DS 11.20 



DYE STAINS OF GENERAL APPLICATION 



297 



in which the medusae are swimming. The 

 rotary movement of the left hand should 

 on no account be discontinued until at 

 least three or four minutes have passed 

 after the addition of the fixative. A safe 

 criterion is that the endodermal canals 

 shall have become opaque through the 

 action of the fixative before the rotary- 

 movement is discontinued. The medusae 

 will be found to settle to the bottom of the 

 bowl in about the same period of time as 

 it takes the mounter to resecure the use of 

 liis left arm. The medusae may then be 

 removed, one at a time with a glass spoon, 

 to a fresh large volume of the fixative in 

 which they should remain overnight, 

 though a period of two or three days in this 

 fixative, will not damage them. 



There now commences, between this 

 time and the time of the final mounting 

 of the medusa in balsam, a continual 

 struggle against the collapse of the delicate 

 mesoglea with the subsequent wrecking of 

 the mount. This collapse is produced by 

 osmotic pressure, and it is sufficient to re- 

 move the medusae to distilled water to 

 ensure spoihng them. It is therefore neces- 

 sary to transfer the medusae first to about 

 a gallon of clean, filtered, sea water, to 

 commence the process of washing, and 

 then, through a quite finely drawn glass 

 tube connected with the fresh water tap, to 

 permit the sea water slowly to be replaced 

 by fresh water. 



It is now necessary to rig up some form 

 of drip mechanism by which the water in 

 which the medusae are resting may be re- 

 placed with increasing strengths of alco- 

 hol by a slow and continuous process. The 

 writer himself uses a 50 milhhter wide- 

 mouth bottle standing upon his bench 

 with a liter aspirator bottle standing on 

 the shelf above. A rubber tube from the 

 aspirator bottle passes to an ordinary 

 pipet inserted through a hole in the cork 

 of the bottle, from which comes a second 

 drainage tube leading to the waste. A 

 screw-controlled pinchcock in the middle 

 of the rubber tube leading from the aspi- 

 rator bottle permits a very fine control of 

 the flow of alcohol which is estabhshed, 

 of course, before the cork is inserted into 

 the 50 milhhter bottle. For replacing, in 

 the first stage, the water used for wasliing 



\\'ith 15% alcohol, a flow of 50 or 60 drops 

 a minute will be found perfectly safe. The 

 flow should be permitted to continue until 

 the whole hter of 15% alcohol has passed 

 out of the aspirator bottle and through the 

 50-milhliter bottle in which are the 

 specimens. 



For the type of process which is now 

 being described it is better to utilize 

 Grenadier's dry powder, the method of 

 preparation for which is given in the ab- 

 breviated formula below (11.21 Grenacher 

 1879), than it is to endeavor to use the 

 prepared solution in 70% alcohol. If this 

 powder is obtainable, or can be made, it is 

 recommended that about 100 millihters 

 of a 2% solution be prepared in 30% al- 

 cohol and run through the aspirator bottle 

 into the specimen bottle in the manner in 

 which the alcohol has already been used. 

 This stain may be permitted to remain in 

 contact with the specimens overnight or 

 for whatever period of time is convenient 

 to the operator. The aspirator bottle is 

 now again filled with a hter of 30% alco- 

 hol to wliich has been added approxi- 

 mately one milhhter of hydrochloric acid. 

 This acid alcohol is now used to flush out 

 the stain from the specimen bottle. Unless 

 a relatively pale pink solution alone re- 

 mains after the passage of the acid alcohol, 

 a further hter will have to be run through. 

 The specimens may be left to differentiate 

 in acid alcohol until inspection with the 

 naked eye shows the mesoglea to be prac- 

 tically free of stain. The internal organs, 

 however, should remain a rather bright 

 pink. It must not be forgotten that the 

 apparent degree of staining will increase 

 greatly when the specimens are cleared. 



The process of dehydration should now 

 be completed using successive liter por- 

 tions of 60% alcohol, 80% alcohol, and 

 95 % alcohol run through by the slow flow 

 method already described. To complete 

 the process of dehydration the author 

 prefers, on a cost basis, to use acetone in 

 place of absolute alcohol, though there is, 

 of course, no reason why alcohol should 

 not be employed. However, it usually is 

 necessary to run through at least two 

 liters of the last dehydrating agent in 

 order to be quite certain that the speci- 

 mens are perfectly dehydrated. 



