358 RESEARCH IN PROTOZOOLOGY 



wise and inverted. The box is then raised from the lid, leaving 

 the slides in the lid, separated from each other by the pieces 

 of pasteboard. All the slides from a box, or any group of them, 

 may now be easily assembled, fastened together by means of a 

 stout rubber band, and given a group label. 



To stain one block of twenty-five slides, place sixty or seventy 

 drops (about 1.3 cc.) of Giemsa stock solution in a clean glass 

 vessel and pour in seventy-five cubic centimeters of water. The 

 pouring in of the water insures sufficient mixing. Then stand the 

 block in the diluted stain and leave for about one hour. Any clean 

 glass or porcelain dish will do, provided it is of such dimensions 

 that the stain will cover the thick films to the top, and not wet 

 the pasteboard slips. 



Dilute the stain immediately before use and use it only once. 

 To dilute the stock Giemsa solution, use distilled water, neutralized 

 or at most but slightly alkaline (pH 7.0 to pH J. 2). Rain water 

 caught in the open (not from a roof), melted snow, or ice made 

 from distilled water, will serve, especially if boiled until free from 

 carbonic acid. In any case, see that the water does not contain free 

 acid. If it is not convenient to make a hydrogen-ion determination, 

 a single indicator, phenol red, neutral red or an alcohohc solution 

 of hematoxylin will serve. One may neutralize with a one per 

 cent solution of sodium or potassium hydroxide or with dilute 

 hydrochloric acid. Distilled water is often acid, and it is well 

 to test the reaction of any water before using it. A tap water 

 not too heavily impregnated with salts may give good results, but 

 salts (especially sodium or magnesium chloride) tend to precipi- 

 tate part of the stain. The sediment remaining in a dish after a 

 previous staining may also precipitate part of the stain. A good 

 practice is to rinse each staining dish immediately after use. 



Occasionally, infusoria become so numerous in a supply of 

 water long kept in the laboratory that they appear in. the thick 

 films and may confuse the inexperienced by their resemblance to 

 parasites. 



Decolorization after staining is not always necessary. A suffi- 

 cient decolorization is usually obtained by setting the block, while 

 the slides are still Wet from the stain, for five minutes in clean 

 water, preferably the same as that used for diluting the stain. 



These staining directions will serve for most cases, but a good 

 deal of latitude is permissible if one observes the essentials — a 



