Methods for Testing Stains 313 



1:2,000,000 for the others). Inoculate each set of the tubes containing broth and 

 diluted dye with the proper culture, using 0.5 ml. of the diluted culture (i.e. ap- 

 proximately 100 organisms) per tube. Mix each tube well and incubate at 37° C. 

 Examine for growth on the 1st, 2nd and 4th days. 



A satisfactory sample should show bacteriostatic action on all three organisms 

 sufficiently like that of the check sample so that the first dilution permitting growth 

 will not be more than two stages apart from it in the above series of final dilutions. 



Fast Gbeen FCF 



Identification: Fast green FCF is the disodium salt of p, p -dibenzyldiethyldi- 

 amino-p"-hydroxytriphenylcarbinol trisulfonic acid anhydride, C37H34O10N2S3- 

 Na2. Identification is by the following method: Dissolve 50 mg. in 250 ml. of 50% 

 alcohol. Dilute 3 ml. of this solution to 200 ml. with 50% alcohol. Read in Beckman 

 spectrophotometer. Absorption maximum 624-625 mju; ratio P— 15/P-[-15 is from 

 1.02 to 1.09. 



Method of Analysis: Dissolve 200 mg. of dye in 200 ml. of distilled water, add 3 g. 

 of sodium acid tartrate, heat to 70° C and titrate with 0.05 N TiCla. The end point 

 varies in color from yellow to reddish brown. The following data are used in cal- 

 culating the percentage of anhydrous dye in the original sample: 



Molecular weight 808.832 



Hydrogen equivalents per mol of dye 2. 



ml. of N/10 TiCls per gram of dye 24.727 



Samples of this stain should contain not less than 85% anhydrous dye. 



Biological Tests: Fast green is tested for use as a cytological counterstain. Sec- 

 tions of root tips or buds are fixed in Flemming's fluid or in craf and are stained in 

 1% aqueous safranin for from 30 to 50 minutes, rinsed in distilled water and differ- 

 entiated with 0.2% fast green in 95% alcohol until the chromatin and nucleoli re- 

 main red. The slides are then passed through absolute alcohol to xylene and moimt- 

 ed in balsam. Chromosomes and other chromatin bodies as well as nucleoli and 

 lignified walls should appear bright red, while the spindles, cellulose walls, and 

 cytoplasm are green. 



Light Gkeen SF Yellowish, C. I. No. 670 



Identification: Light green SF yellowish is the disodium salt of p, p -dibenzyldi- 

 ethyl-diaminotriphenylcarbinol trisulfonic acid anhydride, C37H34N209S3Na2. Iden- 

 tification is by the following method: Dissolve 50 mg. in 250 ml. of distilled water. 

 Dilute 5 ml. of this solution to 200 ml. with distilled water. Read in Beckman 

 spectrophotometer. Absorption maximum 629-634 mfji, ratio P-15/P-J-15 is from 

 1.04 to 1.09. 



Method of Analysis: Dissolve 200 mg. of dye in 200 ml, of distilled water, add 

 10 g. sodium acid tartrate, heat to 70°C. and titrate with 0.05 N TiCU. The end 

 point is a sharp change from light green to yellow. The following data are used in 

 calculating the percentage of anhydrous dye in the original sample: 



