24 



fixation is the quickest, and at the same time is quite satisfactory. 

 After the slide is cooled, or washed, to remove the fixing solutions, 

 it should be placed for a few minutes in a freshly prepared solu- 

 tion (1 per cent) of chlora/^ene, as recommended by Williams, to 

 remove the mucus and proteid material which otherwise blur the 

 field. Other authors (38) have recommended diluting the semen 

 with about twenty volumes of a 0.12 per cent sodium carbonate 

 solution in 0.8 per cent sodium chloride. From this liquid the 

 cells should be centrifuged for several minutes, removed with a 

 pipette, and smeared on the slide. Following this, the slide should 

 be thoroughly washed, preferably for ten minutes in running 

 water, after which it is ready for staining. Numerous methods 

 have been used for this procedure, but the sperms are more or 

 less erratic in their reactions to the dyes, and one must be very 

 careful to use the same method in all samples, in order to obtain 

 uniform results. For quick staining, to bring out gross abnormal- 

 ities of structure, and number of sperms present, one may use 

 Gram's stain, or a light stain with any of the aniline dyes, such 

 a.s fuchsin. To bring out the finer structure, particularly of the 

 head, more careful technic must be employed. 



Carnett and others (38) recommend the following: " The 

 method of staining by iron-hematoxylin, particularly when supple- 

 mented by a cytoplasm stain, has proved, on the whole, the most 

 satisfactory, and possesses the additional advantage of being abso- 

 lutely permanent, a quality that few anilines can boast. The 

 method consisted of treating the fixed object — and here the fixing 

 agent was heat — with a two per cent solution of iron-alum for 

 from two to four hours. The excess of iron-alum was then com- 

 pletely removed 1)y pure water, and the object treated with a solu- 

 tion of hematoxylin (one per cent aqueous) for twelve hours or 

 longer. The cells by this time were perfectly black. However, a 

 1 per cent solution of iron-alum removed the stain from the cyto- 

 pla.sm. leaving the chromatin of the head, the centrosome, and the 

 axial filament a brilliant blue-black. Care must be taken that the 

 preparation is not over-decolorized. After deeolorization a satu- 

 rated aqueous solution of eosin was added for from one to three 

 minutes. This stained the protoplasmic envelope pink, and, unless 

 the envelope is overstained, the view of the inner structures is not 

 impaired in the least." 



AVilliams (17) recommends using two staining solutions, one of 

 alcoholic eosin and fuchsin, the other a diluted methylene blue. 

 The results obtained are, however, more or less erratic, due to 

 the unstable character of the former stain, and the ease with 

 which one may over or under stain. Many beautiful specimens 

 may, nevertheless, be obtained by this method. I have frequently 

 used a fairly quick method, though one not satisfactory in ail 

 cases, which consists of staining for five or six minutes in a satu- 

 rated aqueous solution of fuchsin, washing in water, and counter- 

 staining for a few seconds in a strong solution of methylene blue. 



