APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1976, p. 446-447 Copyright © 1976 American Society for Microbiology
Vol. 31, No. 3 Printed in U.S.A.
Improved Method of Assay for Choline A. E. TANGUAY* AND M. H. BLANCHARD American Cyanamid Company, Pearl River, New York 10965
Received for publication 2 September 1975
A modified assay for choline is described which is shorter, yields more consistent responses, and requires considerably less time, space, and equipment than the existing assays.
The purpose of this paper is to report on improvements in the existing assays for choline (1, 3). The basic procedure is the same as that described for the use of choline assay medium, (3). Modifications were made in the methods used to prepare inocula. Since the assay organism grows poorly on Neurospora culture agar (Difco Laboratories, Detroit, Mich.), cultures that yield increased numbers of conidia are grown on choline assay medium supplemented with 0.2% yeast extract, 0.2% malt extract, 1.5% agar, and 1 ,ug of choline chloride per ml. The medium is adjusted to pH 6.7 to 6.8, and 15 ml is dispensed per culture tube (2.3 by 17.8 cm).
After sterilization, the tubes are cooled in a slanted position. To prepare a stock conidial suspension, several slants are inoculated with 1 loopful of Neurospora crassa ATCC 9277 mycelium per slant. Cultures are incubated for 2 days at 30 C followed by 3 to 5 days at room temperature in the light (2). Conidia are harvested by adding 10 ml of 0.012 M phosphate buffer (pH 7.0) containing 15% glycerol (5, 6) to each slant. A sterile inoculating loop is used to dislodge conidia from the mycelial mat, and the resulting conidial suspension is filtered through two layers of sterile cheesecloth. Adjust this stock conidial suspension with buffered glycerol so that a 10-fold
TABLE 1. Growth response of N. crassa (as measured by weight of dried mold pads) to a freshly prepared inoculum versus a standardized conidial suspension stored at 3 to 5 C Inoculum concn
Fresh 1 loop/100 1 loop/100 1 loop/i00 1 loop/100 1 loop/lOO
ml ml ml ml ml
Conidial suspension 2.0 ml + 10 ml 2.0 ml + 10 ml 2.0 ml + 10 ml 2.0 ml + 10 ml 2.0 ml + 10 ml 2.0 ml + 10 ml 2.0 ml + 10 ml 3.0 3.0 3.0 3.0
ml + 10 ml + 10 ml + 10 ml + 10
Age (weeks)
Incubation (h)
Fresh Fresh Fresh Fresh Fresh Fresh
0.0
0.5
1.0
1.5
2.0
3.0
4.0
112 112 112 88 88
2.5 2.1 2.2 1.1 1.7
5.5 5.9 5.1 3.2 4.0
9.6 9.9 8.0 4.7 5.7
13.9 13.0 10.0 6.7 6.6
17.2 15.8 12.3 9.0 7.6
19.5 17.8 13.7 12.2 9.1
21.3 19.0 15.5 15.1 11.3
64 64 64 64 64 64 64
1.1 1.4 1.1 1.1 1.2 1.3 0.8
5.6
8.3 8.4 8.1 8.1 8.6 8.2 6.5
10.5 10.5 10.2 9.5 10.5 10.5 8.5
12.1
5.1 5.4 4.8 5.2 5.3 4.5
11.9 11.8 11.5 12.4 12.0 10.4
14.5 15.1 15.4 14.9 15.7 14.7 13.2
18.9 19.4 19.3 18.0 19.3 18.9 15.9
1.1 0.8 0.6 0.8
4.1 3.5 4.1 2.8
6.7 6.0 6.7 4.9
8.2 7.9 9.2 6.1
10.1 10.0 10.9 7.1
14.1 13.9 14.0 9.5
17.0 18.4 18.2 12.1
1.1 1.0 0.7
5.5 4.5 3.4
8.7 7.1 5.8
11.1 9.2 7.4
13.8 11.2 9.1
18.1 14.7 12.1
22.8 18.9 15.6
2 3 4 7 8 9
ml ml ml ml
11 12 14
16
64 64 64 64
3.5 ml + 10 ml 3.5 ml + 10 ml 3.5 ml + 10 ml
16 20 24
64 64 64
a
Dilution: Micrograms of choline
Inoculum growth (mg)
per
dish (20 ml). 446
VOL. 31, 1976
dilution in distilled water will read 55 to 60% transmittance on a Lumetron colorimeter at 640 nm. After adjustment, this stock suspension is stored at 3 to 5 C. For use as inocula, an appropriate dilution (usually 1 to 5; Table 1) is made in singlestrength choline medium (equal parts of choline assay medium and water). A series of test standards should be run with each new conidial preparation to determine the amount of inoculum needed to obtain an optimum growth curve. (Table 1). By adjusting the inoculum concentration as the age of the conidial suspension increases, the inoculum can be used for at least 6 months. Responses from using a single conidial source were more consistent than when fresh inocula were prepared for each assay (Table 1). The original method of assay for choline (1, 4), carried out in 125-ml Erlenmeyer flasks, is cumbersome and requires a large amount of bench, autoclaving, and incubator space. The substitution of sterile, disposable plastic petri dishes (100 by 20 or 25 mm) for Erlenmeyer flasks greatly reduces the amount of space needed to run the assay, and more samples can be assayed at one time. The assay is set up in large tubes (2.3 by 17.8 cm) that can be easily sterilized and inoculated before transferring to petri dishes. Both standard and samples are diluted to an estimated potency of 1 pg of choline per ml; 0.0, 0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 ml of standard and 0.5, 1.0, 1.5, and 2.0 ml of sample are pipetted per tube. The volume is adjusted to 10 ml with distilled water. Ten milliliters of choline assay media (supplemented with 3 g of asparagine per liter) are added to each tube, and the assay is steri-
NOTES
447
lized at 121 C for 8 min. Each tube is inoculated with 1 drop of inoculum before transferring test solutions to plates, and the plates are incubated at 30 C. Since light encourages sporulation (2), the assay is incubated in the dark. The larger surface area obtained in petri dishes, along with the more concentrated inoculum and elevated incubation temperature, results in more rapid growth of N. crassa (Table 1). At the end of the incubation period, 1 ml of 33% aqueous formalin solution is added per dish, and the pads are removed. Pads are harvested with an inoculating needle fitted with an L-shaped needle made from a paper clip, dried by pressing against several layers of filter paper, and then rolled into a ball with the fingers and placed on porcelain spot plates. They are dried at 100 C for 2 h, cooled, and weighed. We thank A. Bogert and H. Dubrin for their suggestions in the preparation of this manuscript. LITERATURE CITED 1. Bandelin, F. J. 1949. The microbiological determination of choline. J. Am. Pharm. Assoc. Sci. Ed. 38:304307. 2. Davis, R. H., and F. J. DeSerres. 1970. Genetic and microbiological techniques for Neurospora crussa, p. 91. In S. P. Colowick and N. 0. Kaplan (ed.), Academic Press Inc., New York. 3. Difco 1Laboratories. 1968. Supplementary literature, p. 439-440. Difco Laboratories, Detroit. 4. Horowitz, N. H., and G. W. Beadle, 1943. A microbiological method for the determination of choline by use of a mutant, Neurospora. J. Biol. Chem. 150:325. 5. Tanguay, A. E. 1959. Preservation of microbiological assay organisms by direct freezing. Appl. Microbiol. 7:84-88. 6. Tanguay, A. E., and A. B. Bogert. 1974. Survival of Saccharomyces cerevisiae and Sarcina lutea at refrigerator temperatures. Appl. Microbiol. 27:1175-1176.
Vol. 31, No. 3 Printed in U.S.A.
Improved Method of Assay for Choline A. E. TANGUAY* AND M. H. BLANCHARD American Cyanamid Company, Pearl River, New York 10965
Received for publication 2 September 1975
A modified assay for choline is described which is shorter, yields more consistent responses, and requires considerably less time, space, and equipment than the existing assays.
The purpose of this paper is to report on improvements in the existing assays for choline (1, 3). The basic procedure is the same as that described for the use of choline assay medium, (3). Modifications were made in the methods used to prepare inocula. Since the assay organism grows poorly on Neurospora culture agar (Difco Laboratories, Detroit, Mich.), cultures that yield increased numbers of conidia are grown on choline assay medium supplemented with 0.2% yeast extract, 0.2% malt extract, 1.5% agar, and 1 ,ug of choline chloride per ml. The medium is adjusted to pH 6.7 to 6.8, and 15 ml is dispensed per culture tube (2.3 by 17.8 cm).
After sterilization, the tubes are cooled in a slanted position. To prepare a stock conidial suspension, several slants are inoculated with 1 loopful of Neurospora crassa ATCC 9277 mycelium per slant. Cultures are incubated for 2 days at 30 C followed by 3 to 5 days at room temperature in the light (2). Conidia are harvested by adding 10 ml of 0.012 M phosphate buffer (pH 7.0) containing 15% glycerol (5, 6) to each slant. A sterile inoculating loop is used to dislodge conidia from the mycelial mat, and the resulting conidial suspension is filtered through two layers of sterile cheesecloth. Adjust this stock conidial suspension with buffered glycerol so that a 10-fold
TABLE 1. Growth response of N. crassa (as measured by weight of dried mold pads) to a freshly prepared inoculum versus a standardized conidial suspension stored at 3 to 5 C Inoculum concn
Fresh 1 loop/100 1 loop/100 1 loop/i00 1 loop/100 1 loop/lOO
ml ml ml ml ml
Conidial suspension 2.0 ml + 10 ml 2.0 ml + 10 ml 2.0 ml + 10 ml 2.0 ml + 10 ml 2.0 ml + 10 ml 2.0 ml + 10 ml 2.0 ml + 10 ml 3.0 3.0 3.0 3.0
ml + 10 ml + 10 ml + 10 ml + 10
Age (weeks)
Incubation (h)
Fresh Fresh Fresh Fresh Fresh Fresh
0.0
0.5
1.0
1.5
2.0
3.0
4.0
112 112 112 88 88
2.5 2.1 2.2 1.1 1.7
5.5 5.9 5.1 3.2 4.0
9.6 9.9 8.0 4.7 5.7
13.9 13.0 10.0 6.7 6.6
17.2 15.8 12.3 9.0 7.6
19.5 17.8 13.7 12.2 9.1
21.3 19.0 15.5 15.1 11.3
64 64 64 64 64 64 64
1.1 1.4 1.1 1.1 1.2 1.3 0.8
5.6
8.3 8.4 8.1 8.1 8.6 8.2 6.5
10.5 10.5 10.2 9.5 10.5 10.5 8.5
12.1
5.1 5.4 4.8 5.2 5.3 4.5
11.9 11.8 11.5 12.4 12.0 10.4
14.5 15.1 15.4 14.9 15.7 14.7 13.2
18.9 19.4 19.3 18.0 19.3 18.9 15.9
1.1 0.8 0.6 0.8
4.1 3.5 4.1 2.8
6.7 6.0 6.7 4.9
8.2 7.9 9.2 6.1
10.1 10.0 10.9 7.1
14.1 13.9 14.0 9.5
17.0 18.4 18.2 12.1
1.1 1.0 0.7
5.5 4.5 3.4
8.7 7.1 5.8
11.1 9.2 7.4
13.8 11.2 9.1
18.1 14.7 12.1
22.8 18.9 15.6
2 3 4 7 8 9
ml ml ml ml
11 12 14
16
64 64 64 64
3.5 ml + 10 ml 3.5 ml + 10 ml 3.5 ml + 10 ml
16 20 24
64 64 64
a
Dilution: Micrograms of choline
Inoculum growth (mg)
per
dish (20 ml). 446
VOL. 31, 1976
dilution in distilled water will read 55 to 60% transmittance on a Lumetron colorimeter at 640 nm. After adjustment, this stock suspension is stored at 3 to 5 C. For use as inocula, an appropriate dilution (usually 1 to 5; Table 1) is made in singlestrength choline medium (equal parts of choline assay medium and water). A series of test standards should be run with each new conidial preparation to determine the amount of inoculum needed to obtain an optimum growth curve. (Table 1). By adjusting the inoculum concentration as the age of the conidial suspension increases, the inoculum can be used for at least 6 months. Responses from using a single conidial source were more consistent than when fresh inocula were prepared for each assay (Table 1). The original method of assay for choline (1, 4), carried out in 125-ml Erlenmeyer flasks, is cumbersome and requires a large amount of bench, autoclaving, and incubator space. The substitution of sterile, disposable plastic petri dishes (100 by 20 or 25 mm) for Erlenmeyer flasks greatly reduces the amount of space needed to run the assay, and more samples can be assayed at one time. The assay is set up in large tubes (2.3 by 17.8 cm) that can be easily sterilized and inoculated before transferring to petri dishes. Both standard and samples are diluted to an estimated potency of 1 pg of choline per ml; 0.0, 0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 ml of standard and 0.5, 1.0, 1.5, and 2.0 ml of sample are pipetted per tube. The volume is adjusted to 10 ml with distilled water. Ten milliliters of choline assay media (supplemented with 3 g of asparagine per liter) are added to each tube, and the assay is steri-
NOTES
447
lized at 121 C for 8 min. Each tube is inoculated with 1 drop of inoculum before transferring test solutions to plates, and the plates are incubated at 30 C. Since light encourages sporulation (2), the assay is incubated in the dark. The larger surface area obtained in petri dishes, along with the more concentrated inoculum and elevated incubation temperature, results in more rapid growth of N. crassa (Table 1). At the end of the incubation period, 1 ml of 33% aqueous formalin solution is added per dish, and the pads are removed. Pads are harvested with an inoculating needle fitted with an L-shaped needle made from a paper clip, dried by pressing against several layers of filter paper, and then rolled into a ball with the fingers and placed on porcelain spot plates. They are dried at 100 C for 2 h, cooled, and weighed. We thank A. Bogert and H. Dubrin for their suggestions in the preparation of this manuscript. LITERATURE CITED 1. Bandelin, F. J. 1949. The microbiological determination of choline. J. Am. Pharm. Assoc. Sci. Ed. 38:304307. 2. Davis, R. H., and F. J. DeSerres. 1970. Genetic and microbiological techniques for Neurospora crussa, p. 91. In S. P. Colowick and N. 0. Kaplan (ed.), Academic Press Inc., New York. 3. Difco 1Laboratories. 1968. Supplementary literature, p. 439-440. Difco Laboratories, Detroit. 4. Horowitz, N. H., and G. W. Beadle, 1943. A microbiological method for the determination of choline by use of a mutant, Neurospora. J. Biol. Chem. 150:325. 5. Tanguay, A. E. 1959. Preservation of microbiological assay organisms by direct freezing. Appl. Microbiol. 7:84-88. 6. Tanguay, A. E., and A. B. Bogert. 1974. Survival of Saccharomyces cerevisiae and Sarcina lutea at refrigerator temperatures. Appl. Microbiol. 27:1175-1176.
