COLLECTION, CULTIVATION, OBSERVATION 729 



and make a thin film of it toward one end of the sUde. Make a small 

 thick smear in the center of the other half of the slide. Dry. When 

 thoroughly dry, immerse the thick film part in distilled water and de- 

 haemoglobinize it. Let the slide dry. 



Blood smears must be stained as soon as possible to insure a proper 

 staining, as lapse of time or summer heat will often cause poor stain- 

 ing especially of thick films. Of several blood stains, Giemsa's and 

 Wright's stains are used here. For staining with Giemsa's stain, the 

 thin film is fixed in absolute methyl alcohol for 5 minutes. Rinse well 

 the slide in neutral distilled water. After shaking the stock bottle 

 (obtained from reliable makers) well, dilute it with neutral distilled 

 water in a ratio of one drop of stain to 1-2 cc. of water. Mix the solu- 

 tion and the blood film is placed in it for 0.5-2 hours or longer if 

 needed. Rinse the slide thoroughly in neutral distilled water and 

 wipe off water with a tissue paper from the underside and edges 

 of the slide. Let the slide stand on end to dry. When thoroughly dry, 

 place a drop of xylol and a drop of cedar wood oil (used for immersion 

 objectives) and cover with a coverglass. The mounting medium 

 should be absolutely neutral. Do not use Canada balsam for mount- 

 ing, as acid in it promptly spoils the staining. 



For Wright's stain, fixation is not necessary. With a medicine 

 dropper, cover the dried blood film with drops of undiluted Wright's 

 stain, and let the film stand horizontally for 3-5 minutes; then the 

 same number of drops of neutral distilled water is added to the stain 

 and the whole is left for 10-30 minutes. The stain is then poured off 

 and the film is rinsed in neutral distilled water. Dry. Mount in xylol 

 and cedar wood oil. 



Use of coverglass on a stained blood film is advocated, since a 

 cedar wood oil mounted slide allows the use of dry objectives which 

 in the hand of an experienced worker would give enough magnifica- 

 tion for species determination of Plasmodium, and which will very 

 clearly reveal any trypanosomes present in the film. Furthermore, 

 the film is protected against scratches, and contamination by many 

 objects which may bring about confusion in detecting looked-for 

 organisms. 



Films made from splenic punctures for Leishmania or Trypano- 

 soma are similarly treated and prepared. 



c. Section preparations 



Paraffin sections should be made according to usual histological 

 technique. Fixatives and stains are the same as those mentioned for 

 smear preparations. 



