Göpel P. Evaluation of Mueller-Hinton-agar as a simple medium for the germ tube production of Candida albicans and Candida dubliniensis. Mycoses 2008;51:205-8.  Back to cited text no. 11
Foongladda S, Haouharn P, Sakulmaiwatana P, Chaiprasert A. Comparative evaluation of Candi Select test and conventional methods for identification of Candida albicans in routine clinical isolates. Mycoses 2002;45:75-8.  Back to cited text no. 12
Carrillo-Muñoz AJ, Quindós G, Cárdenes CD, Alonso-Vargas R, Brió S, Arévalo P, et al. Performance of Bacticard Candida compared with the germ tube test for the presumptive identification of Candida albicans. Mycoses 2003;46:467-70.  Back to cited text no. 13
Hilmioglu S, Ilkit M, Badak Z. Comparison of 12 liquid media for germ tube production of Candida albicans and C. tropicalis. Mycoses 2007;50:282-5.  Back to cited text no. 14
Arora DR, Saini S, Aparna, Gupta N. Evaluation of germ tube test in various media. Indian J Pathol Microbiol 2003;46:124-6.  Back to cited text no. 15
Makwana GE, Gadhavi H, Sinha M. Comparison of germ tube production by Candida albicans in various media. NJIRM 2012;3:6-8.  Back to cited text no. 16
Beheshti F, Smith AG, Krause GW. Germ tube and chlamydospore formation by Candida albicans on a new medium. J Clin Microbiol 1975;2:345-8.  Back to cited text no. 17
Warwood NM, Blazevic DJ. Comparison of cream of rice agar and horse serum for differentiating germ tubes of Candida albicans from filaments of Candida tropicalis. J Clin Microbiol 1977;5:501-2.  Back to cited text no. 18
Kim D, Shin WS, Lee KH, Kim K, Young Park J, Koh CM. Rapid differentiation of Candida albicans from other Candida species using its unique germ tube formation at 39 degrees C. Yeast 2002;19:957-62.  Back to cited text no. 19
Mackenzie DW. Serum tube identification of Candida albicans. J Clin Pathol 1962;15:563-5.  Back to cited text no. 20


  [Figure 1]

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