--6 Journal of Applied Microscopy. 



riuid as soon as cut. They will usually sink to the bottom after a short time, 

 especially if they are shaken a little from time to time. If much trouble is expe- 

 rienced in having the objects float on the surface of the killing fluid, they may first 

 be immersed for a ver)- brief moment in 95 per cent, alcohol, and immediately after 

 dropped into the killing fluid. This will cause them to sink. The objects must 

 be kept in the killing fluid from twelve to twenty-four hours. The onion root 

 tips should be left at least twelve hours ,' for larger objects, a proportion- 

 ately longer time. 



2.— WASHING. 



After the tissues have been thoroughly killed and fixed, the next thing neces- 

 sary is to wash out the acid. This may be done by pouring off the acid and filling 

 the bottle with water, and changing from time to time. They should be washed 

 in this way from one to four hours, depending on how often the water is changed. 

 A better way, however, is to set up and use the apparatus described as a conve- 

 nient washing apparatus by the writer.* The water used for washing should 

 be rather pure. If this is not the case, distilled water had better be used. 



3.- -DEHYDRATING AND HARDENING. 



The next step is to remove all water from the tissues, and this must be done 

 very gradually or the tissues will shrink and the protoplasmic contents of the cells 

 will be distorted so that the preparations will be worthless. To remove the water 

 successive grades of alcohol are used. During this process the objects may still 

 be kept in the same bottle. The amount of each grade of alcohol should be 

 sufficient to cover the objects well. The various grades of alcohol should be 

 made up and kept in a special set of bottles. It is best not to use the alcohol 

 more than once for this process. Carr)- them through in the following order : 



1. 10 per cent. Alcohol - - 4 hours. 



2. 25 per cent. " - - 4 to 8 " 



3. 35 per cent. - - - 4 to 8 " 



4. 50 per cent. " - - 4 to 8 " 



5. 7<> per cent. •• - - 48 " 



The objects should be hardened in the 70 per cent, alcohol at least two days, 

 and a longer period is generally better. They may be kept in 70 per cent, alco- 

 hol for several months without injury. 



6. 85 per cent. Alcohol - - 12 hours. 



7. 95 per cent. •• - - 4 to 8 •' 



8. 100 per cent. •• - - 4 to 8 " 



As a general rule, it is convenient to make three changes a day, morning, noon, 

 and night, except the 70 per cent. 



4.— CLEARING. 



The objects must now be put into some fluid which will dissolve paraffin. 

 The best reagent for this purpose is chloroform. 



1. Add one-third chloroform to the absolute alcohol. Let stand from four to 

 eight hours. 



* Descri])ti(m will a]ijiear in Feljniary IoI'knai,. 



