LABORATORY DIAGNOSIS OF PROTOZOAN INFECTIONS 



385 



oil is placed on them for examination. 

 The immersion oil should be removed 

 after the examination has been completed 

 if the slides are not to be thrown away. 

 Many people do this by rubbing the slide 

 with lens paper as tho they were polishing 

 silver, a procedure which removes not 

 only the oil but also many of the blood 

 cells. The following technic, which I 

 first saw demonstrated by Dr. Joseph A. 

 Long, permits one to remove the oil 

 quickly and neatly without disturbing the 

 blood cells. It can also be used for slides 

 which have been covered by a coverslip; 

 by its use, one can remove the oil from a 

 newly mounted slide without also removing 

 either the coverslip or the wet mounting 

 medium beneath it. 



Fold a small piece (about 5 cm square) 

 of lens paper twice so that it is 4 layers 

 thick. Place the lens paper on top of the 

 immersion oil and allow it to take up the 

 oil. Pull if off the slide sideways in a 

 single motion; do not rub. 



Fold a second piece of lens paper like 

 the first. Place a drop of xylene on it. 

 Place the wet lens paper on what remains 

 of the oil. Leave it for a second or two, 

 and then pull it off the slide sideways in 

 a single motion; do not rub. When the 

 xylene has evaporated, the slide will be 

 clean and dry. (Sometimes it is necessary 

 to repeat this second step with a fresh 

 piece of lens paper. ) 



CONCENTRATION OF PROTOZOAN 

 CYSTS FROM FECES 



A number of technics have been devel- 

 oped for the concentration of protozoan 

 cysts and helminth eggs from feces. They 

 are of 2 general types, flotation and sedi- 

 mentation. Each has certain advantages 

 over the other. 



FLOTATION TECHNICS 



These technics make use of solutions 

 of higher specific gravity than protozoan 

 cysts or helminth eggs, but of lower spe- 

 cific gravity than most of the fecal debris. 



When feces are mixed with them, the cysts 

 and eggs will float to the top while most of 

 the fecal material remains at the bottom. 

 Flotation technics are most useful for coc- 

 cidian oocysts, other protozoan cysts, 

 nematode eggs and some tapeworm eggs. 

 They are not satisfactory for trematode, 

 acanthocephalan and other tapeworm eggs. 



Many different solutions have been 

 used, and many variations in technic have 

 been proposed. The methods described 

 here all work satisfactorily. 



Sugar Flotation 



This technic is preferable for general 

 use, but is not satisfactory for protozoan 

 cysts other than those of coccidia. Sugar 

 solution is preferable to sodium chloride, 

 sodium nitrate or other salt solutions ex- 

 cept zinc sulfate. It does not crystallize 

 as readily, and causes less distortion than 

 salt solutions, and it is just as efficient 

 (Levine et al. , 1960). The following tech- 

 nic is a modification of the DCF (direct 

 centrifugal flotation) technic introduced by 

 Lane (1923). 



1. Make a rather heavy suspension of 

 feces in physiological salt solution 

 in a shell vial or other container. 



2. Strain thru 2 layers of cheesecloth 

 into a test tube or centrifuge tube, 

 filling the tube almost half full. 

 The lip of the tube must be smooth, 

 or an air bubble will form under 

 the coverslip following centrifuga- 

 tion (#6 below). 



3. Add an equal volume of Sheather's 

 sugar solution, leaving a small air 

 space at the top. Cover with a 

 plastic coverslip or small piece of 

 card, and invert repeatedly to mix. 



4. Add enough additional Sheather's 

 sugar solution to bring the surface 

 of the liquid barely above the top 

 of the tube. 



5. Cover with a round coverslip. 



6. Centrifuge for 5 minutes. (If a 

 centrifuge is not available, let 

 stand for 45 minutes to 1 hour. ) 



7. Remove the coverslip, place it on 

 a slide, and examine under the 

 microscope. 



