TISSUE CULTURE 353 



room without unnecessary furniture and no dust-collecting ledges. 

 Ensure that the room is free from air currents and not overheated 

 by direct sunlight. Where a considerable number of cultures are 

 to be maintained, it may be convenient to provide the culture 

 room with taps through which superheated steam can be released 

 just before the room is used. Cover the tissue culture table with 

 a sterile black cloth (casement cloth or sateen). A few square 

 blocks of wood about 1 inch thick, beneath the table cloth, are 

 useful as platforms for delicate manipulations. A small Bunsen 

 burner should be handy for the rapid re-sterilisation of instruments 

 and the flaming of flask and tube apertures. A pot of paraffin wax 

 containing about 25 per cent, of vaseline for sealing hanging-drop 

 cultures is kept on a warm plate. Stands for sterile centrifuge 

 tubes in which pipettes may be rested free from bacterial con- 

 tamination are particularly useful (see Fischer, Gewebezuchtung, 

 Miiller and Steinicke, Miinchen, 1930). The operator should work 

 in a sterile gown and wear a surgical mask covering the nose, 

 mouth and hair. 



Four chief methods of explanting tissues are now in general use, 

 and many minor variations of these have been described. 



787. The Hanging-drop Technique. Small explants are set up 

 on a coverslip, which is inverted over a cavity slide and sealed 

 with paraffin wax or vaseline. If fluid medium is used the culture 

 is kept resting on the coverslip and not hanging from its under 

 surface, as in the solid media cultures. These preparations are 

 suitable for cytological work with a limited number of sub-cultures. 



Lay out six to twelve sterile coverslips on a sterile black tile or 

 sheet of xylonite and cover with a large Petri dish. Prepare an 

 equal number of cavity slides by painting a broad band of wax 

 just outside the circumference of the cavity. The thickness of this 

 wax layer will help to raise the inverted culture well away from 

 contact with the cavity of the slide, particularly if the cavity is 

 shallow. Cover these slides with a Petri dish. 



Dissect out the tissue to be cultivated and place it in a small 

 amount of Tyrode solution in a fresh cavity slide. Wash the tissue 

 free from blood corpuscles which degenerate if carried over into 

 the medium, and inhibit growth. Cut the tissue into small 

 explants of equal size with iridectomy knives. The explants 

 should be about 1 to 1-5 mm. in diameter. Do not traumatise the 

 tissue by tearing it with blunt knives or by squeezing it with 

 forceps. If all conditions are favourable the explants will remain 

 translucent like the living, intact embryonic tissues. Sometimes 

 one finds that explants become opaque and whitish in appearance. 

 This striking protoplasmic change appears to be due to injury 

 and possibly to incorrect jpH of the saline solution. If a culture 

 is similarly injured by blunt knives or by undue stretching, it will 

 also show this change instantaneously. Cultures of the opaque 



VADE-MECUM. 12 



