112 CYTOCHEMISTRY OF PROTEINS 



1. Reaction with dinitrofluorobenzene. 



2. Reduction of the nitro groups to NH2 with chromous sulfate. 



3. Diazotisation of the NH2 groups. 



4. Combination with K acid or dithiol. 



5. Combination with silver or lead. 



N0 2 1 N0 2 



PrH -> Pr— /~ ^\N0 2 -> Pr/~ ~\nK 2 



NO 



2 



Pr< >N=N-OH 



NOo SH SH 



SO3H SO3H 



Pr<T ^N=N<^ y Pr<^ ^>N=N 



ch 3 x/V 



OHNH 2 



i t 



Pb Ag Ag 



S0 3 S0 3 



NOo S S 



Pi/ \N=N<^ y Pr<^ 



CH 3 



OHNH 2 



This technique was applied to spermatozoa. The mean opac- 

 ity of the middle piece of bull spermatozoa, before treatment, was 

 0.825. After the treatment via K acid and silver the opacity- 

 was increased to 1.21, and after the treatment via dithiol and 

 lead the scattering power was 1.20. From these increases it was 

 calculated that between 8 percent and 14 percent of the middle 

 piece consisted of material able to react with dinitrofluoroben- 

 zene. 



Potentially, most of the methods outlined above could be modi- 

 fied for use in the electron microscope. But there are many diffi- 

 culties to be overcome, from the points of view of technique of 

 staining, of interpretation, and of accuracy of estimating scatter- 

 ing power. 



