Methods for Testing Stains 303 



Biological Tests: Thionin is tested as a stain for frozen sections, also in Stoughton's 

 method for sections of plant pathological material. For frozen sections the dye is 

 prepared in a 0.5% solution in 20% ethyl alcohol. The sections, immediately after 

 cutting, are immersed in water and placed briefly (1 to 2 minutes) in the stain, then 

 washed and mounted in water for examination. Good differentiation of the nuclei is 

 regarded as the criterion of a satisfactory sample. In the Stoughton method, plant 

 pathologic material is fixed in one of the usual botanical fixatives, paraffin sections 

 are stained for 1 hour in 0.1% thionin in 5% aqueous phenol, dehydrated in succes- 

 sively stronger alcohols and differentiated in a saturated solution of orange G in 

 absolute alcohol for 1 minute. They are then washed in absolute alcohol, cleared in 

 xylene and mounted in balsam. Fungal parasites should be violet to purple; cell 

 walls, yellowish or green; lignified tissue, blue; host nuclei, blue with purple nucleoli; 

 chromosomes, deep blue; and nuclei of the fungal hyphae or spores, deep purple. 



Methylene Blue, C. I. No. 922 



Identification: Methylene blue is tetramethyldiaminodiphenazthionium chloride, 

 (tetramethyl thionin) CieHigNsSCl. This dye is sometimes marketed for textile 

 purposes as the zinc chloride double salt, but the dye in this form is not recom- 

 mended for use as a biological stain. It is identified as follows: Dissolve 50 mg. in 

 250 ml. of distilled water. Dilute 3 ml. of this solution to 200 ml. with distilled 

 water. Read in Beckman spectrophotometer. Absorption maximum 665 ni/j.; ratio 

 P-15/P+15 from 1.37 to 1.70. 



Method of analysis: The dye content is" determined spectrophotmetrically cal- 

 culating from the formula: Percent dye = D-peak X 127. D-peak (color density 

 at peak) is measured on dye solution as described under "Identification". 



For certification, samples of this stain must contain not less than 82% anhydrous 

 dye. 



Biological Tests: Methylene blue is tested for histological and bacteriological 

 staining and as a constituent of Wright's blood stain. As an histological stain it is 

 tested on paraffin sections of tissue fixed in Zenker's fiuid, with phloxine as a coun- 

 erstain, by the procedure given on page IA3- 20 of Staining Procedures. A good 

 sample should show good nuclear staining without removing the phloxine from the 

 cytoplasm. 



As a bacteriological stain it is tested for staining the diphtheria organism and for 

 staining bacteria in milk. For the former purpose smears from a throat culture of a 

 case of diphtheria are stained in three different solutions of the sample under ex- 

 amination; namely Loeffler's formula (methylene blue, 0.3 g.; 95% ethyl alcohol, 

 30 ml.; 0.01% KOH, 100 ml.), alcoholic aqueous methylene blue (same, but with 

 distilled water in place of 0.01% KOH), and also in a 1% aqueous solution of the 

 dye. The solutions are applied to the preparation for a few seconds which is then 

 washed in tap water and examined under the microscope to see if the typical barred 

 or granular structure shows. A good sample should show this typical staining with 

 all three solutions. 



For staining bacteria in milk, 0.01 ml. is placed on a microscopic slide and smeared 

 over an area of 1 sq. cm. with a stiff needle, dried with gentle heat on a level surface, 



