Methods for Testing Stains 317 



Methyl Violet 2B, C. I. No. 680 



Identification: Methyl violet 2B is a mixture of the more highly methylated 

 fuchsins, principally pentamethylpararosanilin chloride, that is, the chloride of 

 pentamethyltriaminotriphenylcarbinol anhydride. For purposes of estimation the 

 formula C24H28N3CI is employed. The following method of identification is used: 

 Dissolve 50 mg. of sample in 250 ml. of 50% alcohol. Dilute 2 ml. of this solution 

 to 200 ml. with 50% alcohol. Read in Beckman spectrophotometer. Absorption 

 maximum 583-587 mn; ratio P-15/P+15 is from 1.07 to 1.18. 



Method of Analysis: Spectrophotometric. Percent dye = D-peak X 207. D-peak 

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



Samples of this stain should contain not less than 75% anhydrous dye. 



Biological Tests: Methyl violet 2B is tested by the same procedure as crystal 

 violet, C. I. No. 681. Results should be the same except that the violet color ob- 

 tained is of a redder hue. 



Crystal Violet, C. I. No. 681 



Identification: Crystal violet is the chloride of hexamethyfpararosanilin, that is, 

 hexamethyltriaminotriphenylcarbinol anhydride, C25H30N3CI. Identification is by 

 the following method: Dissolve 50 mg. in 250 ml. of distilled water. Dilute 2 ml. 

 of this solution to 200 ml. with distilled water. Read in Beckman spectrophotom- 

 eter. Absorption maximum 589-593 mju; ratio P— 15/P+15 is from 1.04 to 1.19. 



Method of Analysis: Spectrophotometric. Percent dye = D-peak X 209. D-peak 

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



For certification, samples of this stain must contain not less than 88% anhydrous 

 dye. 



Biological Tests: Crystal violet is tested in four different procedures: (1) the 

 Gram stain; (2) the Flemming triple stain; (3) followed by iodine in the staining of 

 cytological preparations; (4) for its bacteriostatic action. 



(1) In testing crystal violet for staining bacteria by the Gram technic, the sample 

 is made up in a 0.5% aqueous solution, without the use of anilin oil or any other 

 mordant, and bacterial smears are stained by the following procedure: Crystal or 

 gentian violet 1 minute, tap water 1-5 seconds, Lugol's iodine solution 1 minute, 

 wash in tap water and blot dry, 95% alcohol 30 seconds, 0.25% safranin solution 

 10 seconds, tap water 1-5 seconds. In making the test, a weakly Gram-positive 

 organism and a Gram-negative organism are tested; the former should appear 

 blue, the latter red. 



(2) In testing the sample in the Flemming stain, the following procedure is fol- 

 lowed: Root tips or buds, fixed in Flemming's fluid or craf, are embedded in paraf- 

 fin and sectioned. Sections are stained for 20 minutes to 1 hour in 1% aqueous 

 safranin, rinsed in distilled water (sometimes differentiated in weakly acidulated 

 alcohol and then rinsed in distilled water), stained from 2 to 30 minutes in a 1% 

 solution of the sample being tested, again rinsed in distilled water, dehydrated in 

 95% and absolute alcohol, differentiated in a 0.25% solution of orange G in clove 

 oil about 15, minutes, cleared in xylene and mounted in Canada balsam. A good 



