LABORATORY DIAGNOSIS OF PROTOZOAN INFECTIONS 



379 



for a few species, even longer). If it is 

 not desired to study the sporulated oocysts 

 immediately, the fecal suspension can be 

 transferred to a bottle and stored in the 

 refrigerator. The oocysts will remain 

 alive for several months or, in some spe- 

 cies, as long as a year. 



It is best to sporulate coccidian 

 oocysts before they have been subjected 

 to refrigeration, since in some species 

 (apparently a minority), refrigeration of 

 the unsporulated oocysts prevents subse- 

 quent sporulation altho it does not harm 

 sporulated oocysts. 



MIF (ME RTfflOLATE -IODINE - 

 FORMALDEHYDE) STAIN- 

 PRESERVATION TECHNIC 



This technic was first introduced by 

 Sapero, Lawless and Strome (1951) and 

 improved by Sapero and Lawless (1953). 

 It was designed especially to permit iden- 

 tification of human protozoan trophozoites 

 and cysts, but can also be used for hel- 

 minth eggs and for parasites of domestic 

 animals. It is simple and relatively cheap, 

 permits rapid (almost immediate) wet- 

 fixed staining of the smears, and preserves 

 the parasites so that feces can be collected 

 in the field or by untrained persons and 

 shipped to the laboratory for later diag- 

 nosis. There is no appreciable loss or 

 deterioration of parasites or cellular 

 exudates for 6 months or more. 



A. Direct Examination Technic for Fresh 

 Fecal Specimens . 



1. The MIF stain is composed of tinc- 

 ture of 1:1000 merthiolate No. 99 

 (Lilly), Lugol's solution (5%) and 

 40% formaldehyde solution (USP). 

 Since Lugol's solution is unstable, 

 it should be freshly prepared every 

 3 weeks, and the amount used 

 should be varied with its age. The 

 following amounts (in ml) are rec- 

 ommended: 



2. Place 1 ml of the stain (sufficient 

 for 25 to 30 fecal smears) in a Kahn 

 tube. Place some distilled water in 

 a second tube. Put a small caliber 

 medicine dropper in each tube. 



3. Place 1 drop of distilled water at 1 

 end of a slide. Add a drop of MIF 

 stain. Mix. 



4. Add a small amount of feces and mix. 

 Do not use too much feces, or fixa- 

 tion and staining will be poor. The 

 finished wet smear should be thin 

 enough so that the slide can be tipped 

 on edge without the coverslip sliding. 



5. Add a coverslip and examine at once. 

 If it is desired to examine the slide 

 later, ring it with petrolatum to keep 

 the preparation from drying out. 



B. Collection and Preservation of Fecal 

 Specimens in the Field for Subsequent 

 Examination in the Laboratory . 



1. Prepare the following stock MF 

 solution: 



Store in a brown bottle 



2. Measure 2. 35 ml MF solution into a 

 standard Kahn tube and stopper with 

 a cork. 



3. Measure 0.15 ml of 5% Lugol's so- 

 lution into another Kahn tube and 

 close with a rubber stopper. (Or 

 keep the Lugol's solution in a bottle, 

 and add the proper amount to the 

 MF solution just before adding the 

 feces in step 4 below. ) 



4. At the time the fecal sample is col- 

 lected, pour the MF solution into 

 the Lugol's solution. Within a few 

 seconds , add an amount of feces 

 equal to 2 medium -sized peas (about 

 0.25 g), and mix thoroughly with an 

 applicator stick. Do not use too 

 much feces. Stopper the tube and 

 set aside for future examination. 



5. To examine, draw off a drop of 

 mixed supernatant fluid and feces 

 from the top of the sedimented layer 

 with a medicine dropper and place on 

 a slide. Mix thoroughly, crushing 

 any large particles. Add a cover- 

 slip and examine. 



