STAINING PROTOZOA 45 



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 bacterial 

 preparation is stained by dilute carbol fuchsin or Loffler's methylene 

 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 decoloriz- 

 ing, however, must be lightly done, otherwise the spore will lose its red stain. 



M oiler'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 

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



Agar Jelly Staining Method of H. C. Ross 



Very clear i^% 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 solution 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 albumin fixative 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 % chloroform 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 i to 12 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 thiosulphate 

 to remove the iodine which was used to remove the mercury. Wash in water five 

 minutes, then stain with Giemsa's stain as used in blood-work for one to ten hours. 

 Wash and mount. 



Vital Staining of Protozoa with Neutral Red Solution. As a stock 

 solution one uses a 0.5% aqueous solution of neutral red. 



