98 



always deal willi llic coaguluin and llicrciore modifications 

 had to be iiukIc in llic ordinary sliiining piocesses in order 

 to avoid any trouble from the clot. In many cases, therefore, 

 the clot has to be eliminated. This is sometimes very diffi- 

 cult; nl llu- sanu' tinu', as llu' clot is dissolved, tlie cells 

 may disappear loo, wlicn the s])ecimens are treated further. 

 Some iuvesligalors, such as Lewis and Lewis, have a 

 beaulilul leehnicpie for staining, as tliey do not use j)lasma 

 clol lor cullivalion. As tlie culture work is taken up here 

 from an entirely different point of view, a physiological, 

 the clot is always present and special methods have then 

 to be (level()|)ed in order to follow^ the morphological changes 

 in the cultures during the experimentation. 



The vital staining is made use of a good deal in llie 

 culture work. The methods for doing it are simple and 

 need only a few w^ords derived from experience. The stains 

 used are the usual; neutral red, methylene blue, trypan 

 blue, Janus green, and Janus black and so on. The slain 

 can be added to the i)lasma before coagulation has set in, 

 or before the growth had begun, or it can be applied by 

 opening the culture, after it has already grown for some 

 time, and adding a drop of the staining solution on top af 

 the plasmy clot and resealing the culture immediately after. 

 The concentration of the dyes used is such that the slain 

 is present in the culture medium in about 1—20.000 or 

 40,000. In this eoneenlration most of the dj^es are luirmless 

 to the tissue cells. They can often be cultivated for several 

 passages, in a medium containing the dye. For photograpnic 

 purposes, the vital staining method is very good for obtaining 

 characteristic pictures of living cells. 



The fixation of a tissue culture is done best in Ringer's 

 solution containing 2 o/o formalin. One hour in this solution 

 is sufficient for the fixation. The alcohol or sublimate 

 fixation cannot be applied because the plasma clot becomes 

 very opaque and intransparent. 



