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10 October 2003

Mounting Diatoms for Microscopic Study

by Albert G. Ingalls
Excerpted from "Scientific American's The Amateur Scientist", first published January, 1953.

"For live collecting the amateur should equip himself with a few wide-mouthed jars, a spoon and an ordinary coffee or tea strainer of 40 or 50 mesh. As specimens are taken in the field, the collector records the date, time and location along with other data that will assist in the subsequent identification of the material.

"To prepare diatoms for mounting, the amateur will need: technical sulfuric acid, technical hydrochloric acid, hydrogen peroxide, powdered potassium bichromate, distilled water, strainers of 40 to 50 mesh, two Pyrex beakers holding 80 cubic centimeters a Pyrex custard dish, a conical flask of 125 c.c., an assortment of pint and quart jars, glass stirring rods, half-ounce storage bottles with caps, and clean pipettes.

"The collected material should be transferred to a large jar. Filtered water is added and the material is thoroughly beaten with a glass stirring rod to dislodge the diatoms from foreign objects The organisms should then be strained and permitted to settle; allow an hour per vertical inch of solution. Living diatoms should be processed in a darkish place, because their metabolic processes release tiny bubbles of oxygen which cause the organisms to rise to the surface. Having settled, the diatoms will form a brown layer on the bottom of the jar. The water should then be poured off without losing the specimens. This washing process should be carried out three or four times, particularly when the diatoms are salt-water species. Distilled water should be used wherever the local water supply carries a heavy content of lime.

Upper Row: Marine diatoms. Lower Row: Fresh-water diatoms. Click image to enlarge.

"Part of the material is then transferred with the pipette to the Pyrex beaker to form a layer about an eighth of an inch thick. The beaker should be placed in the custard cup. The excess water is removed with the pipette, leaving the specimens moist but not wet. Next powdered potassium bichromate approximately one third the bulk of the diatoms, is stirred-into the mixture. Then comes an operation which must be performed outdoors or in a window with a strong outward draft, as the reaction produces poisonous fumes. Slowly add approximately five c.c. of technical sulfuric acid to the diatom-bichromate mixture. A violent reaction will follow. Beat down the resulting bubbles with a stirring rod. Should the heat of the reaction break the beaker, the custard cup will prevent the loss of the specimens.

"The material is then transferred to a quart jar and washed with distilled water five times, allowing about an hour of settling time for each vertical inch of water. The diatoms are now ready for separation according to size. This is accomplished by the familiar process of elutriation. The material is transferred to the first of a series of uniform glass containers. Water is added, and after a certain interval, say half an hour, the water is poured off carefully into the second jar of the series. Again at the end of the predetermined interval, the second jar is poured off into the third. This is repeated until the diatoms are separated into the desired sizes. The diatoms are now ready for individual storage bottles, to which a few drops of hydrogen peroxide may be added as a preservative.

"For mounting diatoms, select a cover glass of 12-millimeter diameter and a thickness of .11 to .20 mm. The slide itself should have a thickness of 1 mm. (A supply of cover glasses, slides and mounting cement or 'medium' is available at most optical shops.) Clean the slide and cover glass thoroughly with soap and water. Select a storage bottle containing diatoms and shake it until they are in suspension. With the pipette place a drop of distilled water in the center of the cover glass. If the glass has been cleaned properly, the water will spread to the edge but not overflow. Next add a drop from the storage bottle which holds the diatoms in suspension. The diatoms will spread evenly and settle on the glass. Allow the water to evaporate overnight. Protect the glass from dust.

"You will next need a small hot plate and a bottle of medium. I prefer Hyrax for mounting diatoms. Warm the slide on the hot plate and place a drop of Hyrax on the slide's center. Then remove the slide from the hot plate and place it on a wooden support to prevent rapid cooling. With sharp pointed forceps, pick up the cover glass, invert it so that the diatom side is down and press it gently onto the medium until the fluid reaches the edge of the cover glass The slide is then returned to the hot plate and the Hyrax is brought to a state of vigorous bubbling. As the bubbling slows down, remove the slide to the wooden support. Experience will teach the proper moment of transfer.

"After cooling, the slide is placed under the objective and examined. If the washing and mounting have been performed carefully, the amateur is in for a thrill he will long remember. Diatoms have been called 'nature's jewels,' and man has yet to fashion anything more exquisite.