Organic Ultrastructure of Diatoms: A Possible TEM Technique 

    

Michael A. Gorycki, Ph.D.                      

ABSTRACT

The frustules of diatoms, including fossil forms, make them good subjects for scanning electron microscopy. However, the organic structure of living diatoms is difficult to study by transmission electron microscopy (TEM) because they are too thick, and the frustules, being composed of silica, make them poor candidates for ultra-thin sectioning. The frustules also make the organic material, contained within, difficult to examine by phase contrast microscopy. Since diatoms are useful in the study of water quality, and form part of many food chains, it might be helpful if ultra-thin sections of their organic structuring could be made available for examination. A tentative method is described here that allows a monolayer of whole diatoms to be embedded on a plane in Araldite epoxy and then be exposed to hydrofluoric acid so that the siliceous frustules can be dissolved. A second epoxy may be then allowed to fill the voids created by the hydrofluoric acid. A thin organic layer that may surround the silica structures is retained. This technique allows ultra-thin sections of the organic material of specimens in the embedding plane to be aligned to the cutting plane of a glass knife on the ultramicrotome and cut, stained, and studied by transmission electron microscopy. It presents a comprehensive image of the complete organism.

INTRODUCTION

The study of a living foraminifer, Rosalina floridana (Cushman), (Master’s Thesis New York University, Gorycki, M. A., 1967) involved embedding individuals in Araldite epoxy. The embedded specimens were transected and the calcareous shell wall was dissolved with hydrochloric acid. The wall was replaced with a second plastic so that thin and ultrathin sections of entire individuals could be cut (Gorycki, 1971), (Figs. 1 and 2).  

  

Fig. 1. Low power light micrograph (approx. 225X) of micron-thick section, cut on a glass knife, and stained with methylene blue, of Rosalina floridana showing three largest (youngest) chambers containing some tenuous ectoplasm (ECT), round algal food organisms (Dunaliella parva) (DP), and numerous fine diatoms (Nitschia sp.) (NS). More posterior chambers are filled with endoplasm (dark). Pellicle (P) forms the inner organic chamber walls. Exterior calcareous shell walls, dissolved with hydrochloric acid and replaced with Epon 812, are not visible because phase contrast microscopy could not be employed at this magnification. Specimen was also treated with hydrofluoric acid to dissolve silica of diatoms. All micrographs, except Fig. 3, are from my thesis (Gorycki, 1967), or reproduced in Micropaleontology (Gorycki, 1971).

Ectoplasm, often associated with food organisms, is usually found inside younger chambers, on the outside of the shell, and in the form of anastomosing reticulopodia at a distance from the shell. Older chambers are filled with anterior endoplasm and food organisms. Innermost chambers only contain posterior endoplasm.

 

Fig. 2. High power light micrograph of section of Rosalina floridana, embedded in Araldite epoxy (approx. 1400X), cut on a glass knife. Shell wall, dissolved with hydrochloric acid and replaced with Epon 812, is lighter. Chamber at bottom contains anterior endoplasm and food organisms embedded in Araldite epoxy as is the area outside the shell wall and also filling the pores. Phase contrast microscopy allows differentiation of the two epoxies not seen in Fig. 1. Specimen also treated with hydrofluoric acid.

Early on, it was discovered that the associated food organism, Nitzschia sp., would not allow satisfactorily sectioning of the foram with a glass knife (Fig. 3). 

Fig. 3. Ultramicrograph (approx. 14,000X) of a diatom food organism associated with the foram Rosalina. This first attempt at ultrathin sectioning revealed fragments of the diatom’s siliceous frustules (black) that damaged the glass knife, causing striations and making sections of both it and Rosalina unsuitable for study. Note organic ultrastructure within the specimen’s interior chamber. 

As a result, it was decided to expose the diatoms associated with the embedded forams to hydrofluoric acid to dissolve the frustules. This allowed the glass-knife sectioning and study of the foram and associated diatoms to take place. 

Workers who are interested only in the study of diatoms should ignore the use of hydrochloric acid if there is no evidence of the presence of calcium carbonate in the sample. It is hoped that this first simplification of the embedding technique might be advantageous in the  detailed study of these ubiquitous organisms [1]. 

[1] http://www.tolweb.org/Diatoms/21810

Thin and ultra-thin sections of diatoms included in the anastomosing reticulopod of Rosalina were prepared as an adjunct to the study of that foram because of the use of hydrofluoric acid treatment. Fig. 4 shows a light micrograph of diatoms, with their included organic structure associated with beaded reticulopods and ectoplasm within a chamber of a foram. It is a one-micron section made with a glass knife. Note absence of sectioning artifact because of the solution of the frustules. 

Fig. 4. Ectoplasm of Rosalina floridana containing food organisms (diatoms). Section stained with methylene blue. No sectioning artifact evident. Note beaded appearance of abstricting vesicles of ectoplasmic reticulopod. They are strongly suggestive of a linear configuration (approx. 1400X).

Fig. 5 shows an electron micrograph of Nitzschia sp., with its siliceous frustule dissolved. Some cytoplasm is present inside the shell, which is represented only by its insoluble organic matrix outer coating [2].

[2] www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0061675

Fig. 5. Electron microgram of an ultrathin section of the diatom Nitzschia sp. (NS), which was treated with hydrofluoric acid after embedding in Araldite. Note the organic casing surrounding the former shell wall. The diatom seems to lack an appropriate amount of internal organic ultrastructure, but this may be due to its having being partially digested by the foram. A round algal food organisms (Dunaliella parva) (DP) is present also (approx. 4,500X).

PREPARATION OF DIATOMS FOR ELECTRON MICROSCOPY

As can be seen in Fig. 5, the organic outer casing remains intact which suggests that an in situ study of diatom tissue within the shell might be successfully carried out using a variation of the embedding technique described here. The diatoms may be embedded as a monolayer of individuals in unpolymerized Araldite epoxy. They can be gently pressed between a horizontal sheet of polyethylene (Gorycki, 1966) and a flat, frosted surface of a blank of polymerized epoxy. After polymerization, and removal of the polyethylene, I suggest that the fixed, Araldite-embedded organisms may be exposed by gentle abrasion with a fine, clean, flat whetstone. The abraded surface is then cleaned and exposed to a concentrated hydrofluoric acid bath, washed with distilled water, and exposed to water-soluble Epon 812. See Gorycki, (1971) for a detailed description of the technique. The Epon 812 step is probably necessary because the supporting frustules have been dissolved and may have to be replaced by supportive Epon 812. The specimen is again mounted on a horizontal polyethylene sheet and the Epon 812 polymerized. Owing to the thinness of the specimens, it is possible that the steps involving abrasion and embedment with Epon 812 may not be necessary. Accurate sectioning of the surface layer (containing the diatom material) would have to carefully proceed using a glass knife. It is also uncertain how deep into the prepared specimens that sections may be cut because it is not known to what depth the hydrofluoric acid or the Epon 812 (if employed) penetrates. As noted above, no hydrochloric acid has to be used with these specimens.

Sections would have to be cut parallel to the plane of sectioning on the ultramicrotome [3] (see the sections entitled “Block Face Alignment” and “Oriented Tissue Block Exposure”).

[3] www.geocities.ws/ag526/muscle/muscle.html

The sections would then be exposed to a lead stain and studied. 

CONCLUSIONS

The emphasis of my master’s thesis was the study of living foraminifera. The inclusion of a few diatoms was ancillary. The partially tried technique as described here for diatoms might also be successful in the study of radiolaria. This conclusion is based on the light and electron microscopy images, of forams and associated diatoms, presented here, which show no artifacts or presence of siliceous material. Again, to err on the side of caution, only glass knives should be used.

Questions, comments and criticism are welcomed and may be addressed to me at: Gorycki@yahoo.com

REFERENCES

Gorycki, M. A., 1966, Oriented Embedding of Biological Materials and Accurate Localization for Ultrathin Sectioning: Stain Technology, v. 41, no. 1, p. 37-42.

Gorycki, M. A., October, 1967, Ultrastructure of Living and Fossil Foraminifera; A Microscopical Technique for Testate Microorganisms: Master’s Thesis, New York University.

Gorycki, M. A., 1971, A Compound Embedding Technique for the Microscopical Study of Living and Fossil Foraminifera: Micropaleontology, v. 17, no. 4, p. 492-500.