268 RESEARCH IN PROTOZOOLOGY 



field on a single glass slide. On each slide ten or twenty short 

 straight-line smears parallel to each other may be drawn by using 

 the cut petioles of leaves from separate plants. After the smears 

 dry, the slides are wrapped in paper for transport to the laboratory 

 where fixation is accomplished by immersion in absolute alcohol. 

 Subsequent staining for ten seconds with a few drops of the 

 ordinary anilin gentian violet solution used for bacteria suffices 

 to show the presence of flagellates. The excess of stain is removed 

 by running tap water over the slide, which is then drained. This 

 stain is very quickly applied to large numbers of smears and the 

 flagellates are rendered conspicuous by its use, since it stains 

 the flagella intensely. Smears from infected plants can be dis- 

 tinguished from those from uninfected specimens by the use 

 of a four millimeter or even a sixteen millimeter objective, and 

 a I ox ocular. 



For more careful staining of dried smears Wright's blood stain 

 is used. This staining mixture contains a fixing agent and so 

 may be placed directly on the dried slide, allowed to remain 

 there for one minute, then diluted with an equal part of distilled 

 water. The diluted mixture forms a metallic film on its surface, 

 and is allowed to stand two minutes, after which the slide is rinsed 

 in distilled water and drained. This method of staining dififeren- 

 tiates the organelles very well. The nuclei stain pink, the parabasal 

 bodies deep blue, and the cytoplasm pale blue. The flagella stain 

 with difficulty but appear purplish when visible. If the flagella are 

 not well stained a small portion of a smear may be treated with a 

 drop of anilin gentian violet in addition to the original Wright's 

 stain, thus making the flagella conspicuous on the individuals in 

 part of the sample. This is particularly necessary if measurements 

 are to be made of the total lengths. Field survey slides stained 

 originally with anilin gentian violet, if desired for more careful 

 examination in the laboratory, may be restained with Wright's 

 mixture after destaining with several changes of methyl alcohol. 



For careful study of the finer structures of the flagellates 

 it is necessary to fix them while they are still in moist latex. 

 This may be done by dipping the fresh smears into absolute 

 methyl alcohol, Schaudinn's fluid, Regaud's fluid or some other 

 standard fixing solution. The slides must be immersed promptly 

 to avoid drying at the edges of the smears where the fluid layer is 

 thin. After thorough fixation, and the removal of the fixing agents, 



