FURTHER BIOCHEMICAL METHODS vi^.-Q 



succinate, methyl succinate (Thunberg, 1933), a-glycerophosphate 

 (Green, 1936) and d-glutaric acid (Wcil-Malherbe, 1937). Methyl 

 succinate has not been found in biological material and a-glycero- 

 phosphate and d-glutamate are not extracted with ether, therefore, 

 this method is highly specific for succinic acid (Krebs, 1937). 



Neutral Solvents {acetone and ethyl, butyl and isopropyl alcohols). 

 These solvents are best distilled from a neutral or slightly alkaline 

 fermentation liquor. Acetone, in an aliquot of the distillate, is oxidized 

 with iodine in alkali and excess of the iodine back-titrated with thio- 

 sulfate. (Goodwin 19'-20). The other solvents are not oxidized under 

 these conditions. Isopropyl alcohol can be oxidized by dichromate 

 and orthophosphoric acid to acetone and the latter distilled off and 

 determined as above. Stahly, Osburn and Werkman (1934) show that 

 94% of the acetone is recovered in the distillation. The analytical re- 

 sults should, therefore, be corrected accordingly. These authors state 

 that ethyl alcohol can be entirely oxidized by dichromate to acetic 

 acid, while in the case of butyl alcohol, 89.6% is oxidized to butyric 

 acid and 10.4% to acetic acid. The two acids may then be deter- 

 mined by distillation or by partition. For small quantities of ethyl or 

 butyl alcohol the method of Johnson (1932) may be used. 



Carbon Dioxide. Large quantities of CO2 may be detected by the 

 appearance of gas and its characteristic reactions. Because of its great 

 solubility, however, it can seldom be thus detected, and a measure of 

 the gas evolved is never an accurate determination of the quantity of 

 CO2 produced. 



For accurate results, use should be made of an aeration train of 

 which the essential elements are: a wash tower containing alkali to 

 remove CO2 from incoming air; a flask or other container for the cul- 

 ture; an absorption tower containing a measured amount of standard 

 alkali with beads or other device to break up the stream of air; and an 

 aspirator or pump to force or to pull the air thru the train. When 

 using this method special precautions to avoid contamination should 

 be observed; and no reliance should be placed on results unless tests 

 at the end of the experiment show that the original organism is 

 present in pure culture. 



When a considerable number of cidtures are to be studied simul- 

 taneously, the Eldredge tube (Fig. 1) can be used more conveniently 

 and often with sufficiently accurate results. A satisfactory sized tube 

 is one having a capacity of about 60 ml. in each arm. (These tubes are 

 not as yet listed by supply houses, but arrangements to handle them 

 have been made with the W ill Corp., Rochester, N. Y. and Macalaster 

 Bicknell Co., Washington and Moore Sts., Cambridge, Mass.) 



In using the Eldredge tube, place 20 ml. of the medium in one of the horizontal arms 

 and sterilize. Inoculate and then place in the other arm a measured quantity (usually 15 

 to 25 ml., depending upon the amount of COj expected) of a freshly prepared X/10 

 barium hydroxide sohition. (One may use NaOII or KOII, but the insohdjility of the 

 BaCOj formed makes I5a(OH)j more satisfactory in giving a visual indication of CO3 

 production.) Immediately after inserting the alkali, push the cotton i)lugs down in the 

 tubes and seal, .\fter at least two weeks incubation titrate the barium hydroxide with 

 N/10 HCl or preferably H2SO4. using i)henolphthalein as an indicator. Compute the 

 amount of CO 2 produced from the equation: ml. of Ba(OH)2Xnormality of Ba(0H)2 



