Preparing Diatom Samples: Part 2

Read Part 1, in which Joe Holmes provides an introduction to diatoms—what these microscopic organisms are and their importance.

Upon my arrival in early 2013 as a volunteer in the phycology lab of the Canadian Museum of Nature in Gatineau, Quebec, I learned on the job how to prepare diatom specimens for storage and microscopic study.

An array of diatoms.

Several diatom species, seen through an SEM microscope. Image: Paul Hamilton © Canadian Museum of Nature

Because of all the steps and time involved, processing an individual sample—starting with a dried field sample and ending with it mounted on a microscope slide—may take two days. However, because there are usually dozens of samples to work on, they are processed together in groups of 12. Processing a box of 100 or more may take a week of full-time work, or a couple of months part-time (my speed at one day a week).

Acid Shell Cleaning

The first thing we do when receiving a sample is clean the microscopic shells (valves) of the diatoms of any lingering organic material, much like cleaning out a seashell.

From each sample (wet or dry), material containing thousands to millions of diatoms is added to a glass beaker and marked with the sample’s collection reference number. Powder-like dry material is scraped off filter paper from inside the sample bottles. Twelve samples are normally processed together as a group.

Beakers and bottles on a counter.

Beakers with dry material from sample bottles. Image: Joe Holmes © Canadian Museum of Nature

A small amount of acid mix is carefully squirted into each beaker and boiled on a hotplate for 25–30 minutes at high temperature. Afterwards, the beakers are allowed to cool for 15–20 minutes before the centrifuge process is started. Boiling in acid dissolves and separates diatoms and other organic matter, leaving the cleaned diatom shells, which are made of silica. The remaining particulates are like small pebbles and clay.

Equipment under a fume hood.

Acid-dispensing bottle and heater with boiling beakers. Image: Joe Holmes © Canadian Museum of Nature

Centrifuging Water Mixture

To separate the diatoms from other material, we use a series of deionized water dilutions. (Deionized water has had most of its mineral impurities removed).

Each boiled beaker mix is poured into a corresponding plastic centrifuge tube marked with the sample’s reference number. The tube is then filled within a centimetre of the top with deionized water.

The tubes are placed in a centrifuge and run at 3000 revolutions per minute for 10 minutes, the objective being to force the diatoms down into the cone-shaped bottom of the tube.

A look under the lid of a centrifuge.

Centrifuge with 12 tubes ready to run. Image: Joe Holmes © Canadian Museum of Nature

Once done, most of the liquid mix is removed using a narrow hose attached to a siphoning pump, leaving diatoms and some liquid at the bottom of the tube.

Tubes are then refilled with fresh deionized water and the centrifuge/siphoning/refill procedure is repeated five times to increasingly purify the diatom mix.

After the final run/siphoning, the remnant “button” of diatoms and remaining liquid are shaken from the bottom and poured into a new sample bottle. A metal scraper is often required to get at all the lingering material if it is sticking to the bottom. Lids are labelled with the corresponding reference number.

Each final sample bottle should be about a quarter full of the diatom/water mix. Bottles are placed in boxes of 100 and into cabinet drawers in the wet collection to await microscope-slide creation.

Personal protection is required during the above stages. I wear special neoprene rubber gloves and a face visor when handling any acids. Acid boiling is done under a fume hood that removes toxic vapours. For centrifuge and siphoning work, I use latex gloves. Waste water is disposed of in an environmentally safe manner.

A man wearing protective gear stands in a laboratory.

Joe Holmes working at a fume hood in the lab. Image: Paul Hamilton © Canadian Museum of Nature

Making Light-Microscope Slides

Light microscopes can magnify up to 1600 times and are used to look straight into the layers of diatoms based on fine focusing.

To create the glass slides of diatom samples for use in the microscope, the required samples are selected from the museum’s wet collection. A slide-warming table is laid out with small glass cover-slip blanks corresponding to the sample bottles to be processed. A drop of deionized water is added to each cover slip using a micropipette.

Each sample bottle is gently shaken to better mix the water with diatoms, and then two to three drops of the sample are added to its corresponding cover slip. The same micropipette is cleaned with deionized water between samples to avoid cross contamination.

Cover slips are allowed to dry, leaving a dried diatom smudge on each one.

Glass slide blanks are prepared for each cover slip by recording their reference number on the label portion of the slide (with a pen, label sticker or etching tool).

Next, we need to permanently “glue” the cover slip on top of the glass slide. To do this, a mountant (of similar colour and viscosity as honey) is used to fix each cover slip to its corresponding slide. The museum uses either Naphrax or Hyrax.

One at a time, each slide is heated on a hotplate at 300°C for three to four seconds. A drop of mountant is added to the slide using a glass rod. The corresponding cover slip (smudge side down) is attached to the bubbling mountant using tweezers. Using a small metal tool, air bubbles are gently forced out of the cover slip to make a tight fit with the slide.

Slides are allowed to cool for at least 15 minutes. Excess mountant is scraped off with a razor blade and then cleaned off with a damp cloth.

Once completed, the slides are ready to use under the microscope and can be filed in the museum diatom slide collection by reference number in wooden cabinets with thin metal drawers that hold 20 slides each.

An open cabinet with one drawer pulled out.

A cabinet for filing glass slides. Image: Joe Holmes © Canadian Museum of Nature

Making SEM Discs

A machine called an SEM is used for looking at precise details within diatom. It can magnify up to 100 000 times, but the magnification that we generally use for diatoms is up to 20 000x.

For preparing samples to be viewed by the SEM, a different procedure is used from that for light microscopes. Samples from the wet collection are dried on small pieces of aluminum foil that are then placed on metal discs, rather than drying them on cover slips.

To examine the sample in the SEM, further processing using a vacuum process must be done to apply an ultrathin amount of gold as a conductive coating before a sample disc is ready to use.

Because of the use of gold and initial equipment costs, the SEM is more expensive to use than light microscopes (which the museum has many of).

Data Storage

With both the light microscope and SEM, we have special cameras and software to take diatom photos.

The photos and specimen data are then copied into the museum’s phycology collection database, which is publicly accessible online.

This entry was posted in Collections, Plants and Algae, Tools of the trade and tagged . Bookmark the permalink.

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