[HARRISON & BARLOW] NODULE ORGANISM OF THE LEGUMINOSAE 171 



film is killed and fixed before staining, the mucilage shrinlcs awa.y from 

 the cells, leaving a narrow clear space, giving an appearance as if the 

 cells were capsulated. 



A Stain to demonstrate the nature of the Mucilage. Spread a loop- 

 fiil of a mucilaginous culture from agar or from a liquid culture on a 

 slide, dry in the air, flood an instant with water, and immediately flood 

 with some stain as gentian violet or fuchsin. The mucilage of the air- 

 dried film takes up water and when the stain is added the mucilage con- 

 tracts and assumes certain patterns or figures composed of bands and 

 strands of fine a.nd coarser, intricately interlaced filaments. These are 

 sometimes arranged in wreaths like smoke in form and sometimes in 

 quite regular hexagonal figures, (see photographs). The bacterial cells 

 are assembled along the slime threads and correspond in numbers to 

 the thickness of the slime thread on which they lie. They take the stain, 

 (see photographs 28 and 30). 



Kishalt's Amyl-Gram Stain. This is the same as Gram's stain 

 except that amyl alcohol is used as a decolorising agent instead of ethyl 

 alcohol. Ps. radicicoJa is quickly decolored by Gram's method, but stains 

 deep violet by Kiskalt's stain. This stain is applicable to all cultures 

 of Ps. radicicola and is useful in making preparations for photography 

 from liquid media and especially from the nodules, since the amyl alcohol 

 clears up the background, brings the bacteria into prominence, and ex- 

 hibits their internal structure. (See photographs 15, 16, 26, 32, 33, 34). 



9. Viahility of Pseudomonas radicicola. Some observations on the 

 viability of Ps. radicicola on agar and in liq^uid media are collected in 

 the following table. The cultures were grown a short time at 2^)°C or 

 25°C and were then kept at the temperature of the laboratory. These 

 same cultures were all successfully transferred to various other media 

 more than once in the interval record in the table, and gave a prompt 

 and characteristic growth in favourable media. The transfers recorded 

 in the table were to ash-maltose-agar in all cases and also to ash maltose 

 water in some cases. The growth, morphology and staining reactions 

 were carefully observed and were characteristic of Ps. radicicola as else- 

 where described. The same is true of colonies which developed in plate 

 cultures in ash-maltose-agar made from certain of these cultures, after 

 a lapse of nearly a year and eight innntbs in one case. The limit of via- 

 bilit}' of these culture is not yet known, but this organism will probably 

 live more than two years on favourable agar and in favourable liquid 

 media. The longest time here recorded is tAvo years all but five days. 



The growth was abundant and mucilaginous, the cells were actively 

 motile in hanging drops; stained with alcoholic gentian violet they 

 showed single polar flagella. Occasionally branched forms were seen 

 from agar cultures and were frequent in liquid media. 



