SYLLABUS 119 



Attempts for further purification of the active extracts have in 

 most cases not been made, probably because loss of activity is frequently 

 encountered. 



17a. Supplementary information on receptor spots, by W, Weidel 



Preparation of receptor material 



Bacteria from a 15 1. broth culture grown to near saturation are 

 spun dowTi in a Sharpies Supercentrifuge and resuspended in isotonic 

 Michaelis buffer (sodium veronal sodium acetate-HCl) of pH 8.2 

 (250 cc). A few cc of toluene are added and the suspension is kept at 

 37°C with slight aeration for 24 hours. After a few hours the pH is 

 controlled and readjusted to 8.2 if necessary. Toluene lost by aeration 

 has to be replaced. 



After centrifugation in an angle- centrifuge the clear, yellow super- 

 natant is discarded and the sediment washed thoroughly twice with 

 100% alcohol on the centrifuge. Two washings with 0.85% NaCl 

 follow in order to remove the alcohol. The sediment is resuspended in 

 a solution of trypsin (4 g) in 250 cc of Michaelis buffer of pH 8.2. 



Commercial trypsin is suspended in the amount of water required 

 for dilution of the concentrated buffer mixture, the suspension is 

 slightly acidified with N-HCl (Congo paper faintly purple) and 

 centrifuged. The decanted supernate is then adjusted to pH 8.2 

 wdth N-NaOH, combined with the concentrated buffer mixture and 

 the whole liquid used for resuspension of the bacterial sediment 

 mentioned above. 



Trypsin digestion is carried out at 37°C under toluene for 48 

 hours. After high speed centrifugation (12,000 rpm in Sorvall centri- 

 fuge) the clear supernatant is discarded and the sediment, which usually 

 consists of two layers of different appearance, is resuspended in 125 cc 

 of Michaelis buffer of pH 5.2; 2-3 mg of crystallized lysozyme and a 

 few cc of toleune are added and the mixture is then incubated at 37° C 

 for 24 hours. Following this treatment, the pH of the mixture is 

 switched to 8.2 with N-NaOH and a solution of 2 g trypsin in 50 cc of 

 water, purified as described above, is added. After incubation for 24-48 

 hours under toluene at 37° C some heavy material is removed by low 

 speed centrifugation (3,000 rpm) for 15 minutes. The decanted, grey- 

 ish, strongly light scattering supernatant is thereupon centrifuged at 

 high speed, yielding an almost clear supernatant and a grey paste, which 

 in strong light is completely transparent, showing a yellow-brown color. 

 This material is washed several times on the high speed centrifuge wdth 



