THE OLIVE TUBERCLE: NON-NUTRITIONAL ENVIRONMENT 409 



Non-nutritional Environment. Determine sensitiveness of 

 the organism to Liebig's meat extract, to concentrated beef 

 juice (titrate), to Merck's peptone from flesh, to heat, to freez- 

 ing, to sunlight, to dry air, to sodium chlorid (try 2 per cent 

 first), to sodium hydrate, and to various acids (citric, malic, 

 tartaric, etc.)- Also test effect of various fungicides. What 

 is the optimum temperature for growth? With us it has grown 

 better at 20C. than at 30C. or above. According to Petri, 

 its optimum temperature is about 20C. Are your experiments 

 in conformity with this conclusion? Can you obtain growth on 

 or in any medium at 0C.? Carry on the experiment for several 

 weeks and watch your ice thermostat very carefully. Can you 

 get it to grow at 37C.? What is the thermal death-point? 

 (See Petri's observation.) 



FOR THE DISEASE. Signs. After inoculation, how long be- 

 fore incipient tubercles are visible on leaves and shoots? Exam- 

 ine about every third day. (In my own experiments these 

 growths were seldom clearly visible, as such, earlier than the 

 end of the second week and the tubercles continued to grow for 

 several months.) Describe the gross appearance of the young 

 tubercles; of the old ones; of the effect of the disease on roots, 

 trunk, branches and leaves. Are the tubercles always fissured? 

 Do they contain an unusual amount of tannin, or peroxidase? 



Histology. With a razor or very sharp knife cut slices free- 

 hand of young soft galls (1 to 2 cm. in diameter) and examine 

 under the hand lens. Draw some of them enlarged a few diam- 

 eters. Examine also at once in water under high powers of the 

 microscope in thin, freehand sections. Can you make out the 

 bacteria? Can you see them flood out of the tissues? Contrast 

 with No. XIV. Fix in Carney's fluid, embed in paraffin, and 

 cut in various directions to learn the distribution of the bacterial 

 cavities and the reaction of the tissues. To what is the water- 

 soaked appearance due? Are the bacteria lodged between the 

 cells or in their interior? What tissues are chiefly involved in 

 the overgrowth? Is there action of the organism at a distance? 

 If you have studied crown gall, compare sections of the two 

 tumors, and of stems cut between primary and secondary tumors. 

 Some of our thin sections have been stained with Ziehl's carbol 



