Microscopy 101

Figure 2: Making grooves in aluminum SEM stubs. (a) Vise, stub, metal rule, and scoring to make grooves. (b) Diamond scribe and (c) tip of scribe. (d) Angle of tip and (e) a typical aluminum SEM stub. (f) Groove pattern.

1200 grit silicon carbide paper, wetted, on a hard, flat surface. Te debris was cleaned off with double distilled water (DDW). Ten the stub was securely fastened in a vise mounted on a bench ( Figure 2a). A steel rule was used as a guide to score deep grooves by hand pres- sure using a diamond scribe ( Figures. 2a–2c) at a measured angle of approxi- mately 100° on standard aluminum stubs (Figure 2d–e). Te ruler was then placed at 90° to the previous grooves to produce a grid pattern (Figure 2f). A little prac- tice was required to make approximately evenly spaced grooves between 20 μm and 125 μm wide and about 1 mm or less apart. Holding the scribe vertically resulted in even, deep grooves. Experi- menting with scribing pressure allowed for an optimal groove width for the size of diatoms being studied. Again, with wetted 1200 silicon carbide paper, the stub was gently and quickly sanded to remove high points caused by the scribe. Te stubs were cleaned by brush scrub- bing with soap and water and rinsed well in DDW. Aſter drying, stubs were pol- ished on a folded paper towel backed by a hard surface, or polished to 14,000 grit (1 to 2 μm) with a wet diamond wheel or sheet, then finally cleaned with 100% ethyl alcohol. With a little practice, it took 6–10 minutes to make each stub. Cell deposition procedure. Gen-

Figure 3: (a) Cells of Thalassiosira punctigera strewn on a SEM stub with grooves. (b) No tilting of the SEM stage. In the center of Figure 3a, there are two diatoms of T. punctigera. (c) Ditylum brightwellii cells (arrows) on grids spaced approximately 200–800 μm apart and 70–125 μm wide. No tilting of the SEM stage. (d) A cell of D. brightwellii, from the stub in Figure (c) that fell into a groove valve face upwards. The single arrow- head indicates the location of the cell. The double arrow shows the width of the groove, which is 73 μm wide. Bars in (a) = 75 μm, in (b) = 20 μm, in (c) = 500 μm, in (d) = 20 μm.

2020 January •

erally, frustules are cleaned with either acids or hydrogen peroxide [4]. Differ- ent types of diatoms usually require different cleaning methods. Te robust Coscinodiscus are cleaned with a dif- ferent method than delicate Ditylum spp. Aſter using an applicable cleaning method for the sample being investi- gated, the salts used in cleaning are washed out with DDW, centrifuging between washing, depending on clean- ing method and type of diatom. House- hold bleach tends to produce a lot of salts appearing on stubs and on the speci- mens. So to be safe, especially for SEM, wash with DDW at least 100 mL, even better with 150 ml. If using hydrogen peroxide then less salts and less wash- ing with DDW. With a delicate diatom like Ditylum, it is best to let it settle for over 2 hr., or centrifuge at 1600 rpm for 30–40 min. More robust diatoms like Coscinodiscus can handle 3000 rpm for 30 min. Te frustules settle to the bot- tom of tubes very quickly, most likely


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