42 SPOEOZOA. 



inoculable from one dog to another, but as fatal infections 

 are often obtained, a number of animals will be required. 

 Ticks fed on a dog will remain infective for long periods, and 

 the parasites can thus be maintained in them. Strains of 

 HsRmojproteus columhse can be maintained in pigeons by 

 breeding Lynchia maura in the cages in which they are kept. 

 A fresh pigeon is introduced into the cage from time to time 

 to hand over the infection from fly to fly. Toxoplasma gondii 

 is also inoculable to a variety of laboratory animals. 



Permanent Preparations of Fixed and Stained Protozoa. — 

 Although it is important to study the hving organisms in the 

 first instance, the finer details of structure can only be studied 

 in properly fixed and stained smears or films. Some of the 

 methods of general apphcation are described below. 



Staining under the Cover-slip. — For staining large organisms, 

 such as the Gregarines, the following method is used : — 



Put wax feet at the corners of a square cover-shp, and invert 

 it over a drop of the fluid containing the organisms. The wax 

 feet should hold the cover-sHp firmly to the slide. With 

 a pipette run a Httle fixative at one side of the cover-slip, and 

 draw it through by holding a piece of filter-paper at the opposite 

 side. When the fixative has had time to act, wash it out by 

 substituting another fiuid (alcohol or water, as the case may be) 

 and draw it through with filter-paper in the same manner. 

 The stream should not be so violent as to wash away the 

 organisms, but the substitution should be complete. Then 

 run in the stain, allow it to act, and wash out and differentiate, 

 if necessary controUing the process under the microscope. 

 Dehydrate and then clear in clove-oil or xylol, and run in a very 

 fluid Canada balsam . It is very important to see that the trans- 

 fer from one fiuid to another is not too rapid, as otherwise 

 there is great risk of shrinkage, and also to see that the dehydra- 

 tion is complete. 



The following is an indication of the length of time generally 

 required, but should be regarded as no more than an indication. 

 Fix in Bouin's fluid, 5 min. ; wash in 70 per cent, alcohol, 

 5 min. (several changes) ; stain in borax- carmine, 5 min. or 

 more ; dehydrate with 70 per cent., 90 per cent., and absolute 

 alcohol, 5 min. each, changing the absolute alcohol once or 

 twice ; clear by running in a mixture of clove-oil and absolute 

 alcohol and then pure clove-oil ; mount in Canada balsam 

 by running the same under the cover-shp. 



Preparation of Wet Fixed Films. — Films made on shdes or 

 cover-shps may be fixed with one of the fixing fluids, without 

 being allowed to dry. After fixation the films are washed 

 free of fixative, and stained and mounted Hke sections fixed 

 on slides. The results shown by wet fixed films are far 

 superior to those shown by dried films, described later. If the 



