NITRATES 



231 



NITROPRUSSIDE REACTION 



Some of these are given under Gallo- 

 cyanin, Gallamin Blue and Carbol- 

 Fuchsin. See also the methods of 

 Huber, Johnson and King and buffered 

 thionin (Windle, W. V., Rhines, R. and 

 Rankin, J., Stain Techn., 19-13, 18, 77- 

 86). An apparatus has been devised 

 apparently suitable for obtaining the 

 Absorption Spectra of Nissl bodies. 



Nitrates. Atake frozen sections of fresh 

 tissues. Cover section on a slide with 

 1-2 drops hot 10% "Nitron" in 5% aq. 

 acetic acid. Place in refrigerator 30 

 min. to permit nitrates to crystallize 

 and e.xamine in polarized light. Nitron 

 is 1 , 4-Diphenyl-3 , 5-endanilo-dihydro- 

 triazol. It precipitates nitrates as in- 

 soluble salts (Cramer, G., Zbl. allg. 

 Path., 1940, 74, 241-244). 



Nitrazine — nitrazine yellow, delta dye in- 

 dicator — An acid mono-azo dye sug- 

 gested as substitute for ponceau de 

 xylidine in Masson's Trichrome Stain. 



Nitrazine Yellow, see Nitrazine. 



Nitrocellulose for imbedding. Low 

 viscosity nitrocellulose ("Hercules Pow- 

 der Co.'s R.S. 0.5 second nitro- 

 cellulose") does not require to be washed 

 as in the case of celloidin. First add 

 absolute alcohol, break up lumps and 

 add ether. Use 100 gms. nitrocellulose, 

 100 cc. absolute alcohol and 140 cc. 

 anhydrous ether. For evaporation a 

 large surface is required in proportion 

 to depth. A precision microtome is 

 needed for sectioning blocks after first 

 hardening in 70-80% alcohol. Blocks 

 are cut both dry and wet. Serial 

 sections 4 microns thick are obtainable 

 whereas in celloidin the minimum is 

 about 12 microns. Since low viscosity 

 nitrocellulose (L.V.N.) is more readily 

 dissolved than celloidin by absolute 

 alcohol the use of butyl alcohol between 

 95% alcohol and xylol is suggested 

 (Davenport, H. H. and Swank, R. L., 

 Stain Techn., 1934, 9, 137-140). 



Nitro Dyes. Chromophore-N02. All 

 strongly acid. Aurantia, martius yel- 

 low, picric acid. 



Nitro Reaction to distinguish between pyr- 

 rols and indols. Treat preparation with 

 a mixture of sulphuric and nitric acids 

 (equal parts). Substances containing 

 the benzene ring (and among them 

 indol compounds) are nitrified and 

 recognizable by their canary yellow 

 color whereas the pyrrols are not 

 nitrified (Lison, p. 162). See Lison, 

 L., J. Physiol, et Path. G6n., 1933, 31, 

 82-99). 



Nitrogen. The titrimetric method of 

 Bruel, D., Holter, H., Linderstr0m- 

 Lang, K. and Rozlts, K., C. rend. trav. 

 lab. Carlsberg, S^r. chim., 1946, 25, 

 289-324 is recommended. 



Nltroprusside Reaction for Glutathione. 



1. IVIattei and Dulzetto (Atti. e. rend, 

 della Accad. dei Lincei, 1928, 8, 317). 

 Fix in 20% trichloracetic acid. Treat 

 frozen sections 3-4 min. with a fresh 

 solution of sodium nitroprussiate. After 

 quickly drying expose to NII3 vapor. 

 Freeze solidly with ice or solid CO2. 

 Examine frozen on slide at 5°C. The 

 violet color of sulphydryl rapidly disap- 

 pears. 



2. Joyet-Lavergne (Ph., Bull. d'Hist., 

 1928, 5, 331-349) Method 1 : apply to 

 tissue 1 drop 5% aq. sodium nitroprus- 

 siate, then 1 drop ammonia and examine 

 immediately. Method 2: before apply- 

 ing reagent as above he uses a stimulant 

 10% aq. potassium cyanide, 5 min.; or 

 2% aq. sodium sulphite, 10 min., or sat. 

 ammonium sulphate, 15 min., or tri- 

 chloracetic acid, 2-5 min. Method 3 for 

 fixed tissues: fix several hours in abs. 

 ale. or in formol 15 cc. + physiological 

 saline sol. 75 cc. Tease tissue or make 

 frozen sections. Stimulate with potas- 

 sium cyanide or ammonium sulphate. 

 Then apply reagent. 



3. Giroud and Bulliard (A. and H., 

 Protoplasma, 1933, 19, 381-384). Apply 

 to fresh teased tissues or frozen sections 

 10% aq. sodium nitroprussiate alka- 

 linized by about 2% ammonia. Fix the 

 color by treatment for several seconds 

 with 5% aq. zinc acetate. Dehydrate, 

 clear and mount in balsam in the usual 

 way. The violet color becomes red but 

 lasts some time especially if kept in ice 

 box. The same technique is possible 

 after alcohol fixation. 



Lison (p. 135) has considered the spec- 

 ificity of these reactions and recom- 

 mends analysis given in an article by 

 Rapkine contained in the last edition 

 of Langeron's Precis de Microscopie. 

 For fresh tissues (pieces, smears, frozen 

 sections) (a) Glutathione reduced. Add 

 to tissue on slide 1 drop 5% sodium 

 nltroprusside for plants, 2% for animals. 

 Add_ a reinforcer such as sat. aq. am- 

 monium sulphate or crystals, then drop 

 of ammonia. Red or violet color, (b) 

 Glutathione total. Treat tissue with 10% 

 cyanide of potassium, 5-10 min. Then 

 (a), (c) SH radicals fixed to proteins. 

 10% trichloracetic acid 15 min. Wash 

 in much water. Repeat several times. 

 For fixed tissues avoid employing 

 absolute alcohol or trichloracetic acid. 

 Use instead formol-saline (above). 

 Then follow as for fresh tissues. Fix 

 colors with zinc acetate as described. 



Bourne (G., Austral. J. Exp. Biol. & 

 Med. Sci., 1935, 13, 238-249) puts frozen 

 sections into hot 5% aq. acetic acid 

 30-90 sec. ; drains off the acid ; adds 5% 

 sodium nltroprusside (saturated with 



