304 Biological Stains 



dipped in xylene a few minutes to remove the fat, immersed in 90-95% ethyl alcohol 

 a few minutes to fix the smear to the slide. It is then dipped 2-4 times in methylene 

 blue solution (methylene blue, 0.3 g.; 95% ethyl alcohol, 30 ml., after dissolving, 

 mixed with 100 ml. distilled water), washed briefly in 90-95% ethyl alcohol until 

 the intense blue color changes to a faint tinge. (This decolorizing may be omivted 

 if the staining period has been made briefer, 10-15 seconds, or if a more dilute stain- 

 ing fluid has been employed). The slide is then dried and examined. A good sample 

 should not remove the milk smear from the slide and should show deeply stained 

 bacteria in a light blue background. 



In testing as a constituent of Wright's stain, the same procedure is followed as in 

 the case of eosin. 



Methylene Azure, C. I. No. 923 

 (azure a and azure b) 



Identification: Methylene azure is obtained by the oxidation of methylene blue, 

 which results in a mixture of di-, tri- and tetramethyldiaminodiphenazthionium 

 chlorides in varying proportions. The first-mentioned product, asymmetrical 

 dimethyl thionin or azure A is C14H14N3SCI; the second. Azure B or trimethyl 

 thionin, is C15H16N3SCI. They are identified by the following method: Dissolve 50 

 mg. sample in 250 ml. of distilled water. Dilute 3 ml. of this solution to 200 ml. 

 with distilled water. Read in Beckman spectrophotometer. Absorption maximum 

 of azure A is 622.5-632.5 m/x, of azure B, 647.5-654 m^; the ratio P-15/P+15 for 

 azure A is from 0.94 to 1.05, for azure B is from 0.94 to 1.12. 



Method of Analysis: Dissolve 100 mg. of dye in 175 ml. of distilled water, add 25 

 ml. of pH 4.0 acetate buffer (6 volumes of 50% NaAc '31120 + 4 volumes of glacial 

 acetic acid), heat to boiling, and titrate with 0.05 N TiCla to a yellow endpoint. 



If qualitative examination has shown the dye sample to be almost pure azure A 

 or almost pure azure B, the percentage of anhydrous dye in the original sample may 

 be determined from the following data. If the dye is found to be a mixture, the 

 actual dye content cannot be accurately determined. 



Azure A Azure B 



Molecular weight 291.793 305.819 



Hydrogen equivalents per mol of dye 2. 2. 



ml. of N/10 TiCla per gram of dye 68.541 65.398 



Samples of this stain must contain not less than 55% anhydrous dye. 



Biological Tests: Azure A is tested on paraffin sections of animal tissue fixed in 

 Bouin's or Zenker's fluid by means of Haynes' modification of French's stain. The 

 paraffin is removed by applying each of the following for 3 minutes: xylene, absolute 

 alcohol, 95% ethyl alcohol, and distilled water. The sections are stained for 5 

 minutes in 1.5% aqueous azure A, treated 5-10 seconds with absolute ethyl alcohol, 

 counterstained with a saturated solution of ethyl eosin in clove oil for 30 seconds, 

 then xylene, 10-30 seconds, xylene, two changes of 1-2 minutes each, and mounted 

 in xylene bal=<»m. Good nuclear staining is regarded as the criterion of a satisfactory 

 sample. 



