A NOTE ON A RAPID CLEARING TECHNIQUE OF WIDE APPLICATION

The use of lactophenol in conjunction with cotton blue staining, first described by Klebahn (I9g0), for demonstrating the presence of fungal hyphae in infected plant material is now a well-known technique, and there must be many mycologists who have noticed that under the clearing action of lactophenol plant tissues become almost transparent except for lignified xylem elements. It was this observation which led the writer, in 1939, to develop a rapid clearing technique, using pure lactic acid (B.P.) instead of lactophenol, for the morphological study of a wide range of botanical material. A method of clearing by means of lactic acid, which was similar to this in some respects, was described by Simpson (1929) and has since become widely used in America, though it does not appear to be well known in England. The technique described here was designed primarily for examining the internal vascular structure of the flower and has subsequently been found most useful for examining the arrangement of the sporangia within the sori of ferns; it has also been used to demonstrate the vascular bundles within the fertile spikes of members of the Ophioglossaceae and to show the arrangement of the sporangia within the cones of species of Selaginella. The principal advantages of the technique lie in its rapidity, in the fact that no staining is required, that it may be applied equally to fresh material, spirit material and dried herbarium specimens, and that preparations may be moflnted permanently in the clearing fluid. For studying the vascular structure of large or medium-sized flowers, longitudinal and transverse hand-cut slices of tissue up to 2 mm. in thickness may be used, while small flowers can often be examined whole, and for studying the. contents of a fern sorus it is sufficient to use a portion of the frond without any preliminary dissection. Satisfactory results have been obtained with dried herbarium material, both of ferns and of angiospermous flowers, after soaking out by means of the modified Juel technique described by Tillson & Bamford (I938). Before proceeding 'with the clearing process, it is necessary first to decolorize the tissues. Thus, if fresh material is used, all chlorophyll must be removed. Spirit material, though free from chlorophyll, is often coloured brown, and herbarium material usually contains a similar brown coloration. This can usually be removed by treatment with alkaline hydrogen peroxide (':2o vols.'), though the process may take 24 hr. or more. The clearing process consists of heating the material for a few minutes in pure lactic acid (B.P.) kept at ioo0 C. in a boiling water-bath. The preparation can then be examined in a watch-glass under a binocular microscope, and any dissection which may be required can then be carried out. Examination at this stage will also reveal any air bubbles which there may be within the tissues, and which must be removed under a vacuum pump before the preparation is mounted permanently.