COLLECTION, CULTIVATION, OBSERVATION 727 



Silver-impregnation methods. Since Klein (1926) applied silver 

 nitrate in demonstrating the silver-line system of ciliates, various 

 modifications have been proposed. 



Dry silver method (Klein, 1926). Air-dried cover glass smears are 

 placed for 6-8 minutes in a 2 per cent solution of silver nitrate and 

 thoroughly washed. The smears are exposed to sunlight for 2-8 hours 

 in distilled water in a white porcelain dish, with occasional control 

 under the microscope. The smears are then washed thoroughly and 

 air-dried; finally mounted in Canada balsam. 



Wet silver method (modified after Gelei and Horvath, 1931). The 

 ciliates are fixed in a centrifuge tube for 5-10 minutes in sublimate- 

 formaldehyde solution, composed of saturated corrosive sublimate 

 95 cc. and formaldehyde 5 cc. The specimens are now washed twice 

 in nonchlorinated water and once in distilled water; they are then 

 treated in 1.5-2 per cent solution of silver nitrate for 5-20 minutes. 

 Without washing, the specimens in the tube are exposed to direct 

 sunlight for 10-60 minutes in distilled water, after which the speci- 

 mens are washed 4-6 times in distilled water, one minute each. Pass- 

 ing through a gradually ascending alcohol series and xylol, the speci- 

 mens are mounted in Canada balsam. 



Fontana's method. For staining filamentous structures such as the 

 extruded polar filament of microsporidian spores, this method is the 

 most satisfactory one. After air-drying the smears are fixed for 5 

 minutes in a mixture of formaldehyde, 20 cc; glacial acetic acid, 1 

 cc; and distilled water, 100 cc After washing in running water, the 

 smears are placed in the following mordant composed of equal parts 

 of 5 per cent tannic acid and 1 per cent carbolic acid, for about 2 

 minutes at about 60°C. Wash the smears in water and place them for 

 3-5 minutes in 0.25 per cent solution of silver nitrate warmed to 

 60°C., to which ammonia has been added drop by drop until a gray- 

 ish brow n cloud appeared. Wash thoroughly and air-dry. After pass- 

 ing through 95 per cent and absolute alcohol, and xylol, the smears 

 are mounted in Canada balsam 



b. Blood film preparations 



Thin film. The finger tip or ear lobe is cleaned with 70% alcohol. 

 Prick it with an aseptic blood lancet or a sterilized needle. Wipe off 

 the first drop with gauze and receive the second drop on a clean slide 

 about half an inch from one end (Fig. 336, 1). Use care not to let the 

 slide touch the finger or ear-lobe itself. Quickly bring a second slide, 

 one corner of which had been cut away, to the inner margin of the 

 blood drop (i), and let the blood spread along the edge of the second 



