STAINING OF PROTOZOA 39 



which is heated over a water bath for five to seven minutes. Take the dish contain- 

 ing the preparation off the water bath and as soon as it becomes slightly opalescent 

 as the result of cooling remove the cover-glass preparation and wash thoroughly in 

 water. Then heat a few drops of the ethylamine silver solution upon the mordanted 

 cover preparation until it just steams and the margin appears black. Next wash 

 thoroughly in water and mount. This gives the most satisfactory results of any 

 method I have ever experimented with. 



Spore Staining. The most satisfactory spore staining method is 

 really the negative staining of the spore obtained when a bac- 

 terial preparation is stained by dilute carbol f uchsin or Loffler's methy- 

 lene blue. The spore appears as a highly refractile piece of glass in 

 a colored frame. 



The acid-fast method, as for tubercle bacilli, gives good results. 

 The decolorizing, however, must be lightly done, otherwise the spore 

 will lose its red stain. 



Holler's Method. Fix films and then treat with chloroform for one or two 

 minutes. Wash thoroughly and treat with a 5% solution chromic acid for one 

 minute. Wash in water and then stain as for acid-fast organisms with carbol 

 f uchsin. Use a i% sulphuric acid solution instead of the 3% acid alcohol. 



Agar Jelly Staining Method of H. C. Ross. 



Very clear i 1/2% solution of agar is colored with Unna's polychrome 

 methylene blue, Giemsa's solution, thionin or Gram's solution of iodine. Very 

 thin smears of blood, faeces or gastric content sediment are made and either 

 fixed lightly in the flame or air dried. A drop of the melted colored agar solu- 

 tion is placed on the smeared cover-glass and this is mounted immediately on a 

 clean slide. The preparation is ready for examination in about two minutes. 



The Staining of Protozoa. 



Unless staining albuminous material it is well to add a little blood- 

 serum or white of egg to the preparation about one loopful to a smear. 

 The serum or white of egg is best preserved by the addition of 2 % chloro- 

 form and kept tightly corked. 



Giemsa's Method. Fix moist smears with a fixative made by 

 adding i part of 95% alcohol to 2 parts of saturated aqueous solution 

 of bichloride of mercury. Keep in this solution twelve hours. Now 

 wash for a few seconds in water and then for about five minutes with a 

 dilute Lugol's solution (KI, 2 gm.; Lugol's solution, 3 c.c.; Aqua, 100 

 c.c.). Now wash in water and then in a 0.5% solution of sodium thio- 

 sulphate to remove the iodine which was used to remove the mercury. 



