APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1977, Copyright © 1977 American Society for Microbiology
p.
219-220
Vol. 33, No. 2 Printed in U.S.A.
Rapid Method for Detection of Urea Hydrolysis by Yeasts H. S. RANDHAWA* Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110007, India D. K. PALIWAL
AND
Received for publication 26 July 1976
A method is described for the rapid detection of urea hydrolysis by yeasts, using the Berthelot color reaction. The results could be determined within 30 to 50 min with this method, compared with 8 to 72 h usually required with Christensen urea agar.
Urea hydrolysis has been widely used as a criterion in yeast taxonomy, its greatest application being as a confirmatory test for the recognition of anascosporogenous species (5). The test is carried out on Christensen urea agar as described by Seeliger (3). This communication deals with the application of the Berthelot color reaction, used earlier as a rapid means of detecting urease activity in the Enterobacteriaceae (1), to the yeastlike fungi. MATERIALS AND METHODS The reagents were prepared in deionized water as described below. Sodium phenate. Phenol, 25.0 g, was dissolved in 800 ml of water, mixed with 78 ml of 4.0 N sodium hydroxide solution, and diluted to 1 liter. It was stored in the refrigerator and was used within 1 month unless it turned brown earlier, in which case it had to be discarded. The solution must be brought to room temperature before use (2). Sodium hypochlorite. Calcium hypochlorite (bleaching powder), 20 g, was mixed in a small quantity of water, and the volume was brought to 100 ml. An equal quantity of 30% sodium carbonate solution was added, and the mixture was kept at room temperature for 2 to 3 h with intermittent shaking. It was filtered to obtain a yellowish solution of sodium hypochlorite that was standardized iodometrically (4, 6). The solution was diluted to obtain a nearly 0.06 N sodium hypochlorite solution. Test strains. One hundred and forty-one isolates belonging to 23 species of 8 yeast genera, available from the Fungus Culture Collection of the Vallabhbhai Patel Chest Institute, were tested. Berthelot reaction. A loopful of a 3- to 4-day-old culture of each test strain (Sabouraud glucose agar at 25°C) was suspended in 4 ml of deionized water and was mixed with 1 ml of 1.6% freshly prepared urea solution. The mixture was incubated at 25°C for 20 min, followed by addition of 3 ml of sodium nitroprusside (0. 1%), 2 ml of sodium phenate, and 3 ml of sodium hypochlorite (approximately 0.06 N) in quick succession. Urease-positive strains produced a blue color within 30 min of incubation in darkness at 37°C. The blue complex showed maximum absorbance at 630 nm, and its optical density remained constant thereafter for more than 24 h.
Christensen urea medium. The medium was prepared as described by Seeliger (3) and had the following composition: peptone, 1 g; glucose, 1 g; sodium chloride, 5 g; potassium dihydrogen phosphate, 2 g; phenol red, 0.012 g; and distilled water, 1 liter. The ingredients were dissolved and the pH was adjusted to 6.8. To it was added 20 g of agar, and after dissolution 4.5-ml aliquots of the medium were dispensed into 16-mm cotton-plugged test tubes. These were sterilized for 15 min at 15-lb overpressure, and immediately after autoclaving 0.5 ml of 20% filter-sterilized urea solution was added. After mixing, the tubes were slanted. The test strains were inoculated onto urea agar slants and were incubated at 25°C. The cultures were observed daily up to 5 days. The reactions were recorded as positive after the appearance of a deep pink color.
RESULTS Of 141 yeast isolates tested, urea hydrolysis was detected in 92 by both the Berthelot color reaction and Christensen urea agar (Table 1). No urease activity was detected in the other 49 yeast isolates with either method. With the Berthelot technique, the time required for a positive result, depending upon the test strain, ranged from 30 to 50 min, as against 8 to 72 h with the Christensen procedure. Table 2 provides more detailed data on the time required for detection of urea hydrolysis in 77 strains of Cryptococcus neoformans, using the Berthelot reaction, Christensen urea agar, and Christensen urea broth. DISCUSSION The Berthelot color reaction is a more rapid test than Christensen urea agar for detecting urea hydrolysis by yeasts. The underlying principle in both the procedures is the same; i.e., urease activity is indicated by the liberation of ammonia when the test organism is cultivated with urea as a substrate. On Christensen medium, ammonia production causes a rise in pH, leading to a color change in the indicator phenol red from yellow to pink. In the Berthelot 219
APPL. ENVIRON. MICROBIOL.
PALIWAL AND RANDHAWA
220
TABLE 1. Urea hydrolysis by yeasts as indicated by the Berthelot color reaction and Christensen urea agar
Species
Candida albicans C. claussenii C. guilliermondii C. melinii C. parapsilosis
No. of strains tested
16 2 1 2 7
No. of strains showing positive results with: BerChristhelot tensen color urea test agar 0 0 0 0 0 0 0
TABLE 2. Comparison of time taken for detection of urea hydrolysis by the Berthelot color reaction and the Christensen method, using 77 strains of Cryptococcus neoformans No. of strains showing positive results with:
Time (h)
1 4 8 12 16
Berthelot
Christensen urea agar
Christensen urea broth
color reac-
0 0 0 2 5 8 8 9 22 9 8
0 0 1 12 32 16 6 2 2 2 1 1 1 1
77
tion
C. scottii
1
1
C. tropicalis C. veronae Cryptococcus albidus C. albidus var. diffluens C. neoformans Kloeckera apiculata Pichia etchelsii P. ohmeri
8
0
0 0 0 1 0
1
0
0
1 1 77 1 3
1 1 77 0 0
1 1 77 0 0
P. vini var. vini
1 1
0 0
0 0
Rhodotorula glutinis R. graminis R. rubra Saccharomyces cerevisiae S. chevalieri
2 2 6 1 1
2 2 6 0 0
2 2 6 0 0
S. rouxii
1
0
0
LITERATURE CITED
Sporobolomyces antarcti-
1
1
1
1
1
1
3
0
0
141
92
92
1. Bergquist, L. M., and R. L. Searcy. 1963. Application of Berthelot reaction for rapid detection of urea hydrolysis by Proteus strains. J. Bacteriol. 85:954-955. 2. Fawcett, J. K., and J. E. Scott. 1960. A rapid and precise method for the determination of urea. J. Clin. Pathol. 13:156-159. 3. Seeliger, H. P. R. 1956. Use of a urease test for the screening and identification of cryptococci. J. Bacteriol. 72:127-131. 4. Vogel, A. I. 1961. A text book of quantitative inorganic
cus
S. salmonicolor var. fischerii Torulopsis candida Total
reaction, on the other hand, ammonia forms a blue complex in the presence of sodium phenate and sodium hypochlorite, with sodium nitroprusside serving as a catalyst. ACKNOWLEDGMENTS This work was carried out in part with financial assistance from the Council of Scientific and Industrial Research, New Delhi.
20 24 28 32 36 44 52 60 72
4 1 1
Acknowledgment is also due to V. C. Misra, Mahendra Pal, and T. Kowshik for their cooperation.
analysis including elementary instrumental analysis, 3rd ed., p. 364-365. The English Language Book Society and Longmans Green, London. 5. Walt, J. P. van der. 1970. Criteria and methods used in classification, p. 96-97. In J. Lodder (ed.), The yeasts, 2nd ed. North-Holland, Amsterdam. 6. Williamson, D. H., and J. F. Wilkinson. 1958. The isolation and estimation of the PHB inclusions of Bacillus sp. J. Gen. Microbiol. 19:198-209.
p.
219-220
Vol. 33, No. 2 Printed in U.S.A.
Rapid Method for Detection of Urea Hydrolysis by Yeasts H. S. RANDHAWA* Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110007, India D. K. PALIWAL
AND
Received for publication 26 July 1976
A method is described for the rapid detection of urea hydrolysis by yeasts, using the Berthelot color reaction. The results could be determined within 30 to 50 min with this method, compared with 8 to 72 h usually required with Christensen urea agar.
Urea hydrolysis has been widely used as a criterion in yeast taxonomy, its greatest application being as a confirmatory test for the recognition of anascosporogenous species (5). The test is carried out on Christensen urea agar as described by Seeliger (3). This communication deals with the application of the Berthelot color reaction, used earlier as a rapid means of detecting urease activity in the Enterobacteriaceae (1), to the yeastlike fungi. MATERIALS AND METHODS The reagents were prepared in deionized water as described below. Sodium phenate. Phenol, 25.0 g, was dissolved in 800 ml of water, mixed with 78 ml of 4.0 N sodium hydroxide solution, and diluted to 1 liter. It was stored in the refrigerator and was used within 1 month unless it turned brown earlier, in which case it had to be discarded. The solution must be brought to room temperature before use (2). Sodium hypochlorite. Calcium hypochlorite (bleaching powder), 20 g, was mixed in a small quantity of water, and the volume was brought to 100 ml. An equal quantity of 30% sodium carbonate solution was added, and the mixture was kept at room temperature for 2 to 3 h with intermittent shaking. It was filtered to obtain a yellowish solution of sodium hypochlorite that was standardized iodometrically (4, 6). The solution was diluted to obtain a nearly 0.06 N sodium hypochlorite solution. Test strains. One hundred and forty-one isolates belonging to 23 species of 8 yeast genera, available from the Fungus Culture Collection of the Vallabhbhai Patel Chest Institute, were tested. Berthelot reaction. A loopful of a 3- to 4-day-old culture of each test strain (Sabouraud glucose agar at 25°C) was suspended in 4 ml of deionized water and was mixed with 1 ml of 1.6% freshly prepared urea solution. The mixture was incubated at 25°C for 20 min, followed by addition of 3 ml of sodium nitroprusside (0. 1%), 2 ml of sodium phenate, and 3 ml of sodium hypochlorite (approximately 0.06 N) in quick succession. Urease-positive strains produced a blue color within 30 min of incubation in darkness at 37°C. The blue complex showed maximum absorbance at 630 nm, and its optical density remained constant thereafter for more than 24 h.
Christensen urea medium. The medium was prepared as described by Seeliger (3) and had the following composition: peptone, 1 g; glucose, 1 g; sodium chloride, 5 g; potassium dihydrogen phosphate, 2 g; phenol red, 0.012 g; and distilled water, 1 liter. The ingredients were dissolved and the pH was adjusted to 6.8. To it was added 20 g of agar, and after dissolution 4.5-ml aliquots of the medium were dispensed into 16-mm cotton-plugged test tubes. These were sterilized for 15 min at 15-lb overpressure, and immediately after autoclaving 0.5 ml of 20% filter-sterilized urea solution was added. After mixing, the tubes were slanted. The test strains were inoculated onto urea agar slants and were incubated at 25°C. The cultures were observed daily up to 5 days. The reactions were recorded as positive after the appearance of a deep pink color.
RESULTS Of 141 yeast isolates tested, urea hydrolysis was detected in 92 by both the Berthelot color reaction and Christensen urea agar (Table 1). No urease activity was detected in the other 49 yeast isolates with either method. With the Berthelot technique, the time required for a positive result, depending upon the test strain, ranged from 30 to 50 min, as against 8 to 72 h with the Christensen procedure. Table 2 provides more detailed data on the time required for detection of urea hydrolysis in 77 strains of Cryptococcus neoformans, using the Berthelot reaction, Christensen urea agar, and Christensen urea broth. DISCUSSION The Berthelot color reaction is a more rapid test than Christensen urea agar for detecting urea hydrolysis by yeasts. The underlying principle in both the procedures is the same; i.e., urease activity is indicated by the liberation of ammonia when the test organism is cultivated with urea as a substrate. On Christensen medium, ammonia production causes a rise in pH, leading to a color change in the indicator phenol red from yellow to pink. In the Berthelot 219
APPL. ENVIRON. MICROBIOL.
PALIWAL AND RANDHAWA
220
TABLE 1. Urea hydrolysis by yeasts as indicated by the Berthelot color reaction and Christensen urea agar
Species
Candida albicans C. claussenii C. guilliermondii C. melinii C. parapsilosis
No. of strains tested
16 2 1 2 7
No. of strains showing positive results with: BerChristhelot tensen color urea test agar 0 0 0 0 0 0 0
TABLE 2. Comparison of time taken for detection of urea hydrolysis by the Berthelot color reaction and the Christensen method, using 77 strains of Cryptococcus neoformans No. of strains showing positive results with:
Time (h)
1 4 8 12 16
Berthelot
Christensen urea agar
Christensen urea broth
color reac-
0 0 0 2 5 8 8 9 22 9 8
0 0 1 12 32 16 6 2 2 2 1 1 1 1
77
tion
C. scottii
1
1
C. tropicalis C. veronae Cryptococcus albidus C. albidus var. diffluens C. neoformans Kloeckera apiculata Pichia etchelsii P. ohmeri
8
0
0 0 0 1 0
1
0
0
1 1 77 1 3
1 1 77 0 0
1 1 77 0 0
P. vini var. vini
1 1
0 0
0 0
Rhodotorula glutinis R. graminis R. rubra Saccharomyces cerevisiae S. chevalieri
2 2 6 1 1
2 2 6 0 0
2 2 6 0 0
S. rouxii
1
0
0
LITERATURE CITED
Sporobolomyces antarcti-
1
1
1
1
1
1
3
0
0
141
92
92
1. Bergquist, L. M., and R. L. Searcy. 1963. Application of Berthelot reaction for rapid detection of urea hydrolysis by Proteus strains. J. Bacteriol. 85:954-955. 2. Fawcett, J. K., and J. E. Scott. 1960. A rapid and precise method for the determination of urea. J. Clin. Pathol. 13:156-159. 3. Seeliger, H. P. R. 1956. Use of a urease test for the screening and identification of cryptococci. J. Bacteriol. 72:127-131. 4. Vogel, A. I. 1961. A text book of quantitative inorganic
cus
S. salmonicolor var. fischerii Torulopsis candida Total
reaction, on the other hand, ammonia forms a blue complex in the presence of sodium phenate and sodium hypochlorite, with sodium nitroprusside serving as a catalyst. ACKNOWLEDGMENTS This work was carried out in part with financial assistance from the Council of Scientific and Industrial Research, New Delhi.
20 24 28 32 36 44 52 60 72
4 1 1
Acknowledgment is also due to V. C. Misra, Mahendra Pal, and T. Kowshik for their cooperation.
analysis including elementary instrumental analysis, 3rd ed., p. 364-365. The English Language Book Society and Longmans Green, London. 5. Walt, J. P. van der. 1970. Criteria and methods used in classification, p. 96-97. In J. Lodder (ed.), The yeasts, 2nd ed. North-Holland, Amsterdam. 6. Williamson, D. H., and J. F. Wilkinson. 1958. The isolation and estimation of the PHB inclusions of Bacillus sp. J. Gen. Microbiol. 19:198-209.
