386 



lABORATORY DIAGNOSIS OF PROTOZOAN INFECTIONS 



(If desired, Steps 2 to 4 can be mod- 

 ified by straining the fecal suspension into 

 a second shell vial, mixing with an equal 

 volume of Sheather's sugar solution, and 

 then filling the centrifuge tube with the 

 mixture. ) 



Zinc Sulfate Flotation 



Zinc sulfate solution has the advantage 

 of concentrating the cysts of protozoa such 

 as Entamoeba and Giardia without distor- 

 tion. The following technic is a modifica- 

 tion of that introduced by Faust el al. 

 (1938). 



1. Make a suspension of feces in 

 physiological salt solution in a 

 shell vial or other container. 



2. Strain 4 ml of the suspension thru 

 2 layers of cheesecloth into a test 

 tube or centrifuge tube. The lip 

 of the tube must be smooth. 



3. Add tap water to within 1 cm of the 

 top of the tube. 



4. Mix thoroughly and centrifuge for 

 5 minutes. 



5. Pour off the supernatant fluid. 



6. Add a small amount of zinc sulfate 

 solution and mix with an applica- 

 tor stick. Add more zinc sulfate 

 solution until the tube is almost 

 full, cover with a plastic coverslip 

 or a small piece of card, and in- 

 vert repeatedly to mix. 



7. Add enough additional zinc sulfate 

 solution to bring the surface of 

 the liquid barely above the top of 

 the tube. 



8. Cover with a round coverslip. 



9. Centrifuge for 5 minutes. 



10. Remove the coverslip, place it on 

 a slide, and examine under the 

 microscope. 



SEDIMENTATION TECHNICS 



Sedimentation technics can be used 

 for concentration of protozoan cysts, and 

 are necessary for the concentration of 

 trematode, acanthocephalan and some 

 tapeworm eggs, which sink to the bottom 

 of the solutions used in the flotation tech- 

 nics. A few protozoan cysts such as 

 those of Eimeria leuckarli also sink to 

 the bottom. 



Since they are essentially washing 

 processes, sedimentation technics may 

 not concentrate cysts and eggs as much as 

 flotation technics. Many different sedi- 

 mentation technics have been developed. 

 The two described below appear to be 

 among the best. 



Formalin-Triton-Ether (FTE) 

 Sedimentation Technic 



This technic was introduced by 

 Ritchie (1948) and modified by Maldonado, 

 Acosta-Matienzo and Velez-Herrera (1954). 

 The latter considered it the nearest to an 

 all-round diagnostic procedure, since it is 

 highly effective -for the detection not only 

 of schistosome, hookworm, whipworm and 

 ascarid eggs but also of protozoan cysts. 



1. Mark off a test tube at the 5 ml and 

 6 ml levels. 



2. Place 5 ml of 10% formalin con- 

 taining a drop of Triton NE in the 

 tube. 



3. Add 1 ml of feces. 



4. Break up the feces thoroughly with 

 a wooden applicator. 



5. Strain the suspension thru 4 layers 

 of cheesecloth into a 15 ml conical 

 centrifuge tube. Squeeze the cloth 

 to get out as much liquid as pos- 

 sible. 



6. Add 5 ml of commercial ether to 

 the suspension in the centrifuge 

 tube. Cover the tube with a plastic 

 coverslip and shake vigorously. 



7. Centrifuge (at 2000 r.p.m. in a 

 horizontal centrifuge with a radius 

 from the center to the tip of the 

 tube of 8 inches; if another type of 

 centrifuge is used, change the speed 

 of centrifugation accordingly) for 



1 minute after the centrifuge has 

 reached its terminal speed. 



8. Loosen the plug of detritus at the 

 formalin solution-ether interface 

 with an applicator stick, pour off 

 all the supernatant fluid rapidly, 

 and, holding the tube slightly in- 

 verted, clean its walls carefully 

 with a piece of clean, dry gauze. 

 This is done to prevent the liquid 

 and debris on the walls of the tube 

 from sliding down to the bottom 

 and diluting the sediment. 



