SMEAR PREPARATION OF MONOCYSTIS 99 



If the worm shows satisfactory infection, the remainder of the material from 

 the eye-dropper pipette is placed in a watch glass and diluted with 0.8 per cent 

 sodium chloride until it forms a dispersion about intermediate in thickness 

 between cream and milk. Working as rapidly as possible, as many smears as are 

 required are made from this dilution. The dilution in question will not retain 

 the parasites in good condition for more than about five minutes, but, if insuf- 

 ficient smears have been made in this time, it is easy to take a fresh supply of 

 the seminal fluid from another vesicle and to dilute it in the fresh watch glass. 

 The smears should be made with two slides in the manner described in Chap- 

 ter 7, and each slide should be placed face downward in fixative for three or 

 four minutes before being removed to a coplin jar of distilled water. 



After they have been washed in water, the smears should be transferred to 

 70 per cent alcohol where they can remain until they are ready for staining. 

 Any stain may be used, but it is conventional to employ a hematoxylin mix- 

 ture. The method of staining is easy. The solution is made in accordance with 

 the directions given and diluted to the extent of about 2 per cent with distilled 

 water. The slides are placed in this diluted solution and left until examination 

 under the low power of the microscope shows them to have been ade- 

 quately stained and differentiated. Then they are rinsed exceedingly briefly in 

 distilled water and dried. There is not the slightest necessity to employ any 

 dehydrating agent, such as alcohol, for the smears should be sufficiently thin 

 and the objects in them sufficiently well fixed that drying will not make the 

 least difference. To complete the mount, a drop of the mountant selected is 

 placed in the center of each smear, a coverslip added, and the whole put on 

 a warm plate until it is dry. 



Summary 



1. Make a temporary, thick smear from the seminal vesicle of an earthworm. 

 If plenty of spore cases are present, proceed with step 2; if not, test other 

 worms until a heavily infected specimen is found. 



2. Place two glass rods in a petri dish and lay a slide across them. Pour in just 

 enough fixative to wet the lower surface of the slide. 



3. Dilute the contents of the infected seminal vesicle with 0.8 per cent sodium 

 chloride to the consistency of a thin cream. 



4. Make a smear of cream as described on page 49. 



5. Lay each smear face down on glass rods in fixative for 3 to 4 minutes. Wash 

 and accumulate in a coplin jar of 70 per cent alcohol. 



6. Stain and differentiate smears. Dry in air. Mount in balsam. 



