390 



LABORATORY DIAGNOSIS OF PROTOZOAN INFECTIONS 



CPLM (Cysteine-Peptone-Liver Infusion - 

 Maltose) Medium 



This medium was developed by John- 

 son and Trussell (1943) for Trichomonas, 

 but it can also be used for other enteric 

 protozoa. 



A. Liver infusion . 



1. Mix the following thoroughly, using 

 a Waring blendor if available: 



Bocto liver powder 20 g 



Distilled water 330 ml 



2. Infuse for 1 hour at about 50° C. 



3. Heat with stirring at 80° C for 5 

 minutes to coagulate the protein. 



4. Filter thru a Buchner funnel. 

 About 320 ml of liver infusion are 

 obtained. 



B. Preparation of final medium . 



1. Mix the following, using a Waring 

 blendor if available: 



Cysteine monohydrochloride .... 2. 4 g 



Peptone 32.0 g 



Maltose 1.6g 



Agar 1.6 g 



Ringer's solution 960 ml 



2. 



3. 



4. 

 5. 



6. 



10. 



11. 



Add the liver infusion from A above. 

 Adjust the pH to 7.0 (approximately 

 20 ml of 1.0 N NaOH are needed). 

 Heat to dissolve the agar. 

 Filter thru cotton into a 2000 ml 

 flask. 



Add 0. 7 ml of 0. 5% methylene blue 

 solution. 



Place 300 ml amounts in 500 ml 

 Erlenmeyer flasks. 

 Autoclave for 15 minutes at 15 

 pounds pressure. 



Add 75 ml sterile inactivated serum 

 to each 300 ml flask. 

 Place 7 to 10 ml amounts aseptic- 

 ally in sterile, plugged test tubes. 

 Incubate for 2 days at 37° C to test 

 for sterility before use. 



BGPS (Beef Extract-Glucose-Peptone - 

 Serum) Medium 



This medium was introduced by Fitz- 

 gerald, Hammond and Shupe (1954) for use 

 in the diagnosis of Trilriclionio}ias foelits 

 infections, but it can also be used for other 

 trichomonads. 



1. Mix the following in a 3 liter flask: 



Difco beef extract 3 g 



Glucose 10 g 



Bacto peptone 10 g 



NaCl 1 g 



Agar 0.7 g 



Distilled water 1000 ml 



2. Dissolve by boiling. After cooling, 

 adjust the pH to 7. 4 with 1 . N 

 NaOH solution. 



3. Cover the mouth of the flask with 

 heavy paper and autoclave for 30 

 minutes at 15 pounds pressure. 



4. After cooling, add 20 ml inacti- 

 vated (at 56° C for 30 minutes) 

 beef serum aseptically, and mix 

 thoroughly. 



5. Dispense in 10 ml amounts into 15 

 ml culture tubes. Test for ster- 

 ility by incubating at 37° C for 2 

 days. 



6. Just before inoculation, add 500 to 

 1000 units of penicillin and 0. 5 to 

 1.0 mg of streptomycin to each ml 

 of medium, and mix thoroughly. 



7. Pipette the inoculum on the top of 

 the medium in such a way as to 

 minimize mixing. The trichomo- 

 nads migrate to the bottom of the 

 tube, while yeasts and molds tend 

 to remain near the top. Incubate 

 at 39° C for 3 to 5 days. To ex- 

 amine, remove a sample from the 

 bottom of the tube with a pipette. 



Diamond's Trichomonad Medium 



This medium was introduced by 

 Diamond (1957) for the axenic cultivation 

 of trichomonads. It can be used success- 

 fully for more species than other media. 



