COLLECTION, CULTIVATION, OBSERVATION 895 



enough albuminous substance in the culture fluid. If a small drop of 

 fresh egg-white emulsified in sterile distilled water is smeared on the 

 coverglass very thinly with the tip of a clean finger, before mounting 

 material for smear, more specimens will adhere to and remain on the 

 coverglass upon the completion of the preparation. Let the smear 

 lie horizontally for 5-10 minutes or longer. 



Parasitic Protozoa live in media rich in albuminous substances, 

 and therefore, easily adhere to the coverglass in smear. Make uni- 

 formly thin smears on coverglasses. If the smears are made from 

 dysenteric or fluid stools, they should be fixed almost immediately. 

 Smears made from diarrhoeic or formed stools by emulsifying in 

 warm salt solution, should be left for a few minutes. In any case, do 

 not let the smear become dry except a narrow marginal zone. 



The smears are fixed next. The most commonly used fixative for 

 Protozoa is Schaudinn's fluid. This is made up as follows: 



Cold saturated mercuric 



bichloride (6-7%) 66 cc. 



Absolute or 95% alcohol 33 cc. 



Glacial acetic acid 1 cc. 



The first two can be kept mixed without deterioration, but the acid 

 must be added just before fixation. Fix at room temperature or 

 warmed to 50°C. The fixative is placed in a square Petri dish and the 

 smear is gently dropped on it with the smeared surface facing down- 

 ward. With a little experience, air bubbles can be avoided and make 

 the smear float on the surface of the fixative. After about one minute, 

 turn it around and let it stay on the bottom of the dish for 5 to 10 

 more minutes. In case the smear is too thick, a thin coat of vaseline 

 on the upper side of the coverglass will make it to float. About six 

 coverglass-smears may be fixed in the dish simultaneously. 



The coverglass-smears are now transferred to a Columbia staining 

 jar for coverglasses, containing 50% alcohol for 10 minutes, followed 

 by two changes for similar length of time. Transfer the smears next 

 to 30% alcohol for 5 minutes, and then to a jar with water, which 

 is now placed under gently running tap water for 15 minutes. Rinse 

 them in distilled water and stain. 



Other fixatives frequently used for Protozoa are as follows: 



Bouin's fluid 



Picric acid (saturated) 75 cc. 



Formaldehyde 25 cc. 



Glacial acetic acid 5 cc. 



