DETERMINATION OF CARBOHYDRATES IN BACTERI-OLOGICAL CULTURE MEDIA

CLARENCE F. SCHMIDT, JR.Department of Bacteriology, School of Medicine and Dentistry, University of

Rochester, Rochester, New York

Received for publication, January 28, 1931

I. APPLICATION OF THE FERRICYANIDE REDUCTION METHOD TOQUANTITATIVE DETERMINATION OF GLUCOSE IN PEPTONE WATER

IntroductionIn many quantitative studies of the carbohydrate metabolism

of bacteria, it is more convenient to use small samples than largeones for analysis. On account of this fact, a reliable and accuratemicro-method is more serviceable than a macro-method. At theoutset of an investigation of glucose utilization by bacteria itwas found unexpectedly that the micro-method in common use,based upon the reduction of copper, was subject to errors whichhave not been adequately considered by many who have em-ployed this method. It was necessary, therefore, to reinvestigatemicro-methods for the determination of glucose. The resultsof these studies, and of the application of a ferricyanide reductionmethod for the determination of glucose in peptone water and inbacterial cultures will be recorded in this communication.Among the micro-methods, the volumetric copper reduction

method of Shaffer and Hartmann (1920-21) has been used mostfrequently in bacteriological work for the determination of reduc-ing sugars. Stiles, Peterson and Fred (1926) recommend theuse of the micro-reagent of Shaffer and Hartmann, and presenta table extending from 0.067 to 2.078 mgm., by which glucosemay be determined from the difference in titration values ob-tained. The authors recommend the use of basic lead acetate toprecipitate the protein, using disodium phosphate to remove the

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excess lead. Satisfactory results were obtained in some casesdirectly upon the medium without precipitation of the protein.As a test of the accuracy of the method the recovery of glucoseadded to media was determined. The authors report "theagreement between the calculated and observed values variedbetween 0.01 and 0.04 per cent by the macro method. Recoveryby the micro method was fully as good or better than by themacro method." By this is meant that when 1 per cent or 10mgm. per cubic centimeter of glucose were added the glucosefound was 0.99 per cent or 0.96 per cent (personal communica-tion). However, such a deviation at a concentration of 1 per centor 10 mgm. per cubic centimeter means an error of 1 to 4 per cent,while the same deviation at a concentration of 0.5 per cent or 5mgm. of glucose per cubic centimeter means an error of 2 to 8per cent in the recovery of added glucose.Magee and Smith (1930) using the Shaffer and Hartmann

method for the determination of glucose in a meat infusion broth,report that an accuracy greater than :i:10 per cent cannot beobtained. The most accurate results were obtained directlyupon the medium without precipitation of the protein.

Merrill (1930) reports the use of the Shaffer and Hartmannmethod as recommended by Stiles, Peterson and Fred. Theauthor says (p. 265).

Despite the fact that in making the media the glucose was accuratelyweighed and added in proportion of 1 grammade up to 100 cc. in volumet-ric flasks, values ranging from 0.95 to 1.06 per cent glucose wereobtained by the Shaffer-Hartmann method. It should also be notedthat the same reagents were used throughout these determinations, asufficient quantity being prepared in the beginning for all tests. Thus,the maximum variation between the controls was approximately 10 percent. It seems justifiable to consider this the normal limit of variationof the carbohydrate determination.

Somogyi (1926) reporting a study of various copper reagentsfound that the reaction of the reagent is of extreme importance.He reports, "comparatively small changes in alkalinity produceconsiderable differences in reduction values" (p. 601). The

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changes in alkalinity are considered as affecting the breakdownof the glucose molecule into reactive fragments, rather thanaffecting the oxidizing power of the copper complex. He alsocalls attention to the danger of reoxidation of the cuprous oxideduring the heating period. The reagent recommended is stronglybuffered with a mixture of Na2CO3 and NaHCO3.The results I obtained in attempts to use the copper reduction

micro-method of Stiles, Peterson and Fred (1926) were highlyunsatisfactory. To summarize briefly the results obtained; noreagent could be prepared which would give 100 per cent recoveryof glucose from aqueous solution when the table of Stiles, Petersonand Fred was used to compute results; the recovery of glucoseadded to media was variable at different times with the samereagent and varied with different reagents prepared at differenttimes. Even upon solutions of pure glucose in distilled water,the determinations were subject to considerable variation. Thesevariations could not be controlled by any means. The valuesobtained were closer to theoretical and showed less variation withsmaller amounts of glucose (0.5 mgm. as compared to 1.0 mgm.).For a given concentration of glucose the values obtained showedless variation with a heating time of five minutes than with fifteenminutes. The variations, therefore, have been attributed tothe effect of reoxidation of the cuprous oxide, during either theheating or cooling period.

In view of the complexity of the copper reagents, the knownsusceptibility of cuprous oxide to reoxidation and the irregularresults obtained by various authors, it would seem advisable toreplace the copper reduction method by one not possessing thesedisadvantages. The reduction of ferricyanide by sugars has oftenbeen reported in the literature, but until the present time, as faras is known to me, has not been used for the determination ofreducing sugars in bacteriological media, with the exception ofone article to which reference will be made later.The Hagedorn-Jensen (1923a) ferricyanide method for the

determination of blood sugar has been recommended by manyworkers. The principle of the method has been used by otherswho have extended the range, since the upper limit of the original

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method is 0.385 mgm. Von Issekutz and von Both (1927) havemodified the original method to cover a range from 0.725 to J4.99mgm. of glucose. Bryant (1929) has modified the method forblood sugar determinations. Hanes (1929) has extended therange from 0,2 to 3.8 mgm. Sobotka and Reiner (1930) reportsatisfactory results with the method of Hanes. Gohr (1930) hassuccessfully applied the reduction of ferricyanide to the determi-nation of lactose in milk.The publication of Cianci (1929) did not come to my attention

until the data upon which the present paper is based had beenpractically completed. Cianci has applied the method of vonIssekutz and von Both to the determination of glucose in anammonium tartrate synthetic medium and in a peptone medium.The reduction of ferricyanide by bacterial bodies and by peptonewas noted. The author believes that the determination of acorrection factor for these reducing values gives more satisfactoryresults than any method attempting precipitation. AlthoughCianci is not clear as to the method of determining the correctionfactor and does not give the accuracy of the method, he is thefirst to publish the principle of the use of a correction factor forthe purpose of obtaining greater accuracy in sugar determinationsupon bacteriological media. The results upon synthetic mediawere extremely accurate being within 0.4 to 0.6 per cent oftheoretical.A brief review of the chemical principles upon which the method

is based may be of value. Glucose, or another reducing sub-stance, is heated with potassium ferricyanide in alkaline solution,the reduced ferrocyanide is precipitated by zinc sulphate in acidsolution, while an amount of iodine equivalent to the remainingferricyanide is liberated from potassium iodide and titrated withsodium thiosulphate.

(1) K, [Fe(Cn)sJ + glucose-.K4 (Fe(Cn)g] + oxidation products(2) H4 [Fe(Cn)o] + 2ZnSO4-*Zn2 [Fe(Cn)a] + 2H2,04(3) -21, [Fe(Cn).l + 2HI-*2H4 [Fe(Cn)6I +.I2(4) 2Na2S2,O + I1--Na2,4O, + 2NaI

The reaction is carried to completion in all cases by the presenceof the zinc sulphate which removes the ferrocyanide. A blank

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without glucose is used to determine the amount of ferricyanideoriginally present. It is most convenient to express all values interms of cubic centimeters of sodium thiosulphate. By subtract-ing the amount of thiosulphate solution required for the samplefrom that required for the blank, the number of cubic centimetersof thiosulphate equivalent to the ferricyanide reduced is obtained.Reference to a curve or table prepared from pure glucose solutionswill give the amount of glucose or of reducing substance expressedas glucose, present in the sample.

II. THE FERRICYANIDE REDUCTION METHOD

To permit the use of a 50 cc. burette for titration it was neces-sary to employ less concentrated reagents than those recom-mended by Hanes (1929). The thiosulphatewas reduced to 0.0050normal solution and the concentration of the ferricyanide reagentadjusted so that large volumes of the thiosulphate would not berequired for titration.The modified reagents necessary for the determination are as

follows:1. K3 [Fe(Cn)6], 4.2 grams; Na2CO3 (anhydrous), 10.6 grams.

Dilute to 1000 cc. in a volumetric flask, using distilled water. Ifthe solution is allowed to stand a week before using, the blankdeterminations will be constant, and will remain constant forseveral months. This solution should be stored in a brown glassbottle as it is affected by light.

2. KI, 25 grams; ZnSO4, 50 grams; NaCl, 250 grams. Dilutein a volumetric flask to 1 liter with distilled water. Allow thesolution to stand a few days then filter. This solution must beprotected from light.

3. Five per cent acetic acid made by dilution of glacial aceticacid by volume.

4. Starch indicator: Dissolve 1 gram of soluble starch in 100cc. of hot water. Add 20 grams of NaCl. This solution will keepindefinitely even though exposed to air.

5. Sodium thiosulphate: A tenth normal stock solution isprepared and kept on hand to be diluted for use whenever deter-iinations are to be made. In making the stock solution the

JOURNAL OF BACTERIOLOGY, VOL. 3XII, NO. 1

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addition of 0.1 to 0.2 gram of NaOH per liter of solution will aid inmaintaliing a constant normal factor over a long period of time.The stock solution should be standardized against a solution ofKI03 or K2Cr2O7. A convenient method of standardization isgiven by Hanes (1929). It is most convenient to adjust thestock solution to exactly 0.1000 normal, after which a dilution of25 cc. to 500 or 50 cc. to 1000 will give an exactly 0.0050 normalsolution. It has been found that more satisfactory results areobtained by titrating with a solution exactly 0.0050 normal thanby titrating with one slightly stronger or weaker and convertingto 0.0050 normal by use of the normal factor. It should beemphasized that the normal factor should be used for conversionif the thiosulphate varies by 0.0001 from 0.0050 since such adifference is sufficient to cause an error of about 2 per cent. Afterdilution the thiosulphate should be checked by titration againsta suitable standard. It is well to keep two standard solutionsfor titration with the dilute thiosulphate and to check against eachevery few days.The procedure for the determination is as follows: 1 cc. of the

sample containing between 0.2 and 2 mgm. of glucose is measuredinto a 200 by 25 mm. Pyrex tube. Five cubic centimeters of theferricyanide reagent are added followed by 4 cc. of distilled water,which is used to wash down the sides of the tube. The tubes arestoppered and heated for fifteen minutes in a vigorously boilingwater bath, after which time they are removed and cooled byimmersion in a beaker of cold water. Five cubic centimeters ofthe KI-ZnSO4-NaCl reagent are added followed by 5 cc. of 5 percent acetic acid and the liberated iodine is titrated immediatelywith 0.0050 normal sodium thiosulphate. The starch indicator(3 to 4 drops) is added when a faintly yellow color is still apparent,and the titration is continued to the disappearance of the bluecolor. The endpoint occurs as sharply as in any procedure whichuses the starch-iodine titration although it may be seen a littlebetter by diffuse daylight or artificial light than by direct sunlight.Due to the presence of the precipitate in the tubes it is necessaryto shake them quite vigorously during the titration.The stoppering of the tubes is not necessary to prevent re-

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oxidation, but serves to prevent the entrance of water dropletsduring boiling. The time of cooling has no appreciable effectupon the values obtained, a fact previously observed by Bryant(1929).The blank determination is carried out in the samemanner upon

5 cc. of the ferricyanide reagent plus 5 cc. of water. For anygiven set of determinations the same pipette should be used formeasuring out the ferricyanide reagent. It seems needless tosay that all volumetric glassware, flasks for dilution, pipettesand burette, should be of suitable accuracy for quantitative work.The amount of glucose present in the sample is obtained by

subtracting the cubic centimeters of thiosulphate used for thetitration of the sample from the cubic centimeters of the thiosul-phate required for the blank and by reference to table 1 preparedfrom pure glucose solutions. This table of glucose equivalentsis presented after considerable hesitation in view of the experiencewith tables prepared for copper reagents. The table originallyprepared from more than 100 closely checking determinationshas been repeatedly checked since then in the course of about ayear's work, with many different preparations of the ferricyanidereagent and different samples of glucose. Although the blankdeterminations of two different reagents may differ by as muchas 0.6 to 0.8 cc., the difference between titration values of blankand sample was always found to be the same.The glucose used in the preparation of the table and in all

subsequent work where a desired amount of glucose was added tomedia, was Pfanstiehl c.p. glucose which was kept over sulphuricacid in a vacuum desiccator for two weeks or longer before use.This fact must be recognized,-that the table is based uponanhydrous glucose,-since if glucose containing any moisture isused in making media, the results obtained by the determinationof glucose upon a sample of the medium will indicate less than100 per cent recovery of the added glucose. The use of anhydrousglucose gives an accurate standard to which other values may becompared by the reduction method.As an illustration of the accuracy of the method upon pure

glucose solutions the following example may be cited, in which

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TABLE 1Gluco8e-thioasulphate equivalents

Milligrams glucose equivalent to cubic centimeter thiosulphate difference intitration between sample and blank.

THIOSULPHATE GLUCOSE THIOSULPHATE GLUCOSE TIOSULPHAT] GLUCOSE

cc.

1.01.11.21.31.41.51.61.71.81.92.02.12.22.32.42.52.62.72.82.93.03.13.23.33.43.53.63.73.83.94.04.14.24.34.44.5

mgm.0.1810.1990.2170.2350.2530.2710.2890.3070.3250.3430.3610.3790.3970.4150.4330.4510.4690.4870.5050.5240.5430.5610.5790.5970.6160.6340.6520.6700.6880.7060.7240.7420.7600.7780.7960.814

cc.

4.64.74.84.95.05.15.25.35.45.55.65.75.85.96.06.16.26.36.46.56.66.76.86.97.07.17.27.37.47.57.67.77.87.98.08.1

mgm.0.8320.8500.8680.8860.9040.9220.9400.9590.9770.9961.0141.0321.0501.0681.0861.1041.1221.1401.1581.1761.1941.2121.2301.2491.2671.2851.3031.3211.3391.3571.3751.3931.411.1.4291.4481.466

cc.

8.28.38.48.58.68.78.88.99.09.19.29.39.49.59.69.79.89.910.010.110.210.310.410.510.610.710.810.911.011.111.211.311.411.511.6

mWm.1.4841.5021.5201.5381.5561.5751.5931.6111.6291.6471.6651.6831.7011.7191.7371.7561.7741.7921.8101.8281.8461.8641.8821.9001.9181.9361.9541.9721.9912.0092.0272.0452.0632.0812.099

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a reagent four months old was used. One cubic centimeter of aglucose solution containing 15.15 mgm. of glucose per cubiccentimeter was diluted 1:10 in a volumetric flask. Glucose wasdetermined upon duplicate 1 cc. samples of the dilute solutiongiving 1.502 and 1.511 mgm., average 1.506 mgm., difference fromtheoretical 0.009 mgm. or 0.6 per cent. A second dilution of theoriginal sample gave 1.511 and 1.511 mgm.-difference fromtheoretical, 0.004 mgm. The variation between the averages ofthe two samples is less than 0.3 per cent. It is evident that themethod shows a satisfactory accuracy upon aqueous solutions ofglucose.

III. THE USE OF THE FERRICYANIDE METHOD FOR THE DETERMINA-TION OF GLUCOSE IN PEPTONE WATER

The peptone water used in these experiments was in all casesa 2 per cent aqueous solution of Bacto-peptone plus 0.5 per centNaCl, sterilized by autoclaving at 15 pounds for fifteen minutes.Most of the samples were prepared in the usual manner in themedia kitchen.

Since the method was designed for the determination of glucoseupon 1 cc. quantities of a 1: 10 dilution from an original sampleof 1 cc., the dilutions in experimental work were made in thefollowing manner: Stock solutions of glucose were prepared byaccurately weighing the desired amount and diluting to volumewith 0.5 per cent benzoic acid. It was necessary to add benzoicacid to prevent the growth of molds in the glucose solution keptin the icebox. The benzoic acid had no effect upon the reductionof ferricyanide. One cubic centimeter of the stock solution waspipetted into a 10 cc. volumetric flask, followed by 1 cc. of thepeptone water under investigation. If a precipitant was usedthis was added, then after dilution to volume and mixing, 1 cc.samples were removed for the determination of reducing sub-stance. This procedure provided a 1:10 dilution of the peptonewater containing an amount of glucose dependent upon the con-centration of the stock solution of glucose.The accurate recovery of added amounts of glucose immediately

presented difficulties. In all cases the determinations resulted in

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more than 100 per cent recovery of glucose. The per cent re-covery based upon the amount of glucose added increased as theactual amount of glucose present decreased. This pointed tothe fact that the reduction by peptone was constant. A study ofthe reduction of the ferricyanide reagent by 1 cc. samples ofpeptone water showed that the amount of reduction was equiva-lent to 0.7 to 0.8 mgm. of glucose. If this reduction wereneglected it would mean an error of 7 to 8 per cent on a 10 mgm.sample or 14 to 16 per cent on a 5 mgm. sample. Various precip-itants among which may be mentioned tungstic acid, basiclead acetate and zinc hydroxide, were used in attempts to removethe reducing substance, but in no case could this be done.On the other hand, it was found that the use of a precipitating

agent was of value in the determination of glucose in cultures.Although none of the reducing substance is removed, the valuesupon individual determinations are slightly more constant afterprecipitation, and when the Somogyi (1930) zinc hydroxideprecipitation is used filtrates from quite acid cultures are approxi-mately neutral. Two other advantages of precipitation are theremoval of bacterial substances which may exert an appreciablereducing action, and the fact that samples removed from a cultureat intervals of time and imediately precipitated may be allowedto accumulate before a series of determinations are made. Afterprecipitation of cultures no change in the reduction values takesplace for at least twenty-four hours.The method of precipitation used (except in the preliminary

study of precipitating agents) and recommended is that ofSomogyi (1930). The reagents and procedure are as follows:

(1) 10 per cent ZnSO4-7HsO(2) 0.5 per cent NaOH

The NaOH is so adjusted that 10 cc. of the zinc sulphate solu-tion require from 10.8 to 11.2 of NaOH to produce a permanentpink color with phenolphthalein, the zinc hydroxide being titratedslowly with continuous shaking in the presence of 50 to 70 cc. ofwater.For precipitation, 1 cc. of the sample (either sterile glucose-

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peptone water or culture) is placed in a 10 cc. volumetric flaskfollowed by 1 cc. of the zinc sulphate and 1 cc. of the sodiumhydroxide. After dilution and mixing the sample-may be filteredthrough a Whatman No. 1 filter paper into a small tube. Nosubstances reducing ferricyanide were removed during the courseof filtration.The determination of the reducing substance of peptone could

not be made upon 1.0 cc. samples of a 1:10 dilution because ofthe following phenomena. Although, theoretically, the reductionof 1 cc. samples of a 1:10 dilution should be one-tenth that of 1cc. samples of the original peptone this is not the case. When the

TABLE 2The effect of dilution upon the reducing action of peptone water

MILLIGRAMS AS

TREATMENT GLUCOSE, AVERAGZEXTREMES 4 AVRGDETERMINATIONS

Sl.. ...... l Diluted 1: 10 1.020-1.036 1.030

Undiluted 0.846-0.882 0.864Diluted 1:10 1.440-1.530 1.51

Sample 2 Diluted 1:10 precipi- 1.44 1.44tated tungstic acid

Diluted 1:10 precipi- 1.260-1.620 1.44tated Zn(OH)2

values obtained upon 1:10 dilutions are calculated to the basis ofreducing power per cubic centimeter of original peptone water,the result obtained is about 50 per cent greater than that obtainedupon undiluted peptone water. This is shown in table 2, thevalues of the 1:10 dilution being calculated to the basis of undi-luted peptone water for comparison. It will be noted that thevariations between individual determinations are much greaterthan those obtained upon glucose solutions. The values obtainedupon the undiluted peptone water correspond closely to thoseobtained by subtracting the added amount of glucose from thetotal reducing power as is done in the determination of the reduc-ing constant which will be explained below. Although no attempt

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42 CLARENCE F. SCHMIT, JR.

is made to explain this phenomenon it should be noted that withthe reagents used the values below 0.2 mgm. are more or lessunsatisfactory from the standpoint of accuracy, although withglucose solutions the values obtained tend to be lower ratherthan higher than the theoretical values. The question of thenature of the reducing substance will be considered in thediscussion.In spite of these difficulties it has been found possible to de-

termine glucose in peptone water with what appears to be a

TABLE 3

Reducing constant of peptone waterDetermination of rk of 2 per cent peptone water and the recovery of added

glucose by the use of the average rk.PER CENT

TOTAL REDUCTION rk AVERAGE RECOVERY OFGLUCOSE TOTAL REDUC- REDUCTION ADDED GLUCOSEADDED TION MINUS CORRECTED FOR USING COR-

DupUcats Avenge AADDED GLUCOSE AVERAGE rk RCTED AVERAGEIDuplicatesAverage ~~~~REDUCTION

mgm. mgm/cc.I 5.88- 5.61 5.75 0.75 5.05 101.0

5s.o 5.79- 5.70 5.75 0.75 5.05 101.05.70- 5.52 5.61 0.61 4.91 98.25.79- 5.79 5.79 0.79 5.09 101.8

15.93-15.66 15.79 0.64 15.09 99.615.15 15.83-15.75 15.79 0.64 15.09 99.6

15.83-15.83 15.83 0.68 15.13 99.8

Average.0.70

satisfactory accuracy. This is made possible by the fact that thereducing power of a given sample of peptone water is constant.It is possible then to determine this constant reduction, forwhich the name reducing constant or rk is suggested. By thesubtraction of the rk from the total reducing substance found inthe medium after glucose is added or after growth has occurred,the amount of glucose is obtained.Since this value must be determined upon the sterile medium

before glucose is added the most convenient method for the prepa-ration of a glucose-peptone water medium seems to be as follows:

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the medium is prepared and sterilized in the usual manner, the rkis determined in the manner to be described, then using a 20to 30 per cent solution of glucose sterilized by filtration the desiredamount of glucose is added. A determination of the total reduc-ing substance is made, the rk previously determined is subtracted,and the amount of glucose present is accurately known. Themedim may be adjusted to any desired concentration by this

TABLE 4

The accuracy of recovery of added glucose using a previously determined rk

TOTALRED'UTION, AVERAGE PER CENTPEPTNZSMPLZ GLUCOSE TOTx RzEDUCToN4 AVER- COR- RECOVERYPE]l9PTONE SAMPLE |iADDED DEXTZREIE OF 4 AGE RECTED COB-

DETERMINATIONS FOR rk RLECTED

mgm.

A Not precipitated, rk 10.03 10.59-10.83 10.65 10.05 100.1= 0.6 10.68-10.77 10.70 10.10 100.6

10.50-10.83 10.71 10.11 100.710.68-10.91 10.81 10.21 101.7

B Not precipitated, rk 8.09 8.69- 8.96 8.85 8.15 100.7= 0.7 8.69- 8.88 8.74 8.04 99.3

Precipitated, Zn(OH)2, 8.69- 8.88 8.74 8.04 99.3rk = 0.7 8.51- 8.88 8.79 8.09 100.0

C Not precipitated, rk 8.09 8.78- 8.96 8.91 8.11 100.2= 0.8 8.87- 8.96 8.94 8.14 100.6

5.02 5.79- 5.88 5.83 5.03 100.25.88- 5.88 5.88 5.08 101.2

Precipitated, Zn (OH)2, 10.23 10.86-11.13 10.97 10.17 99.4rk = 0.8 10.86-11.04 10.96 10.16 99.3

10.00 11.86-10.95 10.86 10.06 100.610.77-10.95 10.86 10.06 100.6

method. It is better to add a slight excess of glucose above thedesired amount, and after determining how much is present diluteto the desired concentration with sterile peptone water havingthe same rk. The medium may then be distributed aseptically tosuitable containers. Needless to say, the medium must be care-fully guarded against changes in concentration due to evapora-tion. This procedure is not as laborious as it appears and seemsto be the only way in which it is possible to make accurate glucose

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determinations until some method is found for the precipitationof the reducing substances.The determination of the rk of peptone water is made in the

following manner: 1 cc. of a glucose solution of known concentra-tion is placed in a 10 cc. volumetric flask, 1 cc. of the peptonewater is added and precipitation carried out in the manneroutlined above. The total reducing substance is determinedupon 1 cc. quantities of the filtrate. Subtracting the milligrams

TABLE 5

The effect of the growth of various bacteria upon the non-precipitable reducing sub-stance in peptone water. The recovery of glucose added after growth

PER CENTTOTAL AVERAGE RECOVERY

CULTUREC GLUCOSE TOTAL REDUCTION, REDUC- COR- USINGrk - 0.7 TUBE ADDED DUPLICATES TION, RECTED COR-

AVERAGE FOR rk RECTEDAVERAGE

mor.

B. subtilis f 1 10.00 10.68-10.59 10.63 9.93 99.3..~sutt....... 2 10.00 10.86-10.86 10.86 10.16 101.6

f 1 10.00 10.68-10.68 10.68 9.98 99.8Staph. aureus. '~. . . . . 2 10.00 10.68-10.86 10.77 10.07 100.7

f 1 10.00 10.68-10.95 10.81 10.11 101.1B. aerogenes.{.....l 2 10.00 10.59-10.68 10.63 9.93 99.3

TOTAL REDUCTION,rk - 0.8 EXTREMES OF 4

DETERMINATIONS

01 8.09 8.78- 8.96 8.91 8.11 100.2

B .c 2 8.09 8.78- 8.87 8.85 8.05 99.53 15.15 16.11-16.29 16.20 15.40 101.64 15.15 15.85-16.02 15.95 15.15 10 . O

of glucose added leaves the remainder as the reduction due topeptone expressed in terms of glucose. Several determinationsshould be made and averaged. The effect of the quantity ofglucose added is negligible and may be disregarded when thetotal error of the method i 2 per cent is accepted.The values given in table 3 in milligrams per cubic centimeter

refer to the calculated values of the original sample; the actualdeterminations were one-tenth of these values.

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A large number of determinations have been made to studythe recovery of added glucose from peptone water using the rkcorrection which had previously been determined on the sample.Table 4 gives a condensed suimmary of representative dataobtained by this procedure, showing in all cases that the amountof glucose present in 2 per cent peptone water may be determinedwith an extreme error of 42 per cent between the concentrationsof 5 and 15 mgm. The recovery from 15 to 20 mgm., the upperlimit of the method, is as good. Below 5 mgm. it would seemadvisable to take a 2 cc. sample of the filtrate and after subtrac-tion of twice the reducing constant calculate to the basis of 1 cc.of original sample. Further work is contemplated dealing withthe lower region of the method.A further step necessary to complete the method was to show

that during growth the reducing substance of peptone waterdid not change. Table 5 shows that during the growth of theorganisms used neither an increase nor a decrease of the rk tookplace. These data were obtained by determining the rk in theusual manner, then inoculating with the organisms concernedand determining the rk again after incubation. In all cases therecovery of glucose added after growth and corrected by the rkdetermined upon the sterile medium was within i2 per cent,the accepted limit of the method. The data upon B. coli (table 5)were compiled from work done at different times. The data uponthe other organisms (table 5) were obtained after four days ofincubation. Determinations made, using several members ofthe paratyphoid group, gave the same results.The method has been used successfully in the determination of

the utilization of glucose by various species of bacteria in culturesin peptone water. The results of this study will be reported ina separate communication.

DISCUSSION

In view of the data presented, it is evident that the accurate.determination of glucose cannot be made without the use of acorrection factor for the reducing power of the medium. Whenthis factor is taken into consideration and the method as outlined

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above is used, it is possible to determine glucose within the limitsof -2 per cent upon bacterial cultures in a glucose peptonemedium.Attempts to apply the method to Douglas broth and media

containing meat extract have thus far been unsuccessful becauseaccurate correction factors could not be determined due to thefact that a part of the nonprecipitable reducing substance wasutilized during the growth of the organisms. This factor doesnot enter into the work carried out upon peptone water since ithas been demonstrated that the organisms used neither increasednor decreased the reducing substance in peptone water duringtwenty-four hours or longer of growth. The rk as determinedupon a given sample before and after growth of these organisms,was constant within the limits of error of the method.The nature of the substance or substances present in peptone

water which reduce the ferricyanide reagent, is unknown. At-tempts to isolate and concentrate this substance were unsuccess-ful. The fact that the Molisch reaction was always positive infiltrates which gave reduction, indicates the possibility of thecarbohydrate-like nature of this substance. On the other hand,it is well known that the Molisch reaction is not specific forcarbohydrates and the fact that the organisms used, in theaggregate having a great diversity of fermentative properties,did not appreciably attack the reducing substance indicatesthat it is not of a carbohydrate nature.Furthermore, it is well known that the ferricyanide reagent is

not specific for carbohydrates but is reduced by many othersubstances. Hagedorn and Jensen (1923b) reported that theirreagent was reduced by uric acid and creatinine but not by eitheracetone or ,-oxybutyric acid. Holden (1926) reported that uricacid, creatinine and cystine reduced the ferricyanide reagentwhile glutamic acid and glycine did not. No other substanceswere tried. Flatow (1928) reported the reduction of ferricyanideby uric acid, glutathione and thiasine. It is entirely possiblethat some of these substances may be present in the commercialproducts known as "peptone." However, the presence of none

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CARBOHYDRATES IN CULTURE MEDIA 47

could be conclusively demonstrated although it is highly probablethat some free amino acids are present.

SUIMMARY

A method is presented for the determination of glucose in pep-tone water, and bacterial cultures in this medium. This methodis based upon the reduction of ferricyanide and involves the useof a correction factor for the reducing action of the peptone. Ithas been shown that the method possesses an accuracy of 42per cent.

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BENEDICT, S. R. 1926b The determination of blood sugar. II. Jour. Biol.Chem., 76, 457.

BRYANT, H. L. 1929 A potassium ferricyanide method for the determination ofreducing substances in blood. Jour. Lab. and Clin. Med., 14, 1082.

CIANCI, V. 1929 Dosaggio del Glucosio Nelle Culture di Germi. Boll. dellaSocieta Italiana di Biologia Sperimentale, 4, 1136.

FLATOW, L. 1928 tJber ferricyanometrische Mikromethoden in der Blutanalyse.Biochem. Zeit., 194, 132.

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VON ISSEKUTZ, B., AND VON BOTH, J. 1927 Einfache Methode zur Bestimmungder Glucose in Mengen von 1 bis 15 mg. Biochem. Zeit., 183, 298.

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SHAFFER, P. A., AND HARTMANN, A. F. 1920-21 The iodometric determinationof copper and its use in sugar analysis. II. Methods for the determina-tion of reducing sugars in blood, urine, milk and other solutions. Jour.Biol. Chem., 45, 365.

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SOBOTKA, H., AND REINER, M. 1930 The Hagedorn-Jensen method applied tovarious sugars. Relation of reducing power to configuration. Bio-chem. Jour., 24, 394.

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