[har iisoN A BARLOW] NODULE ORGANISM OF THE LEGUMINOSAE 167 



for a long time, especially if well separated. Surface colonies may 

 attain a diameter of 1 mm. to 2 mm. in five days, and 3 mm. to 4 

 mm. in fifteen days. After some weeks the period of active growth 

 ceases, the colonies retain their general shape, but sink and become 

 thin and flat on account of evaporation of the water from the mucilage 

 in which the cells lie. Under the iidcroscope, the surface colonies 

 appear luore finely granular than the deep colonies. 



We have failed to detect tlie presence of any organism except Ps. 

 raduicola in leguminous nodules. Plate cultures from a nodule in 

 special media and in ordinary gelatin and agar media of the laboratory, 

 develop pure culture of Ps. rodicicola or else remain sterile. It is 

 JTue that other colonies, mostly moulds, occasionally appear, but they 

 ; re almost always surface colonies, although they are never numerous, 

 and are not of the same kinds in plates from the same nodule. Fur- 

 tlier, we have frequently made streak cultures direct from the interior 

 of nodules on inclined gelatin and agar media, and such cultures are 

 negative or else develop pure cultures of Ps. radicicola, according to the 

 medium. 



IsoJaiion of colonies. — The needle is touched into a colony, then 

 thrust into agar medium in a Freudenreich flask. Hanging drop and 

 stain preparations are then made from the same colony. 



6. Growth of Ps. radicicola on ash-maJtose-agar. — The medium 

 n"i,nv be allowed to solidify while the tubes or Freudenreich flasks are 

 standing upright and later are inoculated by thrusting the needle into 

 the middle of the agar. In two or three days at 25° C, there is 

 raised, circular, transparent, wet-shining growth spreading on the sur- 

 fnce from the point of inoculation, and a filiform growth along the 

 needle puncture. This filiform growth increases and many fine fila- 

 ments may radiate from it horizontally in the agar. These filaments 

 nre shorter toward the bottom. 



The surface growth .increases, but maintains its sharp circular 

 outline, and in four to twelve days spreads over the whole surface of 

 t^e agar as a white, partly transparent, thick, mucilaginous or slimy 

 Ifiver, with a wet-shining surface. The growth usually draws out in 

 fi fine thread when touched with a needle. In favourable media the 

 gro-wi;h is copious a.nd in seven days may increase to a depth of 6 mm. 

 over a surface of 10 mm. in diameter. After some months, this layer 

 IS thinner and denser according to the evaporation from its surface, 

 '-nt in Freudenreich flasks, or in tubes sealed with sealing wax, there 

 's little change. Cultures on ash-maltose-agar in Freudenreich flasks 

 'pmain alive for more than a year at room temperature (see viability). 



