Oscillatoria - For most purposes it is best to study Oscillatoria in the living condition. It is readily found in watering-troughs, in stagnant water, on damp earth, and in other habitats. The commonest forms have a deep blue-green or brownish color. It is very easy to keep Oscillatoria all the year in the laboratory. Simply put a little of a desirable form into a gallon glass jar half filled with water. By adding water occasionally to compensate for evaporation, the culture should keep indefinitely. In a jar with a tightly fitting cover we have kept such a culture for years without renewing the water.
For the purposes of identification and herbarium specimens the material may simply be placed on a slip of mica and allowed to dry. When wanted for use, add a drop of water and a coyer, and the mount is ready for examination. After the examination has been made remove the cover, allow the preparation to dry, and then return it to the herbarium.
Good mounts may be made by the Venetian turpentine method. Species of medium size are more satisfactory for a study of the nucleus than the very large species. Fix in a chromo-acetic-osmic solution (1 g. chromic acid, 3 c.c. acetic acid, and 1 c.c. 1 per cent osmic acid). Stain in iron-alum haematoxylin, and follow the Venetian turpentine method. While the nuclei are easily seen in such preparations, still better views can be secured from sections of paraffin material fixed in the same solution or in Flemming's, weaker solution. The section should be about 3( thick. After staining with haematoxylin, stain lightly with orange dissolved in clove oil. In paraffin sections the scattered condition of the material as it appears in thin sections is very annoying. As soon as the material is thoroughly washed in water, arrange it so that the filaments will all have the same general direction. This will enable you to get longitudinal and transverse sections. As you begin with the alcohols, use a Petri dish and lay a slide over the material, and keep it .there until you imbed in paraffin. This will keep the filaments from spreading out too much, and you will be able to get as much on one slide as you would be likely to get on a dozen slides without such precaution.
Tolypothrix.-This form occurs as small tufts, either floating in stagnant water or attached to plants and stones. Some species grow upon damp ground. It furnishes an excellent example of false branching (Fig. 33). Like all small filamentous algae, it may be dried on mica for herbarium purposes. Venetian turpentine mounts and paraffin sections are prepared as in Oscillatoria, Tolypothrix is even better than Oscillatoria for a study of the nucleus.
Scytonema is a similar form which is fairly common. It is often found as a feltlike covering on wet rocks.
In staining forms like Tolypothrix and Scytonema, which have a thick sheath, take care not to obscure the cell contents by staining the sheath too deeply. If the sheath is not stained at all, you may not be able to see the nature of the false branching. Iron-alum haematoxylin, with orange in clove oil for the sheath, is good for sections. Magdala red, with light green for the sheath, is good for Venetian turpentine mounts.
Nostoc - Nostoc is a cosmopolitan form. It occurs on damp earth or floating freely in water. In a fruit can or a battery jar, Nostoc is easily kept year after year in the laboratory. Young specimens are generally in the form of gelatinous nodules, but in older specimens the form may be quite various. It is very easy to make sections, since the gelatinous matrix cuts well and holds the filaments together. Chromoacetic acid is a good fixing agent. Stains which stain the gelatinous matrix make the preparations look untidy, but they show that each filament of the nodule has its own gelatinous sheath. Small nodules may be stained in bulk and be got into Venetian turpentine. Crushed under the cover, they make instructive preparations.
Rivularia - This form is readily found on the underside of the leaves of water-lilies (Nuphar, Nymphaea, etc.), but is also abundant on submerged leaves and stems of other plants. It occurs in the form of translucent, gelatinous nodules of various sizes. Chromo-acetic acid gives beautiful preparations, but good results can also be secured from formalin or picric-acid material.
The most instructive preparations for morphological study can be obtained by the Venetian turpentine method. Stain in iron" haematoxylin and very lightly in erythrosin, the latter stain being used merely to outline the sheath. When ready for mounting, crush a small nodule under a cover-glass. The paraffin method is easily applied, since the gelatinous matrix keeps the filaments in place. Any form of similar habit may be prepared in the same way.
Gloeotrichia - Gloeotrichia, in its later stages, is a freefloating form. In earlier stages it is attached to various submerged aquatic plants. The nodules, when young, are firm like Nostoc, but as they grow older and larger they become hollow and soft. The older forms become so much dissociated that they lose their characteristic form and merely make the fixing fluid look turbid. Allow a drop of such material to spread out and dry upon a slide which has been slightly smeared with albumen fixative. Leave the slide in 95 per cent alcohol 2 or 3 minutes to coagulate the albumen fixative, and then stain in safranin. If the background appears untidy, stain for 24 hours, or longer; you can then extract the stain from the background, and still leave the long spore and some of the other features of the filament well stained. A touch of cyanin will bring out the sheath. Cyanin and erythrosin is a good combination if the material is clean. The firmer nodules may be treated like Nostoc or Rivularia.
Wasserbluthe - Many genera of the Cyanophyceae occur as scums, often iridescent, on the surface of stagnant or quiet water. Some of the commonest forms are Coelosphaerium and Anabaena (Fig. 35). Some of the Chlorophyceae also occur as Wasserbluthe. Where the material is very abundant, it may be collected by simply skimming it off with a wide-mouthed bottle, but where it is rather scarce, it is better to filter the water through bolting silk and finally rinse the algae off into a bottle, adding enough formalin to the water in the bottle to make a 5 per cent solution. The material may be kept here indefinitely, but after a few hours it is ready for use. If the forms are small, like Anabaena, smear a slide lightly with Mayor's albumen fixative, as if for paraffin sections, add a drop of the material and allow it to dry over night or for 24 hours; then immerse the slide in strong alcohol for a few minutes, and then proceed with the staining. Cyanin and erythrosin form a good combination for differentiating the granules. Delafield's haematoxylin, used alone, stains some granules purple and others red. Iron-alum haematoxylin is excellent for heterocysts. With patience, these Wasserbluthe forms may be stained in iron-haematoxylin and brought into Venetian turpentine, from which they will yield much better preparations than can be secured by the dryingdown method.
Sometimes Anabaena, mixed with Gloeothece or Gloeocapsa, occur floating in gelatinous masses which hold together fairly well, so that it is easy to fix in the chromoacetic-osmic solution recommended for Oscillatoria, stain in iron-alum haematoxylin, and follow the Venetian turpentine method.
With such material we have tried a more expeditious method with excellent results. After staining in haematoxylin, we have used a series of alcohols, 2 1/2, 5, 10, 15, 20, 30, 40, 50, 70, 85, 95, and 100 per cent, allowing only 3 or 4 hours for the entire series. Then use mixtures of clove oil and absolute alcohol, beginning with 1 part clove oil to 4 parts alcohol, followed by equal parts clove oil and alcohol, then 3 parts clove oil to 1 of alcohol. At this point, stain in orange dissolved in clove oil. Drain off the stain and transfer to pure clove oil. Then place the material in thin balsam, about 1 part of the balsam used for mounting to 3 parts of xylol. Here the material may be kept indefinitely. Mounts may be made in balsam from this stock.
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