44 SPOROZOA. 



fluid, wash in several changes of 70 per cent, alcohol to remove 

 the mercuric chloride ; then wash in 70 per cent, alcohol to 

 which a few drops of Wiegert's iodine solution have been 

 added ; finally, wash in 70 per cent, alcohol to which a drop 

 of 1 per cent, solution of sodium thiosulphate has been added, 

 in order to remove all traces of iodine. Bring down through 

 successive grades of alcohol mto distilled water if it is desired 

 to use a watery stain ; a stay of a few minutes in each solution 

 will sujB&ce . After fixation with Zenker's fluid, wash successively 

 with distilled water, distilled water with a few drops of iodine 

 solution, distilled water with a trace of 1 per cent, solution 

 of sodium thiosulphate, and distilled water. 



Staining Methods. — The following stains are usually employed 

 for staining Protozoa : — 



Borax-carmine. — Fix in Bouin's fluid for 10 to 20 minutes 

 according to bulk and permeabihty. Wash out in 70 per cent, 

 alcohol (several changes). Stain in borax-carmine till 

 thoroughly penetrated ; 15 minutes are usually enough 

 for small objects. Differentiate in acid alcohol (70 per cent, 

 alcohol to which hydrochloric acid is added to the strength 

 of 1 per cent.),controlhng under the microscope. Dehydrate 

 in 90 per cent, to absolute alcohol. Put in a mixture of clove - 

 oil and absolute alcohol, equal parts. After a few minutes 

 transfer to pure clove-oil, and leave there till cleared. Mount 

 in Canada balsam. 



Delafield's Hematoxylin. — Fix in Schaudinn's fluid. Wash 

 in 30 per cent, alcohol, and bring down through 10 per cent, 

 alcohol to distilled water. Add a few drops of Delafield's 

 hsematoxylin solution to a watch-glass full of distiUed water. 

 Leave in stain for a few minutes to an hour or more according 

 to bulk. Bring up from distilled water through ascending 

 grades of alcohol to 70 per cent, alcohol ; differentiate in 

 70 per cent, acid alcohol. Dehydrate ; clear ; mount. 



Heidenhain's Iron Hsematoxylin. — Fix in Schaudinn's fluid 

 for 10 to 30 minutes, according to the size and permeabihty 

 of the object. Bring down to 30 per cent, and 10 per cent, 

 alcohol to distilled water. Wash out the fixative thoroughly. 

 Transfer to 4 per cent, solution of the violet crystals of iron 

 alum (sulphate of iron and ammonium) in distilled water for 

 \ hour to 12 hours according to the size of the organisms. 

 Stain in Heidenhain's aqueous hsematoxylin solution (about 

 0-5 per cent.) for 30 minutes to several hours. Wash in distilled 

 water. Differentiate in 1 per cent, iron alum solution till 

 the granules in the nuclei are distinct, the films being removed 

 into distilled water and examined on a sHde with the micro- 

 scope from time to time. When differentiation is complete, 

 wash in several changes of distilled water and then for half 

 an hour or more in running tap-water, Counterstain, if 



