The value of the Marchi method for staining tissue stored in formalin for prolonged periods.

This paper reports the demonstration of degenerating myelin by the Marchi method in material kept in formalin for periods ranging from 22 months to over eight years. It has generally been believed that prolonged immersion in formalin renders material useless for investigation by the Marchi method, because of the constant occurrence of extensive artefacts. Fourteen days has usually been considered the maximum period during which tissue may be exposed to the action of formalin before making Marchi preparations in order to obtain clear incontrovertible evidence of myelin degeneration. In Marchi preparations the presence of abundant black pseudoMarchi bodies, or of black staining of normal myelin sheaths, may completely obscure the tissue degeneration. Bertrand wrote in 1930: " Sur toutes les preparations au Marchi existe une fine poussiere de granulations noiratres sans rapport avec un processus degeneratif quelconque, il nous a semble que cet aspect etait particuli6rement marque apr6s une longue fixation au formol, sur un mat6riel ancien. Il est mdme frequent de voir des gaines de myeline normale noircies sur un segment plus ou moins 6xtendu de leur circonference." Swank and Davenport (1934) stated: "Prolonged formalin fixation . . . increased the dust." They also showed that prolonged fixation (for one year) causes staining of the normal myelin sheaths. They considered the artefacts caused by formalin fixation to be the result of prolonged action of that fixative on the lipoids. Later, in 1942, Mettler and Hanada wrote: " The brain should never be allowed to fix in formalin alone for more than 24 hours." These are only examples of numerous similar statements. Occasional reference has been made to satisfactory Marchi preparations obtained after rather longer storage in formalin than usual. Poirier, Ayotte, and Gauthier (1954), in a paper describing a modified form of the Marchi method, showed a photograph of clear degeneration in a monkey after 100 days in formalin. But no worker, to our knowledge, has