1923] 



JENNISON — POTATO BLACKLEG 49 



fications of Shaffer's ('13) original method appear, but these are 

 largely omissions of certain steps which were determined by pre- 

 liminary experimentation to be unnecessary. For instance, acetic 

 acid and colloidal iron were not used in the process because it 

 was determined that the small amounts of albuminous material 

 present in the cultures did not interfere with the determination 

 of the reducing sugars present. The precautions emphasized 

 by Shaffer were observed throughout. The cuprous oxide was 

 titrated immediately upon being dissolved and without removal 

 from the centrifuge tube in which it was precipitated and thrown 

 down. In order to avoid breakage of the centrifuge tubes when 



fe ^ V/X W.V V,V,l*V***^)_, 



these were plunged in the water bath circular wire baskets (pi. 

 2, fig. 5) having wooden bottoms and tops, with holes for 2 



centrifuge tubes, were used. No oxidizable substance other than 

 the sugar was allowed to get into the solution, and the cuprous 

 oxide solvent employed was made from chemically pure sub- 

 stances in order to avoid presence of ferrous iron. This was fur- 

 ther assured by adding a trace of permanganate. 



Procedure. — The procedure outlined in the steps given below 

 was followed in carrying out all experiments in this phase of the 

 work : 



(1) A large volume of Dunham's solution was made up, auto- 

 claved, and then filtered through paper. 



(2) To separate portions of this solution, 1 per cent of the test 

 substances (see list p. 46) was added and dissolved. 



(3) Exactly 10 cc. of each of the 8 nutrient solutions thus prepared 

 were placed in each of 10 special culture tubes ("wasp" tubes, pi. 2, 

 fig. 5). These tubes were large enough to permit dilution of the cul- 

 ture to exactly 60 cc. This was made possible by carefully stan- 

 dardizing each tube and permanently etching the 60-cc. mark on the 

 slender neck. Had these tubes been drawn out and graduated to hold 

 50 cc, calculations would have been simplified. 



(4) The tubes were plugged with cotton in the usual manner and 

 the contents sterilized in the autoclave by exposing for 15 minutes 

 at 15 pounds pressure. One of the preliminary experiments per- 

 formed showed that this method, if carefully controlled, did not 

 hydrolyze the di- and polysaccharides used (table ix). 



(5) Each of the 8 media was inoculated with a loopfid of an 

 invigorated culture of the organism to be tested for its ability to 

 hydrolyze the test substances employed. Two uninoculated "control" 

 tubes of each sugar broth were carried along, incubated, and tested in 

 identically the same manner as the inoculated cultures in each set. 



