Carmine-stained Pectinatella 181 



alcohol. Naturally, they will float, but as soon as they have sunk to the 

 bottom it may be presumed that they are sufficiently rehydrated. Either 

 the cloth-ended tube containing them may be transferred to the dish of 

 stain or the 70 per cent alcohol may be poured out of this tube and stain 

 substituted for it. Two of the advantages of this stain are that it is rela- 

 tively rapid in action— very few specimens will not be stained adequately 

 in 5 to 10 min— and it does not matter how long the materials remain in 

 it. It is, therefore, often convenient to leave the specimens in stain over- 

 night and to start differentiation the next morning. Either they are re- 

 moved to differentiating solution or, alternatively, the stain is poured off 

 and the differentiating solution substituted for it. In the latter case three or 

 four changes will be required, owing to the necessity of leaving some stain 

 in the bottom of the tube to avoid pouring the specimens out with it. 

 Indeed, unless the operator is experienced, it is safer to shake the tube, 

 so as to distribute the specimens thoroughly in the stain, and then to tip 

 everything into a large finger bowl of differentiating solution from which 

 the specimens may be picked out later and transferred to a new tube of 

 differentiator. It is tragically easy, in pouring off the stain, to pour speci- 

 mens with it down the sink. As soon as the stain has been washed off with 

 the differentiating solution, a single specimen should be transferred to a 

 watch glass and examined under a low power of the microscope. It is 

 more than probable that little differentiation will be required, so a simple 

 rinse may be adequate. It is difficult to judge the exact degree of differ- 

 entiation required, and it must be remembered that the object will ap- 

 pear darker after clearing than it does in the differentiating solution. The 

 internal organs should be sharply demarcated when the outer surfaces of 

 the specimen are relatively free from stain. This may be judged in Pectina- 

 tella by placing a coverslip on the specimen and examining one of the 

 branches of the lophophore under the high power of the microscope. 

 Differentiation may be considered complete when only the nuclei in the 

 cells of the lophophore are stained. The specimens are washed in four or 

 five changes of 70 per cent alcohol to remove the acid before they are 

 placed for at least a day in 96 per cent alcohol as the first stage of dehy- 

 dration. Next they should be transferred to two changes of at least 6 hr 

 each in a considerable volume of fresh 96 per cent alcohol and cleared. 

 Absolute alcohol is not necessary if one is using terpineol as a clearing 

 agent. 



There is some danger, if specimens are transferred directly from 96 

 per cent alcohol to a fluid as viscous as terpineol, that they will become 

 distorted by the violent diffusion currents. This may be readily avoided 

 in the following manner. A fairly wide (about 1 in.) glass vial is filled 

 about half full with terpineol. Ninety-six per cent alcohol is poured very 

 carefully down the side of the vial in order to float a layer of alcohol on 

 top of the terpineol. The specimens are now dropped into the alcohol. 



