74 BOTANICAL GAZETTE ^ [January 



impaired by the presence of large masses of free spores, which 

 retain their staining properties for some time after maturing. Later 

 the spore walls seem to become entirely impervious to stains, and as 

 a result when the secondary mycelium develops beyond a slight 

 clouding effect no difficulty is encountered from this source. The 

 best results are obtained when the print is made soon after the 

 mycelium begins to adhere readily to the smeared slide. 



The nature of the killing agent employed was found to have 

 no noticeable effect on the preparation. Flemming's mixtures, 

 both weaker and stronger, picro-formol, picro-acetic, Carnoy's 

 fluid, and 95 per cent alcohol were tried with apparently the same 

 results. Owing to the small diameter of the filaments, the pene- 

 tration is probably so nearly instantaneous that plasmolysis is 

 effectively prevented by nearly any toxic agent capable of readily 

 wetting the material. In order to obviate the necessity of washing, 

 strong alcohol was used almost exclusively. 



Much more depends on the proper choice of a stain. Saffranin, 

 gentian violet, Bismarck brown, and eosin usually fail to bring about 

 a sufficiently deep coloration. Carbol-fuchsin acts powerfully 

 and rapidly, but is poor for purposes of differentiation. Haiden- 

 hain's iron-alum haematoxylin is good for protoplasmic structures. 

 The most satisfactory results were obtained with Delafield's 

 haematoxylin, which if allowed to act for 24 hours, with the proper 

 degree of decolorization, yields deeply stained, clear preparations, 

 showing vacuoles, metachromatic, and nuclear structures, as well 

 as septa, with remarkable distinctness. 



The spores of all species of Actinomyces are developed by a 

 transformation of more or less specialized hyphal branches dis- 

 tinguishable from the sterile hyphae of the aerial mycelium at an 

 early stage in their development. In general, with the exception 

 of such inflated hyphae as are shown in figs. 47, 48, and 106, the 

 diameter of any portion of sterile mycelium is attained at the time 

 it arises through the elongation of the growing filament tip, subse- 

 quent increase in thickness being very slight. The sporogenous 

 branches, however, are in the beginning conspicuously thinner than 

 the axial hyphae from which they are derived. Later, when their 

 final linear extension has been nearly attained, increase in thickness 



