HYDROGEN ION INDICATORS 



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HYDROGEN ION INDICATORS 



exhibit a sharp and pronounced color 

 change in the proper pH range. It 

 should be fairly soluble, readily dif- 

 fusable, 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 dye 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. 



HYDROGEN ION INDICATORS 



Indicator 



Bromphenol blue 

 3.0 N/20 NaOH 



Sodium alizarin 

 Bulphonate 

 (Alizarin red) 

 1% aq. or sat. 

 in saline 



Bromcresol green 

 2.9 N/20 NaOH 



Methyl red 

 7.4 N/20 NaOH 



Chlorphenol red 

 4.7 N/20 NaOH 



Bromcresol purple 

 3.7 N/20 NaOH 



Bromphenol red 

 3.9 n/20 NaOH 



Methyl violet 

 1% aq. or sat. 

 in saline 



Bromthymol blue 

 3.2 N/20 NaOH 



Phenol red 

 5.7 N/20 NaOH 



pH Range and Colors 

 yellow ♦- 3.0 — 4.6 -► blue 



yellow «- 3.8 — 5.0 -► pink 



yellow ■<— 4.0 — 5.6 — ► blue 

 red ♦- 4.2 — 6.3 -+ yellow 



yellow <— 4.8 — red — 6.8 -♦ purple 

 yellow *— 5.4 — 6.6 —» purple 



yellow *- 5.4 — red — 7.0 — ► purple 



blue- violet «- 6.0 — 7.0 -» violet 



yellow <— 6.0 — 7.4 -> blue 



yellow <- 6.6 — 7.8 -» red 

 (6.8 — 8.4) 



Remarks 

 Very strong stain, too far on acid side. 



Very toxic, weak stain. 



Strong stain, persistent, well tolerated. 



Unstable in organism, weak stain, fixes on 

 lipoids. 



Powerful stain, well tolerated. 



Strong stain but rapidly excreted, is toxic 

 and exhibits dichromaticism. 



Very strong stain, well tolerated. 



Weak stain, toxic. 



Weak stain, very toxic to mice, but not for 

 insects. 



Rapid, intense stain, very well tolerated . 



