MICROTECHNIQUE FOR THE SHOOT APEX

THE SHOOT apex is one of the most difficult parts of the plant to prepare for microscopic examination by the paraffin method. There is great variation in cell size, cell wall thickness and in the number and size of vacuoles (Foster, 1938, 1939a, 1939b; Zirkle, 1932). Use of ordinary methods of microtechnique usually causes pronounced undulations in cell walls and much shrinkage of protoplasts. A technique has been developed that has given consistently good results for shoot apices of all vascular plants studied in this laboratory and seems to be equally satisfactory for plant tissues in general. This method introduces several improvements that aid in obtaining gradual and more complete dehydration and infiltration of tissues, lack of which in the past appears to have been the prime cause of artifacts. In addition, experiments with fatty acids, alcohols and other components of killing solutions have been carried out which give some indication of the reagents that should be used for successful histological-cytological fixation in the shoot apex. METHOD.-Terminal buds of Tradescantia fluminensis Well., which were used as test objects in these experiments, were dissected so that all but the youngest leaves were removed, and killed with aspiration in the fixatives to be tested. The majority of alcoholic killing solutions were made according to the formula of Chamberlain (1935): 5 cc. (0.087 GMW) of acetic acid, 5 cc. of commercial formalin (37 per cent formaldehyde) and sufficient 70 per cent ethyl alcohol to make 100 cc. of solution. For the ethyl alcohol was substituted the same volume of methyl, iso-propyl, n-propyl or t-butyl alcohol, and for the acetic acid was substituted the equivalent amount of another fatty acid: 3.7 cc. formic (88 per cent solution), 6.5 cc. propionic, 8.0 cc. butvric, or 9P4 cc. valeric. The only other alcoholic killing agent employed in this work was Juel's. It has been used extensively by the Buder school of anatomists and the formula followed was that given by Korody (1937): 50 per cent ethyl alcohol, 100 cc.; glacial acetic acid, 2 cc.; ZnCl2, 2 g. Not so many variations of this formula were carried out as in Chamberlain's. For acetic acid one of the other fatty acids was substituted in the same molar concentration, and no substitutions were made in the alcohol. Tests of killing solutions were also extended to several well-known aqueous fixing agents by substituting for the acetic acid the same molar concentration of each of the lower fatty acids. The formulae for the ones that were used are given by Chamberlain (1935): chromo-acetic, Licent's, Flemming's stronger, corrosive sublimate-acetic, Bouin's and Bensley's. Regaud's formula (Guyer, 1936) (K2Cr2O7 and formalin) was also employed. Zirkle's (1933) procedures were followed for addi1 Received for publication November 2O, 1940. tional chromic acid mixtures: fatty acid, 0.33 M; chromic acid, 0.25 M; formaldehyde, 1.33 M. With these concentrations combinations were tried with all three components, with each of the fatty acids plus formaldehyde, and the chromic acid with formaldehyde. Washing of the buds was begun after they had remained in the killing solutions for twenty-four hours. For those killed in Chamberlain's formula, washing was carried out for twenty-four hours with three changes of 70 per cent solutions of the corresponding alcohol. Apices killed in Juel's mixtures were washed in three changes of 50 per cent ethyl alcohol for the same period. Distilled water was used in the same manner for washing the buds killed in aqueous solutions. Concentrations of each of the five alcohols used in the dehydration were those recommended by Chamberlain (1935): 21/2, 5, 71/2, 10, 15, 20, 30, 40, 50, 70, 85, 95, 100, 100, 100. One hundred cc. of each of the above concentrations of alcohols were used in 200 cc. capacity, wide-mouthed, cork-stoppered bottles. In the bottoms of the three bottles of absolute alcohol were placed, for the purpose of keeping the alcohol more nearly water-free, 20 g. of either anhydrous CuSO4 or of CaO.2 The powder was allowed to settle and was then covered with a circular, coarse filter paper to prevent stirring. For the buds killed in Chamberlain's formula, dehydration began with 85 per cent of the corresponding alcohol; for those killed in Juel's it began with 70 per cent ethyl alcohol, and for those killed in aqueous solutions it began with 21/2 per cent alcohol. For ease in changing from one bottle to the next and to prevent damage to the tissues through handling, the buds were placed in 7X2 cm. glass vials that had four 1-mm. holes cut in their bases (fig. 14). The perforated vials were left in each dehydrating solution for approximately six hours. At intervals during that period the whole bottles were tilted so that new solution ran into the tops of the vials and over the plant tissues. In changing the vials they were rested upon blotting paper so that most of the adhering solution would not be carried over to dilute the next higher concentration of alcohol. Clearing was also carried out according to the method proposed by Chamberlain (1935) with the following concentrations of C.P. (water-free) xy-