LABORATORY DIAGNOSIS OF PROTOZOAN INFECTIONS 



389 



(Other standard closures for bac- 

 teriologic work can also be used. ) 



3. Place the tubes upright in a vac- 

 uum desiccator. Evacuate the 

 desiccator slowly. As evacuation 

 proceeds, the egg mixture begins 

 to bubble, and within 4 minutes a 

 dense foam of egg begins to climb 

 in the tubes. Stop the evacuation 

 before the cotton plugs become 

 wet, and allow the tubes to remain 

 in the evacuated desiccator for an 

 hour. The purpose of this treat- 

 ment is to remove the dissolved 

 air from the medium. If it is 

 allowed to remain, it will bubble 

 out during subsequent sterilization 

 and coagulation, roughening and 

 pitting the slant surface (Levine 

 and Marquardt, 1954). 



4. Release the vacuum, pack the 

 tubes in baskets, slant them in 

 the autoclave, and inspissate and 

 sterilize them simultaneously at 



15 pounds pressure for 20 minutes. 

 Best results are obtained when no 

 butt of medium is left in the tubes. 

 When this is done, 2 ml of fluid 

 makes a slant about 1. 5 inches 

 long in an 18 x 150 mm tube. 



B. Fluid overlay . 



1. Mix the following aseptically: 



Sterile Ringer's solution 500 ml 



Sterile 10% glucose solution 10 ml 



Sterile serum (horse, rabbit, cow, etc.). 10 ml 



2. Add sufficient fluid overlay to 

 each egg slant to cover the whole 

 slant. Aseptic technic must be 

 used thruout . Incubate at 37 ° C 

 for 2 days prior to inoculation to 

 test for sterility. 



Balamuth's Amoeba Medium 



This medium was developed by Bala- 

 muth (1946) for enteric amoebae, but it 

 can be used for other enteric protozoa as 

 well. 



1. Mix 288 g dehydrated egg yolk with 

 288 ml distilled water and 1000 ml 

 physiological salt solution. Mix 

 with a Waring blendor or similar 

 instrument until the suspension is 

 smooth. 



2. Heat over an open flame in the 

 upper part of a double boiler, 

 stirring constantly, for 5 to 10 

 minutes until coagulation begins. 



3. Continue heating over boiling water 

 in the double boiler for 20 minutes 

 until coagulation is complete. Add 

 160 ml distilled water to replace 

 water lost by evaporation. 



4. Filter thru a muslin bag. When 

 the bag cools, squeeze it gently to 

 obtain the maximum amount of fil- 

 trate. 



5. Add enough physiological salt solu- 

 tion to the filtrate to bring its vol- 

 ume to 1000 ml. 



6. Place 500 ml of filtrate in each of 

 2 Erlenmeyer flasks. Autoclave 

 at 15 pounds pressure for 20 min- 

 utes. 



7. Chill the flasks by refrigeration 

 overnight or in some other way. 



8. Filter while cold thru 2 layers of 

 Whatman qualitative filter paper in 

 a Buchner funnel, using negative 

 pressure. Pour the mixture thru 

 the funnel in small amounts, re- 

 placing the filter paper frequently. 



9. Add an equal volume of Balamuth's 

 buffer solution to the filtrate. 



10. Add 5 ml of crude liver extract 

 (Lilly, No. 408) to each liter of 

 medium. 



11. Dispense in 5 to 7 ml amounts in 

 tubes. 



12. Autoclave at 15 pounds pressure 

 for 20 minutes. 



13. Add a small amount of sterile rice 

 powder to each tube. Incubate for 

 24 hours at 37° C to test for ster- 

 ility. (If desired, the medium can 

 be stored in large flasks in the re- 

 frigerator after autoclaving; it can 

 be kept for a month or more with- 

 out deteriorating, but any sediment 

 which forms should be removed by 

 filtration before use. ) 



