364 Journal of Applied Microscopy. 



agent, but is rather expensive. It causes but little shrinking of the protoplasm 

 and is a time saver if material is to be imbedded in paraffin. With 95 per cent., 

 or with absolute alcohol, objects are generally left in the reagent until needed for 

 use, but such material becomes very brittle. The addition of glycerine is an 

 improvement if material is to be kept long. Acetic acid has been used with 

 alcohols to counteract the tendency to shrink. One of the most successful of the 

 alcohol combinations is 



c. Canioys Fluid. — Absolute alcohol, 6 parts ; chloroform, 3 parts ; glacial 

 acetic acid, 1 part. The penetration of the reagent is excellent, and only a few 

 hours are needed for fixing. Material should be washed in absolute alcohol 

 (perhaps 95 per cent, alcohol would do no harm) until there is no odor of acetic 

 acid. This should not require more than one or two hours. It is better to 

 imbed in paraffin at once, but when this is not convenient the material may be 

 transferred to 85 per cent, alcohol and then to 70 per cent., where it may be left 

 until needed. Cyanin and erythrosin, fuchsin and iodine green, and similar 

 combinations give particularly brilliant staining after this reagent. 



THE CHROMIC ACID GROUP. 



Chromic acid, or solutions with chromic acid as a foundation, are the most 

 generally useful killing and fixing agents yet known to the botanist. A 1 per 

 cent, solution of chromic acid in water gives good results, but it is better to use 

 the chromic in connection with other ingredients, such as acetic acid, formic 

 acid, osmic acid, etc. The proportions of the various ingredients must, for the 

 present at least, be determined by experiment. With favorable objects like fern 

 prothallia, spirogyra, and other things which can be watched while the fixing is 

 taking place, suitable proportions are rather easily determined because specimens, 

 after being placed in the reagent, may be examined at frequent intervals, and 

 combinations which cause plasmolysis may be rejected and different proportions 

 tried until satisfactory results are secured. For example, fern prothallia might 

 be placed in the following solution: chromic acid, '1 gr.; acetic acid, 1 cc; and 

 water 97 cc. If plasmolysis takes place, weaken the chromic or strengthen the 

 acetic, since the chromic has a tendency to produce contraction, and the acetic 

 to cause swelling. Too large a proportion of acetic acid, however, may cause 

 distortion, and hence it would be better to weaken the chromic. In case of fern 

 prothallia, 3 parts chromic, 1 part acetic, and 390 parts water will cause practi- 

 cally no plasmolysis, and the fixing is sufficiently thorough to permit imbedding 

 in paraffin. A combination may be quite satisfactory for fern prothallia and 

 still fail to give good results with spirogyra. For very critical work the most 

 favorable proportions must be determined for the particular plant under investi- 

 gation. When the effect of the reagent cannot be observed directly, it is well to 

 make a free-hand section and thus determine whether plasmolysis is taking place. 

 It is not safe to judge the action of a fixing agent by the appearance of sections 

 cut from material which has been imbedded in paraffin, because shrinking of 

 the cell contents often takes place during the transfer from absolute alcohol to 

 the clearing agent or during infiltration with paraffin, and sometimes during even 

 later processes. When in doubt as to proportions we would suggest 2 cc. 



