82 



EEPOKT OF CHEMICAL LAlSOItATORY 



Recovery of 

 implicated 

 organism from 

 flies and ants 



Production of 

 a gum-like 

 substance 

 " in vitro " 

 by action of 

 bacteria 



find whether sucli an infection was thus capable of being conveyed from tree to tree under 

 ordinary circumstances. Some tlies were caught after being seen apparently feeding on 

 the recently tapped surface of a branch. These were killed and introduced into sterile 

 test-tubes, a few c.c. of sterile water being then added. After being left thus for a few 

 hours, a loopful of the water was introduced into sterile tubes of gelatine media containing 

 sugar. These were then cultivated at 37 C. for a day. In some cases the flies were put 

 direct into the tubes of gelatine. In all cases, cultures were made from the gelatine on 

 plates of saccharose agar, as was done with the gum-bearing branches. Among the colonies 

 obtained from the flies in this way was a number showing the same appearance as those 

 produced by the bipolar staining organism already isolated. A further examination of 

 these colonies revealed the presence of this organism, which showed all the properties of 

 that obtained from the twigs. An examination of some ants obtained from tapped branches 

 also showed the presence of this bacterium. The method of transmission from tree to tree 

 is probably simply mechanical, the legs and wings of the insects picking up small quantities 

 of sap containing the organisms, some of which are conveyed to the exposed surface of 

 the next branch on which the insect alights. 



Having proved the invariable presence of these bacteria in gum-bearing branches, and 

 having also found a ready means for their transmission from one tree to another, the 

 question of the production of gum from the sap of the tree by these organisms had next 

 to be investigated. The best way to do this seemed at first sight to be by growing the 

 organisms in the sterilised sap of the hashdb tree itself. It was found impracticable to 

 obtain this in any quantity by pressure, so the following method was adopted. One or two 

 young trees from which no gum was exuding were cut down and the branches chopped into 

 lengths of about two inches. These pieces were bruised, put in a steriliser with water, and 

 boiled for a short time. The water was then poured ofi' and the extraction repeated, the 

 two extracts being mixed and concentrated. The resulting liquid represented a somewhat 

 diluted sap, but on filtering off the dirt, bark, etc., it was found also to contain a 

 considerable quantity of gum. This liquid was therefore unsuitable as a medium for 

 testing the gum-producing powers of the bacteria, as it would have been very difficult with 

 the means at my disposal to estimate with any degree of accuracy the proportion of gum 

 present before and after the experiment. An artificial medium was therefore used, which 

 had been found suitable for the cultivation of the bacteria in tubes and on plates. This 

 contained 4 per cent, levulose, 1 per cent, glycerine, O'l per cent, asparagin, and O'l per 

 cent, potassium citrate in water. I preferred using a liquid medium to a solid one 

 containing agar in addition to these constituents, as in the latter case there is a danger 

 of some agar getting scraped off when removing the slime produced by the bacteria, and this 

 may give, on hydrolysis, products resembling those derived from gum. 



The above medium, after being sterilised, was inoculated from a pure culture of the 

 bacterium and kept at 37 C. for about a month. The growth was then stopped by heating 

 the flask in the steriliser for half-an-hour. The liquid was then concentrated to half its 

 original volume over a small flame, a few drops of dilute sulphuric acid being added towards 

 the end in order to precipitate any substances of a protein nature which might have been 

 formed by the bacteria. The liquid was then filtered, and to the clear filtrate, on cooling, 

 was added three times its volume of alcohol. The whole was well stirred up and allowed to 

 settle, after which the supernatant liquid was poured ofl", the remainder being filtered and 

 the precipitate washed with 75 per cent, alcohol. This precipitate was redissolved in a little 

 water, brought down again with a large excess of alcohol, filtered off, washed with alcohol 

 and dried. 



