78 



access of oxygen to the point desired being kept in mind. At inter- 

 vals, free hand sections should be examined under the microscope to 

 determine the state of the reaction. If the tissue is mammalian, it 

 should be kept protected from evaporation during this time and 

 warm, as in an incubator. 



If the stain was applied by immersion, a shorter time suffices; 

 up to 15 minutes or so with a subsequent exposure to the air of l /2 

 to 1 hour wet with the dilute solution. 



Small aquatic animals may be immersed in very dilute solu- 

 tions (1/100 to 1/1,000%), the optimum strength and time of im- 

 mersion being experimentally determined. 



In some instances (particularly parasitic worms) the tissue 

 hold the methylene blue in reduced form in spite of exposure to the 

 air and the color is only developed when placed in the fixer (below). 

 In any event it is better to fix the stain earlier than later. 



4. Two methods are standard for preserving tissue stained 

 intra vitam with methylene blue, the first of these (Dogiel's) is suit- 

 able only for such tissue as may be exposed for study by teasing; 

 the second (Bethe's) may be used both for such preparations and 

 those which it is desired to imbed and section. 



Dogiel's Method. Immerse the tissue in a saturated solution of 

 ammonium picrate (orange-yellow needles) for 2 to 24 hours according 

 to the size of the piece, using abundance of fluid. If maceration 

 occurs, Dogiel suggests addition of 1% of 1% osmic acid. Transfer 

 tissue to equal parts of the ammonium picrate solution and glycerin in 

 which the tissue may be preserved, teased, and mounted. 



Bethe's Method. Immerse the tissue in a 5 to 10%aqueous solu- 

 tion of ammonium molybdate for 1 to 24 hours according to the size of 

 the piece, using abundance of the fluid. Trim the tissue as desired, 

 removing all unnecessary parts, dividing it into smaller pieces, etc. 

 Wash in distilled water, changed several times, for 1 to 3 hours. 

 Dehydrate rapidly in 95% and absolute alcohol, 4 to 6 hours, 

 shortening the time if possible. Imbed rapidly in celloidin ( 206) 

 which may be hardened in 67% alcohol and sections cut. 



It may be advisable, particularly in summer work, to have the 

 water and alcohol specially cooled to prevent dissolving of the stain 

 in the alcohol. Tissue already fixed in the ammonium picrate may 

 be refixed in the ammonium molybdate solution. Indeed, this 

 double fixation is recommended by Bethe as particularly suitable for 

 invertebrate material. 



