APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1976, p. 621-622 Copyright ©) 1976 American Society for Microbiology
Vol. 31, No. 4 Printed in U.SA.
Microapplicator and Micropipette for Inoculation of the Embryonated Chicken Egg' WILLIAM C. STEARMAN III, JAMES 0. NORMAN, AND H. DEL VAR PETERSEN Veterinary Toxicology and Entomology Research Laboratory and Southern Region, Agricultural Research Service, College Station, Texas 77840 Received for publication 20 November 1975
Aflatoxin G, was used to test the speed and accuracy of the microapplicator and the micropipette. The 50% lethal dose of both assay systems was approximately the same, and 4.0 gg of G2 had an 85% lethal effect in both systems. The 50% lethal dose of the microapplicator and the micropipette was lower than that of the syringe but, of these, only the micropipette can combine the accuracy of the microapplicator and the speed of the syringe. The Association of Official Analytical Chemists has adopted the procedure of using the fertile chicken egg in the bioassay of aflatoxins (1). In using the embryonating chicken egg as an assay system, we needed a more precise method of inoculation, as opposed to the syringe now used. Aflatoxin G,, obtained from Calbiochem, Los Angeles, Calif., was easily soluble in methanol at a concentration of 1 mg of G2 per 1 ml of methanol. Methanol is relatively nontoxic to the chicken embryo at levels less than 0.05 ml (3). Two methods were studied: (i) using a microapplicator (Instrumentation Specialties Co., Lincoln, Neb.; model M) and (ii) using a micropipette (Oxford sampler micropipetting system, Fisher Scientific Co., Pittsburgh, Pa.). The microapplicator was used with a 0.5-ml syringe and a 22-gauge needle and was calibrated to deliver 0.02 ml with five depressions of the pedal switch when set on approximately 4.20 s. The needle was bent 900, so about 25 mm extended downward after the sharp point had been removed by filing. The fertile white leghorn eggs selected for the test weighed between 52 and 63 g at the onset of incubation and had been incubated for 5 days before inoculation. An egg punch was used to make a hole. Then the egg was brought -ap to the needle, and the needle was allowed to enter the hole and descend downward until the tip of the needle just touched the air cell membrane, but care was taken not to rupture the membrane. One drop of sample was delivered per two depressions of the pedal switch; thus, the needle tip had to be in contact with the membrane. In this manner, the entire dose was delivered to the fertile chicken egg.
A 0.02-ml Oxford micropipette was used, with its standard disposable tip cut approximately at the end of the micropipette; a 23gauge needle was placed on the shortened, disposable pipette tip. A standard 0.005-ml disposable pipette tip was placed over about 5 mm of the 23-gauge needle. The pipette system was then tested for delivery of 0.02 ml of sample. The standard 0.005-ml pipette tip was placed in the hole of the egg between 3 and 5 mm, and the plunger was depressed to deliver the entire dose at once (Fig. 1). The results are tabulated (Table 1). A 50% lethal dose (LDw) of 1.73 jig of G2 per 0.02 ml per egg of methanol was obtained with the microapplicator, whereas an LD5,, of 1.93 ,ug of G2 per 0.02 ml per egg of methanol was obtained with the micropipette. A probit analysis of the two lines of the microapplicator and micropipette indicates that they are parallel and have approximately the same LD values. The LDN,, value in this study is considerably less than the LD,0 of 2.7 ,ug of G2 per egg in a
FIG. 1. (A) shows components used to modify Oxford micropipette; (B) shows the assembled tip (each box in grid is 0.5 by 0.5 inch [ca. 1.27 by 1.27 cm]).
I Address reprint requests to: H. E. Smalley, Director, VTERL, ARS, USDA, P. 0. Drawer GE, College Station,
Tex. 77840.
621
622
NOTES
TABLE 1. LD values for the microapplicator and micropipette in micrograms per 0.02 ml per egg Method LD30 LDa LD70 LD,O LD95 Microapplicator 1.15 1.73 2.62 5.07 7.30 1.24 1.93 2.98 5.98 8.80 Micropipette
APPL. ENVIRON. MICROBIOL.
much as 70% faster than the microapplicator method. The microapplicator method has several disadvantages in that each syringe must be calibrated and the method is relatively slow and tedious. LITERATURE CITED
previous study in which a syringe was used (2). In both methods studied, 4.0 ,ug of G2 per 0.02 ml per egg of methanol proved to be 85% lethal to the chicken embryos, and 5.0 g.g of G2 per 0.02 ml per egg of methanol proved to be 100% lethal. The micropipette appears to be the better instrument. It gives the ease and speed of a syringe, with much greater accuracy. It is as
1. Association of Official Analytical Chemists. 1970. Official methods of analysis, 11th ed. p. 436-438. Association of Official Analytical Chemists, Washington,
D.C. 2. Kraybill, H. F. 1969. The toxicology and epidemiology of mycotoxins. Trop. Geogr. Med. 21:1-18. 3. McLaughlin, J., Jr., J.-P. Marliac, J. M. Verret, M. K. Mutchler, and 0. G. Fitzhugh. 1963. The injection of chemicals into the yolk sac of fertile eggs prior to incubation as a toxicity test. Toxicol. Appl. Pharmacol. 5:760-771.
Vol. 31, No. 4 Printed in U.SA.
Microapplicator and Micropipette for Inoculation of the Embryonated Chicken Egg' WILLIAM C. STEARMAN III, JAMES 0. NORMAN, AND H. DEL VAR PETERSEN Veterinary Toxicology and Entomology Research Laboratory and Southern Region, Agricultural Research Service, College Station, Texas 77840 Received for publication 20 November 1975
Aflatoxin G, was used to test the speed and accuracy of the microapplicator and the micropipette. The 50% lethal dose of both assay systems was approximately the same, and 4.0 gg of G2 had an 85% lethal effect in both systems. The 50% lethal dose of the microapplicator and the micropipette was lower than that of the syringe but, of these, only the micropipette can combine the accuracy of the microapplicator and the speed of the syringe. The Association of Official Analytical Chemists has adopted the procedure of using the fertile chicken egg in the bioassay of aflatoxins (1). In using the embryonating chicken egg as an assay system, we needed a more precise method of inoculation, as opposed to the syringe now used. Aflatoxin G,, obtained from Calbiochem, Los Angeles, Calif., was easily soluble in methanol at a concentration of 1 mg of G2 per 1 ml of methanol. Methanol is relatively nontoxic to the chicken embryo at levels less than 0.05 ml (3). Two methods were studied: (i) using a microapplicator (Instrumentation Specialties Co., Lincoln, Neb.; model M) and (ii) using a micropipette (Oxford sampler micropipetting system, Fisher Scientific Co., Pittsburgh, Pa.). The microapplicator was used with a 0.5-ml syringe and a 22-gauge needle and was calibrated to deliver 0.02 ml with five depressions of the pedal switch when set on approximately 4.20 s. The needle was bent 900, so about 25 mm extended downward after the sharp point had been removed by filing. The fertile white leghorn eggs selected for the test weighed between 52 and 63 g at the onset of incubation and had been incubated for 5 days before inoculation. An egg punch was used to make a hole. Then the egg was brought -ap to the needle, and the needle was allowed to enter the hole and descend downward until the tip of the needle just touched the air cell membrane, but care was taken not to rupture the membrane. One drop of sample was delivered per two depressions of the pedal switch; thus, the needle tip had to be in contact with the membrane. In this manner, the entire dose was delivered to the fertile chicken egg.
A 0.02-ml Oxford micropipette was used, with its standard disposable tip cut approximately at the end of the micropipette; a 23gauge needle was placed on the shortened, disposable pipette tip. A standard 0.005-ml disposable pipette tip was placed over about 5 mm of the 23-gauge needle. The pipette system was then tested for delivery of 0.02 ml of sample. The standard 0.005-ml pipette tip was placed in the hole of the egg between 3 and 5 mm, and the plunger was depressed to deliver the entire dose at once (Fig. 1). The results are tabulated (Table 1). A 50% lethal dose (LDw) of 1.73 jig of G2 per 0.02 ml per egg of methanol was obtained with the microapplicator, whereas an LD5,, of 1.93 ,ug of G2 per 0.02 ml per egg of methanol was obtained with the micropipette. A probit analysis of the two lines of the microapplicator and micropipette indicates that they are parallel and have approximately the same LD values. The LDN,, value in this study is considerably less than the LD,0 of 2.7 ,ug of G2 per egg in a
FIG. 1. (A) shows components used to modify Oxford micropipette; (B) shows the assembled tip (each box in grid is 0.5 by 0.5 inch [ca. 1.27 by 1.27 cm]).
I Address reprint requests to: H. E. Smalley, Director, VTERL, ARS, USDA, P. 0. Drawer GE, College Station,
Tex. 77840.
621
622
NOTES
TABLE 1. LD values for the microapplicator and micropipette in micrograms per 0.02 ml per egg Method LD30 LDa LD70 LD,O LD95 Microapplicator 1.15 1.73 2.62 5.07 7.30 1.24 1.93 2.98 5.98 8.80 Micropipette
APPL. ENVIRON. MICROBIOL.
much as 70% faster than the microapplicator method. The microapplicator method has several disadvantages in that each syringe must be calibrated and the method is relatively slow and tedious. LITERATURE CITED
previous study in which a syringe was used (2). In both methods studied, 4.0 ,ug of G2 per 0.02 ml per egg of methanol proved to be 85% lethal to the chicken embryos, and 5.0 g.g of G2 per 0.02 ml per egg of methanol proved to be 100% lethal. The micropipette appears to be the better instrument. It gives the ease and speed of a syringe, with much greater accuracy. It is as
1. Association of Official Analytical Chemists. 1970. Official methods of analysis, 11th ed. p. 436-438. Association of Official Analytical Chemists, Washington,
D.C. 2. Kraybill, H. F. 1969. The toxicology and epidemiology of mycotoxins. Trop. Geogr. Med. 21:1-18. 3. McLaughlin, J., Jr., J.-P. Marliac, J. M. Verret, M. K. Mutchler, and 0. G. Fitzhugh. 1963. The injection of chemicals into the yolk sac of fertile eggs prior to incubation as a toxicity test. Toxicol. Appl. Pharmacol. 5:760-771.
