Journal of Medical and Veterinary Mycology (1992), 30, 323-326

SHORT COMMUNICATION

A microfermentation test for the rapid identification of yeasts

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T. C. MOLINA 1, S. K. MISHRA I AND D. L. PIERSON 2

~Microbiology Laboratory, Biomedical Operations and Research Branch, KRUG Life Sciences and 2National Aeronautics and Space Administration, Johnson Space Center, Houston, TX 77058, USA (Accepted 27 March 1992)

The accuracy and speed of a microfermentation test (MFF), developed as a supplementary aid to other yeast identification systems, were compared with the conventional fermentation method in identifying 15 yeast species. The MFT significantly reduced the incubation period required to obtain a definitive identification. The method is easy to perform and the media and space requirements are minimal.

Despite tremendous advances in microbial identification technologies, the fermentation test continues to be an important step in the identification of yeasts. Even the stateof-the-art automated systems may require supplementary fermentation tests which must be performed manually. The traditional Wickerham test is probably the most widely used fermentation method to identify yeasts [2, 6]. The method employs an inverted Durham tube for the collection of CO2 and great care must be observed in the initial preparation of the tubes. Generally, this method is cumbersome and time-consuming, often requiring several weeks before final results can be recorded. This preliminary communication describes an easy-to-perform microfermentation test (MFT) that significantly reduces the time required to obtain definitive identification results. Cultures of 15 reference strains representing nine genera of yeasts were obtained from the American Type Culture Collection (Table 1). The rationale for selecting these strains was to include species known to ferment one or more of the sugars to be tested, as well as species which are non-fermenters. Only glucose, galactose, maltose, sucrose, lactose, raffinose and trehalose were selected for the study because these represent carbon sources frequently used in fermentation tests for the routine identification of clinically significant yeasts [1, 4, 5]. All tests were run in triplicate. Test strains were grown for 24-48 h on Sabouraud glucose agar slants, then suspended in sterile distilled water to McFarland turbidity standard No. 2. Standard procedures were used to perform the Wickerham fermentation test [2, 6]. Briefly, the basal medium was prepared by dissolving 7-5 g of peptone (Difco, USA), 4-5 g of yeast extract (Difco, USA) and 0-04 g of bromothymol blue (Aldrich, USA) in 1000 ml distilled water. Two-millilitre aliquots of this solution were dispensed into 125 x 16 mm screw-capped tubes; 50 x 6 mm Durham Correspondence address: T. C. Molina, KRUG Life Sciences, 1290 Hercules Drive, Suite 120, Houston, TX 77058, USA. 323

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Comparative evaluation of microfermentation (MFT) and the conventional Wickerham test for yeast identification Incubation period required to obtain results (days) c

Fermentation results b

Species tested ~'

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Candida albicans

Candida glabrata Candida parapsilosis Candida tropicalis Cryptococcus albidus Cryptococcus laurentii Dekkera interrnedia Hansenula anomala Hansenula californica

ATCC

No.

1

2

3

14053

+

+

2001

+

.

+

22019

+

+

.

13803

+

+

+

34140

.

18803

.

34076

+

.

.

.

. .

4

5

6

-

-

-

. .

.

.

.

.

-

.

+

5-6

2-3

5-6

1-2

+

.

.

.

+

+

+

-

Conventional method M F T

+ .

+

.

7

+

+

+

-

5-6

1-2

5-6

3-4

NF

NF

NF

NF

26-28

3-4

60811

+

+

+

+

10680

+

.

.

.

36534

+

32765

.

.

.

.

.

.

.

NF

NF

9449

.

.

.

.

.

.

.

NF

NF

.

9-10

2-3

7-8

1-2

7-8

1-2

Khty veromyces rnarxianus

var.

marxianus Rhodotorula glutinis

Rhodotorula rubra Saccharomyces cerevisiae Saccharomycopsis lipolytica Trichosporon capitatum

2366

+

9773

.

28576

.

+

-

+

+

+

+

. .

+

-

. .

.

+

+

. .

.

.

-

-

5-6

1-2

NF

NF

NF

NF

Nomenclature as described by Kreger-van R i j [2]. These results are in conformity with the standard descriptions for the tested species [ 2 , 3 ] . glucose; 2 = galactose; 3 = m a l t o s e ; 4 = s u c r o s e ; 5 = l a c t o s e ; 6 = raffinose; 7 = trehalose. = fermentation positive; ( - ) - fermentation negative; N F = non-fermenters. c R e s u l t s based on incubation a t 3 0 ° C . ~' h

1 = (+)

tubes were inverted and placed in the tubes and the units were sterilized. Care was taken to prevent air bubble formation in the Durham tube. Aqueous solutions of the test sugars were prepared at 6% (w:v), except for raffinose, which was used at a concentration of 12%. The sugar solutions were filter-sterilized and 1 ml added to the 2 ml of basal medium in the tube to reach the final carbohydrate concentration of 2%. Final raffinose concentration was 4%. Tubes were inoculated in triplicate with 0.1 ml of the yeast suspension. The caps were loosened and the tubes were incubated at 30°C. The tubes were checked for gas production at 24 h intervals for 28 days. A positive fermentation reaction was indicated by the presence of gas inside the inverted Durham tube. The test was repeated using 25°C for incubation. For the MFT, the same basal medium was used. Carbohydrate solutions were prepared at 8.5% (w:v) concentration except for raffinose which was 17%. Microtitre plates (Falcon 3911 Micro Test, Becton Dickinson, USA) were cut into 8-well strips, labelled and sterilized by ultraviolet irradiation for 6-8 h at a distance of 6 inches from a Blak-ray lamp using two 15-watt ultraviolet tubes (Curtin Matheson Scientific). Fifteen millilitres of the basal medium was mixed with 7-5 ml of the carbohydrate solution and 150 p~l of the resulting mixture was dispensed into each well. Addition of

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~

Bubble in well

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Candida a/b/cans

~

ZiplocboO

~

Vaspar Seal

~ Emptywell

SaccharomycOs Cerovis/oo

FIG. ]. Diagrammaticillustrationof two 8-wellstripscut from a microtitreplate and inoculatedwith (a) Candida albicans, and (b) Saccharomyces cerevisiae. Test carbohydrate was 1 = glucose; 2 = galactose; 3 maltose; 4 = sucrose; 5 = lactose; 6 = raffinose; 7 trehalose, and none in c which served as a control. The inoculated strips were incubated at 30°C, upside down. Note the presence of gas in (a) 1, 2, 3 and 7, and (b) 1, 2, 3, 4 and 6.

50/zl of cell suspension (prepared as described above) brought the final carbohydrate concentration to 2%. A control without a carbon source was included for each yeast. Each well was then sealed with about 25/zl of melted sterile vaspar (50 g paraffin and 50 g petroleum jelly). To avoid trapping air in the wells while sealing, the tip of the pipette used to deliver the melted vaspar was allowed to touch the side of the wall. The sealed plates were placed in 10 × 15 cm Ziploc bags (Curtin Matheson Scientific) with the sealed side down and incubated at 30°C for up to 4 days (Fig. 1). The plates were examined daily; if the wells remained full, the reaction was recorded as negative. Tests were recorded as positive if the wells showed bubbles or if the fluid had leaked out, leaving the wells empty (Fig. 1). This test was also repeated at 25°C. The Vitek automated microbial identification system was used to verify the identity of the test strains. It confirmed the identity of C. albicans, Candida parapsilosis,

Candida tropicalis, Cryptococcus albidus, Cryptococcus laurentii, Hansenula anornala, Rhodotorula glutinis, Rhodotorula rubra, Saccharomycopsis lipolytica and Blastoschizomyces capitatus. Kluyveromyces marxianus var. marxianus was identified as Candida pseudotropicalis (Candida kefyr) which is the imperfect state of this species [2]. Supplementary tests were, however, required to confirm the identity of Candida glabrata and S. cerevisiae. Dekkera intermedia and Hansenula californica

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were not recognized as they were not in the Vitek data base. The fermentation pattern of all strains matched the standard description for those species and the examined carbon sources [2, 3]. In eight of the nine strains that fermented one or more of the tested carbon sources, positive results were obtained in 5-10 days using the Wickerham method. In contrast, the MFT revealed positive results in those strains in 1-4 days (Table 1). In one case, the conventional method required 26-28 days of incubation; by contrast the MFT revealed positive results in just 3-4 days. Both methods produced results faster at 30°C than at 25°C (data not shown). The preliminary data presented here suggest that the MFT is quicker than the conventional Wickerham method for the identification of clinically important yeasts. Even a small amount of CO2 produced during fermentation is easily detected. The amount of media required for each test is reduced from 3 ml to 0.15 ml and the workspace requirements are also minimal. The test is easy to perform and could serve as a useful supplement to yeast identification systems that call for fermentation tests. REFERENCES 1. EMMONS, C. W., BINFORD, C. H., UTZ, J. P 8t. KWON-CHUNG, K. J. 1977. Medical Mycology, 3rd edn, p. 198. Lea & Febiger, Philadelphia. 2. KREGER-VAN RIJ, N. J. W. (Ed) 1984. The Yeasts: A Taxonomic Study. Elsevier Science Publishers, Amsterdam. 3. LODDER, J. (Ed.). 1970. The Yeasts: A Taxonomic Study. North-Holland Publishing Company, Amsterdam. 4. McGINNIS, M. R. 1980. Laboratory Handbook of Medical Mycology. Academic Press, New York. 5. RIPPON,J. W. 1988. Medical Mycology: The Pathogenic Fungi and the PathogenicActinomycetes, 3rd edn, pp. 532-581. W. B. Saunders Company, Philadelphia. 6. WICKERHAM,I. J. 1951. Taxonomy of Yeast. Technical Bulletin No. 1029, United States Department of Agriculture, Washington, DC.

A microfermentation test for the rapid identification of yeasts.

The accuracy and speed of a microfermentation test (MFT), developed as a supplementary aid to other yeast identification systems, were compared with t...
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