Chapter XIII 



139 — 



Vital Staining 



We have tried, by cultures of various plants in nutrient media 

 to which neutral red has been added, to follow under the micro- 

 scope the development of the vacuoles during cellular growth. This 

 has, in addition, made it possible for us to judge the degree of 

 toxicity of this dye. We have been able to obtain cultures of Sapro- 

 legnia, for example, on soy bean bouillon to which 0.001% -0.002% 

 of neutral red or neutral violet have been added. The cultures were 

 grown in Petri dishes (Fig. 88) whose bottoms had a 3 cm. opening 

 covered by a cover slip sealed by asphalt cement (model used at the 

 International Bureau for the Culture of Fungi at Baarn). It suf- 

 ficed to turn the box over and place it under 

 the microscope to be able to follow the devel- 

 opment of the fungus under the oil immer- 

 sion lens and to follow the life history of the 

 vacuoles. Various species of Saprolegnia cul- 

 tivated undere these conditions developed as 

 well as those in the control cultures and fol- 

 lowed out their entire life history from ger- 

 mination of the zoospores to the formation of 

 zoosporangia. Now, during all their develop- 

 ment the dye accumulated in their vacuoles 

 and stained them superbly. In solutions of 

 neutral red beginning with a concentration of 

 0.005%, growth is somewhat retarded. The 

 fungus can stand relatively large doses of 

 neutral red (0.05%-0.06%), although when 

 the concentration of the dye is above a cer- 

 tain paint, it grows only very little. 



With DuFRENOY and Labrousse, we were 

 able to germinate seeds of tobacco in pure 

 culture on Knop's solution to which had been 

 added doses of neutral red from 0.005%- 

 0.02%. We succeeded in doing the same 

 with grains of wheat, barley and lupin seeds, 

 in collaboration either with Obaton or with 

 Gautheret. The seeds germinated normally 

 under these conditions and by examining 



their roots in the Petri dish under a microscope it was possible to 

 see that during the entire growth of the root, neutral red was 

 accumulated in the vacuoles of the meristematic cells, in the dif- 

 ferentiating cells of the cortex, in the root hairs and in the cells 

 of the root cap. Eichhorn found also that roots of Allium Cepa 

 grow perfectly in a solution of neutral red and show entirely 

 normal mitoses. Gautheret for a period of several months kept 

 alive the cells of the root cap of lupin in media to which neutral 

 red had been added. The vacuoles in these cells were stained. In 

 addition it may be recalled that Skupienski succeeded also in 

 obtaining the complete development of one of the Myxomycetes, 

 Didymium nigripes, in a medium to which neutral red was added. 

 The vacuoles were stained during the entire growth of this plant. 



Fig. 89. — Saprolegnia 

 dioica. Zoospores grown 

 on gelatinized soy bean 

 bouillon containing 0.001% 

 neutral red. 1, before ger- 

 mination; vacuolar system 

 consisting of numerous glo- 

 bular bodies stained by the 

 dye. 2, id.; nucleus visible. 

 3, 4, fusion of small vacu- 

 oles into one large one con- 

 taining granules precipi- 

 tated by neutral red. 3, in 

 the germination tube small, 

 spherical, uniformly stained 

 vacuoles are formed which 

 in (5) elongate into fila- 

 ments, form a network and 

 then fuse to form large 

 vacuoles. 



