1 68 Methods of Observing Micro-organisms 



two fluids gradually mix, transfusion currents are formed, and the speci- 

 men is allowed to stand for exactly two minutes longer. It is during this 

 time that the staining takes place. A precipitate usually forms upon the 

 surface of the fluid, so that it must not be poured off, but splashed off 

 by dropping distilled water upon it from a height. The distilled water 

 is added until it no longer shows any color, when the specimen is drained, 

 dried, and mounted in balsam. 



The student may also try staining with hematoxylin and eosin, 

 thionin and eosin, methylene-blue and eosin, or any other dyes, 

 some of which sometimes bring out special details of structure. 

 The protozoa do not show the same reaction to Gram's stain that 

 makes it so useful for differentiating the bacteria. 



STAINING PROTOZOA IN TISSUE 



For this purpose the sections should be embedded in paraffin, 

 cut very thin, and cemented to the slides. 



Ordinary staining with hematoxylin and eosin is rarely of much 

 use. Methylene-blue and eosin is better, but still more useful are 

 the Romanowsky methods, and both the Wright stain and the 

 Marino stain can, with some modification of the time of staining- 

 and washing, be employed with good results. 



Still better and more satisfactory for certain protozoa are the 

 iron-hematoxyhn and the Biondi stain. 



Heidenhain's Iron-hematoxylin* — Fix the tissue, by preference, in. Zenker's 

 solution, though alcohol fixation will do. Embed in paraffin, cut very 

 thin, and fix to the slide. 



1. Stain from three to twelve hours in 2.5 per cent, solution of violet iron- 



alum (sulphate of iron and ammonium). The sections should be 

 stood vertically in the solution, so that no precipitate may form upon 

 them. 



2. Wash quickly in water. 



3. Stain in a 0.5 per cent, ripened alcoholic solution of hematoxylin for 



from twelve to thirty-six hours. 



4. Wash in water. 



5. Differentiate in the iron-alum solution, controlling the results under the 



microscope. The section should be well washed in a large dish of tap 

 water before each examination to stop decolorization. 



6. Wash in running water for a quarter of an hour. 



7. Pass through alcohol, xylol, and mount in xylol balsam. 



A counterstain with Bordeau R. before or with rubin S. after the iron stain 

 is sometimes useful. 



Biondi-Heidenhain Stain.f — The tissues must be fixed in Zenker's or corrosive 

 sublimate solutions. Embed in paraffin, cut very thin, fix to the slide. 



Stain I. Orange G 8 grams 



Water 100 cc. 



II. Acid fuchsin \ 20 grams 



or Rubin S. J 



Water 100 cc. 



III. Methyl-green 8 grams 



Water 100 cc. 



Let the solutions stand for several days, occasionalUy shaking the bottles 

 to make sure that a saturated solution of each is secured. At the 

 end of the time set, mix the solutions in the following proportions: 



* Mallory and Wright, "Pathological Technique," igri, p. 309. 



t Modified from Mallory and Wright, "Pathological Technique," 1911, p. 289. 



