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



(2) Methods for the Demonstration of " Connecting Threads " 

 in the Cell Wall. By Walter Gardiner, M.A., Clare College. 



(Received 7 March, 1898.) 



I have already published in the Proceedings of the Royal 

 Society 1 a brief account of a new method for demonstrating the 

 " connecting threads " which traverse the walls of plant cells. 



In the present paper I propose to make some further remarks 

 upon that method, which may be called the " Kolossow-Safranin 

 method," and also to describe a second means of investigation, 

 which may be briefly described as the " Iodine-Acid- Violet 

 method." 



A. The Kolossow-Safranin Method. 



1. Killing and swelling. In this method the two processes 

 are accomplished simultaneously either with watery Picric acid, 

 Picro-sulphuric acid or Picro-acetic acid, and in certain cases with 

 Sulphuric acid. With refractory tissue, treatment with Picric acid 

 may be supplemented by that of Picro-sulphuric acid. Picro- 

 acetic acid was used with success in the investigation of the 

 tissue of ferns. 



2. Fixing, is effected with Kolossow's mixture of Uranium 

 nitrate and Osmic acid, either of normal strength or diluted with 

 once or twice its own volume of water 2 . Hermann's mixture also 

 gives good results, but Flemming's mixture and fixatives con- 

 taining chromic acid were found to be unsuitable. 



3. Preservation. After fixing, the material may be preserved 

 (apparently indefinitely) in a solution of Thymol water containing 

 •5 grms. Thymol to 1 litre of water, and sections may be taken as 

 required. 



4. Staining, is accomplished either with a saturated aqueous 

 solution of Safranin or a similar solution containing 2 °/ alcohol 

 and 1 °/ aniline. The sections after washing in water are treated 

 for some time with a 2 °/ solution of orange G., which dissolves 

 the dye from the cell wall and leaves the protoplasm and con- 

 necting threads stained. The threads may now be further stained 

 by substitution. In this direction Gentian violet gives generally 

 the most satisfactory results, especially if Gram's method of 

 intensification be employed. Staining with Gentian violet (as 

 with Safranin) affects the whole tissue, but the dye may again 

 be removed from the cell wall by the action of a 5 °/ solution of 

 acid fuchsin. In certain cases, e.g. the endosperm of Hordeum 



1 Gardiner. Roy. Soc. Proc, Vol. xxn. 1897. 



2 Kolossow. Zeit.f. Wiss. Mik.,v.i. 1888. 



