No abstract

I954 reacting 'directly' in the van den Bergh test. Their absorption curves are similar, though not identical, and they differ from that of bilirubin. 3. The visible absorption curves of the azo compounds of these three pigments are indistinguishable. 4. The two direct-reacting pigments of serum occur in small amounts in the urine of patients with obstructive jaundice, and in large quantities in gallbladder bile obtained at necropsy, where they are more abundant than bilirubin. 5. The proportion between the two directreacting pigments of serum is variable. 6. There is evidence that other diazo-positive pigments occur in bile and urine.

Bertrand-Hudson rule; the other exhibits activity resembling that of an extract made from Candida Utili. MATERIALS AND METHODS Organisms. Acetobacter suboxydans, no. 621, was obtained from the American Type Culture Collection, Washington, D.C. It was grown at 300 for 3 days in a yeast-water medium to which 0.3 % (w/v) of KH2PO4 and 2 % (w/v) of glycerol were added (pH about 5.5). The cultures (100 ml. of medium in 500ml. conicalflasks) were aerated by continuous shaking. Yeast water was prepared by boiling 1 lb. of baker's compressed yeast with 400 ml. of water for 30 min., allowing the yeast to settle overnight, filtering the supernatant through kieselguhr and making the volume up to 51. with water. Candida utilis (strain Y 90/77) was obtained through the courtesy of the Northern Regional Research Laboratories, Department of Agriculture, U.S.A. The stock culture was maintained on malt-agar slopes. For experiments the organism was transferred either to the glucose-containing basal medium of Schultz & Atkin (1947) or to a medium having the following composition: 2-0 g. of KH2P04, 0-25 g. of anhydrous CaCl2, 0.5 g. of MgSO4,7H20, 4.5 g. of (NH4)2SO4, 20 g. of mannitol and tap water to 1 1. The cultures were incubated at 30°for 2 days and continuously shaken. Cell-free extracts. The cells ofA. 8uboxydans were harvested and washed three times with water by centrifuging. Washed cells (5-10 g.) were suspended in sufficient 001M-Na2HPO4 solution to make a total volume of 25 ml., and disrupted by treating for 40 min. in a Raytheon 9 kcyc./sec. sonic disintegrator. The cup assembly of the apparatus was set up in a cold room at 2-3°and the internal temperature of the cup kept at 0-1°by circulation of ice-water (1.5 1./min.). The extract was clarified by centrifuging at 20000 g for 30 min. in a centrifuge operating at-5°, and dialysed against water for 12 hr. at 20. The extracts were clear red-brown solutions, * This buffer was made by mixing appropriate volumes of 0-2M aminotrishydroxymethylmethane (tris) solution and 0'1M succinic acid solution.

With the exception of Uyei [1927], who employed the tubercle bacillus, authors have not defined precisely the size of the inoculum, and this is now known to be a critical factor in experiments on bacterial nutrition. Accordingly, we have reinvestigated the growth. requirements of certain Mycobacteria in order to determine the effects of varying the size of inoculum. The use of defined inocula and other rigid conditions has made it possible to demonstrate the stimulant action of certain accessory nutrients in an unequivocal manner. EXPERIMENTAL Organisms. The following species were utilized: Myco.

Although lactic acid is utilized completely during the cultivation of Mycobacterium phlei (Stephenson & Whetham, 1922), it is oxidized incompletely by 'resting cells' (Edson & Hunter, 1947). Extracts of crushed cells and acetone-dried preparations, which retain power to oxidize lactate, lose the ability to oxidize pyruvate and acetate. Since an acetone powder will yield a soluble enzyme, this preparation has provided mean of studying the mechanism of lactic acid breakdown. METHODS Manometric mea8urements. Oxygen uptake and C02 production were determined by the standard methods of Warburg. Substrates (acids as neutral sodium salts) were added after equilibration. Analytical methods. Lactic acid was estimated by the method of Friedemann & Graeser (1933). In this procedure blank titrations of volatile bisulphite-binding material obtained from acetone powders never exceeded 0-3 ml. 0-005M-I2. Acetic acid was detected by the lanthanumiodine test (see Edson & Hunter, 1947); and flavineadenine-dinucleotide by the d-amino acid oxidase test of Warburg & Christian (1938). Materials. l(+)-Lactic acid ([cx15, Zn salt =-8-20) was obtained from the Pfanstiehl Chemical Co., U.S.A. d(-)-Lactic acid ([,]15 , Zn salt = + 8.0°) was prepared by resolution of dl-lactic acid (A.R. (British Drug Houses, Ltd.)) according to the morphine method of Irvine (1906). Catalase was crystallized by the method of Sumner & Dounce (1937) and dissolved in phosphate saline. Cozymase was prepared by the method of Williamson & Green (1940). Acetone powder. The preparation and suspension of the powder have been described (Edson & Hunter, 1947). Acetone-treated bacilli are somewhat altered but retain their acid-fastness. Preparation of a 8oluble enzyme. Because other methods of extraction proved unsatisfactory, 6-5 g. of dry powder were suspended in 40 ml. 0 02M-phosphate buffer, pH 7-4, and ground for 4 hr. in the wet crushing mill of Booth & Green (1938). The gross debris was removed by centrifuging at 3500 r.p.m. for 0 5 hr. The supernatant liquid was brownish yellow and opalescent. Since high-speed centrifugal apparatus was not available, this fluid was diluted with an equal volume of 0-05M-phosphate buffer