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Prevention of cracking of wells in the immunodiffusion agar-plate
BRIEF
NOTES
521
Prevention of cracking of wells in the immunodiffusion
agar-plate
Immunodiffusion tests by means of double diffusion’, immunoelectrophoresis2 and single-radial diffusion3 techniques in agar gel have been widely used in the characterization and identification of antigens and antibodies. The test is based upon the interaction between an antigen and its corresponding antibody, if present, and after several hours or days the subsequent formation of visible immunoprecipitin lines in the agar gel between the wells and troughs where the antigen and antibody were administered. These precipitin lines are visible while the concentration of both reactants is optional or close to that point. However, the precipitin lines are rather faint and cannot be seen distinctly upon macroscopic examination or be photographed directly. Drying the agar plate and staining the reaction more readily distinguishable.
with an appropriate
dye solution makes
After completion of immunoprecipitin reactions in agar, the agar plates are immersed in a large amount of physiological saline or distilled water. The saline or water is changed twice a day and the final washing is carried out overnight with water. In drying the agar plate, an appropriate thickness of filter paper is placed over the surface of the agar and the covered plate is allowed to dry at room temperature. The drying procedure varies depending upon whether rapid or slow drying is desired. For rapid drying, the plate is placed before a hair dryer or under a desk light with a regular electric light bulb. Unfortunately, in many instances the dried agar plate shows cracks around the edges of the wells and sometimes in the troughs. These cracks often occur in small Ouchterlony’s agar plates and intersect the precipitin line area, thereby confusing the reaction pattern. Recently, a technique was found to prevent the formation of cracks during the drying procedure. The filter paper is applied as usual after maximum precipitin reaction has taken place. Using a sharp knife or syringe needle a small hole is made in the wet filter paper at the center of each well or in certain places of the troughs. Holding the plate over a light source facilitates the positioning of the holes in the wells and troughs. It is not necessary for the paper to be cut to the exact size of the wells, a small opening just enough to allow a flow of air is sufficient. Or, if desired, the washed plate can be immersed in clean water and all air bubbles removed by gently shaking the plate. When no bubbles are observed in the wells and troughs, a filter paper is also immersed and the plate covered while in water. Following these procedures, cracking of the agar plate is avoided and preservation of the reaction pattern is assured. This improvement in the agar immunodiffusion tests, although a minor one, is important in the characterization of immunoprecipitin reactions. After the plates are dried the filter paper is carefully removed. If pieces of paper remain on the plate they can be removed by using tap water and rubbing a finger over the surface of the dried agar, or performing this procedure in water. The dried and clean agar plates are stained by appropriate staining dye and destained with 2 to 3”,/, acetic acid solution. The plates are dried at room temperature while held vertically or at a 45” angle. For simple photographic preparation of the immunoprecipitin lines, Contact C&n. C/‘&z.Acta,
21 (1968)
521-522
BRIEF NOTES
522 Printer, size 4 x 5 inches, Yankee @ Model P-45, Photo Calif., can be used. This small photographic instrument
Products,
Inc., Los Angeles,
provides direct contact
with
photographic paper, Velox-Fg, Eastman Kodak Co., Rochester, N.Y. The exposure time can be set for I/Z to I min. After exposure, the paper is immersed in a solution of Dektol Developer, Kodak, for I to 2 min and then fixed with Kodak Fixer for over IO min. The paper is then washed in tap water and dried in an Electric Print Dryer,
Flipper, Model F-I~C, Arkay’s Corporation, Milwaukee, Wise. This study was supported by the American Cancer Society, Grant
number
E-425. Kuakini
Medical Research Institute,
347 North Kuakini Street, Ho~aolulu, Hawaii 96817 (U.S.A.) I 0. OUCHTERLONY, Pvogr. Allergy, 5 (1948) I. 2 J. J.SCHEIDEGGER, Intern. Arch. AZZergy A@. Inwnz~noZ., 7 (rgjj) 3 J. L. FAHEY AND E. RI. MCKELVEY, J. Iwmunol., 04 (1965) 81.
Received
May 13, 1968
Clin.Chim. Acta,21 (1968)521-522
TAKAKI YAMAMOTO NOBUYA HASHIMOTO MITSUO YOKOYAlLlA
103.
NOTES
521
Prevention of cracking of wells in the immunodiffusion
agar-plate
Immunodiffusion tests by means of double diffusion’, immunoelectrophoresis2 and single-radial diffusion3 techniques in agar gel have been widely used in the characterization and identification of antigens and antibodies. The test is based upon the interaction between an antigen and its corresponding antibody, if present, and after several hours or days the subsequent formation of visible immunoprecipitin lines in the agar gel between the wells and troughs where the antigen and antibody were administered. These precipitin lines are visible while the concentration of both reactants is optional or close to that point. However, the precipitin lines are rather faint and cannot be seen distinctly upon macroscopic examination or be photographed directly. Drying the agar plate and staining the reaction more readily distinguishable.
with an appropriate
dye solution makes
After completion of immunoprecipitin reactions in agar, the agar plates are immersed in a large amount of physiological saline or distilled water. The saline or water is changed twice a day and the final washing is carried out overnight with water. In drying the agar plate, an appropriate thickness of filter paper is placed over the surface of the agar and the covered plate is allowed to dry at room temperature. The drying procedure varies depending upon whether rapid or slow drying is desired. For rapid drying, the plate is placed before a hair dryer or under a desk light with a regular electric light bulb. Unfortunately, in many instances the dried agar plate shows cracks around the edges of the wells and sometimes in the troughs. These cracks often occur in small Ouchterlony’s agar plates and intersect the precipitin line area, thereby confusing the reaction pattern. Recently, a technique was found to prevent the formation of cracks during the drying procedure. The filter paper is applied as usual after maximum precipitin reaction has taken place. Using a sharp knife or syringe needle a small hole is made in the wet filter paper at the center of each well or in certain places of the troughs. Holding the plate over a light source facilitates the positioning of the holes in the wells and troughs. It is not necessary for the paper to be cut to the exact size of the wells, a small opening just enough to allow a flow of air is sufficient. Or, if desired, the washed plate can be immersed in clean water and all air bubbles removed by gently shaking the plate. When no bubbles are observed in the wells and troughs, a filter paper is also immersed and the plate covered while in water. Following these procedures, cracking of the agar plate is avoided and preservation of the reaction pattern is assured. This improvement in the agar immunodiffusion tests, although a minor one, is important in the characterization of immunoprecipitin reactions. After the plates are dried the filter paper is carefully removed. If pieces of paper remain on the plate they can be removed by using tap water and rubbing a finger over the surface of the dried agar, or performing this procedure in water. The dried and clean agar plates are stained by appropriate staining dye and destained with 2 to 3”,/, acetic acid solution. The plates are dried at room temperature while held vertically or at a 45” angle. For simple photographic preparation of the immunoprecipitin lines, Contact C&n. C/‘&z.Acta,
21 (1968)
521-522
BRIEF NOTES
522 Printer, size 4 x 5 inches, Yankee @ Model P-45, Photo Calif., can be used. This small photographic instrument
Products,
Inc., Los Angeles,
provides direct contact
with
photographic paper, Velox-Fg, Eastman Kodak Co., Rochester, N.Y. The exposure time can be set for I/Z to I min. After exposure, the paper is immersed in a solution of Dektol Developer, Kodak, for I to 2 min and then fixed with Kodak Fixer for over IO min. The paper is then washed in tap water and dried in an Electric Print Dryer,
Flipper, Model F-I~C, Arkay’s Corporation, Milwaukee, Wise. This study was supported by the American Cancer Society, Grant
number
E-425. Kuakini
Medical Research Institute,
347 North Kuakini Street, Ho~aolulu, Hawaii 96817 (U.S.A.) I 0. OUCHTERLONY, Pvogr. Allergy, 5 (1948) I. 2 J. J.SCHEIDEGGER, Intern. Arch. AZZergy A@. Inwnz~noZ., 7 (rgjj) 3 J. L. FAHEY AND E. RI. MCKELVEY, J. Iwmunol., 04 (1965) 81.
Received
May 13, 1968
Clin.Chim. Acta,21 (1968)521-522
TAKAKI YAMAMOTO NOBUYA HASHIMOTO MITSUO YOKOYAlLlA
103.