«This primer on cleaning and preparation of diatom materials is intended to assist two groups of diatomists in particular: 1) those who take up diatom ...»
How to prepare diatom samples.
Frithjof A.S. Sterrenburg
This "primer" on cleaning and preparation of diatom materials is intended to assist two groups of
diatomists in particular:
1) those who take up diatom studies as part of investigations rooted in some other discipline
(plankton studies, wetlands research etc.)
2) investigators with limited laboratory facilities
Therefore, the overruling criterion has been simplicity. Methods have been selected that yield good results with the minimum of outlay and use the most widely available chemicals only.
These "Diatom Handicrafts" notes will not deal with specialised methods as required for cytological work, for instance.
---------SAMPLE PRETREATMENT Fossil "rocky" material (diatomites) may require specialised treatment and is treated separately
further on. Assume that the sample consists of a gathering like:
- sediment from a pond or a coastal marine mudflat
- scrapings from stones or piles
- leaves of aquatic plants
- harvest from a plankton net The sample will contain - apart from (hopefully) diatoms - mineral debris (sand, mud, silt) and organic matter (from plant debris to small animals). The aim is to remove both as well as possible while losing as few diatoms as possible. Mineral debris is removed by sedimentation, organic matter (including the diatom cell contents) is removed by oxidation. Sedimentation and oxidation will be treated further on.
Samples must be fixed with formalin immediately after collection. Add about a tenth of the sample volume of 40% formalin and swirl.
Throughout the text, the phrase "discard the supernatant" will occur. Do not use a pipette for this, or do not pour off the supernatant, as either action may disturb the sediment and you may lose material. The best way is siphoning with small diameter soft plastic tubing. The speed of draining can be sensitively controlled by pinching the tube.
---------------------------------------------------------------------------------------------Separate the coarse organic debris first. Objects like plant leaves or stems take ages (and gallons of oxidant) to decompose. On the other hand, they cannot be discarded because they may bear rich populations of diatoms (epiphytes!). The same is true for small stones or shells (epilithic species) and even for sand grains (epipsammic species).
Sample pre-treatment aims at detaching the diatoms from such substrata. Procedure:
1) Remove excess water.
If the sample contains a large volume of water, pour the entire gathering - leaves, stems, sand, algae, shells etc. - through a household sieve (plastic, mesh about 1 mm), collecting the "fluid" fraction (which also contains the sand/silt). What remains on the sieve (the solid fraction) goes into a generously sized glass beaker. Let the fluid fraction settle completely (check in direct sunlight, the supernatant should not be "milky"), discard supernatant, resuspend the sediment with just enough water and add to the solid fraction. These steps simply remove excess water and may not always be necessary: if the sample does not contain large plant fragments or floating stems, just let the material settle and discard the excess water.
2) Detach diatoms.
Pour some hydrochloric acid (household quality will do) onto the material. Calcareous matter (limestone, shells) will dissolve with production of foam. Stir and leave until foaming subsides, add some more hydrochloric acid until no foam develops anymore.
Add enough water to cover the sample by a layer of a couple of cms. Heat this gently and let simmer for about half an hour. Beware of fumes. This detaches the diatoms by dissolving mucus.
The process can be assisted by scrubbing the leaves, stems or stones with a plastic toothbrush.
Pour through a plastic sieve and collect the fluid, pour some more water over the residue in the sieve to wash out remaining diatoms. Collect this water too, discard contents of sieve.
3) Remove acid.
Let the fluid settle completely, discard supernatant. Add tap water, mix thoroughly with the sediment, let settle and discard supernatant. Repeat at least twice. This removes the acid and the calcium chloride into which the calcareous matter has been converted. Rinse twice with distilled water.
This pre-treatment procedure ensures that you will not try to oxidize more stuff than is necessary, whilst avoiding major loss of diatoms. You'll always lose a few! At this stage the volume of the sample has become much more manageable and the "raw" material can be stored for further processing if you add formalin. Incineration and mounting (see later) allow quick examination under the microscope.
Not all materials require such pretreatment. The most unfavourable situation has been assumed (leaves, stems). Samples like scrapings from stones, plankton catches or rich harvests of periphyton may not require anything but a rinse in hydrochloric acid just to be sure no calcareous matter is left. Then rinse thoroughly with distilled water and add some formalin.
Type- and other materials from collections require special care. If the material has been oxidised already and is stored in distilled water, slides can be made without further processing. There are
two special cases, however:
1) Tiny fragments of mica with specks of sample.
2) Samples in glass tubes that have dried out and form a hard cake that sticks to the tube.
Museum material is precious and there may be very little of it. Yet, it may be possible to collect specimens from tubes that are on record as being "empty" - and indeed look so! Some concessions may have to be made to cleanliness, the purpose is to at least recover whatever diatoms may be left...
1) Mica fragments.
Place the fragment in a small test tube. Add a few drops of concentrated (30%) hydrogen peroxide. Let stand in sunlight for two days or so or gently heat to about 60° C. for an hour or so (water bath). Swirl gently every now and then. This will detach most of the diatoms from the mica. Take out the mica with pincers and transfer it to another test tube (#2) with some distilled water. Let the fluid in tube #1 settle completely and the supernatant can then be carefully (!) pipetted off, followed by two rinses of distilled water. Check critically (preferably in direct sunlight) that no material sticks to the wall of the tube! If it does, swirl briefly and let settle again.
If the sample is minute, it's easy to lose everything. In that case a slide can be made at this stage, without further rinsing. Some peroxide remains, but this evaporates when you make the slide. To remove any residual organic matter, use incineration (see further on). Note that if the material was of marine origin, rinsing may be unavoidable, to remove the salt.
ESSENTIAL: take out the mica fragment from tube #2, dry it and make a slide of it too! Place the mica on a small blob of mountant on a slide, the surface that carried the material uppermost.
Put another small blob of mountant on a cover-slip, turn over the slip, drop it onto the mica and heat gently for an evenly spread and bubble-free layer of mountant. Apply some pressure (place a small weight on the cover before heating). The microscope image will not be perfect, but I've had a case where the only type specimen present in the entire sample had remained stuck to the mica!
The water that remains in tube #2 is added to tube #1 - it still may contain the single diatom you're looking for....
2) Dried out samples.
The natural inclination is to scrape a bit from the surface. NEVER (!) do this, probably the uppermost layer will merely contain the finest silt or the smallest diatoms in the sample and damage will also ensue.
Procedure: add a few drops of concentrated hydrogen peroxide. This softens the cake and after some time you can re-suspend the sample. Collect your subsample. For both subsample and the original sample replace the peroxide with distilled water (let settle and rinse, repeat 2x), add some formalin for storage.
CAUTION: if the dried-out sample is of "raw" (unoxidized) material, peroxide may lead to foaming, see "Beware of peroxide".
A lively reaction will occur when an oxidant is added to hydrogen peroxide (see further under "oxidation"). But even when used alone, peroxide has nasty surprises in store.
Adding hydrogen peroxide to unoxidized material may result in quite severe foaming and even "brewing up" of the lot. I have had to cope with truly explosive reactions after adding hydrogen peroxide to 40-year-old dried-out mud cakes! The phenomenon appears to be limited to muddy samples (including dirty periphyton) - do some muds contain katalytic minerals?
The most treacherous aspect of peroxide is that "brewing up" may take quite some time (several minutes) to start, when nothing much appears to happen. Then it may chain-react in almost no time to catastrophic intensity. Constantly keep an eye on a peroxide brew and if foaming seems to get out of hand, pour everything QUICKLY into a MUCH larger wide beaker or better yet: a flat dish. NEVER (!) put a stopper on the tube or you may have to collect the remnants of the sample from the ceiling.
To my knowledge, no author has ever issued such a warning while describing the use of peroxide.
- For very small samples, see under "Dry (herbarium) materials".
- Just two methods of oxidation have been selected here. Both yield good results and require the minimum of widely available chemicals. Of course, many more have been described in the literature, but all seem to yield comparable results. Patience is more important than the chemical brew - do not hurry.
- Processing a small portion of the sample, say a layer of a few mm in a 50 ml beaker, is
preferable to treating a larger quantity. Use beakers with a flat bottom and straight (not:
- If hydrogen peroxide is used, refer to "Beware of peroxide" first. Also, use wide beakers or dishes. Glassware must be heatproof (Pyrex etc.) as it becomes boiling hot very quickly.
- All oxidants are corrosive and fumes are toxic.
- Home-cooking of diatom samples knows no immutable laws but asks for flexibility, adapting your procedure to the material in question. Samples differ enormously in "difficulty" !
1) When the sample has settled completely, discard supernatant
2) Add a small quantity of concentrated (30%) hydrogen peroxide
3) Let stand for several minutes. If alarming foaming already occurs, let this subside and only then add a little more peroxide. Repeat until foaming becomes less violent. If no serious foaming has occured several minutes after the first small amount of peroxide has been added, add peroxide until the volume is about 10x that of the original sample.
3) Heat gently (water-bath) for 30 minutes or so (depending on amount of organic dirt).
Constantly watch, foaming can still get out of hand.
4) Take the beaker out of the water-bath and place it on the bench, preferably in a wide dish or on a plate. (If the reaction gets out of hand, you can then save the sample if it boils over).
5) Add a VERY SMALL pinch of finely powdered potassium bichromate, just a speck. A violent reaction will occur, swirl and let subside. Only then add a little bit more of the bichromate. Continue this until the reaction has stopped, the contents of the beaker must now be orange in colour.
6) Let settle completely, discard supernatant, resuspend with ample water and repeat this at least twice.
For plankton catches and other samples with very little organic dirt, steps 1-3 may be sufficient.
Sulphuric acid method:
This has the advantage of not causing violent foaming. Check that all calcareous compounds have been removed first, otherwise the sample will become totally useless because gypsum crystals will form.
1) When sample has settled completely, discard supernatant
2) Add concentrated sulphuric acid (battery acid from garages will do if you have to improvise) until the volume is twice that of the original sample.
3) Add potassium bichromate. In contrast to the H202 method, no special care is necessary as no violent reaction occurs. Just add enough bichromate to make for a saturated solution (a few orange crystals left on the bottom).
4) Let stand for 24 hours or more, or speed up the reaction in a water-bath (60 degrees or so).
Even so, it may take several hours before the sample is clean. The sediment should look greyish and no plant fragments etc. should remain.
5) Let settle completely, discard supernatant and rinse several times as described above.
The sulphuric acid method seems to remove resistant "dirt" somewhat better than the H202 method, mainly because the oxidation reaction is not as abrupt as with peroxide. But again, the principal point is patience, not the chemistry involved.
- After oxidation, sedimentation aims at removing as much of the mineral "dirt" (sand, silt, clay) as possible while losing as few diatoms as possible. Especially with very fine silt/clay, this may be difficult and some concessions may be necessary: the cleaner you want the sample to be, the greater the chance of losses. Attempts at getting "nice, clean" samples may be incompatible with quantitative investigations!
- Especially in sedimentation, you'll have to adapt your procedure to the nature of the sample.
There are no standard time-schedules: some stuff settles in a few minutes, other samples may take hours. The only good method is individual checking, see further.