294 MICROCHEMICAL TESTS 



Waugh {Bio. Chem., vol. 97, 1932). In 1932, Moore and Ray 

 showed that in scurvy the silver nitrate reducing property of the 

 adrenal disappeared. Finally, in July 1933, the Australian 

 investigator, G. Bourne, published the first cytological account of 

 vitamin C. The technique was improved by Giroud and Leblond 

 (Arch. Anat. Micr. 1934, C.R. Ass. Anat. Bruxelles, 1934). They 

 showed that this vitamin was localised in cells in connection with 

 the mitochondria and Golgi apparatus, and the questions thus 

 raised are of prime importance to cytologists. 



G. Bourne has carefully investigated the cytological technique 

 for this vitamin and recommends the following procedure : Fix 

 tissues in a solution of 5 per cent. AgNOg, to 100 c.c. of which 

 5 c.c. of glacial acetic acid have been added (modified solution of 

 Giroud and Leblond). For Protozoa, etc. smears, a few drops of 

 the solution are added to the liquid on the slide with the cells. 

 Examine after ten to fifteen minutes. Or better, isolate organisms 

 in centrifuge, treat en masse with solution, wash in pure glass 

 distilled aq. dest., treat in photographic " hypo." Wash several 

 times in aq. dest. upgrade in ethyl alcohol, clear, imbed and 

 section if necessary. The best results are obtained by carrying out 

 impregnation in darkness. This is important. 



For animal tissues Giroud and Leblond washed out the blood 

 from the dead animal with an isotonic solution of levulose, and 

 injected with acetic acid silver nitrate solution (10 per cent. 

 AgNOg, with the addition of 0*5 c.c. of acetic acid for each 100 c.c. 

 of solution). The silver nitrate was run out with distilled water, 

 followed by injection of " hypo." The hypo, was washed out, 

 pieces of tissue removed and sectioned by the usual wax 

 method. 



A. Bourne's Method for Simultaneous Demonstration of 

 Vitamin C and Fats. Cut fresh tissue by freezing technique. 

 Drop into slightly acid 5 per cent. AgNOg for a few minutes in the 

 dark. Wash in water for a few minutes, pass to Sudan in 90 per 

 cent, alcohol. Mount in glycerin jelly. 



Finally, it should be mentioned that vitamin C may exist in {A) 

 reduced form, reacting readily with AgNOg, and {B) reversibly 

 oxidised form not so reacting. The ordinary technique simply 

 demonstrates form (A). 



To get over this difficulty Bourne exposes pieces of tissue to 

 vapour of glacial acetic acid for some minutes, cuts thin slices and 

 leaves in atmosphere of hydrogen bisulphide for fifteen minutes. 

 This converts form {B) into form [A). AH trace of hydrogen bisul- 

 phide must be removed. This is done in vacuo until most of the 

 HgS has been sucked out of the tissue (fifteen to thirty minutes). 

 The sections are then exposed to a strong stream of nitrogen gas 

 for fifteen minutes. Before concluding that vitamin C is not 

 present the hydrogen bisulphide method should be used. Actually 



