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 ~  Abstract
 ~ Introduction
 ~ Subjects and Methods
 ~ Results
 ~ Discussion
 ~  References
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Year : 2015  |  Volume : 33  |  Issue : 2  |  Page : 293-295
 

A cost-effective carbohydrate fermentation test for yeast using microtitre plate


Department of Microbiology, Mahatma Gandhi Medical College and Research Institute, Pillaiyarkuppam, Pondicherry, India

Date of Submission21-Dec-2013
Date of Acceptance10-Sep-2014
Date of Web Publication10-Apr-2015

Correspondence Address:
A Kali
Department of Microbiology, Mahatma Gandhi Medical College and Research Institute, Pillaiyarkuppam, Pondicherry
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.154884

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 ~ Abstract 

Carbohydrate fermentation test is a well-established technique for speciation of bacteria and fungi. However, long incubation period, cost and procedural complexity may limit its use. Here, we describe a simple modification of conventional carbohydrate fermentation technique using microtitre plate. Thirty-one yeast isolates were identified based on their fermentation property by microtitre plate method and results were compared with conventional method. The modified method had 85.7% sensitivity and 100% specificity. The average time taken to produce positive reaction was 24 hours. When compared with conventional method, modified method reduced turn-around time and cost of processing without significant increase in discordant results.


Keywords: Candida, carbohydrate fermentation test, miniaturized biochemical tests, yeast identification


How to cite this article:
Kali A, Srirangaraj S, Charles P. A cost-effective carbohydrate fermentation test for yeast using microtitre plate. Indian J Med Microbiol 2015;33:293-5

How to cite this URL:
Kali A, Srirangaraj S, Charles P. A cost-effective carbohydrate fermentation test for yeast using microtitre plate. Indian J Med Microbiol [serial online] 2015 [cited 2017 Dec 14];33:293-5. Available from: http://www.ijmm.org/text.asp?2015/33/2/293/154884



 ~ Introduction Top


Pertaining to substantial increase in number of patients with suppressed immunity as a result of human immunodeficiency virus (HIV) infection, advances in cancer chemotherapy and immunosuppressive therapy for organ transplantation, opportunistic yeast infections are becoming increasingly common. Non-albicans candida species have emerged as a significant healthcare problem with the development of resistance to multiple antifungal agents in recent years. Consequently, laboratory identification of yeasts has crucial role in patient-care as well as in research. Carbohydrate fermentation tests are essential for speciation of bacteria and fungi and are particularly relevant in setups where automated identification systems are unavailable. Miniaturization of biochemical tests have been found to be beneficial in reducing the cost of reagents. Although the miniaturized fermentation tests have been developed for bacteria, [1] it has not been standardized for fungi. In this study, we describe a cost-effective in-house simple modification of carbohydrate fermentation test for yeasts.


 ~ Subjects and Methods Top


The objective of this study was to evaluate the performance of miniaturized carbohydrate fermentation test for yeast identification in comparison to conventional identification tests. In this study, a culture collection of 31 clinical yeast isolates, comprising Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida krusei and Cryptococcus neoformans were identified by microscopy, germ tube formation, morphology on cornmeal agar and HiCrome Candida differential agar and tested for carbohydrate fermentation by both microtitre plate method and conventional method. C. albicans ATCC 90028, Candida tropicalis ATCC 750, Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were used for quality control.

The miniaturized fermentation test was carried out in a 96 well (8 × 12 wells) autoclavable polypropylene microtitre plate. The wells of horizontal rows were utilized for testing fermentation reaction of a particular carbohydrate viz. glucose, sucrose, lactose and maltose. 300 μl of basal media containing phenol red indicator and 2% test sugar was added to each well of a horizontal row. The panel was sealed and stored at 4°C. Before use, it was brought to room temperature and 12 vertical columns were labelled with 12 test isolate numbers. Yeast suspension in sterile distilled water equivalent to No. 1 McFarland standard was prepared and one drop of it was inoculated in each well of the vertical column [Figure 1]. The microtitre plate was covered with the lid and incubated at 25-30°C for 7 days. Development of yellow colour was considered as positive result. Same isolates were also tested by conventional sugar fermentation method in 12 × 100 mm glass test tubes containing 3 ml media. In this study, test sugars were prepared as 10% aqueous solutions and were sterilized by free steaming at 100°C. [2] Twenty millilitre and 5 ml of this stock solution were used for testing 24 isolates by conventional and modified sugar fermentation test, respectively. Phenol red indicator was used in both conventional and modified carbohydrate fermentation tests.
Figure 1: Carbohydrate fermentation test for yeast using microtitre plate

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 ~ Results Top


Identification based on microscopy, germ tube formation, morphology on cornmeal agar and HiCrome Candida differential agar had same results as conventional sugar fermentation. Candida albicans was most common isolate (n = 14), followed by Candida tropicalis (n = 12), Candida glabrata (n = 2), Candida parapsilosis (n = 1), Candida krusei (n = 1) and Cryptococcus neoformans (n = 1). Except two discordant results, we found no significant difference in results of conventional and miniaturized sugar fermentation method. Two C. albicans isolates which were sucrose negative in conventional sugar fermentation (no acid and gas formed), displayed positive result (acid produced) in microtitre plate method. There were no discrepancies noted, when only acid production was compared to gas production in interpretation of results of conventional fermentation test. All tests which produced gas also had acid reaction. The sensitivity and specificity of this modified technique was 85.7% and 100%, respectively, when compared with conventional sugar fermentation test with observation of gas production.


 ~ Discussion Top


The principle of fermentation tests is based on detection of acid and gas produced as a result of sugar fermentation. Usually a pH indicator is incorporated in the media to reflect the change in pH in terms of colour change. Unlike bacteria, its use for speciation of fungi has a major limitation. Owing to the slow growth rate of fungi, prolonged incubation up to 3 week is recommended. We found that on an average, the time taken to produce positive reaction was 24 hours for microtitre plate method, in comparison to 72 hours in case of conventional test. This finding is in keeping with other studies where smaller volume of media and a relatively heavy inoculum has been reported to yield a rapid reaction. [3] Although the test in this study was performed with 300 μl of carbohydrate fermentation media, reducing this volume to 100 μl may avoid spillage or contamination during handling. However, this miniaturization from 300 μl to 100 μl needs further evaluation. Aliquoting the sugar solution in smaller volumes and storing these for later use may be helpful in reducing risk of contamination of unutilized sugar solution if stored in a single container. Miniaturized biochemical tests were initially developed for bacterial pathogens. These are also available commercially. Microtitre plate has been used for testing fermentation reactions in  Neisseria More Details and anaerobic bacteria. [1],[4] However, this method has not been tested and standardized for yeasts.

Cost-effective analysis was done and the results are shown in [Table 1]a and b. As per our estimate, this modified technique will reduce the cost of identification when compared with conventional method by 80% (Rs. 39.56 for conventional sugar testing and Rs. 8.17 for the modified method). Although commercial tests (Candifast [5] from ElitechGroup, Puteaux, France and GLABRATA RTT [3] from Fumouze Diagnostics, Levallois Perret, France) with similar methodology are in use for prompt identification of yeasts, the higher costs often limit their application in most mycology laboratories. Hence, this simple in-house modification of carbohydrate fermentation test may be used as an adjunct to the conventional identification tests for rapid identification.
Table 1


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In conclusion, miniaturized carbohydrate fermentation test using microtitre plate may be employed as a cost-effective tool to reduce the turnaround time of yeast identification.

 
 ~ References Top

1.
Kellogg DS Jr, Turner EM. Rapid fermentation confirmation of Neisseria gonorrhoeae. Appl Microbiol 1973;25:550-2.  Back to cited text no. 1
[PUBMED]    
2.
Collee JG, Miles RS, Watt B. Tests for identification of bacteria. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editors. Mackie and McCartney Practical Medical Microbiology. 14 th ed. Chap. 7. London: Churchill Livingstone; 2010. p. 132.  Back to cited text no. 2
    
3.
Freydiere AM, Robert R, Ploton C, Marot-Leblond A, Monerau F, Vandenesch F. Rapid identification of candida glabrata with a new commercial test, GLABRATA RTT. J Clin Microbiol 2003;41:3861-3.  Back to cited text no. 3
    
4.
Stargel D, Thompson FS, Phillips SE, Lombard GL, Dowell VR Jr. Modification of the minitek miniaturized differentiation system for characterization of anaerobic bacteria. J Clin Microbiol 1976;3:291-301.  Back to cited text no. 4
[PUBMED]    
5.
Gundes SG, Gulenc S, Bingol R. Comparative performance of fungichrom I, candifast and API 20C Aux systems in the identification of clinically significant yeasts. J Med Microbiol 2001;50:1105-10.  Back to cited text no. 5
    


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