508 Mr Walter Gardiner, Methods for the Demonstration [Mar. 7, 



treatment. By this means also the rationale of the whole process 

 is considerably elucidated. 



(1) Killing and fixing are in all cases brought about by means 

 of Iodine. Small pieces of tissue are treated either with Iodine 

 water or more usually with a solution of Iodine dissolved in 

 Iodide of Potassium. For delicate tissues of small bulk a strength 

 of 1 or "2 °/o Iodine in "15 or "25 °/ Potassic Iodide in water is 

 sufficient, but for the average tissues a solution containing "5 °/ 

 Iodine in "75 °/ Potassic Iodide in water is necessary for rapid 

 penetration. The tissues may either remain in the Iodine solution 

 as required for cutting or may be preserved in a solution of 

 Thymol water. 



(2) Swelling. Sections having been cut in water are swollen 

 in sulphuric acid. The strength of the acid employed is essen- 

 tially dependent upon the character of the tissue ; 1 — 5 °/ being 

 sufficient for such tissue as that of Char a or the " leaf" of Mnium 

 undidatum, 5 °/ for Viscum album and 30 °/ for Aucuba Japonica. 



Solutions containing Picric or Acetic acid do not give satis- 

 factory results. My experiments appear to show that sulphuric 

 acid is for this particular method the swelling agent par excellence. 



(3) Staining. The sections are first mordanted with Iodine. 

 To this end they are treated for a short time with an Iodine 

 solution in 5 °/ sulphuric acid. The Iodine solution may contain 

 either "5 °/ Iodine in -75 °/ Potassic Iodide, or 1 °/ Iodine in 

 1*25 °/ Potassic Iodide. Meanwhile the staining mixture is 

 prepared, and must be freshly prepared for each occasion. Two 

 stock solutions are made up. The first is a 10 °/ solution of 

 Sulphuric acid and the second is a - 5 % or 1 °/ solution of 

 Pyoktanin or Gentian violet in water. The staining solution is 

 prepared by mixing equal parts (say 5 c.c.) of each, so that what 

 is produced is a '25 or -5 °/ solution of the Violet in 5 % 

 Sulphuric acid. A sufficient quantity having been poured out 

 into a watch-glass, the sections are taken out of the acid Iodine 

 solution, quickly passed through a solution containing 1 °/ o Iodine 

 and "15 % Potassic Iodide in 5 % Sulphuric acid, and then placed 

 in the solution of acid Violet. After some ten minutes have 

 elapsed they are taken out, and washed freely in water and may 

 be forthwith examined. 



(4) Intensifying. If the swelling and staining have been 

 properly conducted it will be found that the protoplasm and 

 "connecting threads" are stained blue, and that the cell wall 

 remains almost uncoloured. It is however a great advantage, 

 and frequently a necessity, to make the staining much more 

 pronounced, so that the threads may be the more easily observed. 



