1358 Journal of Applied Microscopy 



for examination they were removed to a dish of running sea water aerated by a 

 pipette nozzle. Specimens thus treated gave uniformly good results, while those 

 in which this process was omitted did not. Whether the former result was due 

 to the revival of the animal or the oxygenation of the tissues, is hard to say. But 

 the physical condition of the animal seems to play an important part. 



The other method successfully used was to inject a fairly strong solution 

 (1 to 4 per cent.) of methylen blue into the ovarian vein of Molgula;. From 

 thence the fluid reached the heart and was pumped to all parts of the body. A 

 small amount of fluid should be used and great care taken not to allow too much 

 of the body fluid to escape through the hole made by the needle. The per- 

 centage of successful staining by this method is small ; but the results are 

 valuable, because of the use of large specimens. The peripheral system seems 

 to come out especially well by this method. Animals which die as a result of the 

 injecting process give a diffuse staining of tissues that is of no neurological 

 value. This diffuse staining can be recognized at once, by the fact that the whole 

 animal stains a pale greenish blue color ; while in a perfectly successful stain the 

 color is deep blue. 



The use of ammonia is advocated by Apathy in bringing out the stain. He 

 believes that in exposure to the air just before examination, the specimen takes 

 up some ammonia from the atmosphere. Molgulai exposed to the fumes of 

 ammonia gave negative results. The same may be said regarding the exposure 

 of stained tissue to the air. It could not be proven that oxygen was a factor in 

 the bringing out of the stain. 



The fixation of the stain for permanent preservation. — Most of the methods 

 used by investigators {I'ide Arnstein, Apathy, Bethe, Dogeil, Huber. Meyer, 

 Peabody, Retzius, and others) agree in the use of picrate ammonia as a fixing 

 agent. Aqueous solutions are usually employed, and the material is allowed to 

 remain in a saturated solution of ammonium picrate from a few minutes to several 

 hours, or even days, according to the size and permeability of the material. The 

 macerating effect of the fluid is avoided in some cases by the addition of one 

 part to 100 of a 1 per cent, osmic acid solution. Such material may then be 

 either mounted permanently in a solution of saturated ammonium picrate and 

 glycerin in equal parts (rvV/f Meyer, Retzius) ; or in chemically pure glycerin 

 without washing out {inde Dogeil) ; or by the somewhat complicated method of 

 Apathy, when, after fixation in ammonium picrate plus a little (;") drops to 100 c. c.) 

 concentrated ammonia, the material is passed through glycerin ; glycerin and 

 gum arable ; and finally mounted in a solution of gum arable, cane sugar, and 

 water, in equal quantities. I may say, in passing, that with tunicates I found 

 the last named method the least useful. 



By far the best of my results were obtained by the following modification of 

 the methods of iJethe and Apathy : 



Cut out the part to be used, and after examination under a fairly high power 

 of the microscope, remove the successfully stained material to a saturated solu- 

 tion of ammonium picrate in sea water. The pieces of tissue are then immediately 

 removed to a slide, or small dish, where they are left for a few minutes (10 to 20, 

 according to the size of piece), in the following solution : 



