Plant histology: The Venetian Turpentine Method

Just as glycerin jelly is superior to glycerin as a mounting medium, so Venetian turpentine is superior to glycerin jelly. While the method was described by Pfeiffer and Wellheim thirty years ago, it received no recognition in the United States or even in Europe. I made a casual trial of the method when preparing the first edition of this book, but the preparations were such miserable failures that the process did not seem worth mentioning. The method was next brought to my attention during a demonstration in Strasburger's laboratory at Bonn. He was using preparations of Zygnema and Spirogyra, the staining of which surpassed anything I had ever seen. He remarked that it was not worth while to consult the lengthy article, because his preparations had been made by the authors and no one else had made a success of the method. However, when I returned, I made a careful study of the process, and finally learned to use it successfully. The details as given in this paper were too indefinite for practical use, but, after one has learned the method the article can be read with profit.

The great practical advantages of the method are that preparations are as hard and durable as balsam mounts, and that a much greater variety of staining is possible than in case of glycerin mounts.

The principal features of the method are that material is brought from pure glycerin into alcohol, and from alcohol into Venetian turpentine, without passing through xylol or other clearing agent.

After fixing and washing in water and staining in an aqueous stain, e.g., iron-alum haematoxylin, the process is as follows:

1. 10 per cent glycerin until concentrated. 2. Wash the glycerin out thoroughly in 95 per cent alcohol. 3. Complete the dehydration in 100 per cent alcohol. 4. 10 per cent Venetian turpentine in an exsiccator until the turpentine becomes thick enough for mounting. 5. Mount in the Venetian turpentine.

Even after the method became established, there occurred a period of several years during which it was practically impossible to get a Venetian turpentine suitable for histological use. Consequently, it was necessary to resort to glycerin jelly or to try various schemes for bringing material into Canada balsam; but good Venetian turpentines have appeared again and are even more satisfactory than those which established the method in popular favor. We have tested two brands which are giving uniform and excellent results: these are the "Venice Turpentine (True)," sold by the Fuller Morrison Company, of Chicago; and the "Turpentine Venetian" (No. 2605), sold by the National Anilin and Chemical Company, of New York. There are probably other good turpentines and still others are likely to appear. Venetian turpentine is made from the resin of Larix europea. Since this resin is soluble in absolute alcohol, material can be transferred directly from absolute alcohol to a thin solution of the mounting medium, without the intervention of xylol or any other oil, thus eliminating the most dangerous stage in the preparation of filamentous algae and fungi and any forms which are to be mounted whole.

The general outline, just given, is not sufficiently definite for a working introduction. The following concrete examples, describing the use of Venetian turpentine with an aqueous stain, with an alcoholic stain, and with a combination of aqueous and alcoholic stains, will be more practical than general directions. The steps from fixing to mounting, as used with an aqueous stain, will be described first, since this will introduce the method in its least complicated form.

Haidenhain's Iron-Haematoxylin.-Using Spirogyra as a type, proceed as follows:

1. Fix 24 hours in chromo-acetic acid. 1 per cent chromic acid........................100 c.c. Glacial acetic acid..............................3 c.c.

The volume of the fixing agent should be at least 50 times that of the material to be fixed.

2. Wash in running water 10 hours; if running water is not available, 24 hours, changing often. 3. 2 per cent aqueous solution of iron-alum (ammonia sulphate of iron), 2 hours. 4. Wash in running water, 20 minutes. If running water is not available, wash in a large quantity of water and change frequently. 5. Stain over night, or 24 hours, in 1 per cent aqueous solution haematoxylin. 6. Wash in water, 20 minutes. 7. 2 per cent aqueous solution of iron-alum, until the stain is satisfactory. This can be determined only by examining frequently under the microscope. 8. Wash in water, 2 hours. If this washing is not thorough, the continued action of the iron-alum will cause the preparations to fade. 9. Transfer to 10 per cent glycerin, and allow the glycerin to concentrate until it has the consistency of pure glycerin. It is not necessary to use an exsiccator. Merely put the glycerin into shallow dishes, and leave it exposed to the air, but protected from dust. If the material is in Petri dishes or other dishes with a large surface, 3 or 4 days will be sufficient. This process may be hastened by warming, if the temperature does not go above 35 C. If the reduction from 10 per cent glycerin to pure glycerin is accomplished in less than 24 hours, the change in the concentration is so rapid that material is likely to suffer. 10. Wash out the glycerin with 95 per cent alcohol. It will be necessary to change the alcohol several times. From 10 to 20 minutes will be sufficient if the alcohol is changed frequently. This alcohol cannot be used again for the same purpose, but it will be useful in cleaning one's hands and in cleaning dishes which have contained Venetian turpentine. 11. Complete the dehydration in 100 per cent alcohol: 10 minutes should be sufficient. 12. Most failures are now ready to occur,

From the absolute alcohol the material is transferred to a 10 per cent solution of Venetian turpentine in absolute alcohol. The turpentine thickens as the alcohol evaporates, and when it reaches the consistency of pure glycerin the material is ready for mounting. The 10 per cent Venetian turpentine is very sensitive to moisture, and most failures are due to this characteristic; consequently the concentration cannot be allowed to take place with the turpentine exposed to the air of the room. Use an exsiccator. This will not only absorb the moisture from the air, but will soon remove the alcohol from the turpentine mixture. Make an exsiccator as follows: Place a saucer full of soda lime (sodium hydroxide with lime) on a plate of glass, and cover with a bell jar. This is a simple and effective exsiccator. Instead, you may simply scatter soda lime in the bottom of any low museum jar with tight-fitting cover. The tin cans, with tight covers, in which you get your pound of "Improved Vacuum Coffee" make good exsiccators for small amounts of material. You may improvise other forms; the essential thing is to provide a small, air-tight place in which the soda lime may work.

Instead of soda lime you may use fused calcium chloride or the white sticks of sodium hydroxide.

We are now ready for the transfer from absolute alcohol to the 10 per cent Venetian turpentine. Make the transfer quickly. Pour off the absolute alcohol and place the dish, with the material, in the exsiccator; then pour on the 10 per cent turpentine, and immediately put on the cover. This is 'better than to pour on the turpentine and then try to get the dish well placed in the exsiccator.

The greater the surface of soda lime exposed, the more rapid will be the concentration of the Venetian turpentine. The concentration must not be too rapid. Not less than 2 days should be allowed for the concentration of 30 c.c. of the turpentine in an ordinary Minot watch glass.

Great care must be taken not to let any of the soda lime, or other drier, get into the turpentine.

When the lime has become saturated, it may be heated until dry, and then used again. If material is put into an exsiccator with nearly saturated lime, the turpentine becomes milky. If the material is very valuable, wash in absolute alcohol until entirely free from any milky appearance, and start again in 10 per cent turpentine.

As soon as the turpentine has attained the consistency of pure glycerin, it may be exposed to the air without any danger from moisture; but the turpentine would soon become too thick for mounting. If the turpentine has become too thick, thin it with a few drops of absolute alcohol or with 10 per cent or any thin solution of Venetian turpentine.

Mount the material in a few drops of the Venetian turpentine and add a cover. Tapping on the cover with the handle of a needle or scalpel will often separate the filaments so that they are more convenient for examination. Square covers may be used since it is entirely unnecessary to seal the mounts, which are as hard and durable as those mounted in balsam.

Material in the thickened Venetian turpentine, when not needed for immediate mounting, may be put into small bottles. The corks should be of the best quality; otherwise the turpentine will become too thick. While it can be thinned by adding thin turpentine, it is better, for easy mounting, not to let the turpentine become too thick. If the turpentine is only a little too thick, warming it gently will thin it enough for making mounts; but if any material is to be put away, a few drops of absolute alcohol or of a thin Venetian turpentine should be added. Material in Venetian turpentine, well corked and kept in the dark, does not fade or deteriorate in any way.

Magdala Red and Anilin Blue.-Fix in chromo-acetic acid and wash in water, as described in the previous schedule. Transfer from water to 10 per cent glycerin and allow the glycerin to concentrate. It is not necessary to use an exsiccator since there is no danger from moisture in the air. When the glycerin attains the consistency of pure glycerin, wash the glycerin out with 95 per cent alcohol and then proceed with the staining.

1. Stain in Magdala red. At least two Magdala reds are sold by dealers. The one marked "echt" is more expensive, but, in our experience, is inferior' to the one marked simply "Magdala red." Make a 1 per cent solution in 90 per cent alcohol. We use the stain much stronger than recommended by Pfeiffer and Wellheim. The pure stain, allowed to act for 24 hours, does not stain too deeply.

2. Rinse the material for a minute in 90 per cent alcohol.

3. Stain in anilin blue, using a 1 per cent solution in 90 per cent alcohol. We prefer to make a fresh solution every time we have anything to stain. It is not necessary to measure it. A little of the powder-about half the bulk of a grain of wheat-in 30 c.c. of 90 per cent alcohol, will give an efficient solution. The time required for successful staining will vary from 3 to 30 minutes. Do not put all the material into the anilin blue at once, but, by trying a few filaments at a time, find out what the probable periods may be.

4. Rinse off the stain in 90 per cent alcohol, and then treat for a few seconds in acid alcohol (1 very small drop of HC1 to 30 c.c. of 90 per cent alcohol). The acid alcohol fixes and brightens the anilin blue, but extracts the Magdala red. If the anilin blue or the acid alcohol acts for too short a time, the blue will be weak; if they act too long, the red is lost entirely. If the blue overstains too much, wash it out in 95 per cent alcohol. If the red overstains, wait until the mount is finished, and then reduce the red by exposing the slide to direct sunlight.

5. Absolute alcohol, 5 or 6 seconds.

6. Transfer quickly to 10 per cent Venetian turpentine and proceed as in the previous schedule.

The surprising beauty of successful preparations will compensate for whatever failures may occur. Nuclei and pyrenoids should show a brilliant red, while the chromatophores and cytoplasm should be dark blue. The cell walls should show a faint bluish color.

Haidenhain's Iron-Alum Haematoxylin and Eosin.-Follow the schedule for iron-haematoxylin until the glycerin has been washed out in 95 per cent alcohol. Then stain for a minute in a solution of eosin in 95 per cent alcohol. Wash for a minute in 95 per cent alcohol, then a minute in absolute alcohol, and then transfer to the 10 per cent Venetian turpentine.

Haidenhain's Iron-Alum Haematoxylin and Safranin.-Follow the schedule for iron-haematoxylin until the glycerin has been washed out in alcohol, and then add to the 95 per cent alcohol several drops of a solution of safranin in 95 or 100 per cent alcohol and allow the stain to act for 30 minutes or an hour. Then dehydrate in absolute alcohol and transfer to 10 per cent Venetian turpentine.

Other Stains may be used. Aqueous stains should be used before starting with 10 per cent glycerin. Alcoholic stains should be in strong alcohol-about 90 per cent-and should be applied just after washing out the glycerin.

This method is equally good for filamentous fungi and also for the prothallia of Equisetum and ferns, for delicate liverworts and mosses, and similar objects.

If you have a good turpentine, good stains, and avoid moisture, the Venetian turpentine method should not be difficult, and the results with filamentous and unicellular forms and other small objects surpass anything yet secured by other processes.

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