332 Journal of Applied Microscopy. 



METHODS IN PLANT HISTOLOGY. 



Charles J. Chamberlain. 



II. 



THE GENERAL METHOD. 



We shall now consider the routnie of mounting an object in Canada balsam. 

 While the outline refers more particularly to the paraffine method, the principles 

 are general in their application and must be mastered by every one who desires 

 to make tirst-class preparations. Several of the topics, like KiUing and Fixing, 

 Staining, etc., will be treated in detail when considering the various reagents. 



1. Killing and I^ixiug. — Usually the same reagent is used for both killing 

 and fixing. The purpose of a killing agent is to bring the life processes to a 

 sudden termination, while a fixing agent is used to fix the cells and their contents 

 in as nearly the living condition as possible. The fixing consists in so hardening 

 the material that the various elements may retain their natural condition during 

 all the processes which are to follow. This step is one of extreme importance. 

 Take your killing and fixing fluids with you into the field. If you wait until you 

 bring your material to the laboratory you may do some fixing, but it will, in 

 many cases, be too late to do much killing. Always have the material in very 

 small pieces in order that the reagents may act quickly on all parts of the speci- 

 mens. Pieces larger than one-fourth inch cubes should be avoided whenever 

 possible. For very fine work no part of the specimen should require the reagent 

 to penetrate more than one sixteenth of an inch. In general, the volume of the 

 reagent should be ten to twenty times that of the material. The time required 

 for this process varies with the reagent, the character of the tissue, and the size 

 of the piece. About twenty-four hours is a safe period for chromic acid solutions. 

 While a shorter period might sufiice in some cases, even a longer period would 

 probably do no harm. 



2. Washing. — Nearly all fixing agents, except the alcohols, must be washed 

 out from the material as completely as possible before any further steps are 

 taken, because some reagents leave annoying precipitates which must be removed 

 and others interfere with subsequent processes. Aqueous fixing agents with 

 chromic acid as their principal ingredient are washed out with water ; aqueous 

 solutions of corrosive sublimate are also washed out with water, but alcoholic 

 solutions should be washed out with alcohol of about the same strength as the 

 fixing agent ; picric acid, or fixing agents with picric acid as an ingredient, must 

 not be washed out with water, but with alcohol, whether the picric acid be in 

 aqueous or alcoholic solution. Running water is best, and where this is not 

 convenient the water should at least be changed quite frequently. The washing- 

 out process usually takes from twelve to twenty-four hours, but it can be shortened 

 about one-half by keeping the fluid lukewarm. 



3. Hardening and Dehydrating.— Aitex the material has been washed, it is 

 necessary to continue the hardening and also to remove the water. Alcohol 

 is used almost entirely for these purposes. It completes the hardening and 



