294 Biological Stains 



experiments with this diluted titanium solution showed that it could be stored 

 under mineral oil for a normal working day without change, and for 20 hours with 

 approximately a 1-2% decrease in strength. In practice the solution was placed in 

 a burette equipped with a side reservoir, and both surfaces of the solution were 

 covered with J^ inch of mineral oil. A fresh solution was made daily and the solution 

 standardized against eerie sulfate as follows: Exactly 5 ml. of standard eerie sulfate 

 was placed in a 50 ml. Erlenmeyer flask, and 5 ml. of concentrated sulfuric acid 

 added. After adding a drop of ferrous-o-phenanthroline indicator, the solution was 

 titrated with the stored titanium solution. The initial yellow color of the solution 

 passes through a green stage, then nearly colorless, and finally a single drop of 

 Ti"*"*^ produces the orange color of the reduced indicator. The titration is rapid 

 and the endpoint exceedingly sharp. 



Dye Titration with Titanous Chloride 



Minor changes have been made in the dye titration procedure. Smaller amounts 

 of dye are used and a more dilute titanous chloride solution is employed than has 

 been recommended in "Biological Stains, 5th Ed." In the case of most dyes we 

 have preferred a strong acetate bufifer to maintain a more constant pH during the 

 titration, although in some cases the tartrate or bitartrate buffer has been retained. 

 We also prefer to use nitrogen gas washed through two titanium-chloride-safranin 

 bottles to maintain more anaerobic conditions and minimize autoxidation of re- 

 duced dyes during the titration. Following is a general description of the titanous 

 chloride assay as carried out in our laboratory. Specific variations will be noted 

 under the individual dyes. 



The titration is carried out in an ordinary "Mason" jar of about 350 ml. capacity 

 and a 23^ inch diameter mouth. A tight-fitting rubber stopper contains five holes 

 for the accommodation of a gas inlet, gas outlet, the burette extension tip, a ther- 

 mometer and a ground glass sleeve for the shaft of a stirring propeller. The titra- 

 tion jar is mounted on an asbestos covered hot-plate. Water-pumped tank nitrogen 

 is led through two successive gas-washing bottles* containing titanous chloride 

 safranin. The latter solution is made by diluting 40 ml. of 20% titanous chloride and 

 40 ml. of concentrated hydrochloric acid to 500 ml. with distilled water. After 

 filling the gas-washing bottles, approximately 10 mg. of safranin is added to each 

 bottle. This mixture is replaced by fresh solution every two or three days depending 

 on amount of use. 



The weighed dye sample is dissolved in the appropriate solvent in the titration 

 jar, and buffer added. The total volume of dye mixture is usually 200 ml. The 

 solution is stirred, heated if necessary, and the nitrogen passed through the jar for 

 at least fifteen minutes before the titration is started. Like most titrations, the 

 first run locates the approximate endpoint so that a second trial can be rapid and 

 the type of endpoint color change familiar. 



The per cent dye is calculated by the general formula: 



ml. TiCU used X normality of TiCU X mol. wt. of dye X 100 



per cent dye = : — 



Wt. of sample X No. of hydrogen equivalents X 1000 



General Approach to Examination of Individual Stains — Study of the individual 



*Fisher-Milligan Gas Washer Bottles. 



