Section 6 — Cytology 



6.36. Functional Structures in Cytoplasm and 

 Chromosomes of D. hydei Salivary Gland Cells. 



H. D. Berendes (Leiden, The Netherlands). 



Each salivary gland of D. hydei contains 100- 

 150 cells. Larval development at 25°C under 

 optimal food conditions is completed in 121 

 hours, both first and second instar lasting 

 25 hours and the third instar 71 hours. The 

 development of the gland was observed using 

 phase contrast microscopy of whole glands 

 mounted in 0.7 per cent saline at various stages 

 of all instars, and sectioned as well as squashed 

 glands at stages of the third instar and prepupa. 

 Nuclear diameters increase from 4.5 u at hatch- 

 ing, to 55 u. About 20 hours after beginning of 

 the third instar, a differentiation can be seen 

 between proximal and distal part of the glands. 

 First sign of this differentiation was a faster 

 enlargement of the distal nuclei, in connection 

 with a higher polyteny of the chromosomes. The 

 cytoplasm of these distal cells then produces a 

 large number of granules with diameters of 1-2 u. 

 These granules consist of PAS positive material 

 and are not stained by toluidine blue. At the 

 end of the third instar these granules disappear 

 again. At this moment the nuclei of the distal 

 cells have a diameter of 45-55 u whereas the 

 proximal nuclei have a diameter of 28-35 u. 

 During the third instar the cells of the proximal 

 part never contain PAS positive granules, but 

 much smaller particles instead. These particles 

 are positively stained by toluidine blue. They 

 are also present in the distal cells during the third 

 instar. 



The puffing pattern within the five chromo- 

 some arms has been studied in relation to the 

 development of the cells in proximal and distal 

 part of the gland during the third instar. Dif- 

 ferences in puffing pattern between both parts 

 have been found. 



6.37. A Technique for Obtaining Chromosome 

 Preparations of Rat Embryo. M. T. Biola 

 (Fontenay-aux-Roses, France). 



During the examination of chromosome 

 aberrations of the rat embryo after irradiation 

 of the mother, we have determined a technique 

 for obtaining chromosome preparations without 

 tissue culture. 



With this technique we can examine the chro- 

 mosomes of the cells which were in mitosis when 

 the animal was killed and observe the real 

 anomalies of the embryonic cells. 



Embryos are taken, minced into small frag- 



ments which are digested by the combined action 

 of both trypsin and hyaluronidase. 



After 45 min at 37°C, serum is added to inhibit 

 the enzymes. 



The undigested residues are allowed to settle 

 and the cells of the supernatant are carefully 

 removed. The cells are washed with serum and 

 incubated a few hours at 37°C in a nutritive 

 medium, supplemented with colchicine. 



The mitoses are stopped at metaphase. The 

 cells are submitted to hypotonic expansion, they 

 are fixed and spread on slides and air dried. 



With this technique we have been able to 

 obtain metaphase cells with well-spread chro- 

 mosomes allowing good analysis of the caryotype 

 of the rat embryos. 



6.38. Mode of Action of Metal Blocking Agents in 

 Nuclear Dissolution. Fernando Silio and 

 Dolores Angulo (Madrid, Spain). 



Versenate and other chelating substances 

 induce a strong chromatic dissolution and a 

 remarkable increase of cell volume. From the 

 beginning we thought that this action might be 

 due to an indirect process of activation of 

 proteolytic enzymes. In order to confirm this we 

 used cyanide that does not have any chelating 

 action, but is an inhibitor for (heavy metals 

 containing) terminal oxidases. 



The cytological effects obtained with cyanide 

 were equivalent in some aspects to those induced 

 by versenate, which confirms our hypothesis 

 work based on the following facts. 



1. Papain, and other papainlike enzymes, 

 suffer strong activation by cyanide, versenate and 

 other peroxidase inhibitors. Thyolic compounds 

 and many other reducing agents, increase this 

 activation while oxidants inhibit it. 



2. The EDTA presents a much greater affinity 

 for Fe +++ or Cu ++ than for Zn++, which proves 

 its selectivity at low concentrations. The affinity 

 for Ca ++ and Mg is still lower. Therefore it is to 

 be supposed that it will react with Fe before it 

 will do so with Ca and Mg. 



3. As the lysis is already verified at very low 

 concentrations of EDTA or CNK, a very selec- 

 tive action on trace elements may be assumed, 

 rather than on macroelements like Ca or Mg. 



The enormous increase of cell volume ob- 

 served means a great imbibition which may be 

 attributed to a great increase of the osmotic 

 pressure. All this would confirm an intense des- 

 polimerization of macromolecules. 



With coenzyme A we have obtained similar 

 effects. 



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