Non-protein nitrogen. balance, was dissolved in approximately 50 cc. of distilled water. By the method of Folin and Wu. 251

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1 SOME CHEMICAL STUDIES OF COMMERCIAL BACTERIOLOGICAL PEPTONES JAMES G. McALPINE AND GEORGE D. BRIGHAM From the Department of Animal Diseases, Storrs Agricultural Experiment Station Storrs, Connecticut Received for publication May 29, 1928 I. INTRODUCTION In the course of a study of the abortus-melitensis group it was found that certain commercial peptones were better suited to initiate growth and multiplication than others. It was decided, therefore, to make as accurate chemical analyses as present methods will permit of certain well-known commercial brands of peptones in order to determine in what form nitrogen may be best utilized by the members of this bacterial group. II. METHODS Four commercial brands of peptone were employed in this study: Fairchild's, Difco-Bacto, Difco-Proteose and Witte's. One gram of the peptone, accurately weighed on a delicate balance, was dissolved in approximately 50 cc. of distilled water and sterilized in the autoclave for fifteen minutes at 15 pounds extra pressure. The peptone solution was carefully transferred to a 100 cc. volumetric flask and the whole made up to the mark. The following nitrogen determinations were made on aliquot portions of this solution. Five-cubic-centimeter amounts were used in all of the determinations except those for non-protein nitrogen and amino nitrogen by the Sorensen method where 20 cc. and 1 cc. quantities were used respectively. Total nitrogen. method. Non-protein nitrogen. By the Gunning modification of the Kjeldhal By the method of Folin and Wu. 251

2 252 JAMES G. McALPINE AND GEORGE D. BRIGHAM Free-ammonia. By the Van Slyke and Cullen modification of the Folin air current process. Amino-nitrogen. By the Van Slyke and Sorensen methods. The S6rensen results were corrected for free ammonia. The Van Slyke determinations were made on the ammonia-free material. Protein-nitrogen. This was determined by subtracting the non-protein nitrogen from the total nitrogen figure. Polypeptid nitrogen. This nitrogen fraction is represented as the difference between the non-protein nitrogen figure and the sum of the amino and free ammonia nitrogen. The Van Slyke figure was used here for the amino nitrogen. Two different lots of each peptone were analyzed, and all determinations were made in duplicate. The figures obtained from the two different lots of the same brand of peptone were averaged. It was found that the different lots varied only slightly, the greatest variation being found in the two lots of Witte's peptone. III. EXPERIMENTAL The results are given in the accompanying chart in which the different fractions are drawn to scale, with the exception of the free ammonia figures. Owing to the small amount of free ammonia, it was necessary to multiply the scale by ten in this case in order to make the results legible. Total nitrogen. It will be seen that the amounts of total nitrogen do not vary very greatly in these four commercial peptones, Witte, with 152 mgm., is the highest, and Difco-Proteose, with 125 mgm., is the lowest. Non-protein nitrogen. In the next figure on the chart are given the results of the non-protein nitrogen determinations. Witte's peptone which shows the largest amount of total nitrogen has the smallest amount of the non-protein fraction (56.17 mgm.). In the Difco-Bacto peptone the total nitrogen and non-protein nitrogen are not very far from equal. Difco-Proteose peptone has distinctly more non-protein nitrogen (69.3 mgm.) than Witte's, and in the Fairchild peptone, almost three-fourths of the total nitrogen is non-protein (102.5 mgm.).

3 CHEMICAL STUDIES OF BACTERIOLOGICAL PEPTONES 253 Protein-nitrogen. These figures were not obtained by actual determination, but by the subtraction of the non-protein nitrov r T0t./ N Van S(yk 11 ANon-Proten Nl Pkiern N ad to ṾI?r.ee NH, ^P.rpo.tid /V CHART 1 gen figures from the total nitrogen results. The chart shows that the Witte peptone contains the largest amount of protein nitrogen (93.42 mgm.), and that the Difco-Proteose has next to the

4 254 JAMES G. McALPINE AND GEORGE D. BRIGHAM largest (54.6 mgm.), though its protein nitrogen content is less than two-thirds that of the foreign brand. The protein nitrogen in Fairchild peptone is comparatively small (24.2 mgm.) and that in Difco-Bacto is almnost negligible (8.4 mgm.). Polypeptid nitrogen. This fraction also was not obtained by actual determination, but by subtractmg the sum of the amino and free ammonia nitrogen figures from the amount of non-protein nitrogen. Difco-Bacto shows the largest amount of polypeptid nitrogen (82.17 mgm.) and Fairchild's next to the largest (79.57 mgm.). There was correspondingly much less polypeptid nitrogen in the Witte and Difco-Proteose peptones. Amino nitrogen. By the Van Slyke determination the Difco- Bacto, the Difco-Proteose and the Fairchild peptone were found to have about equal amounts of amino nitrogen (approximately 48.0 mgm.), while the Witte peptone contained only about twothirds as much (29.65 mgm.). The Sorensen results are more irregular, indicating perhaps that the di-amino acids were present in greater quantities in some of the brands than in others. Free ammonia. With the exception of the Fairchild peptone, the amount of free ammonia in any of these peptones was almost negligible. It should be remembered that in the chart these amounts are drawn to a scale ten times greater than that used for the other determinations. IV. DISCUSSION The results given in this paper must be regarded merely as preliminary in the main. It is realized that if more lots of each of the peptones had been analyzed the relative amounts of the various fractions might have been expressed in somewhat different figures. However, as previously stated, the only large variations that were found were in the two lots of the Witte peptone. Although the amounts of total nitrogen in all of these peptones appeared to be about equal, the different nitrogen fractions in the various brands differed greatly from each other. It is possible, in a measure at least, to estimate the length of the digestion period and the type of the enzyme employed in the production of peptone, from the above determinations. Relatively long diges-

5 CHEMICAL STUDIES OF BACTERIOLOGICAL PEPTONES 255 tion with trypsin, particularly after continued preliminary treatment with pepsin, will of course yield relatively large amounts of non-protein, particularly amino, nitrogen. On the other hand, digestion with pepsin alone, or followed by a very short tryptic process, will furnish a product high in "protein" (proteose) nitrogen. Practically all of the total nitrogen in the Difco-Bacto product was "non-protein" in character. There was a relatively large amount of polypeptids present, and according to the Van Slyke method the amino nitrogen fraction was high. There appeared to be very little free ammonia and "protein" nitrogen in this brand. The non-protein nitrogen content of Witte's peptone was comparatively small, and was divided about equally between the polypeptid and amino nitrogen fractions. The amount of free ammonia was very small. A large portion of the total nitrogen was of the proteose type. In the Difco-Proteose peptone the total nitrogen was about equally divided between the non-protein nitrogen and the protein nitrogen fractions. About one-third of the non-protein nitrogen was apparently polypeptid and two-thirds amino acid in character. The protein nitrogen was high and the free ammonia was practically negligible. The Fairchild peptone contained a relatively large amount of non-protein nitrogen which was about equally divided between the polypeptid and amino nitrogen (Van Slyke) fractions. The amount of protein nitrogen was small. Evidently digestion is carried on for a longer time in the preparation of this peptone than with Witte and Difco-Proteose peptones. The free ammonia determinations were high. If it may be assumed that the protein nitrogen is practically all proteose in character, the Witte product contains a very large quantity of this product. Difco-Proteose peptone has about half as much as Witte's, while the Fairchild and Difco-Bacto peptones contain relatively small amounts of this nitrogen partition. This is especially interesting in view of the more recent work showing that proteoses appear to be necessary for toxin production. Gibbs

6 256 JAMES G. McALPINE AND GEORGE D. BRIGHAM and Rettger (1927) obtained potent toxins with both the Difco- Proteose and Witte peptone, and they showed that one fraction, evidently proteose in character, was necessary to obtain this result. The results given above corroborate the statement made by Hucker and Carpenter (1927). "The original 'Witte peptone' contained a large amount of proteose and was only slightly digested in comparison with other brands. 'Fairchild's peptone,' on the other hand, is most completely digested. 'Bacto peptone' appears to be intermediate between Witte and Fairchild's." V. SUMMARY 1. Four different brands of commercial peptone were analyzed quantitatively for their various nitrogen fractions. Although the total nitrogen present in all of these different commercial brands was practically the same, there was a marked difference in the amounts of the various nitrogen fractions. 2. Witte and Difco-Proteose peptones contain the largest amount of so-called "protein" nitrogen. It is probable that most of this nitrogen is proteose in character. REFERENCES GIBBS, C. S., AND RETTGER, L. F Some factors governing the production of diphtheria toxin in artificial media. Jour. Immunol., 13, IluCKER, G. J., AND CARPENTER, D. C The relation of hydrolytic decomposition products of protein to bacterial growth. Jour. Inf. Dis., 40,