APPENDIX II 

 METHODS FOR TESTING BIOLOGICAL STAINS 



In the examination of stains submitted to the Biological Stain Commission for 

 certification, procedures for testing them have been worked out for the most com- 

 mon stains and were originally published by Pederson, Conn and Melin (1933-4). 

 They may be regarded as assay methods used in evaluating dyes as biological stains. 



The original methods included determinations of light absorption by means of a 

 visual spectrophotometer; using that instrument, it proved most practical to ex- 

 press the color characteristics of a sample by a simple ratio of color densities at two 

 selected wavelengths. Later, the availability of a photoelectric spectrophotemeter 

 made it possible to characterize stains by complete absorption spectra and by exact 

 location of the absorption maximum. The same type of ratio has been retained, 

 however, as additional characterization. 



It has proved possible to assay (as to actual dye content) by spectrophotometry 

 those stains whose manufacture is apparently sufficiently standardized to result in 

 quite identical spectral characteristics. The color density at the maximmn absorp- 

 tion of such stains is easily related to the dye content as determined by other 

 methods. In the case of stains in which various samples show considerable variation 

 in the position of the absorption peak, it is impossible to establish the spectrometric 

 assay, and it has proved necessary to re-evaluate the titanous chloride method, in 

 which some changes have been made. 



The following pages give the methods in use at the time the present edition of 

 this book is going to press. See Stotz et al. (1950), from which much of the following 

 is quoted. 



GENERAL 



Spectral Characteristics 



A 50 mg. sample of the homogenous (powdered if necessary) stain is weighed to 

 the nearest 0.5 mg. on a Roller-Smith torsion balance. The sample is transferred 

 quantitatively to a 250 ml. volumetric flask and approximately 225 ml. of the 

 appropriate solvent added. The flask is then vibrated on a Boerner shaker^ for 20 

 minutes to insure complete solution of the dye. The solution is made up to the mark 

 with solvent, thoroughly mixed, and an appropriate aliquot taken for further dilu- 

 tion to provide a satisfactory dye concentration for spectrophotometric work. 



Maximum accuracy of weighing and insured complete solution of the dye is of 

 course dispensable if the spectrophotometric assay is not employed. 



Matched Corex cells of 1 cm. light path are employed in the Beckman DU 

 spectrophotometer, the reference cell containing the same solvent as the dye solu- 

 tion. Spectrophotometric readings are taken at least every 5 mju and usually at 

 smaller intervals in the area of the absorption maximum. An actual reading at the 

 exact peak is of course desirable for spectrophotometric assay. 



The older method of expressing the shape of the curve by the ratio of color den- 

 sities at two predetermined wavelengths loses its significance if the exact peak is 

 known, since any differences in the position of the peak necessarily cause a change 



^Arthur H. Thomas Co. 



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