PRELIMINARY REPORT HYDROCYANIC
BY
ON THE PRODUCTION ACID BY BACTERIA.
AND c. c.
OF
B. J. CLAWSON
YOUNG. of Kansas.)
(From
the Department (Received
of Bacteriology, for publication,
University
June 28, 1913.)
Egg culture 41 which had produced HCN spontaneously, and which is mentioned in the paper by Emerson, Cady, and Bailey1 was turned over to us by Dr. Cady for investigation to determine the organism, if any, which caused the generation of hydrocyanic acid. The modified Schijnbein test was used throughout this work to detect the presence of hydrocyanic acid gas. Stained smears were made of the original material in which an almost pure culture of a short rod was seen. Transfers of this material to sterilized whole egg developed hydrocyanic acid in twenty-four hours. From these tubes, the material was plated out on agar and grown at room temperature. After forty-eight hours, colonies appeared which produced a blue-green pigment that was dist.ributed through the agar. Transfers to gelatin showed rapid liquefaction at room temperature. After fortyeight hours, the presence of HCN gas was revealed by the Schonbein test and confirmed by the Prussian blue reaction. The principle characteristics of the organism are as follows: short rod; no spores; slightly pointed at the ends; decidedly motile; Gram negative; liquefies gelatin rapidly at 22’; brown growth on potato; peptonizes milk which becomes slightly alkaline; reduces nitrates to nitrites and ammonia; produces indol; no fermentation or production of gas in lactose, dextrose, saccharose, raffinose, salicin, inulin mannite, or dulcite broth media. It does not reduce neutral red; forms heavy pellicle on broth, and green florescent pigment containing pyocyanin on agar. It will not grow in media which are even slightly acid to sodium alizarin sulplionate.
1 This Jownal, xv, p. 415, 1913.
419
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420
Bacterial
Production
of Hydrocyanic
Acid
From the above we conclude that the organism is Racillus pyocyaneus. No attempt was ma.de in this work to distinguish between Bacillus pyocyaneus and Bacillus JEuoreScens as both have the HCN-producing power. The organism was grown on 50 grams of ‘gelatin in an Erlenmeyer flask at room temperature. After growing for twenty-four hours and testing for HCP\T production, the flask was connected with a U tube containing 2 cc. of a 10 per cent solution of silver nitrate. The air that filtered through the cotton plug was drawn slowly through the flask and U tube for seventytwo hours. The nitrate and precipitate were then transferred to a small distilling flask and strong HCl added. Five cubic centimeters were distilled off into a deceiver buried in freezing cc. killed a chick in less than thirty seconds; old kitten, killed it almost inst,antly. mixture. Of this distillate 0.2 0.4 cc. given to a three-weeks’
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The power which the organism has of producing HCN, is apparently not due to an extracellular enzyme. The organism was grown in gelatin for seventy-two hours at 37”, and had been giving off HCN for forty-eight hours. After filtering through a Berkefeld filter, some of the filtrate was planted again int,o gelatin and incubated, but, gave negative results for HCN. Hydrocyanic acid gas is apparently produced only under aerobic conditions, which led to the belief that the reaction in which HCN was produced was due to oxidation of the proteins. This was subsequently shown to be true. While good tests were always obtained from cultures grown at room temperature (22”), as a rule cultures grown at 37” gave a stronger test for HCN. Different media were used to determine whether or not the ability to produce HCN would be shown in them. Positive tests were obtained at 37” from gelatin, broth, milk, agar, Dunham’s peptone solution, cotton seed meal, and egg, in which several different proteins are involved. The growth of the organism on egg increased the HCN production decidedly. This was true for all strains of pyocyaneus tried. This increase did not diminish when it was subsequently grown on other protein media. Several other strains of B. pyocyaneus were tested for HCN production, all of which returned positive results. They were from the following sources: (1) University of Chicago s’tock culture; (2) egg which had been frozen two and one-half years; (3) soil; (4) l$aw river.
B. J. Clawson
and
C. C. Young
421
The stock culture of B. pyocyaneus from the University of Chicago, when first planted into-gelatin, gave a very weak test for HCN; but after twenty-four to forty-eight hours’ growth in egg, it became a strong producer of hydrocyanic acid. It would seem that the organism, living as a saprophyte, is better prepared to decompose protein substances with the production of HCN, than when living as a parasite. A rabbit was injected intraperitoneally with several cubic centimeters of a twenty-fourhour broth culture of the University of Chicago strain. After three days the rabbit died. An autopsy showed general peritonitis. Transfers made from the heart’s blood gave a pure culture of B. pyocyaneus. This was grown in gelatin, but gave only a faint test for HCN which indicates that the organism living in an animal is reduced in its power to produce HCN. It was then grown for twenty-four hours on sterilized egg, when the test for hydrocyanic acid was one of the strongest obtained. Subsequent transfers from the egg to gelatin showed no diminution of the production of HCN. An attempt to grow the organism on Jordan’s synthetic media2 was unsuccessful. B. Pyocyaneus was not the only organism found which produced HCN from proteins. Miss Myrtle Greenfield, bacteriologist for the State Water Survey, isolated an organism from soil, the name of which has not been determined. It was a strong HCN producer. The characteristics of the organism are as follows: long rod; rounded ends; no spores; very motile; takes all ordinary stains; Gram negative; abundant growth on all ordinary media; growth on agar glistening; orange pigment diffusing rapidly through agar, gelatin, potato and milk; deep colonies in agar fusiform; liquefies gelatin rapidly; b welatin stab infundibuliform. On broth and milk, a delicate pellicle is formed, and the media are colored from the surface down. Milk is gradually peptonized. Litmus is reduced. All media are made slightly alkaline. There is no diastatic action on potato starch. A slight amount of nitrite is formed. The organism grows best at 20” and is aerobic. No acid or gas is formed in dextrose, lactose, saccharose or glycerin broth.
2 Journ.
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of Exp. Med., iv, p. 629, 1899; Jordan:
Botanical
Gazette, xxvii,
p. 9, 1899.
422
Bacterial
Production
of Hydrocyanic
Acid
A culture of Bacillus violaceus from the American Museum of Natural History, was also found to produce HCN in gelatin and egg. It is very possible that other chromogens may have this same property. A number of liquefying organisms were tested and indications of the formation of hydrocyanic acid were obtained although the tests were influenced by ammonia and sulphuretted hydrogen. They will be examined more carefully. There has been much work done on the production of HCN from grain, beans, linseed meal, germinating Sorghum vulgares, and other protein-containing substances. In most cases, the production of HCN is attributed to an enzyme. Apparently all of the workers were using non-sterile material, which could have been easily contaminated by an HCN-producing organism.3 The authors are continuing the investigation with the intention of publishing more complete information concerning the decomposition of protein substances with the production of HCN due to the microorganisms mentioned in the body of the paper.
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3 L. Guignard: HCN in Beans, Recueil actes off. et dot. interessant hyg. Superieur Hyg. Pub. France, 1909; S. J. M. Auld, Jr. : pub., travaux Conseil HCN in Linseed Cake and Other Feeding Stuff, South East Agricultural College, Wye, England, No. 20, pp. 289320, 1911; C. Ravenna (Univ. of Bologna) : HCN from Seeds of Sorghum, Atti R. accad. dei Lincei, xix, II, pp. 356361; HCN from Seeds of Sorghum, Gaz. chim. ital., xli, II, pp. 74-81; C. Ravenna and M. Zamorani (Lab. Agric. Chem.) : Physiological Function of HCN in Plants, Chem. Zentralbl., i, p. 113, 1910; M. J. Offner: de France, xxvii, pp. 342-5; S. J. M. Auld, HCN in Fungi, Bull. sot. mycol. Jr.: Formation of HCN in Linseed Cake and other Food Stuffs, London Board of Agriculture, No. 6, pp. 446-460; No. 8, pp. 657-660, 1912; C. D. Londer: Formation of HCN in Linseed Cake and other Food Stuffs, London Board of Agriculture, No. 11, pp. 904-907, 1911.