BULLETIN 231] WALNUT CULTURE IN CALIFORNIA. 343 



The usual method of securing pure cultures was as follows: A 

 scalpel sterilized by flaming was used to remove the outer blight portion 

 of the young nut. Then some of the diseased interior of the nut was 

 transferred to liquid media, such as acid Dunham solution or acid 

 peptone meat bouillon. About twenty-four hours after inoculation 

 there is usually some evidence of growth and dilution cultures were 

 then made in nutrient agar. In two to five days at a temperature of 

 20 C. the colonies will appear and can then be transferred to potato 

 cylinders or other solid media. The organisms are usually abundant 

 enough in the diseased nuts to plate out directly from the diseased 

 tissue without incubation. 



The preceding table shows that the organism was alive at all seasons 

 of the year in the different diseased tissues. The number of isolations 

 was not at any time very great, but sufficient to show a large number 

 of positive results. The negative results obtained do not mean there 

 was lack of growth in the tubes inoculated with the diseased tissue, but 

 that there was usually some other sort of bacterial growth. There are 

 several kinds of saprophytic organisms that can be found in these old 

 lesions. In the dilution cultures a saprophytic yellow organism was 

 very often present and could not always be distinguished in color or 

 manner of growth from that of the walnut bacteriosis. The difference 

 in growth on potato cylinders, however, readily distinguish the two. 

 Even when lack of growth did occur, too much importance should not 

 be placed upon it, for the walnut blight organism grows more slowly 

 than do the saprophytic organisms and could readily be crowded out 

 by them in artificial cultures. 



A MORE TECHNICAL STUDY Of THE WALNUT ORGANISM. 



Pseudomonas juglandis Pierce. 

 MORPHOLOGY. 



These characteristics were studied in bouillon, agar and potato cul- 

 tures from twenty-four to forty-eight hours' growth, except in old 

 cultures that have been examined for spores. 



Form. The organism has rounded ends and occurs as single rods or 

 often in pairs, and more rarely in chains of several individuals, com- 

 monly four to eight. 



Size. The rods as found in diseased tissue and stained with carbol- 

 fuchsin measure 1.5 to 3.01 microns in length by 0.3 to 0.51 microns in 

 width. 



Staining Reactions. The organism stains readily with the usual bac- 

 teriological stains, carbol-fuchsin, aniline gentian violet, aqueous solu- 

 tion of methylene blue, gentian violet, and by Gram's method. Nothing 

 especially characteristic was observed from the use of the various stains. 



