30 METHODS IN PLANT HISTOLOGY 



A picric-acid combination which has gained some popularity for 

 cytological work is 



Bouin's fluid. — 



Formalin (commercial) 25 c.c. 



Picric acid (saturated solution in water) 75 c.c. 



Glacial acetic acid 5 c.c. 



Fix about 24 hours. Rinse in water for a few minutes to remove the 

 more supei-ficial picric acid, and then complete the washing in 35 per 

 cent or 50 per cent alcohol. There is likely to be some swelling, but 

 spindles of mitotic figures stain well. The formula has given good re- 

 sults with early stages in the female gametophyte of Pinus and would 

 be worth a trial with the embryo sacs of angiosperms. 



CORROSIVE SUBLIMATE 



Corrosive sublimate, or bichloride of mercury, is soluble in water 

 and in alcohol. About 5 g. will make a saturated solution in 100 c.c. of 

 water. It is very much more soluble in alcohol, but for practical pur- 

 poses 5 g. in 100 c.c. of 50 per cent alcohol may be regarded as a 

 favorable solution. Corrosive sublimate used alone does not give as 

 good results as when mixed with acetic acid, chloroform, or picric acid. 

 Fixing is very rapid, the material being fixed almost as soon as it is 

 penetrated by the fluid. Material which is at all transparent, like some 

 ovules and the endosperm of gymnosperms before the formation of 

 starch, becomes opaque as soon as fixed, and so the time needed for 

 fixing is easily determined. From 10 minutes to 30 minutes should be 

 sufficient for onion root-tips or lily ovaries. Smaller or larger objects 

 require shorter or longer periods. When used hot (85° C), the fixing 

 is much more rapid. While a few minutes' fixing may be sufficient, we 

 let the reagent reach the boiling-point, then remove the flame, and 

 just as soon as the bubbling ceases, put the material in and leave it 

 until the liquid becomes cool. It may be left for 20 minutes, or even 

 30 minutes, without any damage. 



Wash out aqueous solutions with water and alcoholic solutions with 

 alcohol. In either case, the washing must be very thorough, since 

 preparations from incompletely washed material are sure to be dis- 

 figured by crystals of corrosive sublimate. After material fixed in the 

 aqueous solution has been washed in water for an hour, add a little of 

 the iodine solution used in testing for starch. The liquid will turn 

 brownish or amber-colored, and then clear up; add a little more, until 

 the liquid fails to clear up completely, a very slight amber remaining 



