Consequently, the authors decided to investigate the various A STUDY OF METHODS FOR THE DETERMINATION OF

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1 A STUDY OF METHODS FOR THE DETERMINATION OF REDUCING SUGARS IN BACTERIAL CULTURES COLORIMETRIC METHODS DOROTHEA KLEMME AND CHARLES F. POE Division of Sanitary Chemistry, Department of Chemistry, University of Colorado, Boulder, Colorado Received for publication, April 12, 1935 In a study of the metabolic products formed by the colon and aerogenes groups of bacteria in sugar media, it became necessary to determine accurately the amounts of residual sugar in the culture-media. A search of the literature revealed that only two methods had been used for the determination of reducing sugars in bacteriological media. Stiles, Peterson, and Fred (1926) tried the Shaffer-Hartmann method (1921) and found it to be satisfactory for the determination of glucose in bacteriological media, both before and after clarification with lead acetate. Magee and Smith (1930), however, found the Shaffer-Hartmann method to be inaccurate when applied to media containing meat infusions. These investigators likewise found the Benedict copper method (1926) to give unsatisfactory results. Consequently, the authors decided to investigate the various calorimetric methods as to their availability for use in the analysis of reducing sugars present in broths used for the growth of bacterial cultures. The results of this investigation are reported in the present communication. The medium used for the growth of the different cultures contained 8 grams of Bacto-Nutrient broth and 10 grams of glucose to the liter. The reaction was adjusted to a ph of 7, and no calcium carbonate was added. The cultures used were members of the Escherichia and Aerobacter groups of bacteria. The medium was placed in 500-cc. florence flasks, 200 cc. to the flask. After sterilization for twenty minutes at a pressure of 15 1 JOURNAL OF BACTERIOLOGY, VOL. 32. NO. 1

2 2 DOROTHEA KLEMME AND CHARLES F. POE pounds, the medium was stored at 4VC. until used. Inoculations were made from actively growing cultures of each organism. The medium used for growing these cultures was of the same composition as that used in the flasks. All incubations were carried out at 370C. TABLE 1 Amounts of glucose found after organisms were grown in 1 per cent glucose medium (Results expressed in milligrams per 100 cc. medium) TIMEOF LWIS- SUMNER KINGSBURY DNTO FOLIN-WU BENEDICT FOLIN GROWTH BENEDICT PHENOL a. E. coli (average 7 strains) hours Original 1,070 1, ,009 1, b. E. communior (average 7 strains) Original 1,063 1, ,002 1, , c. A. aerogenes (average 7 strains) Original 1,082 1, ,009 1, , The colorimetric methods used were the Lewis-Benedict (1915) method as modified by Willaman and Davidson (1924), Benedict (1928), Folin-Wu and Folin (1929), Sumner (1925), Kingsbury (1927), and the 2:4 dinitrophenol method proposed by Poe and Edson (1932). Table la contains the average results obtained for the determination of glucose after 7 different strains of Escherichia coli

3 REDUCING SUGARS IN BACTERIAL CULTURES had been separately grown in the medium. Table lb gives similar results when 7 strains of Escherichia communior were used; and table lc lists the results for 7 strains of Aerobacter aerogenes. Duplicate determinations were run on the medium for some of. the cultures. Satisfactory checks were obtained in each case. In a number of cases, it was noted that the results obtained by certain methods were abnormally high. This fact was especially TABLE 2 Reducing substances present in peptone broth during the growth of E. coli and A. aerogenes (Results expressed in milligrams per 100 cc. medium) LEW ZI S-MNER K NGSBURY DINITRO- FOLIN-WU BENEDICT FOLIN TIME OF GROWTH BENEDICT SUNRKNSUYPHENOL E. coli (average 3 strains) hours Original A. aerogenes (average 3 strains) Original true with the Lewis-Benedict method. A series of experiments were made to determine whether or not any reducing substances were present in the original medium or were formed when the organisms were grown in the broth to which no sugar had been added. The broth used was prepared by adding 8 grams of Bacto-Nutrient broth to 1 liter of water. The results of these experiments are given in table 2. It will be noted that the Lewis-Benedict method showed 3

4 4 DOROTHEA KLEMME AND CHARLES F. POE considerable reducing substances when Escherichia coli and Aerobacter aerogenes were grown in plain broth. The Folin and Folin-Wu methods showed some reducing substances. Little or no increase in reducing substances was shown by the following methods: Sumner, Kingsbury, Benedict, and the Dinitrophenol. Apparently, enough reducing substances are originally present in the broth and are formed during the growth of the bacteria to cause some error when the Lewis-Benedict, Folin, and Folin-Wu methods are used. A point in question concerned the amount of glucose which could be recovered by analysis when different amounts of this sugar were added to the medium. The medium which originally contained 1 per cent glucose was checked by analysis with each of the colorimetric methods. After 0.5 per cent glucose had been added, similar determinations were made. Tests were also made by adding 0.5 per cent glucose to the cultures after fermentation had taken place for various lengths of time. The average results for ten determinations are given in table 3. The organisms used in determinations 1 to 5 were E. coli; in determinations 6 to 10, A. aerogenes (table 4). Magee and Smith (1930) could not recover, quantitatively, added glucose when measured with the Benedict method. The average results as given in table 3 show a fairly satisfactory recovery of glucose for all of the methods except the Folin method. The data in this table do not present the whole story. In table 4 is listed the percentage recovery for the determination with each of the ten cultures. The average percentage recovery for Benedict's method (95.8) is fairly satisfactory; however, the recovery given in the individual determinations varies greatly. For this method, there is a spread of over 32 per cent-from 83.0 to per cent. The spreads for the determinations by the other methods are not so great as in Benedict's method and are as follows: Folin 22.0 per cent; Lewis-Benedict, 19 per cent; Kingsbury, 13.2 per cent; Dinitrophenol, 7.4 per cent; Folin-Wu, 6.2 per cent; and Sumner, 5.2 per cent. The Folin-Wu, Sumner, and DimItrophenol methods, therefore, gave a satisfactory recovery of glucose.

5 REDUCING SUGARS IN BACTERIAL CULTURES 5 Check determinations in the same sample of culture medium gave fairly satisfactory results for all the methods except the TABLE 3 Recovery of added glucose (Results expressed in milligrams per 100 cc. medium*) GLUCOSE FOUND TOTAL AMOUNT THEORETICAL OF ADDED PER CENT METHOD Or* *nal After AMOUNT OF GLUCOSE RECOVERY meiurn 0.5 gram GLUCOSE RECOVERED Lewis-Benedict ,235 1, Sumner ,293 1, Kingsbury ,197 1, Dinitrophenol ,237 1, Folin-Wu ,232 1, Benedict ,200 1, Folin ,173 1, * The data are the average of the results obtained when glucose was added to the media in which ten different organisms were grown. Percentage recovery of NUMBER OF DETERMINA- TION Average... LEWIS- BENEDICT TABLE 4 added glucose from media in which ten different cultures were grown SUMNER KINGSBURY DINITRO- FOLIN-WU BENEDICT PHENOL FOLIN Benedict. In this method the two check determinations would sometimes show as much as gram difference on the basis of 0.50 gram of sugar. For the Folin and Lewis-Benedict method, I

6 6 DOROTHEA IKLEMME AND CHARLES F. POE the maximum difference was 0.03 gram; for the Kingsbury and Folin-Wu, 0.02 gram; and for the Sumner and Dinitrophenol, gram. The effect of dilution was tried on the recovery of added glucose. With each method, dilutions of 1:1, 1:2, and 1:4 were tried. The dilution within the above-mentioned limits did not affect the accuracy of the methods. All experiments up to this point were made on media which had not been clarified. A number of the cultures, especially the aerogenes cultures, produced considerable cloudiness in the broth. Several agents were used to clarify the media before analyses were made for glucose. The results obtained in the analysis of glucose in the clarified medium were compared with the amount of glucose obtained in the unclarified medium. A brief summary of the methods of clarification follows: Starch: Ordinary potato starch was washed several times with water and then dried. To 100 cc. of the diluted medium (1:10) were added 3 grams of starch. The mixture was shaken for ten minutes and then filtered. Carbon, Norite: To 100 cc. of the diluted medium was added 1 gram of norite decolorizing carbon, and this mixture was allowed to stand with frequent shaking for ten minutes. The mixture was then filtered. Fuller's Earth: Three grams of fuller's earth were added to 100 cc. of the diluted medium and allowed to stand for ten minutes with frequent shaking. The mixture was then filtered. Mercuric Nitrate: To 10 cc. of the medium was added 1 cc. of mercuric nitrate solution (10 grams of mercury and 8 cc. concentrated HNO3 were heated on a hot plate, cooled, and added to 8 cc. concentrated HNOa. To this were added 3 cc. of 5 per cent NaOH, after which the solution was made up to 100 cc. and stored in the dark). The mixture was shaken frequently during ten minutes, after which it was diluted to 100 cc. and filtered. The filtrate was made slightly alkaline to litmus with sodium bicarbonate. The mixture was filtered again. Alumina Cream: To 10 cc. of the medium were added about 50 cc. of water and 5 cc. of almina cream, and the whole mixture was diluted to Prepared according to Leach. See Food Inspection and Analysis, page 610, 4ed., 1920.

7 REDUCING SUGARS IN BACTERIAL CULTURES 7 cc. The mixture was shaken and allowed to stand ten minutes; then it was filtered. Lead Subacetate Solution: One cubic centi- TABLE 5 The effect of clarifying agents on the determination of glucose in medium in which organisms are grown (Results expressed in milligrams per 100 cc. medium) Mto~~~~~~ 4 5 ~~~~~~00 0 ~~~ e E 4z d X. F 0 ~~~~ a. aeo. e (aerg of 3 stais 0 ~ ~ 0 a. A. aerogenes (average of 3 strains) Lewis-Benedict Sumner Kingsbury Dinitrophenol Folin-Wu Benedict Folin b. E. coli (average of 3 strains) Lewis-Benedict Sumner Kingsbury Dinitrophenol Folin-Wu Benedict Folin c. E. communior (average of 3 strains) Lewis-Benedict Sumner Kingsbury Dinitrophenol Folin-Wu Benedict Folin meter of 30 per cent solution of Horn's anhydrous lead subacetate was added to 10 cc. of medium and diluted to 100 cc. The solution was allowed to stand ten minutes and was then filtered.

8 8 DOROTHEA KLEMME AND CHARLES F. POE The filtrate, after being treated with anhydrous sodium carbonate until no more lead precipitated, was filtered. Dry Lead Subacetate: To 100 cc. of the diluted medium were added 3 grams of Horn's anhydrous lead subacetate. The mixture was freed from the excess lead with sodium carbonate. The results obtained after using the different above-mentioned clarifying agents are given in table 5. The results in table 5 show that starch, fuller's earth, norite carbon, and lead subacetate are satisfactory as clarifying agents for bacteriological media. A solution of mercuric nitrate when used to clarify the media causes a very low percentage of glucose to be recovered. It was not satisfactory with any of the colorimetric methods. Alumina cream proved to be a satisfactory clarifying agent in all of the methods except the Lewis-Benedict. A study of table 5 together with the previous tables indicates that the Sumner, the Folin-Wu, and the Dinitrophenol methods are the most accurate ones for the determination of residual sugar in bacterial cultures. The Lewis-Benedict method gives results which are too high. In some cases the Kingsbury and the Benedict methods give results slightly too low. The recovery of added glucose with the Lewis-Benedict, the Kingsbury, the Benedict, and the Folin methods is not satisfactory. A number of the clarifying agents are suited for use when the calorimetric methods are to be used for determining the sugar content. All of the agents tried except mercuric nitrate and alumina cream are satisfactory with all of the methods. Apparently, however, accurate results may be obtained without clarification. SUMMARY 1. Seven colorimetric methods have been investigated for use in determining reducing sugars in bacterial culture-media. 2. The accuracy of the sugar determination, when clarifying agents are used, has been investigated. REFERENCES BENEDICT, S. R Jour. Biol. Chem., 68, 759. BENEDICT, S. R Jour. Biol. Chem., 76, 457. FOLIN, Jour. Biol. Chem., 82, 83.

9 REDUCING SUGARS IN BACTERIAL CULTURES 9 KINGSBURY, F. B Jour. Biol. Chem., 75, 241. LEWIS, R. C., AND BENEDICT, S. R Jour. Biol. Chem., 20,61. MAGEE, M. C., AND SMITH, H. G Jour. Bact., 19, 125. POE, C. F., AND EDSON, F. G Jour. Ind. and Eng. Chem., Anal. Ed., 4, 300. SHAFFER, P. A., AND HARTMANN, A. E Jour. Biol. Chem., 45, 349, 365. STILES, H. R., PETERSON, W. H., AND FRED, E. B Jour. Bact., 12, 427. SUMNER, J. B Jour. Biol. Chem., 65, 393. WILLAMAN, J. J., AND DAVIDSON, F. R J. Agric. Research, 28, 479. Downloaded from on September 19, 2018 by guest