160 



Journal of Applied Microscopy. 



growing on the potatoes. For this pur- 

 pose a cover-glass is placed on the table. 

 or, better, on a short board, and a drop 

 of water not much larger than a good 

 sized pinhead is placed in the middle. By 

 means of the sterile platinum wire, a 

 portion of the growth is transferred to 

 the droplet of water. The latter should 

 now be distinctly cloudy. Again sterilize 

 the wire by heating in a flame and when 

 cold spread out the droplet of water over 

 the surface of the cover-glass. The wa- 

 ter evaporates rapidly, if the drop is 

 small, and leaves a perfectly even resi- 

 due distributed over the entire surface. 

 Should the drop be large, it will drj'^ 

 slowly and leave unsightly " shore 

 lines." 



As soon as the water has evaporated 

 the material is fixed to the cover-glass 

 by means of heat. For this purpose the 

 cover-glass is taken up in the forceps, 

 specimen side up, and is touched once or 

 twice to a Bunsen or alcohol flame. This 

 little operation requires judgment and 

 care. The cover-glass must become 

 heated suflficiently to fix the specimen, 

 so that it will not wash off when subse- 

 quently treated with the dye and with 

 water. On the other hand, too much 

 heating will destroy the organism and 

 render it incapable of taking up the dye. 

 The best rule to follow is to touch the 

 cover-glass once or twice to the flame 

 and then bring it at once in contact 

 with the end of the finger. If the cover- 

 glass is so hot that the finger must be 

 withdrawn at once, it indicates that the 

 specimen is fixed and that it is not ne- 

 cessary to heat any more. The cover- 

 glass, with the specimen side still 

 turned up, is held in the forceps and a 

 drop or two of water is placed on the 

 specimen. Then two or three drops of 

 the dilute gentian violet or fuchsin are 

 added and allowed to act for one-quarter 

 to one-half minute. The cover-glass is 

 then washed perfectly clean of dye by 

 being held under a tap, or by rinsing in 

 one or two glasses of water. It is then 

 touched edgewise to a piece of filter or 

 blotting paper in order to drain off the 

 excess of water, and is then placed on 

 the paper with the specimen side turned 

 up. By gentle rotation of the cover- 

 glass, the lower surface becomes per- 

 fectly dry. A clean glass slide is now 

 brought down over the cover-glass, the 

 upper or specimen surface of which is 

 still moist. The cover-glass now adheres 

 to the slide by means of the thin layer 

 of liquid. The specimen is now turned 

 over so that the cover-glass rests on the 

 slide. It is now examined under the mi- 

 croscope with the one-eighth-inch or one- 

 twelfth-inch objective. When making this 

 examination a thin film of water should 

 be between the slide and cover-glass, so 



that the under side of the specimen 

 should be moistened by the water. The 

 bacteria should be stained a deep violet 

 or a deep red, according to the dye em- 

 ployed. Failure to take the stain prop- 

 erly may be due to over-heating while 

 fixing the specimen, or to a weak dye, 

 or to a too short exposure to the dye. By 

 repeated trials, the exact conditions 

 necessary can be ascertained and then 

 followed without any difficulty. 



In the method as described above, the 

 dye is not added direct to the cover- 

 glass, but to a drop of water which is 

 first placed upon it. This little deviation 

 from the process, as ordinarily described, 

 serves to prevent over-staining and also 

 deposition of coloring matter. 



The color can be forced into the speci- 

 m.en, thus making it stand out more 

 sharply than would otherwise be the 

 case, by holding the cover-glass with the 

 dye on it over a low Bunsen or alcohol 

 flame till vapors begin to rise. The 

 specimen is then washed as before. 



Instead of heating the dye on the 

 cover-glass, it may be heated before use. 

 For this purpose the bottle of dye is 

 placed on an iron plate, and this is 

 heated by a low flame. Fig. "3 shows 

 such an iron plate as used in the au- 

 thor's laboratory, slipped under the 

 flange of the ordinary water-bath. 



When it is desirable to preserve a 

 stained specimen, this should be floated 

 from the glass slide by the addition of 

 a drop or two of water to the edge of the 

 cover-glass. The latter should then be 

 placed, specimen side turned up, under 

 a watch-glass or tumbler until perfectly 

 dry. A drop of Canada balsam is then 

 placed in the center of a clean slide and 

 the dried cover-glass, if necessary, 

 waved once or twice over the flame, is 

 inverted and brought down on the bal- 

 sam with the specimen side turned down. 

 On gentle pressure, aided by gentle 

 heating of the slide if need be, the bal- 

 sam will spread out evenly under the 

 cover-glass. 



The entire process of simply staining 

 can be briefly summarized as follows: 



Clean cover-glass. 



Spread specimen. 



Dry in air. 



Fix in flame. 



Add drop of water. 



Add dilute-dye one-quarter to one-half 

 minute. 



Wash in water. 



Examine in water. 



Dry in air. 



Mount in balsam. 



University of Michigan. 



{To be continued.) 



