Preparation of Staining Solutions and the Schedule for their Use

The lead citrate and lead hydroxide solutions normally employed in electron microscopy suffer from the drawback that if used in the ordinary atmosphere their alkaline nature permits the formation of lead carbonate crystals. We have eliminated this problem by using an acid lead stain—with results we think are the equal of those achieved with lead hydroxide or citrate.

Although we cannot find in reference works the reason why uranyl salts are used, we presumed their use stems from the fact that insoluble uranyl compounds would be opaque to electrons. Being a heavy metal, uranium would react with protein and thus become localised. In an electron beam the uranyl-protein compounds would therefore be recognisable as the darker areas in a clear field. If this were so, one might expect an increase in the concentration of the uranyl salt to lead to better staining. From some trials we did, it seemed quite definite that the concentration of uranyl salt had a marked effect on the efficacy of the staining. Methods already employed by electron microscopists use uranyl acetate; but this has a limited solubility in water compared with the nitrate. We therefore chose to use uranyl nitrate.

To escape the formation of lead carbonate crystals, one has to use an acidic lead salt solution. We decided to work with lead nitrate since solutions of this in water are normally acidic without pH adjustment—sufficiently so to exclude the possibility of lead carbonate formation. Initially, we stained sections in a solution of uranyl nitrate and then in a solution of lead nitrate. The results were very heartening—sufficiently so to indicate that these chemicals could be relied on to give us consistent results. Because the nitrates of these two elements are very soluble in water and the common anion effect would not cause precipitation if both were mixed together, we combined the two into one stain solution. Only analytical grade reagents were used.

We also examined the effect of pH on the staining and seemed to get the best effect when the pH was brought to 1.5 with ammonia—using a glass electrode to measure this adjustment.

The staining time is ½ an hour at 35°C.

The following advantages accrue from using this mixed stain. There is no precipitation of lead carbonate (or any other chemical) while using this stain mixture. All staining is done on the bench top in an ordinary atmosphere without page 4 taking any extra precautions other than excluding atmospheric dust from the grids at all times. The overall staining time is shorter with this combined stain.

By using this stain from which lead carbonate cannot be precipitated and filtering all solutions that come into contact with the grid, we find it commonplace to be able to photograph large areas of a section at low magnification (see Plate 1). One is not forced to pick areas whose suitability is dictated by the absence of lead carbonate crystals or other inert material, because now the whole of the grid is usable. In Plate 2, note the freedom from microcrystals originating from the stain. Because of the nature of our stain we find that a high magnification study of an area viewed at low magnification can be made with confidence, knowing that this type of artefact will be absent. Where we did get contamination, we found it coming from the forceps—which we now clean regularly with '400' emery paper.

Up to this point the main fixative used has been osmic acid, although glutaral-dehyde has been tried on several occasions—producing results in staining comparable with those obtained after osmic fixation. We do not know if fixatives other than these will permit a similar quality of staining.

We wish to thank Mr. Alan Hoverd for preparing the accompanying figure. The microscope used throughout this work is a Zeiss EM9A.

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