LABORATORY DIAGNOSIS OF PROTOZOAN INFECTIONS 



387 



9. Add a drop of physiological salt 

 solution to the sediment to facil- 

 itate its removal. 

 10. Take up the sediment with a pipette 

 (a Stoll pipette works well), place 

 on a slide, add a coverslip, and 

 examine under the microscope. 



MIFC (Merthiolate-Iodine- Formaldehyde 

 Concentration) Technic 



This technic was introduced by Blagg 

 et cd. (1955) as a modification of the MIF 

 preservative stain. They found that the 

 MIFC technic was positive for protozoan 

 trophozoites in 74% of 110 positive human 

 fecal specimens as compared with 55% 

 for the MIF direct smear; it was positive 

 for 92% of 226 specimens containing pro- 

 tozoan cysts, as compared with 58% pos- 

 itive with the MIF direct smear. 



1. Prepare an MIF presei'ved fecal 

 specimen as described above 



(p. 379). 



2. When ready to examine, shake the 

 specimen vigorously for 5 seconds. 



3. Strain thru 2 layers of wet surgi- 

 cal gauze into a 15 ml centrifuge 

 tube. 



4. Add 4 ml cold (refrigerated) ether 

 to the centrifuge tube, insert a 

 rubber stopper, and shake vigor- 

 ously. If ether remains on top 

 after shaking, add 1 ml tap water 

 and shake again. 



5. Remove the stopper and let stand 

 for 2 minutes. 



6. Centrifuge 1 minute at 1600 r.p.m. 

 Four layers will appear in the tube: 

 (a) an ether layer on top, (b) a 

 plug of fecal detritus, (c) an MIF 

 layer, (d) the sediment containing 

 protozoa and helminth eggs on the 

 bottom . 



7. Loosen the fecal plug by ringing 

 with an applicator stick. 



8. Quickly but carefully pour off all 

 but the bottom layer of sediment. 



9. Mix the sediment thoroughly, pour 

 a drop on a slide, cover with 

 coverslip, and examine. 



PROTOZOAN CULTURE MEDIA 



NNN (Novy, MacNeal and Nicolle) Medium 



This medium was developed for the 

 cultivation of Leisli))iaiiia, but it can also 

 be used for trypanosomes of the lewisi 

 group. 



1. Measure or weigh out: 



Sodium chloride 6 g 



Agar 14 g 



Distilled water 900 ml 



2. Mix, bring to the boiling point, 

 and place in bacteriologic culture 

 tubes in 5 ml amounts. Sterilize 

 in the autoclave. This is the 

 medium base, and can be stored 

 in the refrigerator. 



3. To use, melt the agar in the tubes 

 and cool to 48° C. Add to each 

 tube 1/3 of its volume of sterile, 

 defibrinated rabbit blood. Mix 

 thoroughly by rolling the tube be- 

 tween the palms of the hands. 



4. Place the tube on a slant without 

 leaving a butt of medium at the 

 bottom, and allow to solidify. 

 This is best done in the refrigera- 

 tor or in ice, since more water of 

 condensation is obtained in this 

 way. (The protozoa develop best 

 in the water of condensation at the 

 bottom of the slant. ) 



5. Seal the tubes to prevent the water 

 of condensation from evaporating, 

 and incubate at 37° C for 24 hours 

 to test for sterility before inocu- 

 lating. 



6. Inoculate suspected material into 

 the condensation water and incu- 

 bate at 22 to 24° C. Transfer 

 cultures every week or two. 



Weinman's Trypanosome Medium 



This medium was developed by Wein- 

 man (1946) for the cultivation of Trypatio- 

 so)}ia gauibiense and T. rliodesiense. It 

 can also be used for other trypanosomes o 



