308 Biological Stains 



hand, cresyl echt violet (Grubler) is a dye of unknown constitution. The method of 

 dentification is: Dissolve 50 mg. in 250 ml. of 50% alcohol. Dilute 5 ml. of this 

 solution to 200 ral. with 50% alcohol. Read in Beckman spectrophotometer. In the 

 case of the National Aniline product, the absorption maximum is 624-630 m^t; the 

 ratio P-15/P4-15 is from 0.99 to 1.05. Cresyl echt violet (Griibler), on the other 

 hand has absorption maxima at 550 m^t and 585 mfj., the ratio P-15/P+15 for first 

 maximum is 1.10. 



Method of Analysis: No method has yet been worked out for the German product. 

 In the case of the National Aniline product: Dissolve 100 mg. dye in 200 ml. of 50% 

 alcohol, add 10 g. sodium acid tartrate, heat to boiling and titrate with 0.05 A^ TiCla 

 to a greenish-yellow endpoint. The following data are used in calculating the per- 

 centage of anhydrous dye in the sample: 



Molecular weight 339.815 



Hydrogen equivalents per mol of dye 2. 



ml. of N/10 TiCla per gram of dye 58.856 



Samples of this stain should contain not less than 88% anhydrous dye. 



Biological Tests. Cresyl violet is tested on celloidin sections of central nervous 

 tissue fixed in 10% formalin or 95% ethyl alcohol. The sections are placed (either 

 with or without previous removal of the celloidin by means of equal parts of abso- 

 lute alcohol and ether) in 0.25% aqueous cresyl violet and heated until steaming. 

 After washing well in distilled water, they are differentiated from one to several 

 minutes in 95% ethyl alcohol, then for a few seconds longer in Gothard's solution, 

 and finally through two or more changes of the 95% alcohol, controlling the results 

 under a microscope and returning to Gothard's solution if necessary. If celloidin 

 was not removed before staining, it is now removed with equal parts of ether and 

 absolute ethyl alcohol. The sections are dehydrated in two changes of absolute 

 alcohol, cleared in xylene and mounted in balsam. A satisfactory sample should 

 show nerve cells and the nuclei of other cells. The cytoplasm of some glia cells 

 and fibroblasts may be faintly stained. The background should be clear and 

 colorless. 



Neutral Red, C. I. No. 825 



Identification: Neutral red is aminodimethylaminotoluphenazonium chloride, 

 C15H17N4CI. The method of identification is: Dissolve 50 mg. in 250 ml. of 50% 

 alcohol containing 1.25 ml. glacial acetic acid. Dilute 5 ml. of this solution to 200 ml. 

 with 50% alcohol containing 1 ml. of glacial acetic acid. Read in Beckman spectro- 

 photometer. Absorption maximum 539-542 m^i; ratio P— 15/P+15 is from 1.00-106. 



Method of Analysis: Spectrophotometric. Percent dye = D-peak X 129. D-peak 

 (color density at peak) measured on dye solution described under "Identification". 



For certification, samples of this stain must contain not less than 50% anhydrous 

 dye. 



Biological Tests: Neutral red is tested for two purposes: vital and supravital stain- 

 ing of protozoa; differential staining of living cells of blood and connective tissues. 



In testing for the first purpose, not only color intensity but relative toxicity and 

 the stability of aqueous solutions in sunlight are determined. Paramecium and ery- 



