92 
Barratt . — The Origin of the 
This observation was directly opposed to the generally accepted idea of 
the origin of the tissues of Hippuris put forward by Sanio ( 2 ), according to 
which the endodermis was derived from the innermost periblem layer. 
Since Schoute’s observation has so far received no independent con- 
firmation, a re-examination of the critical case of Hippuris seemed desirable, 
and material was therefore collected in the spring of 1914 and 1915. 
Schoute’s methods of investigation were closely followed. Stem apices were 
selected from shoots of various sizes, care being expended to choose only 
straight ones. These were embedded and cut in series of transverse sections 
up to within a distance of about 100 n from the apex. The block was then 
rotated at right angles, and the remainder of the apex cut in a longitudinal 
series. 
The advantage of this method is apparent. A median longitudinal 
section through the apex shows very clearly the distinction between 
the periblem and plerome, which is made evident by the small number and 
extreme regularity of the layers of periblem cells which form a series 
of caps covering the central column of plerome, in which the cells are 
less regularly arranged. In transverse 'sections the distinction is not so 
clear in this region, and the use therefore of longitudinal sections through the 
extreme apex of a stem of which the lower part is cut transversely, enables 
one to determine the exact number of periblem layers concerned in the 
origin of the mature tissues. 
It is thus possible to trace with certainty the critical layer, the innermost 
of the periblem, and to identify the structure derived from it. 
In order to be quite certain about the position of the delicate cell-walls 
of the developing tissues, it is necessary to clear away the cell-contents, 
and this can only be done satisfactorily by the use of eau de javelle on 
the sections. The clearing agents failed to penetrate the fixed material 
in bulk, even after the lapse of several days ; on the other hand, the fresh 
materia], though sufficiently cleared, was unsatisfactory because the cell-walls 
were also affected, losing their original firm contour, and in some cases 
actually breaking down. In order to be able to use eau de javelle on serial 
sections, it was necessary to employ a fixative other than albumen ; water 
alone, collodion, and collodion with clove oil were tried, and a mixture of the 
latter in the proportion of 1 : 3 was found most satisfactory. With this fixa- 
tive it is necessary, first, to float out the sections on water and then to transfer 
them to a slide smeared with the mixture. It is essential to have absolutely 
clean slides and to use fixative recently made up. 
Of the many different stains employed, polychrome methylene blue 
gave the best results. It is, however, difficult to keep sufficient stain in the 
sections owing to its tendency to wash out in alcohol, unless the sections are 
mordanted after staining by placing them for a few minutes in a 10 per cent, 
solution of ammonium molybdate. 
