CARMINE-STAINED PECTINATELLA 87 



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 relatively rapid in 

 action — very few specimens will not be stained adequately in 5 to 10 minutes 

 —and it does not matter how long the materials remain in it. It is, therefore, 

 often convenient to leave the specimens in stain overnight and to start differ- 

 entiation the next morning. Either they are removed to differentiating solution 

 or, alternatively, the stain is poured off and the differentiating solution substi- 

 tuted 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 quite 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 specimens 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 that a simple 

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

 tion required, and it must be remembered that the object will appear 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 Pectinatella by placing a 

 coverslip on the specimen and examining one of the branches of the lopho- 

 phore under the high power of the microscope. Differentiation may be con- 

 sidered 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 dehydration. Next they should be transferred 

 to two changes of at least six hours 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 

 through the violent diffusion currents. This may be readily avoided in the 

 following manner. A fairly wide (about an inch) glass vial is filled about half 

 full with terpineol. Ninety-six per cent alcohol is poured very carefijlly down 

 the side of the vial (or on a spoon held in the vial in the manner of a bartender 



