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LABORATORY DIAGNOSIS OF PROTOZOAN INFECTIONS 



1. The base medium is Difco nutrient 

 agar (1. 5%), which consists of: 



Beef extract 3 g 



Bacto peptone 5 g 



Sodium chloride 8 g 



Agar 15 g 



31 R 



Dissolve in 1 liter distilled water, 

 bring to pH 7. 3, sterilize by auto- 

 claving. 



2. To prepare the culture medium, 

 heat the base medium to melt the 

 agar. Before it has resolidified, 

 add the following aseptically to 

 each 75 ml of the base: 



Citroted human plasma previously 



inactivated at 56 C for 30 minutes . 12. 5 mi 

 Human red cells 12.5 ml 



3. Dispense in Kolle flasks or slanted 

 in test tubes. Stopper with rubber 

 corks or seal with Parafilm to re- 

 tard drying. Store in the refriger- 

 ator until used. 



4. Inoculate with suspected material 

 and incubate at room temperature. 

 The trypanosomes grow on the 

 surface as small, rounded, color- 

 less, transparent, slightly raised, 

 glistening, moist-appearing colon- 

 ies 1 to 2 mm in diameter; they 

 are detectable in 5 to 10 days or, 

 exceptionally, in 3 to 4 weeks. 



Tobie, von Brand and Mehlman's 

 Trypanosome Medium 



This medium was developed by Tobie, 

 von Brand and Mehlman (1950) for African 

 trypanosomes. It consists of a solid slant 

 with a liquid overlay. 



1. Solid slant . Measure or weigh out: 



Bacto-beef (Difco) l.Sg 



Bacto-peptone (Difco) 2. 5 g 



Bacto-agar (Difco) 7. 5 g 



NaCl 4.0 g 



Distilled water 500 ml 



Mix the ingredients, dissolve by 

 bringing to the boiling point, adjust 

 to pH 7. 2 to 7. 4 with NaOH, and 



3. 



4. 



autoclave at 15 lbs. pressure for 

 20 minutes. 



Cool to 45° C, and add 1 part of 

 inactivated, citrated rabbit blood 

 to each 3 parts of the above base. 

 Place 5 ml amounts in test tubes, 

 slant, and allow to cool. If de- 

 sired, 25 ml amounts may be 

 placed in flasks. 



Fluid overlay (Locke's solution ). 

 Measure or weigh out: 



NaCl 8. g 



KCl 0.2g 



CaClj 0. 2 g 



KH2PO4 0. 3 g 



Glucose 2. 5 g 



Distilled water 1000 ml 



Autoclave at 15 pounds pressure 



for 20 minutes. 



Place 2 ml of the liquid overlay in 



each tube containing 5 ml of the 



base (or 10 to 15 ml in each flask), 



using aseptic technic. 



Inoculate with suspected material 



and incubate at 24 to 25° C for 10 



to 14 days. 



RES (Ringer's- Egg-Serum) Medium for 

 Enteric Protozoa 



This medium was first introduced by 

 Boeck and Drbohlav (1925). Many differ- 

 ent modifications have been proposed which 

 are as useful as the one described below. 

 The serum may be replaced by egg albu- 

 men, for instance, or the Ringer's solu- 

 tion by Locke's solution. 



The medium is essentially a coagu- 

 lated egg slant overlaid with a fluid nutri- 

 ent solution. 



A. Egg slant . 



1. Mix 12. 5 ml Ringer's solution 

 with each egg used. For best re- 

 sults, mix in a Waring blendor for 

 30 seconds. If a blendor is not 

 used, filter the mixture thru 

 cheesecloth. 



2. Place 2 ml amounts of the mix- 

 ture in cotton-stoppered test tubes. 



