ANIMAI, PARASITES INOCULATED INTO PLANTS. 175 



The paper is stated to be a preliminary one. For his experiments the author took three bacteria, 

 viz, the bacillus of the Siberian plague, the bacillus of typhoid fever, and Bacillus prodigiosus. The 

 plants inoculated were Triticum vulgare, Agapanthus, Polygonum fagopyrum, Trifolium pratense, 

 Sambucus, Hyacinthus, and Tulipa. Heinz's results with his B. hyacinthi-septicus were known to 

 lyominsky. 



Two ways were chosen for investigating this subject : 



(i) Seeds were planted on infected soil; (2) plants were inoculated by puncture, especially on 

 the leaves. The surfaces of the seeds were sterilized by washing in soap and water and then in mer- 

 curic chloride i :iooo. They were afterwards left for half an hour in 1 15000 HgCl^ or for one hour 

 in I :io,ooo HgCl^. Seeds of wheat thus sterilized on their surfaces were treated in two ways. In the 

 one case, they were plunged into colonies of the above named bacteria, then laid in a tin box on 

 steriUzed soil, and covered with about an inch of soil. This little box was then put on a glass plate 

 and covered with a bell-jar, the upper opening in which was covered with cotton. From time to 

 time sterile water was added. The seeds germinated in 5 to 30 days, the room temperature being 

 25° to 27° Celsius. In the other case, the germinations were sometimes made on moist sterilized 

 cotton, but more often on boiled potatoes prepared as for bacterial cultures, except that to them 

 was added a little of the following solution : water, 1,000; potassium nitrate, i ; potassium sulphate, 

 0.25; monopotassium phosphate, 0.25; magnesium sulphate, 0.25. A httle ferrum phosphate in 

 powder was also added. After sterilizing this medium the wheat was put in. This method enables 

 one to decide whether the seed and substratum have been properly sterilized. Then, after a few 

 days, the microbes were introduced on the end of a platinum needle. 



The inoculations on the leaves were made into very young plants and into older ones. The very 

 young plants were germinated in soil or in cotton, and when the green parts had reached a height of 

 2 to 5 cm. the specified microbes were inoculated by means of a needle. On adult plants the inocula- 

 tions were made with a platinum needle shoved flat-wise between the upper and lower surface of the 

 blade, after first washing the leaves in i :iooo HgCl^ and drying them in sterilized cotton. The sur- 

 face of the wound was covered with collodion. Leaves of plants were also dipped into sterilized 

 water to which the microbes had been added. After 3 to 42 days the inoculated leaves were examined 

 microscopically. Cultures were also made from them and inoculated into animals. Leaves to be 

 examined were hardened in alcohol. Bacteria in the growing tissues were stained by the methods 

 in use for animal tissues. Three hundred experiments were made. The author's conclusions are as 

 follows : 



(i) Disease creating microbes [animal pathogenic bacteria] may find conditions for their existence in tissues of 

 the higher plants. 



(2) The uninjured cuticle prevents the entrance of bacteria. 



(3) Mechanical injuries of the leaves and stems of growing plants afford an opportunity for the entrance of bacteria 

 into the tissues. 



(4) The bacillus of Siberian plague [Aplanobacter anthracis], the bacillus of typhoid fever, and the B. prodigiosus 

 can multiply and form colonies inside of living plants. 



(s) In artificial inoculations these three bacilli multiply not only at the point of inoculation but spread into the 

 neighboring parts. 



(6) Although these three bacilli spread from the point of inoculation to adjacent parts of the tissue, they do not 

 extend widely, that is, the whole organ or the whole plant is not infected by artificial inoculation. 



(7) The part of the leaf injured by the microbes sometimes may be identified macroscopically. The injured 

 spot in the leaf differs from the healthy part by being lighter green. Sometimes on the part injured by Bacillus pro- 

 digiosus brick-red spots or stripes are noticed along the track of the injury. [Probably a host-reaction.] 



(8) The disease-creating microbes spread in the plant by way of the intercellular passages, and the size of the 

 microbe is of great importance in this regard. The smaller it is, the easier it spreads in the tissue. For this reason 

 B. prodigiosus spreads farther than the others. 



(9) The author cordd not observe that motility in any way favored its spread. 



(10) The walls of the cells do not absolutely prevent the entrance of the microbes into the cell. 



(11) The protoplasm of the cell may afford a medium for the growth of the microbes. 



(12) The dead and dry tissue does not afford a good medium but the dead and juicy cells afford a very convenient 

 soil for their development. The microbes were also alive in the living cells but preferred the dead ones. 



(13) The bacillus of the Siberian plague multiplies vigorously during the first few days in the leaves of Agapanthus 

 and grows out into a thread. At the end of the first week there is an inclination to form spores, which in course of 

 time becomes more distinct. Many spore chains were visible on the eighteenth day, together with separate spores and 

 nonsporiferous threads. Filaments and spores occur not only at the point of inoculation but also between the healthy 

 cells of the spongy tissue of the leaf, and in the cells themselves. Some of these threads stain well with gentian violet, 

 others do not. Those which do not stain with gentian violet, stain afterward with carmine (double stain) and are 

 refractive. Still others stain only in parts, or are not stained, and look like bright, pale, drawn threads. Slides made 

 42 days after inoculation still showed numerous vegetative forms of the plague bacillus, together with spores and spore- 

 bearing threads. 



