314 STATE HORTICULTURAL SOCIETY. 



as well as bacteria which always find a lodgment in and upon dead plant 

 tissiTe. Since all these forms are microscopic the separation involves a 

 method of procedure familiar only to specialists, and as such beautiful 

 results were reached in the separation of this fungus it suggested a 

 graphic presentation of the method in connection with the study. The 

 method used was the same as that which KocH developed so admirably 

 for the separation of bacteria, and consists in the dilution of the organisms 

 in several quantities of a warm liquid substance which, when spread out 

 in a thin layer and cooled, solidifies and holds each germ firmly fixed at 

 one point in the dilution. This substance is usually some gelatinous base, 

 as gelatine, or agar-agar, containing beef broth and peptone to furnish 

 food for the organisms. In a few days after cooling the dilutions, in the 

 thin layer each germ, by growth, has produced a colony which can be seen 

 with the unaided eye. 



Three glass tubes containing a small quantity of liquid nutrient agar-agar 

 were placed in a water bath at 48'' centigrade. This temjjerature is 

 sufficient to keep the agar liquid, while it is not hot enough to kill the 

 organisms. Now several thin shavings through the fungus pustules on the 

 stem of the privet were transferred to tube No. 1. This was shaken gently 

 to distribute the germs evenly through the liquid. Now a small quantity 

 of the liquid in No. 1 containing the germs was transferred to tube No. 2, 

 making the second dilution, and from No. 2 to No. 3, making the third 

 dilution. Experience enables one to judge quite accurately in making the 

 dilutions so that we estimate the dilution sufficient to cause each germ to 

 lie separately at different points in the liquid agar, at least in dilution 

 No.3. ' 



Each of these dilutions was then poured into a Petrie dish,* and allowed 

 to cool in a thin layer over the bottom. No germs could then be seen in 

 the agar, since they are microscopic and lie singly. The dishes were piled 

 away for a few days. During this time each germ grew and produced a 

 colony which was visible to the unaided eye. The plates or dish cultures 

 were now photographed natural size and the result is reproduced in Plate 

 1. In No. 8 it will be seen that nearly all of the colonies are separate.^ 

 The snowflake-like colonies are those of the desired fungus. The small, 

 compact, circular ones are those of bacteria. One large compact colony is 

 that of a common fungus. 



In Nos. 2 and 1 the fungus colonies are crowded, and have not made such 

 good growth. The colonies of bacteria are more numerous also, and it 

 would be very difficult to obtain a pure culture of the fungus in either of 

 those dilutions. If the dilutions were not numbered it would be an easy 

 thing to determine their number from the size and number of the colonies. 

 The very large compact colony in No. 2 is that of a motile bacterium. 



Pure culture of the anthracnose. — Pure cultures of the fungus could 

 now be started by transplanting with a flamed platinum needle portions of 

 the fungus colonies from No. 3 into a culture tube of nutrient agar. The 

 photograph was taken after these plantings were made which accounts for 

 the broken appearance of some of the colonies. 



From the point of inoculation in the culture tube, where the transplant- 

 ing was made, the fungus threads grow out through the upper surface of 

 the agar radiating in all directions. In a few days minute black bodies 

 appear seated here and there upon the mycelium. These resemble the 



* A Petrie dielt is composed of two shallow glass vessels, one abont three inches in diameter which 

 serves as the bottom, the other of a little greater diameter, which is inverted over the first one for a cover. 



