554 THE CULTIVATION OF TISSUE " IN VITRO." 



the drop of plasma from touching the bottom. The tissue is quickly 

 placed on a coverslip, 2 drops of plasma from the paraffined pipette 

 ( 1039) are added and evenly and thinly spread around the tissue ; 

 this must be done before coagulation occurs. If the plasma is not 

 spread evenly the tissue-culture will grow in many planes, and will 

 be less easy to observe, manipulate, and to fix and stain. When 

 the plasma is spread out the cover glass is inverted over a hollow 

 slide of suitable depth ; a little sterile vaseline may be placed at 

 each side of the cell to assist adhesion preparatory to waxing down. 

 The latter process is done by brushing molten paraffin around the 

 edge of the coverslip, and on the slide, to prevent drying. Im- 

 mediately this has been done the preparation is transferred to an 

 incubator. Carrell and Burrows use a small portable electric 

 incubator which is used for carrying the finished cultures to a bigger 

 incubator in the observation room. 



For the large plate cultures the same technique is used. Tissue may 

 be rapidly removed, cut into very small fragments, suspended in 

 Ringer, and then spread on the cover of a flat glass -covered (Gabrits- 

 chewski) box, and covered with plasma. The Gabritschewski boxes 

 after several days' incubation (three to five days), are opened, and the 

 plasmatic jelly cut out into blocks and preserved. 



Or, instead of using Gabritschewski boxes, one may make the culture 

 on large black plates, which must then be placed in glass boxes with 

 cotton sponges soaked in water, in order to preserve the proper hu- 

 midity. The boxes are then carefully sealed with paraffin and kept in 

 such a position that the fluid products of the culture may drain to the 

 bottom. 



1043. Subculturing is generally difficult, the technique of culti- 

 vating of tissue cells in series being far from perfect. One extirpates 

 a piece of the primary culture at its most active period, and transfers 

 it to a fresh medium, growth often, but not always, beginning anew. 

 Tertiary cultures are made in the same way. CARREL and BURROWS 

 (Journ. Exper. Med., xiii, 1911) find that a very good way is to cut 

 out the middle of the old culture around the original piece of tissue, 

 and then fill up the space with new medium. The old cells grow 

 into the new plasma. 



1044. Fixation and Staining of Cultures. CARREL and BURROWS 

 (Journ. Exper. Med., 1911) remove the cover-glass to which the 

 culture is adherent, and immerse in corrosive sublimate, acetic acid, 

 or formalin, or the various preparations of chrome salts. After- 

 wards the culture is stained in heematoxylin of Benda, Heidengain 

 or Weigert. 



Dr. A. Drew informs me that he has found that the best fixatives 



