VARIATIONS IN MORPHOLOGY OF BOVINE TUBERCLE BACILLI.

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the occurrence of the filamentous forms in contact with the paraffin; for, according to Coppen-Jones, the conditions best suited for the production of filamentous and branched forms are a favorable medium and an abundance of oxygen. No doubt the paraffin also protects the surface of the egg from evaporation and consequent drying and thus preserves the moisture so essential to the growth of the tubercle. bacillus. It must be confessed, however, that this explanation does not appear wholly satisfactory, but none other has suggested itself.

(1) WOLBACH AND ERNST.

REFERENCES.

Observations on the Morphology of Bacillus Tuberculosis from Human and Bovine Sources. The Journal of Medical Research, Vol. X, No. 3, Dec., 1903, p. 313.

(2) DORSET, M.

The Use of Eggs as a Medium for the Cultivation of Bacillus tuberculosis. American Medicine, April 5, 1902, p. 555.

(3) SMITH, THEOBALD.

A Comparative Study of Bovine Tubercle Bacilli and of Human Bacilli from Sputum. Journal of Experimental Medicine, Vol. 1II, 1898, p. 451.

(4) COPPEN-JONES.

Ueber die Morphologie und Systematische Stellung des Tuberkelpilzes und über die Kolbenbildung bei Actinomykose und Tuberculose. Centralbl. f. Bacteriologie, Bd. XVII, 1895, p. 1.

H. Doc. 743, 58-2- -8

INFLUENCE OF FORMALDEHYDE ON THE DIGESTIVE.

ENZYMES.

By T. M. PRICE, PH. D.,

Assistant in Biochemic Division, Bureau of Animal Industry.

This study of the influence of formaldehyde on the digestive enzymes was undertaken with a view of determining for formaldehyde the strength of solutions necessary to cause interference in the action of rennet, pepsin, pancreatin, steapsin, ptyalin, amylopsin, and galactase, in vitro, and what strength would at the same time prevent bacterial development.

The work was done with milk, because milk is, of all foods, the one which most readily undergoes decomposition, and consequently is extensively treated with preservatives, and also because milk is a food on which the activity of most of the digestive enzymes may be determined. Fresh extracts of the various glands from healthy animals were used to furnish the enzymes which could thus be obtained.

Experiment 1.-Action of formaldehyde on calf's rennet.

The rennet was obtained from the mucous membrane of a calf's stomach by digesting it for twenty-four hours at room temperature in 200 c. c. of a 0.2 per cent hydrochloric-acid solution, filtered, and carefully neutralized. One cubic centimeter of neutralized liquid was used to coagulate 10 c. c. of milk. Formaline was added to 100 c. c. of fresh milk in flasks in the following proportions:

TABLE I.-Showing strength of formaldehyde solution added.

The flasks containing the milk and formaldehyde were corked and set aside at room temperature. Fresh milk was always used in the control. At various intervals 10 c. c. of milk was taken out of each

a Formaline is a 40 per cent solution of formaldehyde gas in water.

flask and placed in test tubes, 1 c. c. of rennet solution added, and the tubes placed in the incubator at 40° C.

TABLE II.—Showing the results of Experiment 1.

These results show that formaldehyde added to milk in the proportion of 1:2,500 does not interfere with the rennet's coagulation, while the proportion of 1:1,875 retards the coagulation, and the proportion of 1:500 renders the milk incapable of being coagulated in eighteen. hours.

Experiment 2.-Action of formaldehyde on pepsin.

The pepsin used was in the form of artificial gastric juice prepared from the stomach of a pig. The stomach was opened, emptied of the contents, and then the surface cleaned with a wet sponge. The mucous membrane was removed from all but the pyloric end of the organ; it was then freed from a portion of the water which was adhering to it by pressure between dry cloths and minced. The finely divided mucous membrane was then placed in 2 liters of dilute hydrochloric acid, containing 6 c. c. of concentrated hydrochloric acid per liter, and the mixture digested in the incubator at 40° C. for a day. The liquid was then filtered through paper, and 50 c. c. added to 200 c. c. of a 0.1 per cent hydrochloric acid solution; 10 c. c. of this solution was added to 1 c. c. of milk treated as in Table I and set aside in incubator at 40° C.

It has been demonstrated by Hammarsten and other workers1-2 that milk, when subjected to the action of pepsin hydrochloric acid, is broken up into calcium paracasein and a small amount of albumose-like albumin. The paracasein is precipitated, and may be recognized after digesting the milk for a short time, the paracasein separating and finally decomposing with the formation of paranuclein. Any foreign matter that interferes with the formation of paranuclein interferes with the digestion of the milk. This feature was taken to determine when the formaldehyde affected the digestibility of the milk. It was found that upon the addition of formaldehyde in the proportion

of 1:50 the pepsin digestion was retarded, while in a stronger solution, or in the proportion of 1:25, the digestion was materially interfered with; and in the proportion of 1:125 or less, the digestion was normal with the control.

Experiment 3.-Action of formaldehyde on pancreatin.

Two hundred grams of finely chopped fresh pancreas, free from fat was digested in a liter of water for twenty-four hours, filtered, and the solution made up to twice its volume with a 2 per cent solution of sodium carbonate; 10 c. c. of this mixture was added to 1 c. c. of milk, as in Experiment 2, placed in the incubator at 40° C. and tested every two hours until the control gave no precipitate when saturated with magnesium sulphate, thus showing that all the proteids were in the form of pepton. At the end of eighteen hours the control showed the absence of all proteids that were precipitated by magnesium sulphates, and the tubes containing formaldehyde in the proportion of 1:2,000 or less showed the same, while in the tubes containing formaldehyde in the proportion of 1:1,500 or more the digestion was interfered with.

Experiment 4.-Action of formaldehyde on steapsin.

One hundred grams of finely chopped pancreas free from fat was mixed with 1 liter of a solution of glycerine containing 900 c. c. of glycerine and 100 c. c. of a 1 per cent sodium carbonate solution. This mixture was allowed to stand at room temperature for three days, filtered through linen, and the filtrate used for its fat-splitting enzyme; 2 c. c. of this extract was added to 1 gram of neutral fat obtained from cream by shaking with alkaline ether and then washing well with water. To this neutral fat solution were added varying proportions of formaldehyde. To the tubes containing 1 gram of the fat and 2 c. c. of the extract 10 c. c. of water was added, the tubes shaken in a milk shaker, set aside at 40° C., and then shaken every half hour for twelve hours at this temperature. Their acidity was then determined, using phenolphthalein as indicator. It was found that formaldehyde in the proportion of 1:35 prevented the action of steapsin, and in the proportion of 1:50 the action of steapsin was retarded, while weaker solutions had no effect on the activity of the enzyme.

The enzymes used in the preceding experiments practically are the ones whose activity, if interfered with, would affect the digestibility of milk, but formaldehyde is often used as a preservative for food containing a large amount of starch. The enzymes, ptyalin and amylopsin, were studied with regard to their activity with starch when the starch was in the presence of formaldehyde solutions of varying strengths.

Experiment 5.-Action of formaldehyde on ptyalin.

One gram of

One

The filtered saliva was used for the ptyalin enzyme. potato starch, which had been thoroughly washed with dilute hydro