NINHYDRIN REACTION 



171 



NITRO REACTION 



above. To be sure that u secoiidu,ry im- 

 pregnation or adsorption of the coloring 

 has not taken place, the following test 

 may be executed : A small weight (some 

 milligrams) of a pure amino acid, such 

 as glycine, is dissolved in distilled 

 water; an equal volume of phosphate 

 buffer of pH 6.98 and a few drops of 0.4% 

 ninhydrin solution are added; it is 

 boiled slowly and cooled for 20-30 

 minutes. The ninhydrin employed 

 must be completely consumed — by addi- 

 tion of more amino acid solution. The 

 colored liquid of this reaction is now 

 used to immerse the pieces, with boiling, 

 etc., as for a ninhydrin reaction. If 

 then a certain structure shows a colora- 

 tion, this means that an absorption or 

 adsorption has taken place and a posi- 

 tive niuiiydrin reaction in the same 

 structure does not necessarily demon- 

 strate a proteic or amino acid nature." 

 Nissl Bodies (Tigroid bodies, chromophile 

 granules, chromidia, etc.) are masses 

 of basophilic material easily demon- 

 strable in the cytoplasm of most nerve 

 cells after a wide variety of fixations. 

 Certain types of nerve cells are char- 

 acterized by the shape, number, size 

 and distribution of their Nissl bodies. 

 Since, moreover, the Nissl bodies ap- 

 pear at a definite stage in the develop- 

 ment of the cells and undergo distinctive 

 modifications in physiological and path- 

 ological conditions there can be no 

 question that they represent material 

 present in vivo although they cannot 

 be distinguished as such in living nerve 

 cells. Bensley, R. R. and Gersh, I., 

 Anat. Rec, 1933, 47, 217-237 claim that 

 their discovery of well-formed Nissl 

 bodies, stainable with toluidin blue, in 

 sections of tissues frozen in liquid air 

 and dehydrated in vacuo while still 

 frozen is proof of the presence of Nissl 

 bodies in the living state. Wiemann, 

 W., Zeit. f . d. ges. Neurol, u. Psychiat., 

 1925, 98, 347-404 appears to have made 

 ultraviolet photomicrographs of Nissl 

 bodies, and a dense ash, revealed by 

 microincineration (Scott, G. H., Proc. 

 Soc. Exp. Biol. & Med., 1940, 44, 397- 

 398), corresponds with them topo- 

 graphically. 



The influence of fixation on the shape 

 (and perhaps to a slight degree on the 

 distribution) of Nissl bodies in nerve 

 cells has never been clearly defined. 

 It is known that the Nissl bodies are 

 much more pronounced after fixation in 

 95% alcohol, Zenker's fluid and Car- 

 noy's fluid than they are after fixation 

 in osmic acid, Altmann's fluid and 

 Regaud's fluid. Fixatives of the first 

 group also result in more stainable 

 particles in the nucleoplasm than those 



of the second. For other details see 

 Hopkins, A. E., Anat. Rec, 1924, 28, 

 157-163. Influence of staining is also 

 a factor to be reckoned with Ijecause 

 of the striking difference in appearance 

 of Nissl bodies when intensely and 

 lightly colored. There are many 

 methods from which to make a choice. 

 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., 1943, 18, 77- 

 86). An apparatus has been devised 

 apparently suitable for obtaining the 

 Absorption Spectra of Nissl bodies. 



Nitrates. Make 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 crj^stallize 

 and examine in polarized light. Nitron 

 is diphenyl-endo-anilo-dihydriazole. It 

 precipitates nitrates as insoluble 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-NO;. All 

 strongly acid. Aurantia, martins 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 



