318 Biological Stains 



stain should show red chromosomes and nucleoli, while metabolic chromatin should 

 stain a deep purple on an orange cytoplasm. 



(3) In the test with iodine, paraffin sections of root tips or buds (fixed in Nava- 

 shin's fluid or one of its modifications) are stained for 3 to 10 minutes in a 1% aque- 

 ous solution of the dye, rinsed in distilled water, and treated for 15 to 50 seconds in 

 equal parts of 1% iodine in 80% alcohol and 1% potassium iodide in 80% alcohol. 

 They are next rinsed in absolute alcohol and differentiated in clove oil and cleared 

 in xylene. By this technic, chromatin and nucleoli should stand out a deep violet 

 on a colorless protoplasm. 



(4) In testing for its bacteriostatic action, the same methods are followed as in the 

 case of brilliant green (p. 312) except that the dilutions for the spore-former should 

 be 1:1,000,000 to 1:40,000,000 and for the others 1:5,000 to 1:250,000. 



Methyl Green, (Ethyl Green) C. I. No. 685 



Identification: This methyl green is the zinc chloride double salt of ethylhexa- 

 methylpararosanilin chlorobromide, C27H35N3ClBr-|-ZnCl2. Identification is by the 

 following method: Dissolve 50 mg. in 250 ml. of distilled water. Dilute 10 ml. of 

 this solution to 200 ml. with distilled water. Read in Beckman spectrophotometer. 

 Absorption maximum 630-631 m/x; ratio P-15/P4-15 is from 0.91 to 0.95. Dilute 

 solution just prior to reading because of instability of solution. Take peak readings 

 first then ratio points as rapidly as possible. 



Method of Analysis: Dissolve 200 mg. of dye in 100 ml. of 95% alcohol, add 75 

 ml. of distilled water and 25 ml. of 30% sodium tartrate solution, heat to boiling 

 and titrate with 0.05 N TiCla to a greenish-yellow endpoint. The following data are 

 used to calculate the percentage of anhydrous dye in the sample: 



Molecular weight 652.241 



Hydrogen equivalents per mol of dye 2. 



ml. of N/10 TiCla per gram of dye 30.617 



For certification, samples of this stain must contain not less than 65% anhydrous 

 dye. 



Biological Tests: Methyl green is tested in staining gonorrhoeal smears by the 

 Pappenheim-Saathof stain. The smears from pus are prepared and dried in the air. 

 They are stained 15 seconds without heat in the methyl-green-pyronin stain 

 (methyl green, 1 g.; pyronin Y or B, 0.25 g.; 95% ethyl alcohol, 5 ml.; glycerol, 20 

 ml.; 2% aqueous phenol, 100 ml.) washed in distilled water, blotted dry and ex- 

 amined. The cocci should be bright red and the nuclei green (not purplish). 



It is also tested as counterstain for Bismarck brown by the procedure given on 

 page 303. With a good sample, all the nuclei of the cells stain green. 



Acid Fuchsin, C. I. No. 692 



Identification: Acid fuchsin should be the disodiima salt of the trisulfonic acid of 

 rosanilin or pararosanilin or mixtures of these two. Some samples may contain the 

 mono or disodium salts of the disulfonic acid or the trisodium salt of the trisulfonic 

 acid, but if such is the case they are not nearly as satisfactory as those which con- 



