Monocystis 



SMEAR PREPARATIONS FROM FLUID MATERIAL 



73 



ply of Schauclinn's fixative (Chapter IS, 

 F 3000.0000 Schaudiiiu 1893), an ade- 

 quate supply of clean glass slides, an eye- 

 dropper-type pipet, some 0.8% sodium 

 chloride, and some cophn jars of distilled 

 water. Enough fixative is poured into the 

 petri dish so that when a sHde is laid on 

 the pair of glass rods, its lower, but 7}ot its 

 upper surface will be in contact with the 

 fixative. This level is best established with 

 a plain glass slide before the smears are 

 started. 



The seminal vesicle of the earthworm is 

 slit and a drop of the contained fluid re- 

 moved with the pipet. This pipet is then 

 used to smear a relatively thick layer of 

 the material on the center of one of the 

 clean shdes and, before it has time to dry, 

 this slide is laid face down in tlie fixative 

 for about two minutes. The sUde is then 

 removed, rinsed under the tap, and ex- 

 amined under a high power of the micro- 

 scope after a covershp has been placed 

 over the smear. It is rather difficult to 

 see the trophozoite stages in an unstained 

 preparation, but no difficulty will be ex- 

 perienced in picking out the spore cases 

 (pseudo-navicellae) owing to their rela- 

 tively high index of refraction. It may be 

 taken that adequate numbers of the para- 

 sites are present if not less than three or 

 four of these spore cases occur within the 

 field of a four-millimeter objective in a 

 thick smear of this nature. 



As soon as a satisfactorily infected worm 

 has been found, the remainder of the 

 material from the pipet is placed in a 

 watch glass and diluted with 0.8% sodium 

 chloride until it forms a dispersion about 

 intermediate in thickness between cream 

 and milk. As many smears as are required 

 are made from this cream as rapidly as 

 possible. The dilution in question will not 

 retain the parasites in good condition for 

 more than about five minutes, but if in- 

 sufficient 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 a fresh watch glass. The 



cream should be spread with two slides in 

 the manner described above, and each 

 shde placed face downward in Schaudinn's 

 fixative for three or four minutes before 

 being removed to a cophn jar of distilled 

 water. 



After having been washed in water the 

 smears should be transferred to 70% 

 alcohol where they can remain until they 

 are ready for staining. Any stain may be 

 used but it is conventional to employ a 

 hematoxylin mixture. The author prefers 

 to use the old "triacid" stain of Biondi 

 which is given in Chapter 20 under the 

 heading DS 13.33 Biondi (1888). The ad- 

 vantage of this solution is that the orange 

 G is picked up by the cases of the sporo- 

 cysts, while the trophozoites are red with 

 clear green nuclei. Nuclei of the sperma- 

 tozoa and spermatids of the earthworm 

 occupy so much of the cells in which they 

 are found that they give the whole stain 

 a greenish cast. This green background 

 shows up the red trophozoites and the 

 brilliant orange sporozoites. 



The method of staining is easy. The 

 solution, made in accordance with the 

 directions given, is diluted to the extent 

 of about 2% with distilled water. The 

 slides are then placed in this diluted solu- 

 tion and left until examination under the 

 low power of the microscope shows them 

 to have been adequately stained and 

 differentiated. They are then briefly rinsed 

 in distilled water and dried. There is no 

 necessity to use any dehydrating agent, 

 such as alcohol, which will interfere with 

 the staining, because the smears should be 

 sufficiently thin and the objects in them 

 suflticiently well fixed that drying will not 

 distort them. To complete the mount, a 

 drop of the mountant selected is added 

 and a coverslip applied. Balsam may be 

 used, but the writer considers it to have 

 too high a refractive index, and for that 

 reason prefers one of the "neutral" 

 mountants, based on gum sandarac, the 

 formulas for which are given in Chapter 26 

 under the heading M 23.1. 



