Procedures are given for the preliminary preparation of bulky samples, cleaning recent and fossil diatomaceous material, and the preparation of select and strewn diatom sides.

Keywords Diatoms, recent and fossil, cleaning methods, select and strew slide preparation.

Introduction

This paper is presented in two sections: first a description of the procedure for mounting a selected diatom or making a strewn mount and second, an appendix which includes the details of apparatus, materials and treatment methods under the following headings:

1.	A method of reducing the bulk of material prior to cleaning.
2.	Cleaning freshly gathered diatoms.
3.	Cleaning Oamaru earth and other similar diatomites.
4.	Treatment for the removal of flocculence in diatom samples.
5.	Diatom fixatives for select mounts.
6.	Cleaning slides and cover slips.
7.	Preparing slides for selecting diatoms from sample material.
8.	Preparing cell slides for thick diatom valves.
9.	A “seeker” for picking individual diatoms.

In the following procedures the appendices cited above will be referred to when appropriate, by their prefix numbers only.

Select Mounting

The following procedure is best carried out under a stereo microscope; a zoom instrument being preferable. Having found a diatom valve on a prepared slide (7) which is required for mounting, proceed as follows:

On the near side of sample slide (7) is placed a second dust free slide so that it is touching, and is line with the first slide. A cleaned 10mm cover circle (6) is taken up with tweezers in the right hand and held edgewise between thumb and second finger of the left hand.

Next the tip of the little finger of the right hand is dipped into distilled water and thoroughly rubbed dry on a dust free cloth. The left hand holding the cover is now held close to a spot lamp above the work area so as to drive off the moisture which should take 5 to 10 seconds to clear. The cover is now held near bench level in such a way as to reflect the spot lamp upon its surface, while with the right hand a toothpick is carefully dipped into a diatom fixative (5a-b). The tip of the toothpick is just touched near the edge of the cover so as to deposit the smallest possible drop. The tip of the cleaned little finger of the right hand is now very gently touched to the drop and lightly drawn across the central portion of the cover. This will require much practice, and when correctly done will show as a very thin smear, just visible under the spot lamp, when the cover is correctly manipulated.

The “seeker” (9) is now taken up in the right hand and carefully touched to the diatom required. A careful sweeping action is usually the most successful, and when fixed to the bristle it is possible to manipulate the seeker while studying the valve surface. It is desirable to mount the valve with the inside uppermost so that it will be seen from the outside in the finished mount. This also prevents the valve edges from being sealed in the fixative and trapping air. Sometimes it is necessary to deposit the valve and re-lift it so as to have it in a good attitude for final deposition on the cover. When the valve is suitably in position on the bristle end it is lifted vertically clear of the slide and held there while, with the thumb and forefinger of the left hand, the slide and cover is pushed forward until the cover is in the field of view. This is where the infocus zoom system is a great advantage, for the cover circle can be made to almost fill the field of view, thus allowing the diatom to be placed centrally on the cover with great accuracy.

The bristle end is now very carefully lowered to the centre of the cover and manipulated so as to place the valve level on the gum deposit; again considerable practice and patience is required to master this step. If too much gum (5a) has been deposited the diatom will be swamped and rendered almost invisible. This also results in gum residue usually remaining in the valve pores, often producing artefacts in the finished mount that can be mis-interpreted as valve structures; particularly in the case of the disciforms as the residue usually forms concentric circles. However, if gum (5b) is used, swamping does not occur because the smear dries before the diatom is deposited upon it; and is finally fixed by gently breathing on the cover. Gum (5a) remains wet until dried at a high temperature. When the diatom has been successfully deposited on the cover, the whole area can be inspected for dust particles and these can be swept away with the bristle before the cover with its slide is transferred to a slide heater for final preparation. A half petri dish should be placed over the slide and cover to prevent dust falling upon it.

I have designed and constructed a special micro slide heater utilising the sole plate of a domestic iron. This has an electronically controlled and switched temperature range allowing very accurate control, without overshoot; permitting the boiling point of any solvent such as xylene, to be closely approached without fear of causing bubble formation while hardening the medium, which in this case is naphrax (Fleming, 1954). A temperature of 142 degrees Celsius is chosen for two reasons: It is very near the B.P. of xylene (143 deg. C.) and is ideal for clearing the film of fixing gum (5a).

A cleaned slide (6) to be used for the final mount, is placed on the heater, behind the one bearing the cover circle. Upon the centre of the cleaned slide is placed a small drop of naphrax. It is preferable to use naphrax containing very little xylene which allows the mounts to harden more quickly. This naphrax is quite hard when cold but becomes fluid after standing on the heater for a while. A little xylene may be added occasionally to replace that evaporated away.

Before the cover is placed in the naphrax on the clean slide it is necessary to flood the cover and diatom with a drop of “imbibing solution” which consists of a very thin solution of naphrax in xylene. This page 13 displaces the air from the pores of the diatom valve and largely prevents the formation of air bubbles in the final mount.

The imbibing solution is applied to the cover with a fine pipette and when the solution starts to evaporate away the cover is carefully pushed with a toothpick towards the edge of the slide so that it slightly overhangs the edge nearest to the prepared slide. This allows it to be carefully picked up with tweezers, turned over, and carefully lowered into the naphrax on the prepared slide. The slide can be taken off the heater after a minute or two, allowed to cool and then checked under the microscope. If satisfactory it can be put aside to be returned to the heater when the batch of slides is completed. The hardening should take 10 to 12 hours, when the heater can be turned off and the slides gradually cooled. Any bubbles that have not dispersed in the heating process can be driven off with extra heat from a spirit lamp, taking care to distribute the heat evenly to avoid cracking the slide.

It will be found convenient to make a small stand to take the bottles of the substances used in the above procedures. A base can be made of thick particle board about 110 × 200mm drilled with four holes the exact size of the small bottles used: Two small phials about 18mm diameter, one for the imbiding solution and one for xylene in which a small glass rod is placed after each application of naphrax. This xylene will in time form further imbibing solution from the small amount of naphrax dissolved in it after each application. Two larger holes are drilled to accommodate the diatom fixative bottle and the naphrax bottle when not in use. A hole about 6mm diameter is useful for holding a few toothpicks, and a small bracket can be fitted to hold tweezers and a pipette. The base can be fitted with rubber feet and coated with clear varnish to protect it from soiling.

Strew Mounting

The cleaned sample is checked for density by examining a temporary water mount under phase contrast. The sample can then be concentrated by decanting, or diluted with distilled water. In many cases samples will contain a little ammonia to prevent clumping; or a preservative to prevent the formation of fungus. It is necessary to remove any additive by further washing otherwise an evaporation ring and-or crystals will appear around the edge of the dried strew. An absolutely clean sample, slowly dried, will not leave a residue.

When the sample has been cleaned ready for use, some cleaned 16mm, No. 1½ cover circles are set out on a small strip of wood for easy handling. It is vital that the covers be absolutely clean (6), otherwise the material will not disperse evenly over the cover when deposited on it. The material is thoroughly shaken and a pipette partly filled. This is held several inches above the centre of the cover and a drop or two is allowed to fall. If the cover is clean it should be completely filled like a convex lens. If the liquid does not reach the edges evenly all around it can be made to do so by touching the end of the pipette into the drop and drawing the material towards the edge where it should remain.

The covers are now placed aside, under a perspex canopy to dry as slowly as possible. When dry they can be inspected under a stereo microscope and any dust particles or other debris can be removed. When the strewn material seems satisfactory the cover is placed on a slide upon the heater at 142 degrees C, while a cleaned slide, also on the slide heater, is prepared to receive it. A drop of naphrax is deposited on the slide centre page 14 and a drop of imbibing solution is pipetted onto the strewn cover. The cover is then carefully slid with a toothpick to the edge of the slide where it can be carefully lifted with tweezers, turned over and deposited onto the naphrax. With strewn mounts it is usually necessary to apply heat from a spirit flame to disperse bubbles. When this has been done the slide can be cooled and inspected before returning to the slide heater to harden for 10 to 12 hours.

Appendix

1. A method of reducing the bulk of material prior to cleaning

A lot of sample material collected from tidal inlets, estuaries, etc, contains a large quantity of muddy sludge, sand, and other debris which makes cleaning prolonged and tedious. The following method is very successful in largely overcoming this problem:

The sample is first tipped from the collecting jar into a large beaker of fresh water which tends to kill the diatoms etc, and allows access to marine grasses, filamentous algae, etc. which may be shaken about vigorously and squeezed out to dislodge any adherent diatoms.

The mixture is then allowed to settle for several days when a lot of the water may be siphoned away. The next procedure is to pass the material in small quantities through a sieve, such as a petrol filter fitted into a large plastic funnel held under a running tap. A large container should be placed in the sink to take the material passing through the sieve. Large debris can be removed with tweezers from the material remaining in the sieve before it is washed into a beaker. Sieving should be done in stages, a small quantity at a time, followed by a reverse flushing of the sieve under the tap. This will prevent the sieve from becoming over-loaded and blocked. Upon inspection under the microscope, the heavy fraction may be found to contain no diatoms, but it will certainly separate a lot of debris from the main sample.

The above procedure is repeated a second time using a 38 micron sieve, which will give a good separation of the smaller forms, and the material remaining in the sieve is carefully flushed into a beaker. The whole procedure effectively frees the sample of salt and considerably reduces the bulk of material for final treatment as well as giving a good indication of the diatom species present. The two fractions can be concentrated and treated separately in the final cleaning described under heading 2.

Mud samples of marine estuaries etc, must always be washed in distilled water before further treatment. It has the property of holding clay particles in suspension, which can then be removed by siphoning or decanting. If calcareous water is used any clay particles present will be precipitated with the diatoms.

2. Cleaning freshly gathered diatoms.

The method used is that of Swatman (1941) revised by Hendey (1974). The method given in these references has been found very satisfactory and for several reasons is preferable to the more usual and vigorous mineral acid treatment: It has a more gentle action on the weakly siliceous forms thus preserving some of the species normally destroyed by boiling in acids.

Also it is not a hazardous operation, requiring no heating or giving off dangerous fumes. The cleaning is found in every way as effective as mineral acid treatment.

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3. Cleaning Oamaru earth and other similar diatomites

The following method is that of A. J. Doig (personal communication) and has proved very satisfactory: In a suitable enamel dish place some of the material, broken to convenient sized lumps, to a depth of 12 to 18mm. Add an approximately equal quantity of sodium acetate crystals and heat in a water bath until the crystals liquefy. A few drops of water may be added as the acetate begins to melt. Stand dish aside until quite cold, then drop in a small crystal of acetate. The liquid, which should just cover the lumps of diatomite, will immediately solidify, with the generation of slight heat. Leave for an hour or more for the action to become complete within the lumps of diatomite, then again heat in a water bath. As the acetate melts, a certain amount of material will be seen to have broken down. Again cool and crystallize, and repeat the process until the diatomite has all broken down into mud. Now wash out into a 1/2 litre beaker of warm water, and allow to settle for at least one hour but preferably two. Siphon off the water to just above the sediment, refill and repeat above process at least eight times to remove all traces of acetate.

Oamaru earths may be divided into two groups, the siliceous and the calcareous. The older siliceous deposits, represented by Cormack, Allan's, Bairns and Lower Papakaio, underlie layers of volcanic rock, and contain little or no lime. The slightly more recent calcareous deposits, represented by Jacksons, William's Bluff, Troublesome Gully, Totara and Upper Papakaio, underlie deposits of limestone and contain lime in the form of shell fragments and foramenifera. This lime must be removed before treatment is begun. With calcareous material, after washing out the sodium acetate, pour in hydrochloric acid, in small quantities, until effervescence ceases. This must be removed, along with the calcium chloride formed by reaction with the lime; another series of at least six changes of water being required. The material will now be ready for treatment, which is described in 7 stages.

(1) In a “Pyrex” glass flask, or better still, a Pyrex ovenware dish, concentrate about 6mm of sediment, add about 25mm of commercial nitric acid, diluted 1:1, and boil for 20 to 30 minutes. Unless a fume closet is available, this operation must be carried out in the open air to avoid harmful acid fumes.

After boiling for 20 to 30 minutes, extinguish the stove and allow the sediment to settle. Now carefully pour off the greater part of the acid, and replace with dilute hydrochloric acid, again boil for 20 to 30 minutes. After cooling, pour the contents of the flask or dish into a 1/2 litre beaker of cold water. This must be changed at least six times to remove all traces of acids, allowing at least one hour for settling between changes. Care must be taken not to disturb the sediment when decanting or siphoning, which is preferable. When acid is removed, the material is ready for the second process:

(2) Place material is an enamel vessel with 300ml of water, add 1/2 teaspoon of sodium bicarbonate, and boil for 20 minutes. If a glass beaker is used, “bumping” may cause trouble. After boiling, transfer to a 1 litre beaker of water, allow to settle for two hours, decant or siphon, and repeat settling and siphoning seven or eight times to remove soda. The water will at first be turbid with fine earthy matter, of which a lot is removed during this stage. When the soda is washed out, the material is ready for the third process.

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(3) Return material to the enamel vessel and add a teaspoonful of lux flakes and boil for 20 minutes. Heat must be applied carefully when approaching boiling point, to avoid sudden frothing over. After boiling pour into a one litre beaker of hot water and allow to settle for at least two hours. Diatoms and heavy material will sink, light earthy matter and fine fragments remain suspended in the denser soapy water. After settling, siphon off the soapy water, not going too close to the sediment, and replace with water nearly boiling. If a frothy scum of earthy matter forms on the surface after filling, allow this to remain a few minutes, then stir with a fork or spoon, washing the scum off the sides of the beaker. This will cause sediment held by soap bubbles to sink, but most of it will sink as the water cools. After drawing off each change of water, wipe the rim of soap from the sides of the beaker with a cloth. Repeated changes of hot water must be given until no more soap comes away. If the material is now examined it should show a few free diatoms, but will otherwise look much the same as before treatment.

(4) Again concentrate in a flask and boil again in nitric acid. After the acid has been removed as described in section (1):

(5) Boil again in soap and wash out soap as described in section (3).

(6) After the soap has been partly removed by four or five changes of water, return material to the enamel vessel, bring to the boil, and add about 1/2 teaspoon of sodium bicarbonate carefully and in very small quantities, as much frothing takes place on the addition of soda. Boil for 15 minutes, and wash out with repeated changes of hot water as for the removal of soap. Much earthy matter will be removed with these changes of water, and if now examined, the material should show a number of clean diatoms, sponge spicules, radiolaria, and clean sand, but a certain amount of earthy lumps and flakes will still be present.

(7) Repeat the soap-soda treatment until the material is clean.

A spread of fully cleaned Oamaru earth should show clean sand, diatoms and diatom fragments, sponge spicules and spongiolyths, radiolaria, and nothing else. If flakes or lumps of earthy matter are present, the material is not clean.

Most samples contain a large amount of sand. The heavier sand may be removed by whirling the mixture in a beaker. A teaspoonful at a time in an 800ml beaker is about the right amount. The beaker is whirled around to get the “whirlpool” effect, left to stand for a moment until the sand builds up in the centre, and the water is siphoned off at the edge of the beaker. Only 12mm of water over the sand and diatom mixture should be used at a time for best results. Repeated whirlings in this manner will remove the greater part of the sand. Very fine sand may be removed by sieving material through a 38 micron sieve, but very small diatoms will be lost unless the sievings are kept for examination.

Note: When cleaning large quantities of material, it is best to give one soap treatment before initial boiling in acid. This will reduce the bulk considerably.

4. Treatment for the removal of flocculence in diatom samples

A 5 per cent solution of sodium hydroxide is made in distilled water. To every 5cc of diatom suspension add No More than two drops of the 5 per cent solution. Bring almost to the boil and simmer for no more than one minute. Add cold water, neutralise with several drops of sulphuric acid, bring to the boil for a few minutes and wash sample in the usual way. Unless flocculence is very bad, one treatment should be sufficient. It should also remove the crystals which are sometimes seen in strewn mounts.

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5. Diatom fixatives for select mounts

Several fixatives (gums) have been used sucessfully. The first was that used by A. J. Doig (personal communication); and the second was recommended by Brun which is preferable because it has nearly the same R.I. as glass and is invisible in the finished mount, even under phase contrast.

(a) Egg albumen 50% and Glycerine 50%.

Mix together by stirring fresh egg albumen with an equal amount of glycerine. Filter through the fastest paper (Whatmans No. 1); change the filter paper regularly and pour remaining deposit from one to another. When the mixture becomes too thick for further filtration discard and proceed again with a fresh mixture until sufficient fixative is filtered (10ml is ample). Add 1% chlorbutol and the mixture will keep indefinitely.

(b) Gum tragacanth powder, alcohol (methylated spirit), distilled water.

The powdered gum is made into a paste by carefully adding small quantities of alcohol from a pipette and stirring to prevent lumping. Warm distilled water is then very carefully added while stirring. A saturated solution is made and filtered while warm. Alternatively a thin solution of the gum can be more easily filtered and finally concentrated over a water bath. A little alcohol should be added to prevent mould formation.

6. Cleaning slides and cover slips

The necessity for absolutely clean slides and covers for critical work cannot be over emphasised. “Decon 90”, a laboratory glassware cleaner, has been used with excellent results. A 15 per cent solution in distilled water is prepared and kept in a wide mouth, screw-capped jar. The amount of soaking required depends on the degree of soiling; 1/2 to 24 hours being recommended.

However, with the “pre-cleaned” or “select” slides and covers it is usually sufficient to immerse for 30 to 60 seconds followed by a rinse in distilled water. Drying is done with a soft cloth or handkerchief and finally with tissue, while holding to a strong light source to inspect for dust particles and absolute cleanliness before using.

The following mixture is suitable for very dirty slides and covers which may remain immersed until required, when they are drained, washed in running water for about 10 minutes, rinsed in distilled water and dried as described above:

Slides and covers can also be immersed in glacial acetic acid for cleaning until required. They are then removed, drained and finally rinsed and dried as described above.

7. Preparing slides for selecting diatoms from sample material

Obtain a number of reject slides and with fine carborundum paste grind one side of each slide so as to produce a frosted surface. This is best done by sandwiching a small amount of paste between two slides and gently grinding together until evenly frosted. The process can be checked by washing, drying and inspecting under a light source.

The purpose of the frosted surface is that when a strew of cleaned diatom sample is slowly dried upon its surface, the irregularities produced by the grinding prevents the diatoms from sticking down as they usually do on an ordinary slide.

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The next step is to divide the prepared slides into grid squares which aids in scanning the material. The marking can be done with a carbide tipped scriber or a diamond pencil. The squares should be of such a size that their edges are within the field of view of the stereo microscope at a suitable magnification to the worker.

It is necessary to use top illumination and this tends to refract the light from the diatoms making them easier to see; a very shallow angle of illumination gives the best definition. A matt-black stage plate should be used for best results.

Many preparations can be stored away on frosted slides for long periods and they can be picked from when specimens are required for select mounts. It may be necessary to gently heat the preparations before using so as to drive off any moisture which tends to stick the diatoms down. In some cases however, the small amount of moisture can actually aid the removal of certain valves that may stick slightly to a perfectly dry surface.

8. Preparing cell slides for thick diatom valves

Slides are ringed on a turntable to make a circle of the same diameter as the cover circle to be used. Purple shellac ringing cement, soluble in alcohol, is quite good and withstands temperatures of 100 to 120 degrees C before releasing particles into the mount. Gold size soluble in xylene provides a better ring although it is harder to see when aligning the cover circle upon it. It is not so conspicuous in the finished mount; it withstands a much higher temperature without dispersing particles, and contary to expectations, it does not dissolve in the xylene remaining in the naphrax mounting medium while the mount is hardening.

The slides are prepared in convenient batches and placed in a warming cupboard for about five days to dry slowly. After hardening the ring is slotted in four places to allow air bubbles and surplus medium to flow out.This is accomplished by immersing the slide in a shallow flat dish of water prior to slotting. This prevents the shellac chips from adhering to the slide while cutting which is best done with a “gem” razor blade.

The technique has been very successful for mounts of large valves of Oamaru fossil forms. Providing slides of 1 to 1.2mm thickness are used no trouble will be experienced in focussing the substage condenser and reasonable optical sectioning with a 20 to 40x apochromatic objective is still possible.

9. A “seeker” for picking individual diatoms

The handle: This is made from 6mm square stock brass about 100mm long. It is drilled centrally in one end, parallel to its length, to a depth of 12mm with a 3mm hole. Another hole is drilled 6mm in from the end previously drilled, centrally on one of the sides so as to intercept the end hole. This hole is tapped to take a small grub screw and the diameter of the hole must suit a threading tap of the screw size chosen.

The seeker bristles: These are taken from a well worn paint brush which ensures that the bristles have well-rounded ends, which are essential for diatom manipulation. The bristles are cut about 25mm and a batch is selected under the stereo microscope to see that the bristle end is not split or coated with old paint. Next some pieces of insulated sleeving commonly known as “spaghetti” in the electronics trade, and having an outside diameter of slightly less than 3mm, are cut 25mm long. A small quantity of “Araldite” epoxy resin is now mixed and the cut end of each bristle is dipped into the mixture and worked into the end of the spaghetti pieces, page 19 taking the glue in with it, to a depth of about 12mm. These bristles are now inserted into holes drilled into a small board prepared to receive 10 or so at a time. When hardened, one of the bristles is screwed into the end of the handle ready for use. The others are stored away for future use. Some bristles will be found better for picking up diatoms than others and by exchanging one for another, an ideal one should be found.

Acknowledgment

Thanks are due to Dr Vivienne Cassie, Botany Division substation, DSIR, Auckland, who encouraged me to submit my methods for publication. I also thank Mrs E.M. Doig of Oamaru, for permission to publish the late A. J. Doig's methods of Oamaru diatomite preparation.