STAINING AND OTHER METHODS FOR ENHANCING THE OBSERVATION OF CELLULOSE LACQUER ROCK PEELS.

BY KEITH W. ABINERI, UK.

42 West Borough, Wimborne, Dorset BH21 1NQ, UK
Tel. 01202 885547



Introduction.

With the previously discussed unstained cellulose lacquer rock peels, a small area of the rock surface (ca. 2 cm2) was prepared initially by grinding, flatting and polishing with abrasive papers Nos. 320, 600 and 1200 respectively. Between each stage the rock surface was rinsed thoroughly with distilled water. Then generally 2% hydrochloric acid was used to etch the rock surface i.e. two or three drops of the acid were spread over the area and allowed to react for about 12 - 15 seconds, followed by rinsing with distilled water. Finally, before applying the cellulose lacquer, the rock surface was treated with a few drops of acetone (propanone) to remove the last traces of water. These carefully prepared unstained peels could then be examined using brightfield, darkfield, phase contrast, parallel polarizing plates (PPL) or crossed polarizing plates (XPL) types of illumination. With my Nikon substage condenser it was possible also to use darkfield illumination combined with XPL, to enhance the observation and photography of the walls of the chambers in minute Upper Chalk Foraminifera skeletons, or tests.

However during late 1986 and early l987 the first experiments were completed by the writer to combine the cellulose lacquer rock peel technique with a well established method for staining carbonate rock material. This latter method was published in 1984 by A.E.Adams, W. S. MacKenzie and C. Guilford1, in their excellent "Atlas of sedimentary rocks under the microscope" in the References and refers to the staining of cellulose acetate peels. The very high resolution obtained with the cellulose lacquer unstained peels, encouraged me to use this method of staining with some modifications.

Modifications to produce stained cellulose lacquer rock peels.

In place of the simple acid etching solution the following is used :-

Solution (A):
10 cm3 0.5 Molar Hydrochloric Acid.
20 cm3 Distilled Water.
36 mg Alizarin Red S.

This solution may be held in stock as it is quite stable. Just before staining the rock surface, 4.0 x 10-2 grams of potassium ferricyanide, K3Fe(CN)6 is added to 5 cm3 of solution (A) and dissolved. This mixture will stain many rock samples since only a few drops are needed for about 2 cm2 of each rock surface, but it should be used as soon as possible.

The quantities of Alizarin Red S and potassium ferricyanide can be estimated in the absence of an accurate chemical balance. For this purpose the following approximate "pack densities" might be useful:-

Alizarin Red S 0.5 grams/cm3.
Potassium Ferricyanide 1.5 grams/cm3.

The 0.5 Molar hydrochloric solution can be obtained from chemical suppliers such as B.D.H., who stock also Alizarin Red S and potassium ferricyanide. All these chemicals must be handled with care to avoid contact with skin or eyes.

After the normal grinding, flatting, polishing and thorough rinsing with distilled water, the 2 cm2 area of the dried rock surface is treated with a few drops of the combined etching and staining solution using a small pipette, or dropper. With carbonate rocks or calcareous microfossils or nannofossils in clays or shales, the staining period can be from 20 seconds and 60 seconds, depending on the nature of the rock and practical experience. During this period the solution on the rock surface should be very gently agitated, using the dropper pipette. Now the stained surface must be very thoroughly rinsed with distilled water to remove all traces of the reagents. No detergent must be used.

After drying, acetone2 is applied to remove the last traces of water. Finally the cellulose lacquer is applied and used to prepare the peel and finished microscope slide in the usual manner. During these latter stages avoid touching the stained rock surface.

The following staining colours are obtained.

Pure Calcite3 : Pink to Red-Brown.
Ferroan Calcite4 : Mauve to Deep Blue depending on the Iron (II) content.

The intensity of these colours depends also on grain size. With smaller grain size the intensity is greater. Also :-

Pure Dolomite5 : No stain.
Ferroan Dolomite6 : Very Pale Blue to Green.
Silica7 and Silicates8 : No stain.

The chemistry of this staining procedure will be discussed in a later article.



The six accompanied illustrations and notes cover the following aspects :-

Figures (1) to (4) inclusive : Examples of stained cellulose lacquer rock peels.

Figure (5) : An example of a stained cellulose lacquer peel as seen with darkfield illumination.

Figure 6 : An example of an unstained cellulose lacquer peel as seen with crossed polarizing plates (XPL).

Figures (1) to (4) show individual differential staining of nannofossils and aid in their detection.

Figure (5) shows the effect of oblique illumination on the appearance of a stained cellulose lacquer peel.

Figure (6) shows numerous minute optical figures of coccoliths and other calcareous nannofossils9 embedded in transparent kerogen10 organic residues, which have been removed from the rock surface by the peeling process.


Examples of stained cellulose lacquer rock peels i.e. Figures (1) to (4).

Figure (1). Differentially stained marine nannofossils (coccoliths and coccolithophores) in a layer of coccolithic limestone, from the Upper Jurassic, Upper Kimmeridge Clay, Freshwater Steps Stone Band. Map Reference SY.943.772. Freshwater Steps, west of Hounstout Cliff, Dorset coast. Objective N.A. = 0.65. Total field area of the image ca. 210 microns X 150 microns.


Figure (1) is an example of both replica and removed thin layer type of images. It shows an area of "golden" coloured coccoliths which are really not stained, because they are buried in transparent kerogen, which gives them this appearance. The pink areas and nannofossils are due to pure calcite composition. The dark blue areas and nannofossils indicate ferroan calcite composition. The black objects suggest fusain11 (carbon) or pyrites grains12. Figure (1) implies the occurrence of an algal "bloom" of coccolithophore marine phytoplankton in remote Kimmeridgian times, with associated mixed aerobic13 and anaerobic14 conditions on a minute scale. The picture was obtained with brightfield illumination and PPL.

 

Figure (2). This picture was derived from the same stained cellulose lacquer rock peel as used with Figure (1), but covering a different area with increased magnification. Here oil-immersion objective N.A. = 1.25 was used. The total field area of the image = ca. 90 microns X 70 microns.


Here again Figure (2) is an example of both replica and removed thin layer type of images. It was obtained with brightfield illumination.

The conclusions from examination of Figure (2) are similar to those from Figure (1). It should be noted that the staining of these peels does produce some damage to the nannofossils by the action of the acid (dilute hydrochloric) on the calcareous material. This results in the loss of some clarity with some highly magnified stained images, when compared with similar unstained peel images. However the complex staining on such a minute scale on Figure (2), does appear to represent an equally complex chemistry (or biochemistry) of these largely biogenic sediments.

Figure (3). Here we have larger nannofossils on a complex background of numerous much smaller nannofossils, many of which are embedded in transparent kerogen, and are unstained "golden" coccoliths. This picture is from the Upper Jurassic, Upper Kimmeridge Clay, Freshwater Steps Stone Band. Map Reference : SY. 943.772. Freshwater Steps, west of Hounstout Cliff, Dorset coast. Objective N.A. = 0.65. Total field area of the image = ca. 270 microns X 200 microns.

Figure (3) was obtained with brightfield illumination and PPL. It shows land forest swamp debris mixed with marine nannofossils and microfossils, typical of Kimmeridgian times.

It is a further example of both replica and removed thin layer type of images. The staining on this peel is uneven as is shown on this small area, probably due to kerogen and other organic residues present in the sediment. The prominently red stained calcisphere15 (shown right) is interesting with its outer pale green layer. This suggests a pure calcite interior with an outer layer of ferroan dolomite. The slow change of calcite (diagenesis) to dolomite has occurred in a number of stone bands in the Dorset Kimmeridge Clay. This may have biological origins. The further conversion to ferroan dolomite suggests some anaerobic conditions in this particular case, since minute quantities of the reduced form of iron (II) appear to have displaced calcium in the carbonate rock, in the nannofossil. Elsewhere on this peel calcispheres have been found showing a deep blue interior, indicative of ferroan calcite. These are further evidence for anaerobic conditions on a minute scale. The presence of fusain and pyrites buried in the kerogen on Figure (3) suggests another reason for the anaerobic environment.

A further largely buried object lies in the central area of Figure (3). This is covered with coccoliths and other nannofossils and has a diameter of circa. 58 microns. It is believed to be a marine microfossil, a Dinoflagellate Cyst16. Many species of these are found in the various Kimmeridge Clay beds, often buried in the peeled kerogen layers. They are best studied by complete separation from the rock by palynology techniques using concentrated hydrofluoric acid to remove all the mineral contents and preparing a microscope slide of the organic residues only.

Figure (4). Here we have a minute part of a stained cellulose lacquer peel from the Cretaceous, Upper Chalk, Actinocamax Quadratus Zone. Map Reference SY.851.802. West of Arish Mell on the cliff, Dorset coast. Oil-immersion objective N.A. = 1.25. Total field area of the image 80 microns X 60 microns.


It shows the probable cell-division of a Chalk Calcisphere on a background of numerous Chalk Coccoliths, some of which are damaged or partly buried. The differential staining is interesting and appears to be due to differences in texture or grain size. Furthermore the complete lack of any blue staining implies the absence of any Ferroan Calcite and therefore the predominatingly aerobic environment. We may deduce from this that the Upper Chalk was deposited in shallow open seas in the presence of abundant oxygen, which stimulated the photosynthesis for the growth of coccolithophores in the marine plankton.

On Figure (4) the diameters of the two dividing calcispheres are about 11 - 14 microns. This picture was obtained with brightfield illumination and one polarizing plate above the objective.


Darkfield illumination used with a stained cellulose lacquer peel.

Figure (5). Here we have a small part of a stained cellulose lacquer peel from the Lower Jurassic, Lower Lias, Belemnite Marls, Charmouthian. Map Reference : SY.380.927. East of Charmouth, Dorset coast. Objective N.A. = 0.25. Total field area of the image = circa. 1100 microns X 820 microns.

It is another case of both replica and removed layer type of images. The dark-field form of illumination blocks the light from direct transmission though the peel and enhances oblique lighting. This accentuates the blue staining.

Objects are seen by virtue of scattered light. The largest object on Figure (5) is the section (transverse) of a small Crinoid stem17. This has a diameter of circa. 840 microns. Darkfield illumination enhances its internal structure. The blue colour is thought to be due to the inclusion of Ferroan Calcite, indicating an anaerobic environment. The black and brown mottled area is believed to be due to microcrystals of Pyrites, another indication of anaerobic conditions. Crinoids are a small species of the Echinodermata and are found also in remote times in limestones of the Carboniferous period.

Darkfield illumination used with stained cellulose lacquer peels shows many interesting aspects. It often tends to increase the contrast in the images and can help to distinguish between dark wood fusain and pyrities, which so often occur together.


Crossed Polarizing Plates (XPL) used with an unstained cellulose lacquer peel.

Figure (6). Here we have two minute parts of an unstained lacquer peel, observed under XPL, to show optical figures of calcareous nannofossils embedded in transparent kerogen and other organic residues. The peel is from the Upper Jurassic, Upper Kimmeridge Clay, Freshwater Steps Stone Band. Map Reference : SY. 943.772. Freshwater Steps, west of Hounstout Cliff, Dorset coast. Objective N.A. = 0.65. The total field area of the images = ca. 120 microns X 80 microns and ca. 130 microns X 120 microns.


It shows removed material only in the form of real calcareous nannofossils which produce optical figures under XPL. Under this type of illumination no replicas are seen. The white optical figures indicate uncovered calcareous nannofossils, whereas the pale yellow optical figures are embedded in transparent kerogen, (cf. the "golden" coccoliths shown on Figures (l), (2) and (3)). Clearly most of the optical figures on Figure (6) are derived from the coccolith calcite plates which have come from the breakdown of the numerous coccolithophore unicellular plants (marine phytoplankton). These microscopic optical figures are an important characteristic of the crystalline structure of the calcite in the coccoliths. This technique can be used to study a large variety of coccolithic limestones, throughout geological time.


Notes and References.

1. "Atlas of sedimentary rocks under the microscope". This excellent full-colour handbook by A.E. Adams, W.S. MacKenzie and C. Guilford 1984, published by Longman, has an appendix on the staining of acetate-peels.

2. Acetone (propanone) : a very volatile solvent completely miscible with water.

3. Pure Calcite : crystalline calcium carbonate.

4. Ferroan Calcite : crystalline calcium carbonate with a very small proportion of the calcium displaced by iron(II) in the crystal lattice. This occurs under anaerobic environments.

5. Pure Dolomite : a compounded carbonate of calcium and magnesium.

6. Ferroan Dolomite : Dolomite with a very small proportion of calcium displaced by iron(II).

7. Silica : Silicon dioxide in various forms e.g. quartz grains, sand grains etc.

8. Silicates : frequent ingredients of numerous rock minerals.

9. Calcareous nannofossils : Nannofossils largely composed of calcium carbonate. Many forms belong to the Coccolithophyceae.

10. Kerogen : A solid complex organic material which yields petroleum type hydrocarbons under heat and pressure.

11. Fusain : carbonaceous material derived from decaying vegetation or wood.

12. Pyrites : a most widespread sulphide of iron. A sure sign of anaerobic sediments.

13. Aerobic : in the presence of oxygen.

14. Anaerobic : in the absence of oxygen.

15. Calcispheres : Minute hollow microfossils and nannofossils of calcareous composition. Found frequently in chalk and limestone sediments. They have existed in differing forms since the Devonian Period (circa. 380 million years ago).

16. Dinoflagellate Cysts : a common group of microfossils, which have existed since early Jurassic times (circa. 213 million years ago).

17. Crinoids : These are microscopic animals of the Echinodermata group. They are species Crinoidea and their fossils occur frequently in marine limestones.

Editor's note: Some of the quality of the author's original 35mm slides is lost in the scanned and compressed web images. Comments to the author are welcomed, who can be contacted at the above address or comments can be passed on via the Micscape Editor, see contact on magazine index.

 

Article prepared for the Web by David Walker.

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