HYDROGExX ION INDICATORS 



160 



HYDROGEN ION INDICATORS 



19-27) employed phenol red to estimate 

 alterations in pH in the skin of tarred 

 mice during carcinogenesis. Chambers 

 and his colleagues have added pH indi- 

 cators to tissue cultures (R., Proc. Roy. 

 Soc, B, 1932, 110, 120-124) and have 

 injected them directly into individual 

 living cells (McClung, pp. 62-109). 

 The most enthusiastic investigator tx) 

 employ the phthalein and sulphon- 

 phthalein indicators is Rous (P., Sci- 

 ence, 1924, 60, 363: J.A.M.A., 1925, 85, 

 33-35, and many articles in J. Exp. 

 Med., 1925 to 1927). The literature is 

 extensive but scattered. There are 

 brief reviews by Rous (P., J. Exp. Med., 

 1925, 41, 379-411) and von Mollendorf 

 (W., Ergebn. Physiol., 1920, 18, 141- 

 306). See W. M. Clark in Simmons and 

 Centzkow 161-171. 



It is well to question the dependa- 

 bility of data upon pH of living material 

 as apparently indicated by vital staining 

 methods. Consider the ideal require- 

 ments for such a vita,l stain. It should 

 exhibit a sharp and pronounced color 

 change in the proper pH range. It 

 should be fairly soluble, readily dif- 

 f usable, strongly colored, of low toxicity 

 and stable in the organism (not readily 

 oxidized or reduced or precipitated by 

 tissue electrolytes). Of the many indi- 

 cators employed in analytical chemistry, 

 only a few meet these requirements. 

 Certain errors are to be guarded against 

 in their use. The "salt error" and 

 "protein error" are unavoidably pres- 

 ent. In the application of these vital 

 stains changes may take place that will 

 themselves cause a pH change. Among 

 them anesthesia, trauma, loss of carbon 

 dioxide from exposed tissues, interfer- 

 ence with blood supply, and postmortem 

 change deserve special mention. How- 

 ever crude though the methods may be, 

 these dye indicators are of value in pre- 

 liminary experiments or where no better 

 procedure is applicable. 



The indicator dyes of most promise are 

 certain of the phthalein and sulphon- 

 phthalein compounds. They are gen- 

 erally quite soluble, highly diffusable, 

 show marked color shifts and are fairly 

 constant in composition. The dye 

 solutions diffuse quickly when injected, 

 and quickly appear in the urine and 

 stools. For these reasons, fairly large 

 doses given intraperitoneally are more 

 suitable than subcutaneous injections. 

 But it is doubtful, according to Cham- 

 bers (personal communication), whether 

 the more soluble dyes actually penetrate 

 the walls of most cells. 



The following selection of indicators 

 is based upon the reports of Rous and 

 others, and upon experiments with mice 

 carried out at The Barnard Free Skin 

 and Cancer Hospital. Their chemical 

 names can be found in The Merck Index 

 or in any good textbook of chemistry. 

 Some are to be used in 1% aq. solutions, 

 others in sat. solutions in physiological 

 saline, litmus in either aqueous or agar 

 solution (Rous, P., J. Exp. Med., 1925, 

 41, 379), while the remainder, which 

 are acidic (the sulphonphthaleins and 

 methyl red), require to be converted to 

 their corresponding sodium salts be- 

 cause the latter are more soluble in 

 water. Consequently the proper equiv- 

 alent of sodium hydroxide must be 

 reacted with each compound. Rub up 

 0.1 gm. of the dry dye in a mortar 

 (agate, preferably) with the volume of 

 N/20 sodium hydroxide solution given 

 in cc. below the dj'^e in the table. Filter, 

 wash out the mortar with several small 

 portions of saline (0.9% NaCl) and make 

 all to a volume of 10 cc. For a mouse, 

 0.5-2.0 cc. of the dye solution should 

 be injected intraperitoneally. 



It should be emphasized that wher- 

 ever possible, the glass electrode should 

 be employed for direct measurement of 

 pH in biological fluids rather than indi- 

 cators. Micro electrodes, including 

 injection types, have been developed. 



Indicator 



Bromphenol blue* 

 3.0 N/20 NaOH 



Sodium alizarin 

 sulphonate 

 (Alizarin red) 

 1% aq. or sat. 

 in aaline 



Bromcresol green* 

 2.9 N/20 NaOH 



Methyl red* 

 7.4 N/20 NaOH 



HYDROGEN ION INDICATORS 



pH Range and Colors Value as Vital Stain 



yeUow*— 3.0 — 4.6— » blue Very strong stain, too far on acid side. 



yellow «- 3.8 — 5.0 -► pink 



yellow <— 4.0 — 5.6 — ► blue 

 red «- 4.2 — 6.3 -♦ yellow 



Very toxic, weak stain. 



Strong stain, persistent, well tolerated. 



Unstable in organism, weak stain, fixes on 

 lipoids. 



