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The basal portion of each stem was stored in 70% ethyl alcohol (ETOH).
Before sectioning the tissues were transferred to 50:50 mixture of glycerine:
95% ETOH and infiltrated overnight in a vacuum oven at room
temperature. Whole cross sections of the stems were sectioned on a sliding rnicrotome and the sections were microwave stained for one minute at low
setting with a 03% aqueous Safranin solution. Subsequent work with the microwave technique in this laboratory suggests 20 sec at high setting (700 watts) is also effective. After microwaving the sections were dehydrated to 95
% ETOH, stained for a few seconds with 0.5% alcoholic Fast Green, dehydrated through xylene and mounted in resin (Berlyn and Miksche 1976). Photomacrographs were made of the whole cross-sections and
photomicrographs were made of the xylem nearest the cambium at 250X. This last formed xylem was quite uniform in visual appearance and the cells of the treated plants were dearly larger in appearance than those of the
controL This was quantitatively corroborated by the low coefficients of variation in the data and by the sharp contrast in the magnitude of the means between the treated and untreated plants.
Image analysis was performed through the use of ImageMeasure (M.icrosciences, Inc., Federal Way, WA, 98003, U.S.A.). The photomicrographs were entered into the computer system through a COHLJ coupled charge device (CCD) video camera mounted on a 50 mm macro lens. On each of the 18 photomicrographs of the xylem all the complete cells in the field of view were measured with respect to cell walls (radial and tangential thickness) and lumens (radial and tangential diameter, lumen area). A densitometxy module was used to determine percent cell wall and lumen area of the whole photomicrograph. The photomicrographs were covered with a pull down transparent plastic (acetate) sheet and every cell measured was numbered so that every cell can be relocated for further study if need be. About six hundred cells were measured on each cross-section for a total of ca. 10,800 cells with five different attributes for each cell for .a total data volume of 54,000 measures. The technique takes Ca. one minute per~ cell to get all five
measurements including lumen area. The cell wall and lumen diameters take about the same time as manual methods but with image analysis the data are recorded directly into a computer file for statistical analysis and this saves transfer time and transfer errors. Calculation of lumen area is much faster with the image analysis system as compared to a polar plartimeter.
 

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