36 CULTURE MEDIA 



normal sodium carbonate solution and 20 to 25 grams agar. Prepare as for nutrient 

 agar and sterilize. To i part of this one-quarter strength meat infusion nutrient 

 agar, when melted and cooled down to 6oC., add twice its volume of defibrinated 

 rabbit's blood. This medium is the standard one for the culture of certain trypano- 

 somes 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 extract 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 22C. 



ROW'S H^EMOGLOBINIZED 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 consisting 

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

 SoC. 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. The material is inoculated 

 at the bottom of the tube with a capillary pipette. Incubation at 37C. 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. The tubes of Noguchi and Bronfenbrenner are shown 

 in Fig. 6. Bronfenbrenner uses a iH% a 8 ar instead of the 2% used by Noguchi. 



M'Leod and Soga have simplified Noguchi's method as follows: Take a test-tube 

 and fit a perforated rubber stopper which can be pushed down the tube. A piece 

 of glass tubing is passed through the stopper to project slightly into the test-tube. 

 The other end of the glass tube is drawn out into a capillary tube and bent over at 

 an acute angle. The test-tube is filled to K or % of its depth with neutral bouillon. 

 This is freshly boiled and when cool a piece of sterile tissue is dropped in. A strip 

 of sterile gauze is drawn through a glass bead and soaked in the material it is desired 

 to culture and dropped into the bottom of the tube alongside the fragment of sterile 

 tissue. Ascitic fluid is then run in to a point which would be reached by the bottom 

 of the rubber stopper. As quickly as possible push in the stopper and when the 

 fluid appears in the capillary tube seal off the end in a small flame. Material for 

 study can be obtained afterward by breaking off the capillary tip and introducing 

 a capillary pipette. 



