LABORATORY DIAGNOSIS OF PROTOZOAN INFECTIONS 



391 



1. Mix the following: 



TrypticQse (BBL) 2.0 g 



Yeast extract l.Og 



Maltose O.Sg 



L-cysteine hydrochloride 0. 1 g 



Ascorbic acid 0.02 g 



KjHPO^ 0. 08 g 



KH2PO4 0. 08 g 



Distilled water 90 ml 



Adjust the pH to 6. 8-7. with 1 N 

 NaOH for all trichomonads except 

 T. vaginalis ; for this species, ad- 

 just the pH to 6. with 1 N HCl. 

 Add 0.05 g agar. 

 Autoclave for 10 minutes at 15 

 pounds pressure. 



Cool to 48° C, and add the follow- 

 ing: 



7. 



Sheep serum (inactivated at 56 C for 



30 min. ) 10 ml 



Potassium penicillin G 100, 000 units 



Streptomycin sulfate 0. 1 g 



(The penicillin and streptomycin 

 can be made up in 1 ml distilled 

 water beforehand. ) 

 Place 5 ml amounts of the medium 

 aseptically in sterile, stoppered 

 test tubes. Store in the refriger- 

 ator up to 14 days or longer. 

 Prior to inoculation, incubate the 

 tubes at 35. 5° C for 1 hour. 



All the trichomonads which Diamond 

 (1957) cultivated except T. gallinarum 

 and T. gallinarum -like species grew well 

 at 35. 5° C; the latter grew better at 38. 5° 

 C. 



It has been found in the author's lab- 

 oratory that the phosphates are not neces- 

 sary for the growth of T. foetus, T. suis, 

 T. gallittae, T. gallinarum and several 

 other species. 



RSS (Ringer' s-Serum-Starch) Medium 

 for Balantidium 



The following medium is slightly mod- 

 ified from that introduced by Rees (1927) 

 for the cultivation of Balantidium colt. 



1. Add 25 ml of horse, rabbit or 

 bovine serum aseptically to 500 ml 

 of sterile Ringer's solution. 



2. Tube in 8 ml amounts, using asep- 

 tic technic. 



3. To each tube add a 5 mm loop of 

 rice starch which has been ster- 

 ilized in a large test tube for 30 

 minutes at 15 lb pressure. 



4. Incubate at 37° C for 48 hours to 

 test for sterility. Store in the re- 

 frigerator. 



5. Before inoculation, warm the tubes 

 to 37° C by placing them in the in- 

 cubator. Incubate at 37 ° . The 

 protozoa grow in the bottom of the 

 tube. 



FORMULAE 

 Physiological Salt Solution 



NaCl 8. 5 g 



Distilled water 1000 ml 



D'Antoni's Iodine Solution 



Powdered iodine l-Sg 



1% aqueous KI solution 100 ml 



Allow to stand 4 days before use. This is 

 the stock solution and contains an excess 

 of iodine. Filter small amounts before 

 use. If tightly stoppered, the filtered so- 

 lution will keep 4 weeks before too much 

 iodine has volatilized for use. 



Lugol's Iodine Solution 



Potassium iodide 10 g 



Powdered iodine 5 g 



Distilled water 100 ml 



Dissolve the potassium iodide in the water 

 before adding the iodine. 



Mayer's Albumen Fixative 



Put the whites of several new-laid 

 eggs in a shallow dish. Whip them a little 

 with a fork or wire egg beater, 2 or 3 

 dozen strokes being sufficient. Do not 

 beat them until they are white and stiff. 



