We believe that the cellulose acetate method is destined to prove as successful with hard woody tissues as the Venetian turpentine method has proved with unicellular and filamentous forms; but, it must be confessed that investigators have been unsuccessful in their experience with cellulose acetate. Nevertheless, refractory material, like oak and even harder woods, has yielded thin transverse sections. Cellulose acetate does not injure the finer details of structure, and, on that account, will be superior to hydrofluoric acid, when students have solved the present uncertainties in regard to it. We have such confidence in the value of the method that we are quoting, in full, Mrs. Williamson's short account in the Annals of Botany for January, 1921, which is the only published information upon the subject.
In order to prepare sections of hard vegetable structures it is essential that some method should be devised by which the structure is not only embedded but softened, so that sections can be cut easily and smoothly. After various methods had been tried, the cellulose acetate method successfully used by Dr. Kernel for embedding and' sectioning the fabric of aeroplane wings was used. It was discovered that this method not only embedded hard vegetable structures, but also softened them so that sections are easily obtained. It proved best to use cellulose acetate of French manufacture made from pure cellulose, as the viscosity is more uniform than that of English manufacture, which is obtained from the cellulose of wood.
In the preliminary experiments pieces of oak and beech, cut into halfinch cubes, were passed through strengths of alcohol, then placed in pure acetone for two hours and finally into a 12 per cent solution of cellulose acetate in acetone. There they were left for two months and excellent sections were obtained. Further experiments showed that the passage through alcohols was unnecessary. In the final experiments the pieces of wood were placed in water and the air removed from them, after which they were put into pure acetone for 1 to 2 hours and finally into the solution of cellulose acetate. It was found that the length of time of immersion in the solution of cellulose acetate necessary for softening the tissues varied with the hardness of the wood, the minimum time for soft woods being two days; for woods such as oak and beech, at least six days are required. Experiments were tried with sal(Shorea robusta) and Pyingadu (Xylia dolabriformis), one of the Indian ironwoods, which is extremely hard. After fourteen days in the cellulose acetate solution it was possible to obtain transverse sections of these hard woods. The cellulose acetate solution is therefore capable of softening even the hardest wood in a relatively short time.
In order to stain sections-either hand or microtome-obtained by this method, it is necessary to wash them in pure acetone for 1 to 2 minutes to remove the cellulose acetate, wash in alcohol 1 to 2 minutes, and pass on to the stains selected. Various staining methods for cell walls-such as anilin chloride, methylene blue, and Congo red, ammoniacal fuchsin and Kleinenberg's haematoxylin, etc.-were tried with success. A comparison with stained sections of untreated wood revealed no differences. Delicate tissues in the wood and hyphae of fungi infecting the wood also stain well and are unaffected by the treatment.
A satisfactory method of preparing sections of hard vegetable structures is therefore supplied by the use of a 12 per cent solution of cellulose acetate in pure acetone for softening and embedding.-H. S. WILLIAMSON, Imperial College of Science and Technology.
Various brands of cellulose acetate behave differently. Cellulose acetate obtained from wood is unsatisfactory. We found that cellulose acetate made from photographic films was also unsatisfactory.
In making the solution, use 12 g. of cellulose acetate to 100 c.c. of pure acetone.
It would be worth while to try to adapt this method to the paraffin method.
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