470 RESEARCH IN PROTOZOOLOGY 



water ^- is added drop by drop until a metallic scum appears upon 

 the surface of the mixture. This diluted solution is allowed to re- 

 main for from two to four minutes. At the end of this period, the 

 slide is rinsed in distilled water and is dried in air. If overstained, 

 it may be held in running water for a few minutes. A precipitate 

 of stain may be removed by immersing the slide for an instant in 

 a jar of the stock stain. This will dissolve the precipitate without 

 decolorizing the stain. Inasmuch as different batches of stain fre- 

 quently behave very dift'erently on various kinds of organisms, the 

 time intervals given above will have to be determined for each 

 stain with each species of protozoa stained. 



In using Giemsa's stain the him is fixed with any of the or- 

 dinary fixatives (absolute methyl alcohol is most commonly em- 

 ployed) and is then left in a one in ten dilution of the stock stain 

 in tap water ^^ for from ten minutes to two hours depending upon 

 the material and particular batch of stain. Overstained films can 

 be nicely decolorized by holding them in running tap water for a 

 few moments. The film is dried in air.^^ 



See Chapters XXVIII and XXXV for special uses of the Ro- 

 manowsky stains on plant flagellates and malarial parasites. 



Sections. Tissue containing protozoa may be sectioned following 

 the standard histological procedures for that technique. These 

 processes are so well known that they are not given in detail here. 

 Briefly, the material must first be "fixed" and hardened, washed 

 to remove the excess fixing agent, dehydrated in alcohol, cleared 



^^ The pH of the diluent should be within the range of 6.0 to 6.6. 

 A suitable buffered solution for dilution purpose is given 

 below. It has a pH of 6.4. 



KH2PO4 6.63 gms. 



Na2HP04 2.56 gms. 



Distilled water 1000.00 c.c. 



"If the local supply of tap water is for one reason or another unsuitable 

 for this purpose, distilled water which has been slightly alkalinized by 

 standing for some time with a small quantity of calcium carbonate is quite 

 satisfactory. 



"If possible, when drying films which have been stained by some one of 

 the Romanowsky techniques, an air blast should be used in order to dry the 

 film as quickly and uniformly as possible. It not infrequently happens that 

 in a film which has been left flat on the table or even tilted on edge to drain, 

 areas of differing staining intensities are apparent when the slide has 

 dried. The reason for that is that the drying has not been uniform, and that 

 decolorization has progressed further in one area than in another. 



