164 Methods of Observing Micro-organisms 



Mix solutions A and B and preserve in a well-closed bottle. Place solution C 

 in a bottle with a pipette stopper. When the staining is to be done, one pours 

 rs to 20 cc. of the A B mixture into a glass-stoppered test-tube and adds 2 or 3 

 drops of solution C. A precipitate forms, but quickly dissolves on shaking. 

 More of solution C is added, and the tube shaken untU the solution becomes 

 brown and clouded and one can see a fine precipitate in a thin layer of the fluid. 

 The fluid is next filtered several times through the same filter and caught in the 

 same glass until it will remain clear for several minutes. Then it is poured on the 

 filter a last time and 4 or 5 drops allowed to fall upon each of the prepared cover- 

 glasses. In a short time a sheen is observed upon the surface of the fluid on the 

 cover-glasses, showing that a fine precipitate has formed. When this has 

 occurred, a little experience will show when the proper moment arrives to throw 

 off the fluid and wash the cover in distilled water. It is the precipitate that 

 clings to the flagella and renders them distinctly visible. If no precipitate occurs, 

 the flagella will not be seen. 



L. Smith* offers the following modification of Newman's methodf 

 as being a simple and excellent method of staining flagella: The 

 material and cover-glasses are prepared with care as for the fore- 

 going methods, after which one proceeds as follows: 



1. Transfer a loopful of the baciUary emulsion to the clean slide or cover- 



glass and allow it to dry in the air. 



2. Expose to a nuld degree of heat, holding the glass in the fingers — this is 



rather drying than actual heating. 



3. AUow the stain to drop from a filter upon the film and remain in contact 



five to ten minutes. 

 The formula for the stain is 



I. Tannic acid i gram 



Potassium alum i " 



Distilled water 40 cc. 



Dissolve by shaking or allow to stand overnight in the incubator. 



II. "Night blue"t 0.5 gram 



95 per cent, or absolute alcohol 20.0 cc. 



Mix I and II thoroughly and remove the heavy precipitate by filtration. 

 If not used at once, drop from a filter upon the film. The stain does 

 not keep more than a few days. 



4. Wash carefully but thoroughly in water. 



5. Apply a saturated aqueous solution of gentian violet for about two min- 



utes to stain the bodies of the bacteria. 



6. Wash thoroughly in water, dry with smooth blotting-paper, and mount 



in balsam. 

 To secure a perfectly clean background for photomicrography, it 'is best to 

 stain on a slide. The stain is then poured into a Petri dish, the slide inverted, 

 the end of the slide used to push aside the film on the surface of the stain, and the 

 film then immersed downward, one end of the slide supported, during staining, 

 on a match-stick or bit of glass rod. In this way the adherence of the precipitate 

 to the slide can be avoided. 



THE OBSERVATION OF LIVING PROTOZOA 



When protozoa are to be examined in transparent fluids, such as 

 pond-water or culture fluids in which they have been artificially 

 nourished, use can be made of a "live-box" or of the "hanging drop." 

 Ordinarily, however, the organisms to be examined are contained 

 in blood, in pus, in sputum, in feces, or in some other more or less 

 opaque fluid, of which an extremely thin layer must be prepared in 



* "Jour. Med. Research," 1901, vi, p. 34r. ' 



t "Bacteria," John Murray, London, 2d edition. 

 t James Strong & Son, Glasgow and Manchester. 



