NICOTINIC ACID 



170 



NINHYDRIN REACTION 



Nicotinic Acid. Preliminary detection of it 

 or its amide by fluoresence microscopy 

 (Hirt, A. and Wimroer, K., Klin. Woch- 

 nesdir., 1939, 18, 705-767). Lasting 

 yellow fluorescence. 



Night Blue (CI, 731), a basic dye of light 

 fastness 4 gives beautiful blue-violet 

 coloration of plant tissues but fades 

 (Emig, p. 52). 



Nigrosin, water soluble (CI, 865) — gray 

 R, B, BB, indulin black, silver gray, 

 steel gray — Commission Certified. This 

 is a mixture. It has been used as a 

 counterstain for neutral red in colora- 

 tion of Nissl bodies by Bean, R. J., 

 Stain Techn., 1927, 2, 56-59, as a nega- 

 tive stain for bacteria, Treponema, etc. 

 See Picro-Nigrosin. 



Nile Blue A, see Nile Blue Sulphate. 



Nile Blue Sulphate (C 1. 913)— Nile Blue A 

 — This is an important oxazin dye for 

 which purity tests have been estab- 

 lished (Conn, p. 270). It was intro- 

 duced by Lorrain Smith as a fat stain. 

 Briefly the method is to stain fresh 

 tissues, or frozen sections of formalin 

 fixed tissues, for 10-20 min. in a cone, 

 aq. solution of Nile blue sulphate, to 

 differentiate in 1% aq. acetic acid, 

 wash in water and mount in glycerin. 

 He thought that the neutral fats {glycer- 

 ides) were thereby colored red and the 

 fatty acids blue, but Kaufmann and 

 Lehmann (C. and E., Virchow's Arch, 

 f. Path. Auat. und Physiol., 1926, 261, 

 623-648) came to the conclusion that the 

 method was valueless. However Lisson 

 (p. 202) was unimpressed by their 

 evidence. In his opinion the rose (or 

 red) color does signify the presence of 

 a nonsaturated glyceride whereas the 

 blue color is of no significance because of 

 its lack of specificity. He reported 

 that some mixtures of free fatty acids 

 remain uncolored; for those containing 

 saturated fatty acids non -coloration is 

 the rule; while some others, not con- 

 taining fatty acids, are colored. See 

 Lipids, tabular analysis. 



Nile Pink, fat stain prepared from nile 

 blue sulphate by boiling with dilute 

 sulphuric acid (Rettie, T., J. Path. & 

 Bact., 1931,34, 595-596). 



Ninhydrin Reaction. Berg's (W., Pfluger's 

 Arch., 1926, 214, 243-249) directions: 

 Fix tissues in 10% formalin, wash in 

 water. Boil section for 1 min. in 2 cc. 

 0.2% ninhydrin. Wash, mount in glyc- 

 erin or glycerin jelly. Amino acids, 

 polypeptidps and proteins blue or violet. 

 Romieu (M., Bull. d'Hist. Appl., 1925, 

 2, 185-191) employs a strong solution 

 heated less. See Giroud (A., Proto- 

 plasma, 1929, 7, 72-98). 



Details are given by Serra, J. A., 

 Stain Techn., 1946, 21, 5-18. He ad- 



vises that the tissue first be hardened 

 by fixation for an unspecified time in 2 

 parts 95% alcohol and 1 part commercial 

 formalin (40% formaldehyde) plus 

 "some drops" of glacial acetic acid in 

 10 cc. of the mixture. After this it is 

 well washed in running water and in aq. 

 dest. before the frozen sections are 

 made. He also gives a method for 

 paraffin sections. 



The reaction consists of immersing 

 the sections or fresh materials in equal 

 volumes of 0.4% aq. triketo-hydrinden- 

 hydrate (ninhydrin) and phosphate 

 buffer pH 6.98. The ninhydrin solution 

 must be freshly prepared and the phos- 

 phate buffer not too concentrated. For 

 the latter he suggests 6 cc. M/15 solu- 

 tion secondary sodium phosphate 

 (11.1876 gm. Na2HP04-2H20 per liter) 

 and 4 cc. M/lo primary potassium phos- 

 phate (9.078 gm. KH2PO4 per liter). 

 The reaction is carried out in a covered 

 glass container placed on a boiling 

 water bath. This is allowed to stand 

 1-2 min. in the vapor after it has 

 reached the boiling point. A blue, or 

 violet, color developing while hot or 

 after cooling indicates the presence of 

 amino acids, fre, or bound in peptides, 

 or proteins. 



For microscopic examination mount 

 in pure glycerin squeezing if necessary. 

 The edges can be cemented by using a 

 mixture of 80 gm. coUophonium and 

 20 gms. heated lanolin as reconxmended 

 bj^ Romeis but they must be studied the 

 same day for the color fades quickly. 



Serra cai-efuUy states that the reac- 

 tion is given, not only by all amino 

 acids except proline and hydroxypro- 

 line, by peptides and proteins but also 

 by other compounds such as amines, 

 aldehydes, sugars with free aldehyde or 

 keto groups and by ammonia and am- 

 monium salts. "However, with com- 

 pounds other than amino acids and 

 proteides, the reaction is much less 

 sensitive and sometimes it gives a more 

 reddish color. In general it is easy to 

 exclude the possibility of these com- 

 pounds being present, by their solubil- 

 ity and localization. It must also be 

 remembered that the intensity of the 

 ninhydrin reaction varies according to 

 the nature of the amino acid and the 

 binding of this in the peptides. 



"The coloring formed during the 

 reaction can diffuse and be absorbed 

 by several cell structures. This com- 

 monly happens when the heating is 

 exaggerated and- when compounds easily 

 soluble are present, for instance after a 

 weak fixation. It is, therefore, recom- 

 mended to employ fixatives which 

 harden the tissues, as we have said 



