Journal of Applied Microscopy. 439 



different in its chemical composition from either the cellulose or the lignified 



wall. 



According to Fischer (1897), stains indicate physical but not chemical com- 

 position. Fischer experimented with substances of known chemical composition. 

 Egg albumin was shaken until small granules were secured. These were fixed 

 with the usual fixing agents, and then stained with Delafield's haematoxylin. 

 The extremely small granules stained red, while the larger ones became purple. 

 Since the granules are all alike in chemical composition, Fischer concluded that 

 the difference in staining must be due to physical differences. With safranin, 

 followed by gentian-violet, the larger granules stain red and the smaller violet ; 

 if, however, the gentian-violet be used first, then treated with acid alcohol and 

 followed by safranin, the larger granules take the red and the smaller the gentian- 

 violet. In root tips similar results were obtained. Safranin followed by gentian- 

 violet stained chromosomes red, and spindle fibers violet, while gentian-violet 

 followed by safranin, stained the chromosomes violet, and the spindle red. One 

 often reads that chromosomes owe their strong staining capacity to nuclein, and 

 especially to the phosphorus, but, according to Fischer, this is shown to be 

 unfounded, since albumin gives similar results and yet contains no phosphorus, ■ 

 and is not chemically allied to nuclein. Delafield's haematoxylin is one of the 

 so-called nuclear stains. The nuclei of animals and plants stain deeply with 

 this reagent, but cellulose membranes, the dense protoplasm of embryonic cells, 

 the pyrenoids of green algae, and many other structures resemble nuclei in their 

 staining. The most critical work on this subject has been done by those who- 

 are investigating the structure of the Cyanophyceae and Bacteria, to determine 

 whether these forms have nuclei or not. Biitschli claims that the granules which 

 stain red with haematoxylin are to be identified with chromatin, while Fischer,, 

 whose results have just been given, claims that staining indicates merely physical 

 differences. The subject can not yet be regarded as settled, but whatever may 

 be true in regard to these conflicting theories, all agree that stains are of the 

 highest importance in differentiating structures, and in bringing out details, 

 which would otherwise be invisible. 



PRACTICAL HINTS ON STAINING. 



In later papers specific directions will be given for making a series of 

 preparations ranging from the lowest algae to the flowering plants, but a few 

 suggestions will be made here. 



The number of stains in the catalogues is becoming so great that it is im- 

 possible to become proficient in the use of all of them. It is far better to 

 master a few of the most valuable stains than to do indifferent work with many. 

 The beginner, especially if rather unacquainted with the details of plant 

 structure, may believe that he has an excellent preparation when it is really a 

 bad, or at most an indifferent, one. To illustrate, let us suppose that a pollen- 

 mother-cell in the spirem stage has been stained with cyanin and erythrosin. A 

 preparation in Avhich the cell merely shows a differentiation into nucleus and 

 cytoplasm, must be classed as bad ; if the nucleus shows a definitely outlined 

 spirem thread, the preparation is better, but is still only indifferent ; if the 



