160 METHODS OF CULTURE AND ISOLATION 



used for isolating species or varieties. Another example would be the 

 investigation of the organisms in the coating of decayed grapes in the 

 fall. A drop of sterile water would have to be placed on the cover 

 slip and a grape pressed into it. This method is very convenient, re- 

 quires no special media and facilitates microscopic examinations of 

 the organisms. Such a method lends itself to photomicrography. In 

 fact, Lindner 1 secured such illustrations with which to illustrate his 

 book on fermentation microorganisms. The colonies develop very 

 slowly, which lessens the danger of impure cultures. 



Methods for Investigating the Cytology of Yeasts : At this time 

 we shall refer to the technique of histology, especially those methods 

 which are of much service. The first step in the cytological examina- 

 tion 2 of yeasts should be a microscopical study of the cells colored 

 with neutral red. To do this, the living cells should be placed in an 

 aqueous solution (1-10,000). The protoplasm and the nucleus will 

 remain uncolored; only the less vital parts of the cell will fix the dye, 

 that is, the vacuole and the metachromatic corpuscles contained in 

 it. The dye will diffuse into the vacuole and stain it very lightly. 



When careful observations have been made on living cells, with 

 and without coloration, one may undertake deeper investigations in 

 cells which have been fixed with many of the available substances. 

 The most convenient procedure for this is to cut out a portion of the 

 gelatin or carrot upon which the yeast is developing and place it in 

 the fixing bath. When fixation is completed, the yeast may then be 

 placed on a cover slip, upon which has been spread a layer of gelatin 

 to make it stick. The mount may then be plunged into a staining bath. 

 The fixing and staining will vary, depending upon whether one wishes 

 to study the nucleus or the other contents of the cell. 



For investigations on the nucleus, Guilliermond recommends fixa- 

 tion in Bouin's picroformol solution or Perenyi's solution, which have 

 the following composition. 



Picroformol solution: 



Saturated picric acid : 75 parts 



Glacial acetic acid 5 " 



Formol 20 " 



1 Lindner, P. Atlas der mikroskopischen Grundlagen der Garungskunde. 

 2nd Edition, P. Parey, Berlin, 1910. 



2 Guilliermond, A. Recherches cytologiques sur les levures et quelques moisis- 

 sures a form levures, these doctorate es sciences (Resume in Revue generate de 

 Botan. 15, 1902). Recherches sur la germination des spores et sur la conjugaison 

 dans les levures. Rev. generale de Botanique, 17, 1905. Remarques sur les 

 re cents travaux parus sur la cytologie des levures et quelques nouvelles observa- 

 tions sur le groupe de champignons. Cent. Bakt. 26, 1910. 



