1. Freshly prepare a gold solution from an ampoule of gold chloride crystals by adding 1 mL of a 1% aqueous gold chloride solution to 79 mL of distilled water.
2. Prepare the reducing mixture with 4 mL of 1% trisodium citrate dihydrate, 2 mL of 1% tannic acid, 2 mL of 25 mM potassium carbonate and distilled water to make 20 mL.
3. Warm the solutions to 60°C and quickly add the reducing mixture to the gold solution while stirring. The temperature is critical at this stage. Evidence of sol formation is the red color of the mixture.
4. After the sol has formed, heat the mixture to boiling, and then cool. According to Slot and Geuze (4), the quantities stated here should produce 4 nm (±11.7%) particles. For 6 nm (±7.3%) particles, add 0.5 mL of potassium carbonate to the sodium citrate. The potassium carbonate counteracts the pH effect of the tannic acid. At less than 0.5 mL, the tannic acid has no effect on the pH and may be omitted. Therefore, for 8.2nm (±6.9%) particles, add 0.125 mL of tannic acid to the sodium citrate, and for 11.5 nm (±6.3%) particles, add 0.03 mL
of tannic acid. The sol forms within seconds if a high amount of tannic acid has been added or will take up to 60 min if the tannic acid has been omitted. A calibration curve can be constructed to enable a rapid identification of the amounts of reducing agent required to produce a given gold particle size. The electron micrograph in Fig. 1 shows a Formvar grid preparation of 14-nm gold particles routinely prepared in the author's laboratory using the method of Slot and Geuze (5).
5. Dry a small aliquot of the gold onto a Formvar/carbon-coated 400-mesh copper grid.
6. Wash away any salts in the preparation by floating the grid, specimen-side down, on distilled water.
7. View the grid at 125,000x under the transmission electron microscope. Electron micrographs should be taken and scanned into either a monochrome or full-color image analyser for morphometric analysis. It is also possible to attach the image analysis system directly to the electron microscope and so eliminate the intermediary time-consuming photographic stage. Even the simplest of image analysis programs available nowadays will be able to give a rapid evaluation of the gold particle size, shape, distribution, and the level of aggregation. In a good preparation, at least 80-85% of gold particles should be unclustered singlets. If the size distribution is unacceptably high, the preparation can be purified by centrifugation over a continuous sucrose or glycerol density gradient (5).
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