246 GUIDANCE IN MAKING PERMANENT PREPARATIONS 



(2) Ethyl alcohol. Animals may be transferred gradually 

 or directly to concentrations of alcohol varying from 1 to 8 

 per cent. 



For finer cytological work it is well to avoid any use of 

 anesthetics. 



Fixing. — This is necessary in order to keep the cells and 

 tissues as nearly as possible in their natural position, shape, 

 and structure, and in order that the protoplasm composing 

 them may be kept in condition to stain satisfactorily. 



In selecting a fixing agent remember: 



1. If the material is highly irritable and contractile, it 

 will have to be killed practically instantly with hot solutions, 

 or be previously narcotized. 



2. If there is much lime, an agent that contains much 

 acid should not be used, as the lime will be dissolved and 

 the bubbles of gas are likely to tear or distort tissues. 



3. Where rapid fixation is desirable, as in expanded hy- 

 droids and the like, hot Bouin's picroformol or hot sublimate- 

 acetic is preferable. Where the tissue, or the animal, is not 

 especially muscular, or liable to contraction, any of the fluids 

 may be used. The time that objects should be left in the 

 killing solution varies, approximately, directly as their size. 

 Three minutes will suffice for killing hydroids in Bouin's 

 picroformol or sublimate-acetic. 



Washing. — All objects must be thoroughly washed, after 

 using most killing agents. With most small objects alcohol 

 is preferable, but if the object is large this is too expensive. 

 In general the material used for washing will depend on the 

 fixative employed. See Guyer. 



Dehydrating and Staining. — From water, all objects 

 should be placed successively in 35 per cent, 50 per cent, and 

 70 per cent alcohol, five to fifteen minutes in each for small 

 objects, such as protozoa or individual hydroids. In subse- 

 quent changes from one grade to another allow about the 

 same time. All tissues killed in a corrosive sublimate mix- 

 ture should now be treated with a weak solution of iodine, 



