30 CULTURE MEDIA 



fibrinated rabbit's blood. This medium is the standard one for the culture of cer- 

 tain trypanosomes and other protozoa. Under the designation N.N.N. medium 

 (Nicolle Novy MacNeal) Nicolle has modified the medium so that there is only salt 

 and agar in the base to which the blood is added instead of one containing meat ex- 

 tract and peptone. It is the Hb which seems essential in the culture of various 

 protozoa. Rogers used citrated salt solution, which was slightly acidified with citric 

 acid, in his culturing of Leishmania from the splenic blood of cases of kala azar. 

 Incubation at 22 C. 



ROW'S H^MOGLOBINIZED SALINE MEDIUM. 



Take 10 c.c. blood from rabbit's heart or arm vein of man, defibrinate the blood 

 and then add 10 volumes of distilled water to lake the cells (liberation of Hb). One 

 volume of this laked blood solution is added to two volumes of sterile 1.2% salt 

 solution. 



CULTURE MEDIA FOR TREPONEMATA. 



I. NOGUCHI formerly first inoculated material containing treponemata into the 

 testicle of rabbits, obtaining by this procedure a pure culture, after a few transfers 

 to the testicles of other rabbits. He now grows the organism directly from serum 

 from a chancre. Test-tubes 2 by 20 cm. are filled with 15 c.c. of a medium consist- 

 ing of 2 parts of 2% slightly alkaline agar to which when melted and cooled down 

 to 50 C. is added i part of ascitic or hydrocele fluid. At the bottom of the medium 

 in the tube is placed a fragment of fresh sterile tissue, preferably a piece of rabbit's 

 kidney or testicle. After the medium solidifies a layer of sterile paraffin oil is run 

 in so that it covers the solid medium to a depth of 3 cm. Tne material is inoculated 

 at the bottom of the tube with a capillary pipette. Incubation at 37 C. is carried 

 on for two weeks. The tissue acts by removing any oxygen that may be present in 

 the depths of the medium. Anaerobiosis is a necessary condition. Many specimens 

 of ascitic fluid are unsuited. 



II. Serum Agar of Muhlens and Hofmaim. Fill sterile test-tubes one-third 

 full with horse serum. This is sterilized on three successive days at 55 C. Then 

 add an equal amount of a 3% agar containing 0.5% glucose which has been melted 

 down and cooled to 50 C. The mixed serum agar is then kept at 55 C. for two hours. 

 Such tubes are inoculated as for ascitic agar rabbit tissue media and incubated under 

 anaerobic conditions, preferably in a flask from which the air has been exhausted and 

 the remaining oxygen absorbed as shown in the anaerobic bottle described and 

 illustrated in Fig. 7. 



WELLMAN'S PLACENTAL AGAR. 



Fresh human placenta is thoroughly ground up in a meat chopper, after first 

 washing out the blood by running sterile salt solution through the attached vessels. 

 To each kilo of the macerated placental tissue is added i liter of distilled water. 

 This mixture is allowed to infuse for forty-eight hours at refrigerator temperature, 

 after which it is passed through a No. N Berkefeld which has been previously tested 



