As a rule, protoplasmic connections are not likely to be seen in an ordinary preparation. It used to be thought that the rather large protoplasmic strands seen at the sieve plates of the pumpkin and other Cucurbitaceae were exaggerated examples of protoplasmic continuity; but, as a matter of fact, the large strands do not extend entirely through the plate. The real continuity, through the middle lamella, is scanty and hard to demonstrate.
Very satisfactory material for the demonstration of the connecting strands is furnished by the seeds of the Japanese persimmon, Diospyros Kaki. Fix in formalin alcohol (10 c.c. formalin to 50 c.c. of 70 per cent alcohol) or in a mixture of equal parts 70 per cent alcohol and glycerin. The Philippine Diospyros discolor is even better.
Good views of the strands are most abundant in sections cut parallel with the flat surface of the seed. No imbedding is either necessary or desirable. Clamp the seed in the microtome directly, or fasten it to a wooden block with cellulose acetate (easily made by dissolving a photographic film in acetone; of course the emulsion should be removed), with celloidin, or even with glue. Cellulose acetate seems to be the best. Cut sections 8 to 12( thick; place them in ether several hours to remove any fatty substances; remove the ether with absolute alcohol; transfer to 95 per cent alcohol, then to 50 per cent alcohol, and then to water. Stain in iron-alum haematoxylin. The following schedule for Diospyros Kaki will introduce the method.
1. Fix in formalin alcohol or in glycerin alcohol 4 or 5 days. 2. Cut sections 8 to 12( thick. 3. Chloroform or ether, several hours. 4. Absolute alcohol, 95 per cent and 50 per cent alcohol, 10 minutes each. 5. Wash in water, 5 minutes. 6. Iron alum, 4 per cent, 8 to 24 hours. 7. Wash in water, 30 minutes. 8. Stain in 1/2 per cent haematoxylin 24 hours. 9. Wash in water. At this stage, examine carefully, because it may not be necessary to reduce the stain in iron-alum. If the connections are not too deeply stained, simply dehydrate, clear, and mount in balsam.
After the first 5 stages, a strong stain in Delafield's haematoxylin, 10 to 24 hours, followed by a very weak hydrochloric acid, has given good results. A sharp stain in crystal violet, differentiated with orange in clove oil, often fails, but sometimes succeeds; and, when successful, the connections stand out beautifully.
The endosperm of Phytelephas (vegetable ivory), of dates and many other palms, and probably most hard endosperms, will show the connections by the methods just described; but in many cases it is necessary to resort to special methods in order to demonstrate the continuity. In these special methods a reagent is used which causes the membranes to swell before the stain is applied. It is only by such an exaggeration that the more delicate connections can be shown.
Put thin sections of fresh material into a mixture of equal parts of sulphuric acid and water; and allow the reagent to act for 2 to 10 seconds. Wash the acid out thoroughly in water and stain in anilin blue. According to Gardiner, this stain should be made by adding 1 g. of the dry stain to 100 c.c. of a saturated solution of picric acid in 50 per cent alcohol. The staining solution is then washed out in water, and the sections are mounted in glycerin. The sections may be dehydrated, cleared in clove oil, and mounted in balsam. The anilin blue may be used in 50 per cent alcohol acidulated with a few drops of acetic acid.
Chloroiodide of zinc may be used instead of sulphuric acid. Treat the fresh sections for 2 hours with the iodine and potassium-iodide solution used in testing for starch; then treat about 12 hours with chloroiodide of zinc. Wash in water and stain in anilin blue. Examine in glycerin.
Meyer's pyoktanin method is one of the best. The reagents are as follows: 1. Iodine, potassium iodide solution: iodine 1 part, potassium iodide 1 part, water 200 parts. 2. Sulphuric acid 1 part, water 3 parts; this mixture to be saturated with iodine. 3. Pyoktanin coeruleum 1 g., water 30 c.c. This pyoktanin is a very pure methyl violet obtained from E. Merck in Darmstadt.
Put sections of the date seed into a watch glass full of the first solution, and allow it to act for a few minutes; then mount in a drop of the solution. The connections will be only very faintly stained, showing a slightly yellowish color. At the edge of the cover, add a drop of the second solution. The preparation will darken a little. Then allow a small drop of the third solution to run under the cover. Allow the stain to act for about 3 minutes. Then plunge the whole preparation into water. The action should be stopped before the entire section has become blue. Now wash the section quickly. If there are annoying, granular precipitates, remove them with a soft brush. Mount in glycerin. The membrane should be a clear blue, while the protoplast and connections should be a blue black.
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