Block Intensification of Neurones Stained with Cobalt Sulphide: A Method for Destaining and Enhanced Contrast

One problem with the block intensification method is that, in at least some tissues, there is a marked tendency for thin nerve trunks to become darkened even if they have not been stained with cobalt. In addition, if intensification is allowed to proceed for a prolonged period, as is often necessary to reveal fine processes containing only small amounts of cobalt sulphide, the surface of the tissue can become stained and opaque. A method is described here by which such over-stained preparations can be de-stained to the required degree using a mixture of potassium ferricyanide and sodium thiosulphate. A solution containing these reagents is well known to photographers as Farmer ‘s reducer. When used on over-darkened photographic negatives, silver is oxidized to silver ferrocyanide. Silver ferrocyanide is a yellowish compound which is insoluble in water, but is soluble in sodium thiosulphate. The action of Farmer ‘s reducer is termed subtractive in that it removes similar amounts of silver from regions of differing density on the negative. For this reason, its action is most marked where the density of silver grains is lowest, and contrast is thereby enhanced.

The destaining method described here has been used on the ventral nerve cords of both the cockroach, Periplaneta americana, and the cricket, Acheta domesticus. Preparations were stained with cobalt sulphide both by back-filling (Iles & Mulloney, 1971) and by intracellular injection from microelectrodes (Pitman et al. 1972). Preparations were then treated according to the protocol given in Table 1. Although intensification (stage 7, Table 1) was carried out in the dark, the progress of the reaction was followed by occasionally observing the tissue under a binocular microscope. Intensification was arrested when the ganglia had turned brown by transferring the preparation to 5% gum acacia in 30% ethanol (stage 8, Table 1). When preparations had been intensified and brought to 50% ethanol, they were immersed in destaining solution containing both 1·25% sodium thiosulphate and 1% potassium ferricyanide in 50% ethanol. (Potassium ferricyanide should be dissolved in the sodium thiosulphate solution just before use.) Destaining was observed under a binocular microscope and terminated when desired (normally after 2–5 min) by washing the preparation for several minutes in 50% ethanol. The tissue was then taken through an ascending ethanol series and cleared in either oil of cedarwood or creosote. If preferred, preparations can be dehydrated and cleared immediately after intensification. Then, following initial observation, the tissue can be brought back to 50% ethanol, destained and returned to the clearing agent for further study. This permits a more precise judgement of the extent of destaining desired. When handling tissue in the destaining solution, care must be taken to avoid contamination by salts dissolved from steel instruments, since this can cause a bluish staining of the preparations. Forceps made of corrosionresistant material can be used to overcome this.

Fig. 1 shows the effects of intensification and partial destaining on the appearance of a cockroach metathoracic ganglion. Initially, the neurone was only lightly stained and little of its structure could be discerned (Fig. IA). After intensification alone, superficial staining of the ganglion masked most of the neuronal branching (Fig. 1 B). Partial destaining made the morphology of the neurone clearly visible (Fig. 1C). Destaining starts at the surface of preparations and proceeds inwards. For this reason, the method is especially effective in removing excessive surface stain. Furthermore, because the connectives and nerve trunks of these preparations are thinner than the ganglia, they become destained most rapidly and this is again advantageous since they are usually darkest. Since the destaining solution removes amounts of silver more or less independent of the initial silver density, the effect is more marked in less heavily stained structures, so enhancing the contrast between nerve fibres which were originally stained with cobalt and the surrounding tissue. Finally, since destaining can be continuously monitored, a fine degree of control over the final produce is possible. Conditions can be optimized to suit different requirements by adjusting the concentration of potassium ferricyanide in the destaining solution.

I wish to thank Dr J. J. Wine for his critical reading of the manuscript and the Science Research Council for financial support.