386 Journal of Applied Microscopy. 



limit. This in nowise interferes with the normal activities of the organism, yet 

 effectively restricts the locomotory actions almost to any degree desired, and 

 allows, in most cases, the vital activities quite easy of observation. It has the 

 further advantage that fresh water may be added at any time without special 

 danger of losing the specimen. 



Intra vitam staining may be satisfactorily done with many of the Protozoa, 

 and with a variety of stains, among which methylen blue, methylen green, methyl 

 violet, in very dilute solutions, give good results. Congo red has been recom- 

 mended, and is said to not affect the specimen deleteriously even when used in 

 strong watery solutions. Of course, in this method of staining the object to be 

 attained is the differential condition of the organism in life, and therefore it must 

 be kept under fairly constant observation in order to distinguish the varying 

 effects at varying states of activity, as they approach their maximum or decline. 

 But at no time must there be expected the clearness and sharpness of nuclear 

 definition, etc., as may be secured with the use of one of the specific nuclear 

 stains applied in the usual way. 



Killing, Fixing, etc. — In keeping with the last suggestion, that the best results 

 can only be realized by the application of the more usual histological, or rather 

 c^tological methods, attention will most naturally be directed along that line. 

 And, as in almost any similar research, so here, the first requisite will be material 

 in great abundance. For, as will be seen later, the very operations will neces- 

 sarily involve the waste or loss of considerable numbers of specimens. It may, 

 however, be noted in this connection that even isolated specimens may be killed, 

 stained, and finally mounted directly upon the slide. But since, in any case, one 

 of the fundamental factors consists in the killing and fixing of the organism as 

 nearly instantaneously as practicable in order that the least possible distortion 

 may result, this may as well be considered in this connection. For this purpose 

 recourse must usually be had to an active poison, which while killing will leave 

 the cell as little altered in form and character as possible. To do this by intro- 

 ducing the reagent under the cover-glass and bringing it quickly into contact with 

 all portions of the organism is much more difficult than by the process of plunging 

 a whole mass of specimens at once into a quantity of the reagent. However, it 

 may be done, of course. In my own experience one of the best reagents for this 

 is Lang's fluid, hot, and applied to the edge of the cover and drawn under with 

 blotting paper applied to the opposite edge. This must be followed by a removal 

 of the excess of the reagent by washing it out with water applied in a similar way. 

 And then, furthermore, the application of the appropriate stain must be similarly 

 made, its excess removed, dehydration by the use of alcohols of increasing 

 strength, clearing, and finally mounting in the desired medium, usually balsam. 

 As will be seen, such a method must be at best a somewhat difficult one, 

 and, except under special necessity, hardly worth the time and trouble 

 involved. 



The method sometimes advocated of killing specimens on the slide or cover, 

 after the method commonly suggested for the fixing of bacteria or blood 

 corpuscles, namely, of killing over the flame of a lamp or burner, and drying by 

 desiccation, I have found absolutely worthless for any of the more complex Pro- 



