TISSUE CULTURE 357 



circulation of a limited volume of fluid medium which can be 

 changed at intervals of a few days. This new technique is specially 

 to be recommended for the maintenance of large numbers of 

 cultures for prolonged periods. 



792. Cultures from Non-Sterile Tissues. Several workers have 

 attempted to sterilise tissues before explantation by ultra-violet 

 radiation. Light and all forms of radiation appear to have a 

 harmful effect on living tissues and Chlopkow {ibid., x, 1930, 

 p. 299) recommends, for adult rabbit intestine, the repeated 

 washing of explants in sterile saline solution (Ringer). He also 

 suggests washing in 1 in 40,000 Rivanol solution. 



793. The Fixation and Staining of Tissue Cultures. Per- 

 manent stained preparations of hanging-drop cultures are readily 

 made from the original coverslip on which the tissue has been 

 growing. Flask cultures are generally sectioned in series. Cultures 

 in fluid media are best fixed in the vapour of 2 per cent, osmic acid, 

 iodine or formol, followed by almost any appropriate staining. 

 Cultures in solid plasma medium require more care in staining 

 owing to the strong affinity of fibrin for most histological and 

 cytological stains. When the last sub-culture is made, the medium 

 should be spread as thinly as possible on the coverslip before it 

 clots. Washing in Tyrode solution for half an hour in the incubator 

 before fixation may be followed by almost any fixative and stain 

 without obscuring the cells in darkly stained plasma. With 

 practice it is possible to dissect away the peripheral layers of a 

 fixed plasma clot without damaging the culture, by using fine 

 glass micro-needles and a dissecting microscope. This technique 

 is useful when highly mordanting fixatives such as Regaud or 

 Champy's fluids are employed. 



Fixation for ten minutes in Zenker-formol and staining with 

 Heidenhain's iron haematoxylin is particularly successful for 

 routine preparations. Wallbach (Archiv. f. exper. Zellforsch., x, 

 1931, p. 383) recommends Heidenhain's " Susa " fixation for 

 vitally stained cultures. Giemsa and most other blood stains may 

 be used and the silver impregnation of fibres by routine methods 

 can also be employed. The Feulgen reaction (see Cowdry, Science, 

 Ixviii, 1928, p. 138) stains the nuclei of cells in the zone of new 

 growth and in the explant with a brilliance equal to that of 

 sectioned tissues. The period of fixation for cultures can, in most 

 instances, be considerably shortened and fixatives containing high 

 concentrations of chromic acid and mercuric chloride are best 

 diluted. 



794. Serial Sectioning of Tissue Cultures. Maximow {Arch. f. 

 mikr. Anat., xcvi, 1922, p. 494) has described a short celloidin 

 imbedding technique for cultures. Care should be taken in 

 paraffin imbedding to expose the preparation only for very short 

 periods in frequent changes of wax. Prolonged immersion in 



