PYOCTANIN AND FLUORESCIN 509 



that produced pyocyanin or fluorescin only, and some that were completely achromo- 

 genic. Fordos (1860) obtained pyocyanin in long blue crystals from a solution in 

 chloroform. Wasserzug (1887) showed that its formation was prevented by several 

 substances, such as 5 per cent. KNO3, 8 per cent. KCIO3, 5 per cent, ammonium 

 tartrate, 5 per cent. NaCl, and by many disinfectants not strong enough to inhibit 

 the growth of the organism. Jordan (1899) studied 7 strains of Ps. pyocyanea ; 

 1 strain produced pyocyanin only, 5 both pyocyanin and fluorescin, and 1 fluor- 

 escin only. He found that the fluorescent pigment required for its formation both 

 phosphate and sulphate, while neither of these substances was necessary for the 

 production of pyocyanin. Both pigments are formed in suitable synthetic media. 

 In old cultures a black pigment sometimes appears ; this appears to be an oxida- 

 tion product of pyocyanin. A yellowish-brown pigment, which may also be found 

 in cultures, appears to be an oxidation product of the fluorescent substance. 

 Boland (1899), who worked with a solution of pyocyanin in chloroform, found 

 that it became yellow if exposed to sunlight. Apparently the chloroform was 

 broken up, and chlorine set free, which oxidized the pyocyanin to pyoxanthose. 

 He showed that pyocyanin was largely dissolved by HCl, which turned it red, and 

 pyoxanthose by 33 per cent. H2SO4, which turned it reddish-yellow. Turfitt (1936) 

 found that the production of both pigments was favoured by 1 per cent, glycerol, 

 of pyocyanin by 5 per cent, glycerol, and of fluorescin by asparagin. 



More recently Wrede (1930) has determined the constitution oi pyocyanin, and 

 shown that it can be synthesized by the organism from lactic acid and salts. 

 Chemically he regards it an entirely new type of dye, containing two pentavalent 

 nitrogen atoms, but this requires confirmation (Michaelis 1935). It affords, more- 

 over, the first instance in which phenazine derivatives have been found in nature. 

 Its empirical formula is C26H20N4O2. Wrede states that it dissolves poorly in cold, 

 but readily in warm, water, as well as in chloroform, nitrobenzol, pyridine, and 

 phenol. It is fairly resistant to acids and forms with them red-coloured salts. 

 To alkali, on the other hand, in the presence of oxygen, it is much less resistant 

 and is rapidly broken down. 



Turfitt (1937) obtained a preparation of fluorescin by growth of Pseudomonas 

 in synthetic liquid media and adsorption of the pigment on to " suma-carb," 

 followed by electro-dialysis. The final product, which had an ash value of less 

 than 0-4 per cent., was an amorphous greenish-brown powder, readily soluble in 

 water, phenol, and acetic acid, but not in other organic solvents. A dilute aqueous 

 alkaline solution showed a green fluorescence, becoming colourless and non-fluores- 

 cent on acidification. More concentrated alkaline solutions had a red colour and 

 exhibited an intense green fluorescence. In alkaline solution the pigment showed 

 a well-defined absorption band with its maximum at 410 // ; in acid solution 

 the band was less evident and was nearer the shorter wave-lengths. The empirical 

 formula was found to be C4H7O2N. 



Summarizing, we may say that Ps. pyocyanea forms two pigments : (1) Pyo- 

 cyanin ; a bluish-green pigment, soluble in both chloroform and water, from which 

 it can be obtained in long blue crystals. For its production neither phosphate 

 nor sulphate is required. (2) Fluorescin ; a greenish-yellow fluorescent pigment, 

 soluble in water but not in chloroform. For its production both phosphate and 

 sulphate are required. In old cultures it may be oxidized to a yellowish-brown 

 pigment. Ps. pyocyanea forms pyocyanin and fluorescin ; Ps. fluorescens forms 

 only fluorescin. Both pigments are themselves oxidation products of colourless 



