TRIETHYL PHOSPHATE 



359 



TRIPHEXYLTKTRAZOLIUM 

 CHLORIDE 



ponceau 2 R, 1.0 gm.; light green S F, 

 yellowish, 0.45 gm.; orange G, 0.75 gm.; 

 phosphotungstic acid C.P., 1.5 gm.; 

 phosphomolybdic acid, C.P., 1.5 gm.; 

 glacial acetic acid, 3.0 cc; ethyl ale, 

 50% up to 300 cc. Add acetic to alcohol 

 and put 50 cc. in each of 4 beakers. In 

 first dissolve acid fuchsin and ponceau, 

 in second light green, in third orange 

 and phosphotungstic acid, and in fourth 

 phosphomolybdic acid (the last named 

 by slight warming). Mix and use bal- 

 ance of alcohol to wash out contents of 

 beakers adding them to mixture. Stain 

 keeps well; can be obtained from Will 

 Corporation, Rochester, N. Y. See 

 colored plate by the author. 



Triethyl Phosphate in dehydration. Nelsen, 

 O. E., Stain Tech., 1945, 20. 131-132. 

 recommends the use of this compound 

 (C2H6)3P04) in histological technique, 

 as it displaces water in tissues readily 

 without shrinkage or distortion. Since 

 tissues may be transferred directly into 

 it from water, the tedious alcohol dis- 

 placement series in the paraffin tech- 

 nique is unnecessary. It is soluble in 

 the alcohols, benzene, ether, chloroform 

 and xylol. Nelsen reports excellent 

 results with smears following the tri- 

 ethyl phosphate method. Following 

 fixation and subsequent staining with 

 Feulgen, the smears are first transferred 

 to equal parts of water and triethyl 

 phosphate, then to triethyl phosphate 

 and finally into xylene before mounting. 

 Fast green may be dissolved in it if 

 counterstaining is desired. 



Trimethylcarbinol, see Tertiary Butyl 

 Alcohol. 



Triphenyltetrazolium Chloride, A Valuable 

 Reagent for Studies of Reducing Ac- 

 tivity in Living Organisms — Written 

 by Dr. Robertson Pratt, University 

 of California College of Pharmacy, 

 San Francisco. January 29, 1951 — In 

 aqueous solutions, 2,3,5-triphenyltet- 

 razolium chloride (TPTC) is colorless, 

 diffusible, and readily transported 

 through plant and animal tissues; but 

 in the presence of living tissue or of 

 some other reducing agents, an in- 

 soluble red formazan is precipitated 

 from solutions of TPTC. This is the 

 basis for use of this compound in histo- 

 physiology to determine sites of de- 

 hydrogenase or other reducing activity 

 in living organisms, including cultures 

 of bacteria (Pratt and Dufrenoy, Stain 

 Technol., 1948,23,137). The compound 

 has practical applications also outside 

 of the field of research (see below). 



TPTC has been used successfully in 

 concentrations ranging from 0.05 to 1.0% 

 in distilled water or in buffers within 

 the biologic range of pH values. The 



optimum concentration depends largely 

 on the kind and location of tissue under 

 investigation. Studying sites of re- 

 ducing activity in stalks of sugar cane, 

 where the compound had to be trans- 

 ported through a considerable distance 

 of vascular tissue and some adsorption 

 undoubtedly occurred on the way, 

 Dufrenov and Pratt (Am. J. Bot., 

 1948, 35', 333) found a 0.5% solution 

 satisfactory for demonstrating that 

 dehydrogenase activity was most pro- 

 nounced in the plasmodesmata and in 

 lipidic parts of the cytoplasm. 



In other investigations Pratt and 

 Dufrenoy (Antibiotics, 1949, Phila- 

 delphia, Lippincott; J. Bact., 1949, 57, 

 9) found 0.1% solutions of TPTC 

 ideal for studying the efTects of peni- 

 cillin and other antibiotics on the 

 dehydrogenase systems of bacteria 

 (Huddleson and Baltzer, Science, 1950, 

 112,651). 



Reduction of colorless TPTC to the 

 red, insoluble, formazan occurs over a 

 wide range of pH values. Reducing 

 sugars do not interfere with the reac- 

 tion in the normal biologic range of 

 hydrogen ion concentrations, since 

 they do not reduce TPTC below pH 

 11.0 (Mattson et al., Science, 1947, 

 106, 294). The apparent redox poten- 

 tial of 2,3,5-triphenyltetrazolium chlo- 

 ride is about —0.08 volt (Jerchel and 

 Mohle, Ber. deutsch. chem. Ges., 1944, 

 77-B, 591). Thus the compound can 

 act as an electron acceptor in reactions 

 involving dehydrogenases of pyridine 

 nucleotides (Jensen et al.. Science, 

 1951,113,65). 



Other tetrazolium compounds and 

 several derivatives of 2,3,5-triphenyl- 

 tetrazolium chloride have been used 

 in the same way as TPTC. One of the 

 most useful of these is 4,4'-bis(3,5- 

 diphenyl-2-tetrazolium)-biphenyl di- 

 chloride which is colorless in aqueous 

 solution and upon reduction by living 

 cells yields an insoluble, violet or 

 mauve formazan. Other agents which 

 may be used similarly are the 2,5- 

 diphenyl-3(p-iodophenyl), the 2,3-di- 

 (p-iodophenyl)-5 phenj'l, and the 

 2- (p-iodophenyl) -3- (p-nitrophenyl) -5- 

 phenyltetrazolium compounds. 



Applications of TPTC and similar 

 compounds outside the fields of histo- 

 physiologic and bacteriologic research 

 are in the practical testing of viability 

 of seeds, grains and yeasts; in the dairy 

 industry, for performing the Brucella 

 ring test (Wood, Science, 1950, 112, 

 86); and in the field of antibiotics, for 

 rapid microbiologic assaying of prepa- 

 rations of penicillin and other similar 

 agents. Triphenyltetrazolium chlo- 



