THE CROWN GALL: CULTURAL CHARACTERS 457 



stages as well as later ones) ; growth in nitrate bouillon ; Cohn's 

 solution; Uschinsky's solution; milk, litmus milk; behavior in 

 peptone water in fermentation tubes with various sugars and 

 alcohols. Is there any clouding in the closed end? Try also 

 various plant juices in fermentation tubes. What acids are 

 produced? Test for formic and acetic. Use flasks of river 

 water with 1 per cent peptone, 1 per cent dextrose and a little 

 calcium carbonate. Kxamine at the end of 2 weeks, (> weeks and 

 3 months. According to the chemists, aldehyd, alcohol and ace- 

 tone are also produced. Determine its nitrogen nutrition. 



Non-nutritional Environment. Maximum, minimum and 

 optimum temperatures for growth. Thermal death-point in 

 peptone bouillon (10 minutes exposure). Effect of sunlight, of 

 dry air, of freezing, of salted bouillon, of chloroform in bouillon, 

 of acids, of alkali, of germicides. Determine conditions under 

 which the involution forms are produced. Add various dilute 

 organic acids (1 part acid to 9 parts water) in small quantity 

 (5, 10, lo and 20 drops to each 10 cc.) to 24-hour agar and bouil- 

 lon cultures (holding check tubes), and compare for changes in 

 structure of the organism, i.e., appearance of involution forms 

 (Y-bodies) after 24, 48, 72, etc., hours. Pour plates the 3d, 5th 

 and 10th days, from both checks and treated tubes, using care- 

 fully measured quantities of the fluids. Any reduction in num- 

 ber of organisms in the acidified tubes? Any retardation in 

 development of colonies on plates poured from such tubes? 



Oxidases (?) and peroxidases are said to be much more abun- 

 dant in the galled tissue than in the normal tissue. Can you 

 verify these statements? (See Bull. 213, p. 173.) Does this 

 fact have any pathological significance? Is the formation by 

 the micro-organism of acids and alkalies of pathological signifi- 

 cance? (Consult Jour, of Ayr. Research, Jan. 29, 1917.) 



How many distinct strains are there of the crown-gall or- 

 ganism? I do not call every isolation of an organism a strain, 

 but only such as possess distinct morphological, cultural, 

 pathogenic or other characteristics. The two strains I am most 

 familiar with are the Paris daisy strain and the Hop strain, but 

 there are others. Jensen in Denmark isolated a strain from the 

 Paris daisy which is unlike our strain in its serological reaction, 



