Diatomaceae of Philadelphia/Appendix
APPENDIX
Collection and Preparation of Diatoms
It is assumed that every student of the Diatomaceæ has a general knowledge of the collection, preparation, mounting and examination of material. For the novice, however, the following methods, used by the author for many years, may be of service.
Collection of Fresh-water Material.—The yellow film on the inside of aquaria always contains small species. Stems of water-plants near the shores of ponds and the submerged roots, the brownish coating of rocks in streams and water-falls, fountains, and water-troughs, are prolific. At all times of the year, some diatoms may be found in a thin layer upon the mud of rivers or creeks. In the spring, brown patches of mud, filled with bubbles, floating near the shore in ponds, or coming down with the current in rivers, are rich in various forms. Within the limits assigned to our district, I have made collections in the following localities: Schuylkill River, including the region near Fairmount Dam, several reservoirs and the water-supply; the Wissahickon and Fairmount Park, Darby, Crum and Ridley Creeks, the Neshaminy and the Brandywine; meadow pools and rivulets near the city; the upper Delaware, the Water Gap and numerous cascades northward; the Shawangunk Mountains and the Poconos; many parts of New Jersey along the coast; the Pine Barren region, the Hammonton, Atsion and Kirkwood Ponds and the swamps near Atco.
In the collection of fresh-water material, it is well to be provided with a number of small bottles. Take a handful of the water-plants or algæ, and squeeze the material into the bottles, or, lacking a bottle, wrap it in paper. With a small forceps it is possible to detach minute quantities of a pure gathering which may not need further preparation beyond burning to a red heat on the cover-glass before mounting. A malacca cane, with extending rod to which may be screwed a bottle, net, spoon or hook, is useful on a long trip. If it is impossible to separate the thin film of diatoms from the mud in the bed of streams, dip up the surface mud with one bottle, allow to settle a few minutes, then pour off the supernatant liquid, which will be comparatively free from sand, into another bottle. It must be confessed, however, that the mud in streams near Philadelphia contains a large quantity of fine mica which, in some instances, it is impossible to remove.
Collection of Marine Material.—Shell scrapings, the stomachs of fish, marine algæ, especially the brown and red algæ, the hulls of vessels, mud from anchors and dredgings, are all sources which may prove valuable. In the sand ripples, after the tide recedes, a yellowish-brown deposit will be noticed. This should be taken up carefully with a spoon and placed in a bottle; the sand will settle at once and a very pure gathering will be held in suspension in the water. Such collections may be made along the entire coast of New Jersey on sunny days in summer. In salt meadows near Absecon and Hackensack, large quantities of diatoms, including Pleurosigma, may be obtained in the yellow scum floating on the surface.
The Blue Clay Deposit.—The blue clay occurs as a pre- or post-glacial deposit in the bed of the ancient Delaware River, and, at depths varying usually from fifteen to forty feet below the surface, has been obtained from artesian wells at Pavonia, Pensauken and Gloucester, N. J., also at Port Penn on the Delaware, and especially from the dredgings made by the removal of Smith's Island opposite the city. In the city proper, it may be stated briefly that material may be found in a stratum of very light blue clay at a depth varying from twenty to sixty feet in many places south of Arch St. east of Broad St., and also along the beds of ancient rivulets near Tioga St., at Sixteenth St., and in certain other places which were probably subject to tidal overflow. One of the best collections was made along the bank of the Schuylkill at the east end of Walnut St. Bridge, at a depth of thirteen feet below the surface. Excavations for the Reading Terminal and the Subway and several buildings, as the Bingham House, have furnished numerous specimens.
Cleaning the Material.—Some gatherings may be so pure as to be ready for mounting when treated with dilute alcohol and oil of cloves. If, when gathered, the diatoms are immersed in a saturated solution of picric acid for several days, they may be stained with carmine or methylene blue, or whatever may be required to emphasize the contents of the frustules, including the endochrome and the pyrenoids. After staining, pass as rapidly as expedient through the treatment with dilute alcohol and oil of cloves, and mount in benzol balsam, avoiding heat. A hot solution of mercuric bichloride is sometimes used for the preservation of the endochrome, although washing is needed before mounting. For the particular stain considered best for certain details of structure, it will be advisable to consult works on Micro-Chemistry or Heinzerling (l. c.). The stains of most importance are carmine, methylene blue, hæmatoxylin, gold chloride and Bismarck brown.
Whatever method may be used in staining, the identification of forms is impossible, in most cases, unless the valves are carefully cleaned and the cell-contents destroyed. For this purpose provide a casserole holding from five to eight ounces, an iron tripod stand with alcohol lamp, several six-inch test-tubes, preferably those with a standard base, fitted with pure rubber corks. Take the material as free from twigs, dead leaves, sand, and other matter as possible, place it in the casserole, and add about the same quantity of nitric acid. Boil for twenty minutes and then add about half a teaspoonful of powdered bichromate of potash, stirring with a glass rod. Then take a beaker-glass partly filled with water and pour into it slowly the liquid which has been allowed to cool a short time, whirling the casserole to cause the concentration of sand in the centre. Allow the material to settle for half an hour or longer, according to the amount of diatoms and their size. Pour off the water, add more water, and place in a test-tube. Repeat the decantation, shaking the test-tube, closed with a rubber cork, vigorously each time. From time to time whirl the diatoms in the casserole and throw away the sand collected in the centre. By repeating the decantation, shaking and whirling, the deposit will be found to consist almost entirely of diatoms. It may be necessary to repeat the boiling in the acid and bichromate. If, however, any detritus other than sand is noted, boil in sulphuric acid and add from time to time minute pinches of powdered chlorate of potash, being careful to protect the eyes by holding a piece of glass before them; otherwise the explosions which occur are likely to throw some of the boiling acid into the eyes and destroy the sight. The material, when clean, should be white or, in the case of Synedra, yellowish. It is quite easy to construct a box fitted with the proper apparatus for boiling and provided with a glass door for observation, and a method of introducing the chlorate of potash through a small aperture or tube. The box may be placed in the garden or fastened outside of a window so that the poisonous fumes may be carried off.
An excellent method, in the case of larger forms, is to boil the material already cleaned by the acid in water to which a few shavings of coarse brown soap are added. The difference in density will hold in suspension any flocculent matter, and while many of the smaller forms will not settle, the others will be perfectly cleaned. When satisfied with the cleaning, preserve the stock material in part alcohol and, in using, pour into a smaller bottle the amount required, replace the dilute alcohol with distilled water, and mount as directed. It often happens that gatherings are made consisting almost entirely of sand. Attempts at cleaning in the usual way will cause the loss of nearly all of the diatoms. In this case, after the material has been treated with acid until nothing remains but sand and a few diatoms, the mechanical finger must be used.
In the cleaning of marine deposits, various methods may be required. In the case of partly siliceous species, washing in pure water repeatedly is all that can be done. The larger and heavier diatoms may be separated from the sand by elutriation or by whirling in a casserole, by rocking in a shallow dish the shape of a watch crystal, or by pouring slowly over a strip of plate-glass at least two feet in length inclined at an angle of thirty degrees. The sand will cling to the glass, while the greater portion of the diatoms will run off. Where particles of shells or foraminifera are present, a preliminary boiling in hydrochloric acid is advisable. In all marine gatherings, the salt should first be washed out before proceeding with the cleaning.
For hardened masses of clay and for fossil deposits, it is necessary to boil in carbonate of soda and follow with the acid treatment. Citric acid and acetate of potash used alternately in boiling may be tried. Soaking for a time in acetate of potash and allowing the material to deliquesce for a week before further process, has proved successful in some instances. The repetition of several methods and the gentle breaking of the harder masses with the point of a needle will disintegrate almost any diatomaceous earth, but, as a last resort for refractory deposits, boil in pure water, add a piece of caustic potash about the size of a pea, continue the boiling not more than thirty seconds longer, and pour instantly into dilute hydrochloric acid; otherwise the diatoms will be destroyed. Afterwards proceed with the usual treatment.
Slides and Covers.—Take half an ounce of No. 1 covers, circles, and place them in a wide-mouthed bottle. Add a portion of the following mixture (Dr. Carl Seiler's formula):
| Bichromate of potash | 2 oz. |
| Sulphuric acid | 3 fl. oz. |
| Water | 25 fl. oz. |
Shake the bottle in order that the surfaces of the covers may be fully exposed to the action of the acid, and set aside for several hours. Decant the solution, add water repeatedly until all traces of the mixture are removed, and keep the circles in the bottle in fifty-per cent. alcohol. When needed, take out a circle with forceps and dry on a linen cloth.
The slides may be treated in the same way, or they may be easily prepared by immersion in a solution of washing soda, and then washed and dried. This process may be used in cleaning the balsam or styrax from old slides.
Preparation of Strewn Mounts.—Place several covers on the mounting stand. With a dipping tube, cover each circle with distilled water, and add a small drop of the prepared diatoms, being careful to avoid any vibration of the stand. Heat the stand until small bubbles begin to appear, remove the lamp, and allow the water to evaporate. If the above method is carefully followed, the diatoms will be deposited in an even layer, provided the material is not too dense. Take a slide, centre it, and place a small amount of styrax on the centre. Invert the prepared cover, and gently place it upon the styrax. Heat the slide on the mounting stand until the styrax bubbles and then allow to cool. If bubbles still remain, heat again until they disappear. It is well to mount several slides more than required, as some may be imperfect.
Preparation of Selected Mounts.—Take a slide, place a minute quantity of beeswax on two places at a distance apart nearly equal to the diameter of the cover used. Place a cover on the wax and press it down flat, or sufficiently to keep it in position. Dip a fine needle into the following cement:
| Glacial acetic acid | 12 drachms |
| Gelatine | 2 drachms |
| Alcohol | 1 drachm |
This is made by adding the acid to the gelatine in a water-bath and then the alcohol, and filtering. Apply the moistened needle to the centre of the cover and spread as small a quantity as possible in a thin layer. Now place the slide upon the turn table, centre it with respect to the position of the gelatine, and with the finest sable brush draw a circle about a tenth of an inch in diameter around the gelatine in water-color (Windsor), blue or vermilion, or in India ink. Instead of the water-color, a circle of tin-foil the size of the cover and pierced with a hole in the centre may be used, but the colored circle is to be preferred, as, when brought into view, it indicates exactly the focus required for observing the diatom.
The bottle containing the cleaned material, which has been kept in water and alcohol, should be refilled with distilled water and well shaken, when a small portion may be taken up with a dipping tube and evenly distributed over a portion of a slide and then dried. By the use of a mechanical finger, fitted with a small piece of finely spun glass attached by wax to the holder of the finger, when the microscope is focussed until the glass thread touches the diatom selected, it will adhere to the thread. Raise the body of the microscope, remove the slide containing the spread material, or move it to another part of the stage, and place the slide with the prepared cover in the same position. Now carefully lower the body-tube of the instrument until the diatom rests upon the gelatine, breathe gently upon it, remove the cover from the slide, invert it over another slide containing a drop of styrax and proceed by heating to mount as before. The size of the diatom, the amount of gelatine, and several other factors, will enter into the question of success or failure. I have, however, employed the above method and have mounted thousands of slides of selected diatoms successfully. It is necessary to avoid any air current which will cause the diatom to fall from the thread. On very cold days the glass thread sometimes becomes electrified and the diatoms will not stick; on sultry days in August in our locality the diatoms will stick too closely.
By the same method, slides of arranged diatoms can be made using a glass circle properly marked with lines in the eye-piece. Care should be taken to use glass threads more or less in proportion to the size of the diatoms. A cat's whisker is preferred by some to the glass thread. It has the advantage of not breaking, but unless it is quite short it is too flexible. If the point of the thread becomes covered with gelatine, lower it into a minute drop of water upon a separate slide, and by moving it about it will be cleaned. The diatom itself may be washed in the same way, if it is not too small.
Instruments Required.—For collecting, in order to determine the quality of the find, any simple lens of fifteen to twenty diameters is sufficient. A Stanhope is quite useful although difficult to obtain, while an achromatic triplet of sufficient power will probably be all that is necessary. For selecting with the mechanical finger, an objective of two-thirds-inch focus is the most convenient, but for determining species a one-fifth-inch is needed, an immersion objective being essential for minute forms.
No particular form of microscope is required. Any instrument having standard parts, inclination of the body to the axis, a sub-stage condenser and movable stage, will prove serviceable in nearly all investigations. For critical work, measurement of striæ and location of specimens on the slide, the large models of Bausch and Lomb leave nothing to be desired. One smaller instrument may be used for rapid examination and for selection with the mechanical finger. If the stage is supplied with a vernier, the diatoms can be located rapidly and recorded for future reference. The Zentmayer Army Hospital stand with mechanical stage is excellent. The Continental stands, convenient for laboratory work, especially in the examination of bacteria, are not so serviceable as the larger stands of American and English make. The stand especially designed by Dr. Henri Van Heurck, the celebrated Belgian naturalist, is, without doubt, admirably suited to the investigation of the Diatomaceæ. In the form of the Circuit Stage as made by Watson and Sons, of London, supplied with proper condenser and mechanical stage with vernier attachment, it has been used in the preparation of the present work with much satisfaction.
The drawings have all been made with an Abbé camera lucida, a 3 mm. objective and a No. 10 eye-piece, producing a magnification of about 800 diameters. All illustrations are from actual specimens in my cabinet or, in a few instances, from slides sent me by friends. In the measurement of striæ and puncta, the number in ten microns is stated, and will be found to be approximately correct in most of the drawings, except when the number is in excess of twenty in ten microns, in which case it is impossible to represent the markings accurately on figures of the magnification adopted. All drawings are from specimens in this locality, except in a few cases mentioned in the text.