New Method of Examining Normal and Diseased Tissues. 147 



ill effect on the animal, no more than - 5 e.c. of the same solution should 

 be used for intravascular work. In the latter case coloration sets in speedily, 

 increases up to the second day after injection, but j.a,pidly fades after the 

 fourth day, in any case quicker than when gradual absorption of the staining 

 fluid takes place through the lymphatic channels. 



As for isamin-blue, the maximum dose for intravascular injection should 

 not exceed 03 of a 1-per-cent. solution by a single drop, if its general toxic 

 effect on the animal or the danger of widely-spread thrombosis is to be 

 avoided. Where distinct and delicate granule staining is required the 

 preference should be given to isamin-blue. 



It is undoubtedly safest and best for histological study^to inject the 

 staining fluids subcutaneously. Injections of 1 c.c. of a 1-per-cent. solution 

 per 20 grm. of the animal's body-weight may be repeated many times once 

 a week. In some cases I have gone up to 15 injections, withouttdoing any 

 harm to the animal. Naturally, the vital coloration then becomes most 

 intense and histological details very distinct. For bigger animals, such as 

 the rabbit, dog, and ape, where larger and more frequent doses of the 

 staining fluid are needed, intraperitoneal injections are preferable to 

 subcutaneous, the dose being determined by the animal's weight. It is 

 safe, as far as isamin- and trypan-blue are concerned, to abide by the 

 standard, 1 c.c. of a 1-per-cent. solution per 20 grm. of the animal's weigh 



Both isamin- and trypan-blue allow of fixation by means of a 10-per-cent. 

 formalin solution (best applied from the beating heart of the anaesthetised 

 animal), but it is only in tissues stained by trypan-blue that the ordinary 

 processes of histological technique are applicable. Alcohol extracts isamin- 

 blue also after lengthy fixation of tissues in formalin solution. But even 

 after vital staining with trypan-blue it is advisable to carry along a small 

 quantity of formalin in the dehydrating fluids. When these precautions 

 are taken, durable histological specimens of vitally stained preparations 

 may be obtained, which recall in every detail the conditions prevalent in life. 



As a rule I use sections cut by the freezing microtome from specimens 

 that have been fixed in a 10-per-cent. formalin solution for not less than 

 48 hours. The specimens are then transferred to a slide and allowed to 

 dry until the section appears firmly fixed to the slide. Then the process 

 of counter-staining, dehydration and mounting is rapidly performed. As 

 to counter-staining, I have recently determined that, besides the alum- 

 carmine stain I first advised, other methods likewise yield excellent results. 



For studies of connective tissue reactions it is well to combine the vital 

 stain with Pappenheim-Unna's pyrronin methyl-green solution, inasmuch 

 as this combination permits of differentiating between the vitally-stained 



L 2 



