fluorescent, · trodes were inserted through openings madein the shoulder of the flasks. these...

THE METHOD OF ELECTRICAL CONDUCTIVITY INSTUDIES ON BACTERIAL METABOLISM'

JAMES B. ALLISON, JOHN A. ANDERSON, AND WILLIAM H. COLE

Bureau of Biological Research, Rutgers University2

Received for publication April 14, 1938

The electrical conductivity of solutions depends upon the con-centrations and the mobilities of charged particles of varyingsizes. A culture medium in which bacteria are living and dyingis a heterogeneous system, the composition and complexity ofwhich are constantly changing. Substances in true and incolloidal solution are being chemically disintegrated and syn-thesized. The initial and final compositions may be well-known,but the intermediate reactions and the mechanisms involvedin many cases are not understood. Several difficult problemsare therefore presented in attempting to evaluate conductivitymeasurements in relation to bacterial metabolism. Parsonsand Sturges (1926a, b) have reported a definite correlation be-tween conductivity and the amount of ammonia and aminonitrogen produced by putrefactive anaerobes. This is the onlydirect relationship between conductivity and some product ofmetabolism which has been established so far. Others haveshown, however, (Sierakowski and LQczyeka, 1933 and papersquoted therein) that there is always some relationship betweenchange in electrical conductivity and metabolic processes eventhough the direct cause for the alteration in electrical fieldstrength may be obscure. Further information on such cor-relations has been sought. Experiments have been done, there-fore, with two different aerobic bacteria which produce ammonia,and one which produces lactic acid. It is believed that the data

1 Read before the New York City Branch of the Society of American Bac-teriologists, May 11, 1937.

2 The writers are indebted to Messrs. Darwin Vexler, Arthur E. Orloff, andOscar Beder for assistance in determinations and in many other ways.

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J. B. ALLISON, J. A. ANDERSON AND W. H. COLE

presented show the value of conductance measurements indescribing, identifying, and interpreting the intermediary metab-olism of these organisms.

DESCRIPTION OF ORGANISMS

One of the ammonia-forming organisms was Pseudomonasfluorescent, recently isolated from milk. It conformed in allrespects to the type description in Bergey's Manual of Deter-minative Bacteriology.The other ammonia-forming organism was a lipolytic bacte-

rium isolated from rancid cream by Anderson (Anderson andHardenbergh, 1932). Since this is undoubtedly a new organismthe name Achromobacter lipidis, N. Sp., is proposed because ofits pronounced fat-splitting ability. The organism is a gram-negative, non-spore-forming rod, about 0.4 by 1.6 microns, oc-curing singly and in pairs. It appears to be non-motile or verysluggishly motile. Flagella have not been detected. Agarcolonies are about four or five millimeters in diameter afterforty-eight hours of incubation at 25-280C., after which theyslowly increase in size for several days. These colonies arecircular with an entire edge, smooth, glossy, moist, and finelygranular. With age they become coarsely granular and developradial striations which result in a more or less undulate margin.Young colonies are white and beautifully opalescent. They donot darken with age. The agar slant growth is abundant,glistening and echinulate. Agar stab cultures produce anabundant surface growth with a delicate subsurface growth,diminishing rapidly with depth. In broth the upper portionof the tube first becomes turbid and a slight surface film devel-ops; later the turbidity spreads throughout the entire tube anda characteristic slimy white precipitate accumulates. An al-kaline reaction due to ammonia is produced in broth. Anabundant moist white growth occurs on potato slants. Gelatinis not liquified by freshly isolated cultures, but cultures whichhave been grown on meat extract agar for some months maycause a slight liquification of gelatin in ten days. No indol isproduced in Dunham's solution. Nitrates are readily reduced

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CONDUCTIVITY IN METABOLISM STUDIES

to nitrites. The organism is incapable of fermenting glycerol,glucose, galactose, fructose, sucrose, lactose, maltose, raffinose,inulin, starch, dulcitol, and mannitol. Litmus milk is slowlyreduced and then becomes alkaline, commencing at the surface.The alkaline reaction is due, in part at least, to ammonia pro-duction. Colonies on blood agar plates are not larger than onplain agar and no hemolysis of blood cells or change in hemo-globin occurs. The outstanding characteristic of this organismis its marked lipolytic action, even at low temperatures. Freshlyisolated cultures grow best at 28-29oC., fail to grow above34-350C., and show a growth after several days at 2-4oC.The lactic-acid forming organism studied was Lactobacillus

odontolyticus obtained from the culture collection of E. R. Squibband Company through the courtesy of Dr. G. F. Leonard.

MATERIALS AND METHODS

The conductivity cells made from short-necked flasks variedin capacity from -250 cc. to 1000 cc. The platinized elec-trodes were inserted through openings made in the shoulder ofthe flasks. These electrodes varied in size and in separationaccording to the requirements of the experiment. Anotheropening was made near the top of the flasks so that samplescould be drawn from time to time for chemical analysis. Theslide wire in a Leeds and Northrop student potentiometer wasused as the Wheatstone bridge. The instrument was shielded,and grounded through a Wagner Ground. The 1000-cyclecurrent was supplied by a calibrated audio-oscillator. Thebridge was balanced by obtaining a minimum sound throughtelephones. The resistance was accurately measured to 0.1 ohmand the capacitance to 0.001 microfarad. The cell constantswere checked before and after each run using N/50 KCl solutionas a reference solution.Each medium was sterilized in a conductivity cell and the

whole was brought to the temperature of the incubator beforeinoculation. The temperature of the incubator was kept op-timum for each organism.pH determinations on milk inoculated with L. odontolyticus

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574 J. B. ALLISON, J. A. ANDERSON AND W. H. COLE

were made by the quinhydrone electrode. The pH of one-per-cent peptone solutions inoculated with the lipolytic organismwas determined colorimetrically. The pH of milk inoculatedwith P. fluorescens was determined by the glass electrode de-veloped by MacInnes (MacInnes and Belcher, 1933). VanSlyke's micro-amino-nitrogen apparatus (Van Slyke, 1912)was used to determine amino nitrogen. Care was taken toremove all the ammonia from the sample before analyzing foramino nitrogen since it has been shown that results obtainedwithout the removal of ammonia are 18 to 50 per cent too high,depending upon the temperature and the size of sample (Parsonsand Sturges, 1926c). Ammonia was removed by Folin's aerationmethod (Folin and MacCullum, 1912) as well as by Van Slyke'svacuum distillation method (Van Slyke, 1911). The ammoniawas analyzed colorimetrically after Nesslerization. The CO2in the media was determined manometrically using Van Slyke'sapparatus (Van Slyke, 1917). Bacterial populations werecounted by the standard agar-plate method. There was noevidence of contamination.

EXPERIMENTS ON PROTEOLYSIS

The data obtained from the analyses of skim milk inoculatedwith Pseudomonas fluorescent are listed in table 1. After aninitial adjustment, increase in specific conductivity was foundto be proportional to the amount of ammonia formed. Thisfact is illustrated in figure 1 where the line drawn through theexperimental points is described by the following equation:-

(NH,) = 6.08 X 108 AC + 2 (1)where (NHs) refers to the concentration (millimols per liter)of ammonia formed and A C to the increase in specific con-ductivity of the medium. A similar proportionality was re-ported by Parsons and Sturges (1926a, b) in their work on con-ductivity and ammonia production by certain putrefactiveanerobes. Their proportionality constant was, however, some-what greater, which means that an increase in conductivityrepresented the synthesis of more ammonia in their media

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CONDUCTIVITY IN METABOLISM STUDIES 575

inoculated with putrefactive anerobes than in the skim milkmedium inoculated with P. fluorescens. They also found thisproportionality between ammonia and increase in specific con-ductivity to apply from the origin. The data presented hereshow a change in mechanism which prevents the line from goingthrough the origin and results in the addition of the constant 2.

TABLE 1Data obtained from the analyses of skim milk inoculated with Pseudomonas

fluorescentTemperature 28 4-0.5oC.

TIME

hours0

23477094119141170192216240265336384432456504552574600

SPECIFIC CON-DUCTIVITYX 103

6.586.666.676.857.117.387.618.008.378.709.189.5410.3210.9811.6612.1012.4812.9513.1213.30

AMMONIA

iniUimolsper liter0.00.17651.9454.235.717.128.6510.1012.3513.0518.2317.6527.2031.2034.1035.9036.4042.041.441.6

AMINO NITRO-GEN

miUimol.per liter13.614.419.933.532.3

33.751.351.162.364.064.273.280.5105.195.4114.8109.0114.1132.0

* See text for method of calculation.

CARBON DIOX-IDE

miUimol.per lite0.421.052.185

5.38

6.947.499.15

12.4419.1921.6528.8032.8839,6544'.5047.5054.70

NH,*co0

0.000.281.10

1.15

1.321.421.41

1.461.451.461.201.100.920.950.880.76

pH

6.166.206.24

6.546.606.626.757.05

7.067.02

7.27

7.307.407.377.42

The data obtained from the analyses of a peptone mediuminoculated with Achromobacter lipidis are listed in table 2. Themedium was a one-per-cent solution of Difco peptone in distilledwater. Although the quantity of ammonia formed in the pep-tone medium was less than that formed in skim milk inoculatedwith P. fluorescens, there is still a direct proportionality between

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the increase in specific conductivity and millimols of ammonia.This direct proportionality, which holds from the origin, isillustrated in figure 2. It will be noted that there is a definitebreak in the mechanism of the reaction after the specific con-ductivity has been increased to approximately 0.64 x 10-3 mhos.

40-

30X107:

LJ00-J

10 0

1.0 2.0 3.0 4.0 5.0 6.0AC.GX Io,

FIG. 1. Increase in specific conductivity (AC X 108) of skim milk inoculatedwith Pseudomonas fluorescens plotted against the concentration of ammonia(NH3) expressed in millimols per liter.

This sudden increase in ammonia has been noted qualitativelyin other samples of peptone media inoculated with this organism.Such a change in the mechanism of reaction would be interestingto study in detail, especially if it should prove to be character-istic of this organism.

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The equation which describes the first straight line drawn infigure 2 is

(NH3) = 10.86 X 10 A C (2)

TABLE 2Data obtained from the analyses of I per cent peptone medium inoculated with

Achromobacter lipidis, n. sp.Temperature 28 ±0.30C.

TIME

hours01018243032425668768092104116125130134140143160168201209258264307

SPECIFIC CON-DUCTIVITYX 103

0.91440.92220.94140.95660.9802

1.03631.07871.11681.15371.18151.21281.24161.27181.32391.34611.35851.3930

1.5345

1.64041.62911.6369

1.6739

AMMONIA

millimolsper liter

0

1.461.77

3.483.233.174.40

4.695.005.16

5.55

12.5012.0012.91

15.26

CARBON DIOX-IDE

msUimolsper liter

0.415

1.04

1.66

2.483.2863.73

4.944.804.70

5.58

6.00

6.24

6.35

5.68* See text for method of calculation.

NH,*CO

0

0.700.61

0.760.730.74

0.90

0.95

2.14

2.17

2.90

BACTERIA

milionsper cc.

0.25671.83679.333

118.7

134.3141.7241.7263.0235.3279.0378.0

392.33446.0

395.7365.7

363.7

330.0

pH

7.15

7.20

7.42

7.55

7.607.657.75

7.88

7.92

8.12

8.28

where (NH3) represents the millimols of ammonia per liter andA C the increase in specific conductivity. Although less ammo-nia is produced in the experiment described for this organism

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Li

-i

Lia-

-I0

-

z

0.1 0.2 0.3 OA 0.5 0.6 0.7

AC X I03FIG. 2. Increase in specific conductivity (AC X 103) of a one per cent peptone

medium inoculated with Achromobacter lipidie, n. 8p., plotted against the con-centration of ammonia (NHj) expressed in millimols per liter.

cc

1- ~~~~~~~0J 150

Xi 0a.

0U, 0J0I 100 0

-I

z50 -

z

1.0 2.0 3.0 4.0 5.0 6.0AC X l03

FIG. 3. Increase in specific conductivity (AC X 103) of skim milk inoculatedwith Pseudomonas fluorescerw plotted against the concentration of ammoniaplus the increase in concentration of amino nitrogen (NH, + NH,) expressed inmillimols per liter.

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than that described for P. fluorescens, the proportionality con-stant is higher.

Parsons and Sturges (1926a, b) also found that the increasein conductivity was proportional to the increase in the nitrogencalculated from a formol titration; in other words to the increasein ammonia plus the increase in amino nitrogen. The dataobtained on P. fluorescens shown in figure 3 illustrate such aproportionality. The equation for the line is

(NH3) + (NH2) = 20.0 X 103 A C + 16 (3)

where (NH3) refers to the millimols of ammonia per liter and(NH2) represents the increase in millimols of amino nitrogen perliter. An initial change in mechanism is apparent here, as wellas in the curve for ammonia, since the line in figure 3 does notpass through the origin.

DISCUSSION OF RESULTS ON PROTEOLYSIS

Free carboxyl and free amino groups appear in approximatelyequal amounts as a protein is hydrolyzed. Changes in con-ductivity resulting from enzymatic hydrolysis of a protein mustbe correlated in some way with the appearance of these polargroups in solution. Northrop (1919-1920) showed that whilethe liberation of carboxyl groups through hydrolysis of eggalbumin with pepsin would increase conductivity, the liberationof free amino groups would decrease conductivity because someof the free acid would be bound by these basic groups. Thus,increase in conductivity would not necessarily parallel increasein hydrolysis of the protein. Baernstein (1928) in a similarstudy demonstrated, however, that there were two regions ofpH where conductivity increased in direct proportion to theincrease in amino nitrogen produced through enzymatic hy-drolysis of albumin. One region, pH 1.38 or less, is quite acidwhile the other region, pH 7.36, is on the basic side of the iso-electric point of many amino acids and polypeptides. He pointsout that his results are consistent with the "Zwitterionen"theory of Bjerrum (1923). Thus, in solutions sufficiently acid,

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the conductivity will be influenced by the concentration ofcations formed.The problem of studying proteolysis by living bacteria is not

as simple, however, as one which involves the enzyme alone.Bacteria, for example, not only hydrolyze proteins but alsodeaminize some of the amino groups which are liberated. Am-monia, as well as amino nitrogen, appears in solution. A certainunknown quantity of this nitrogen derived from protein isutilized by the bacteria in the synthesis of protein and othernitrogenous derivatives. The sum, then, of the ammonia plusthe amino nitrogen (equation 3) in the medium will be less thanthe amount of nitrogen actually liberated through the hydrolysisof the proteins. Waksman and Starkey (1932) have pointedout that nitrogen may also be lost from the culture medium asammonia if there is a marked increase in alkalinity. There was,however, no loss in total nitrogen in the skim milk culture ofP. fluorescent, since the total nitrogen of the cultures remainedthe same as the controls. The quantities, therefore, of ammoniaand amino nitrogen must be correlated in some way with eachother, with the degree of hydrolysis, and with specific con-ductivity.According to equations 1 and 3 it is possible to deduce that

there is a direct proportionality between the increase in millimolsof amino nitrogen and the increase in specific conductivity.The pH range is near that which can be predicted from Baern-stein's work to give a direct proportionality between aminonitrogen and increase in conductivity. Incidentally the nar-rowness of this change in pH makes negligible any effect of anincrease in (OH-) on conductivity. Parsons and Sturges (1926)conclude that the increase in conductivity of their cultures ofputrefactive anerobes can be accounted for entirely by theincrease in concentration of ammonium salts. They believethat compounds containing amino nitrogen, such as the aminoacids, polypeptides, etc., would not contribute much to con-ductivity.

It appears from our analysis, however, that either ammonia

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or amino nitrogen is directly proportional to specific conduc-tivity. Further, it is possible to deduce from the data presentedhere on P. fluorescent that the change in concentration ofammonia is directly proportional to the change in aminonitrogen, or

d(NH3)/d(NH2) = K = 0.437 (4)

It follows that the rate of ammonia formation is 43.7 per cent ofthe rate of amino-nitrogen formation or,

d(NHI)/dt = 0.437d(NH2)/dt (5)

The production and activity of the enzymes involved in thecatalysis of hydrolytic and synthetic processes which yieldammonia and amino nitrogen must be closely correlated. Equa-tion 5 can be interpreted to mean that constant proportions ofamino acids are formed and deaminized, as well as utilized innitrogen metabolism, by these organisms. This does not mean,however, that ammonia and amino nitrogen production bymicroorganisms will always bear this close relationship. Forexample, Waksman and Lomanitz (1925) have indicated thatsome microorganisms tend to accumulate amino acids in theearly stages of proteolysis. Later, the amino acids are brokendown and axnmonia nitrogen predominates. Possibly somesuch change as the latter explains the break in the curve shownin figure 2. Other possibilities suggest themselves but theyare all correlated with the growth and development of theorganism itself. Thus, a complete understanding of proteolysisinvolves a knowledge of the intermediary metabolism of eachorganism.

Bacterial metabolism refers to the coordinated chemicalreactions which take place in the complex heterogeneous systemof bacteria and medium. The mechanism of coordination islargely unknown although some kind of a relationship betweenvariables such as ammonia, CO2, (H+), numbers of bacteria,specific conductivity, etc., must exist. There is, for example,some significance to the ammonia/carbon-dioxide ratios. These

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ratios are listed in tables 1 and 2 and they are calculated bydividing the millimols of ammonia produced per liter, by theincrease in millimols of carbon dioxide in the medium at anytime. In the early hours of growth of P. fluorescent, carbondioxide production is greater than ammonia production; i.e.,the ammonia/carbon-dioxide ratio is less than unity. As thegrowth in population proceeds, this ratio gradually increases invalue until it has reached approximately 1.4 at 160 hours whichis almost the maximum. The maximum ratio shows that forevery mol of CO2 held in the medium, almost one and one-halfmols of ammonia are produced. The increase in the ammonia/carbon-dioxide ratio with time can be interpreted to mean eitherthat the organisms are utilizing sources of carbon richer andricher in nitrogen or that less and less nitrogen is required by thepopulation of bacteria for their synthetic needs. As the popu-lation matures, the mechanism of metabolism becomes moreconstant and a maximum ratio is established. The decline inthis ratio which begins at approximately 400 hours would meanthat the organisms are more and more using carbon compoundspoor in nitrogen as a source of energy. Compounds, for example,which could accumulate from the metabolism of amino acidsduring the growth of the population might be utilized at this time.The ammonia/carbon-dioxide ratios listed in Table 2 are

essentially constant from 42 hours until a slight rise occurs at130 hours. This can be interpreted to mean a constant mecha-nism of metabolism over this period. The slight increase in theratio at 130 hours leads to a greater rise to a new value over2.0, slightly above the ratio calculated for ammonium carbonate.This is a change in mechanism of reaction which is in accord withthe change illustrated in figure 2. It is possible that the largeincrease in ammonia is some function of the change in concen-tration of enzymes secreted by the bacteria which are involvedin converting amino groups into ammonia.These speculations emphasize the fact that if the mechanisms

of coordination between these variables were known, then themeasurement of a single variable such as specific conductivitywould permit an integration of the whole process of metabolism.

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CONDUCTIVITY IN METABOLISM STUDIES 583

Further studies need to be made to establish these mechanismsof coordination.

EXPERIMENTS WITH L. ODONTOLYTICUS

The data obtained in the study of the souring of skim milkthrough the metabolism of L. odontolyticus are listed in table 3and shown graphically in figure 4. Conductivity cell no. 1 was

TABLE 3Data obtained from the analyses of skim milk inoculated with

Lactobacillus odontolyticusTemperature 37.65 :410.150C. Cell S1 contained 250 cc. of the medium and

it was not shaken during the period of analysis. Cell #2 contained 750 cc. of themedium and it was shaken before every determination. Samples for pH deter-minations were withdrawn from Cell #2.

TIME

hours024681012141618202224

SPECIFIC CON-DUCTIVITYX 10', CELL 1

7.7057.7747.8637.9698.0868.2978.4508.7378.9789.1779.3679.5649.775

SPECIFICCONDUCTIV-ITY X 10',CELL 2

7.6147.777.8097.9168.028.2748.5258.6878.889.089.3359.7069.797

pH

6.476.456.396.276.236.105.955.715.575.535.395.225.05

TIMM

hours2628303234363840424446485557

SPECIIC CON-DUCTIVITYX 10', CELL 1

10.02410.1810.30510.41510.54510.69510.72810.73610.76110.81510.83810.85410.9410.92

SPECIFICCONDUCTIV-ITY X 10',czLL 2

9.94810.10510.17510.20510.21510.23510.22510.22510.22510.22510.23510.25

pR

4.964.804.684.634.524.404.314.204.104.114.053.93

not shaken during the experiment. Cell no. 2 was shakenbefore each determination. The result was that after the curdformed it remained firm and unbroken in cell no. 1, but wasbroken and settled around the electrodes in no. 2. Conductivitychanged in both flasks, as illustrated in figure 4, at approximatelythe same rate up to the point where the curd began to form.Where the curd was not broken, in cell no. 1, the conductivity

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continued to rise. No further change in conductivity tookplace in cell no. 2 after the curd was broken and allowed to settlearound the electrodes. These facts suggest that in the formercase, where the curd remained unbroken, a colloidal gel wasformed so that minute particles of casein did not hinder thepassage of a current of electricity between the two electrodes incell no. 1 as much as in cell no. 2.

iil 3.1

0

x

110 4.1

1-~~~~~~~~~~~~~~~~~~3

08 9 5.1

(n8 ~~~~~~~~~~~~~~6.1

20 40TIME IN. HOURS

FIG. 4. Time in hours plotted against specific conductivity and pH of skimmilk inoculated with Lactobacillus odontolyttcus. e represent determinations ofspecific conductivity in conductivity cell no. 1 which was not disturbed through-out the experiment. (D represent determinations of specific conductivity inconductivity cell no. 2 which was shaken before each determination. The opencircles 0 represent pH determinations made on samples taken from cell no. 2.

Conductivity changes in this system before the curd is formedare primarily due to the increase in the concentration of thehydrogen ion. Other factors, however, play a role in this in-crease in electrical field strength of the medium. Probably themost important of these other factors is the acid-binding capacityof the casein. Thus, as the lactic acid is formed through the

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decomposition of lactose, a certain number of hydrogen ionswill be removed from solution by combining with the protein.The protein will gradually lose its charge until at the isoelectricpoint the casein separates from solution. The net result is anapproximate linear relationship between specific conductivity andpH up to the isoelectric point.The value of conductivity measurements in the investigation

of the metabolism of lactic-acid-forming bacteria is obvious.Further study should yield information concerning the physicalchemistry of the reaction in milk as well as establish the rate ofacid formation as it is correlated with other variables that arealso a result of the metabolism of these organisms. Conduc-tivity measurements could be used also to advantage in a com-parative study of the rate of acid formation between differentlactic acid forming organisms. A preliminary analysis, forexample, has shown no difference in the rate of formation oflactic acid by Lactobacillus bulgaricus and Lactobacillus odontoly-ticus using change in conductivity as a measure of acid formation.

SUMMARY

An ammonia-forming lipolytic organism isolated from creamis named Achromobacter lipidis, n. sp., and is described. Spe-cific conductivity is directly proportional to the production ofammonia by Pseudomonas fluorescens grown in skim milk andby Achromobacter lipidis, n. sp., grown on one-per-cent peptonemedium. The processes which result in the formation of ammo-nia and amino nitrogen in skim milk inoculated with Pseudomonasfluorescens are interdependent; the rate of ammonia formationbeing 43.7 per cent of the rate of amino-nitrogen formation.The ratio between increase in ammonia and increase in carbondioxide is an aid in the interpretation of the mechanism of me-tabolism. Measurements made on skim milk inoculated withLactobacillus odontolyticus demonstrate that specific conductivityis a linear function of decreasing pH until the isoelectric pointof the casein is reached. A constant specific conductivity isapproached rapidly if the curd precipitates around the elec-trodes while the approach is slower if a colloidal gell is formed.

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REFERENCESALLISON, J. B., ANDERSON, J. A. AND COLE, W. H. 1937 The method of elec-

trical conductivity applied to studies on bacterial metabolism. (Abst.)Jour. Bact., 33: 645-646.

ANDERSON, J. A. AND HARDENBERGE, J. G. 1932 The deterioration of creamby a lipolytic bacterium. Jour. Bact., 23: 59.

BAERNSTEIN, J. D. 1928 The conductivity method and proteolysis. II. Aninterpretation of the conductivity changes. Jour. Biol. Chem., 78:481-493.

BERGEY, D. H. ET AL. 1934 Bergey's Manual of Determinative Bacteriology.Williams & Wilkins Co., Baltimore, Md. 4th ed.

BJERRUM, N. 1923 Die konstitution der ampholyte, besonders der amino-sauren, und ihre dissoziationskonstanten. Z. physik. chem., 104:147-173.

FOLIN, 0. AND MACALLUM, A. B. 1912 On the determination of ammonia inurine. Jour. Biol. Chem., 11: 523-525.

MACINNES, D. A. AND BELCHER, D. 1933 A durable glass electrode. Ind. Eng.Chem. Anal. Ed. 5:199-200.

NORTHROP, J. H. 1919-20 The combination of enzyme and substrate. I. Amethod for the quantitative determination of pepsin. II. The effectof the hydrogen ion concentration. Jour. Gen. Physiol., 2: 113-122.

PARSONS, L. B. AND STURGES, W. S. 1926a The possibilities of the conductivitymethod as applied to studies of bacterial metabolism. Jour. Bact.,11: 177-188.

PARSONS, L. B. AND STURGES, W. S. 1926b Conductivity as applied to studiesof bacterial metabolism. II. Parallelism between ammonia and con-ductivity in nutrient gelatin cultures of putrefactive anaerobes. Jour.Bact., 12: 267-272.

PARSONS, L. B. AND STURGES, W. S. 1926c The magnitude of the error due toammonia and to salts in the Van Slyke amino nitrogen procedure ascommonly applied in studies of bacterial metabolism. Jour. Bact.,11: 165-175.

SIERAKOWSKI, S. AND LECZYCKA, E. 1933 Aenderungen der elektrischen leit-fiahigkeit un Bakterienkulturen. Zentr. Bakt. Parasitk. und Infek-tionsk., 127: 486-492.

VAN SLYKE, D. D. 1911 The analysis of proteins by determination of thechemical groups characteristic of the different amino acids. Jour.Biol. Chem., 10: 15-47.

VAN SLYKE, D. D. 1912 The quantitative determination of aliphatic aminogroups. Jour. Biol. Chem., 12: 275-284.

VAN SLYKE, D. D. 1917 Studies of acidosis. II. A method for the determina-tion of carbon dioxide and carbonate in solution. Jour. Biol. Chem.,30: 347-368.

WAKSMAN, S. A. AND LOMANITZ, S. 1925 Contribution to the chemistry ofdecomposition of proteins and amino acids by various groups of micro-organisms. Jour. Agri. Res., 30: 263-281.

WAxSMAN, S. A. AND STARKEY, R. L. 1932 The decomposition of proteins bymicroorganisms with particular reference to purified vegetable pro-teins. Jour. Bact. 23: 405-428.

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fluorescent, · trodes were inserted through openings madein the shoulder of the flasks. these...

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