In order to find a suitable softener as a pretreatment to an acetocarmine squash technique on Fucus various fixatives and postfixation agents were investigated. None of the fixatives tried (cold and hot 1:3 acetic alcohol, cold and hot acetic acid, cold Carnoy B, Karpechenko, and 1% chromacetic) was an effective softener. The most suitable softening technique was found to be immersion of small pieces of fixed material in a 1M solution of a monovalent metal salt (e.g., lithium chloride) for 15 minutes, followed by an equal time in tap water. The material is then squashed in water under a coverslip and irrigated with acetocarmine containing a trace of ferric acetate, no separate mordant being used. A study was made of the relative abilities of mono-, di- and trivalent metals to soften Fucus cells. It is suggested that the marked softening effect of monovalent metals results from the greater solubility of their alginates.
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Leonard V. Evans (1963) studied this question.