SECTION TWO 



Note: This can be prepared by simply dissolving 

 one Michrome buffer tablet in lOO ml. of distilled 

 water. 



D. Saline solution (0-85% sodium chloride) 



E. Neutral 10% formalin 



Technique: 



1. Pipette 0-667 ml. ^^^^ ^^ solutions A, B, and C into a 

 centrifuge tube. 



2. Remove 0-05 ml. of tumour ascites from peritoneal cavity 

 of host animals with a glass capillary tube, and immediately place 

 the fluid into the centrifuge tube containing the measured amounts 

 of solutions A, B, and C, as prepared at step i above. 



3. Shake the centrifuge tube gently to suspend tumour cells 

 diffusely in the medium. 



4. Incubate at 37° C. for two hours. 



5. Centrifuge for three minutes at 700 revolutions per minute. 



6. Decant the supernatant fluid. 



7. Wash the precipitate with solution D. 



8. (Optional) Add 2 ml. of solution E, as fixative, to the 

 precipitate in the centrifuge tube and stir with a glass rod. 



9. (Optional) After ten minutes' fixation, centrifuge for three 

 minutes at 700 revolutions per minute. 



Note: Fixation inhibits enzymatic activity. 



10. Using scrupulously clean slides, prepare smears of the 

 precipitate, or make squash preparations, with one drop each of 

 the precipitate and glycerine, and seal edges of the coverslips with 

 soft paraffin wax, petroleum jelly or Laktoseal. 



Results: 



Succinic dehydrogenase activity is demonstrated by the purple 

 pigment which is deposited intracellularly. 



Notes: 



(a) The author (Hirono) states that any lipid droplets present 

 intracellularly were always stained, and that when tumour cells 

 were incubated in a medium lacking sodium succinate, crystalline 

 precipitation was not observed. 



(b) For more detailed information and photomicrographs 

 readers are referred to the original paper. 



Reference: Hirono, I. (1957). 



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