TEE BEHAVIOR OF THE GRAPHITE ELECTRODE INELECTRO-CHEMICAL CELLS

I The Determination of the Oxidation- Reduction Potential of Sterile Culture Media By Means of the Graphite Electrode

II Oxidation-Reduction Studies in Relation to the Growth and Differentiation of the Species of the Genus Brucella

III The Preparation and ITse of the Platinized- Graphite Electrode

ByC. D # Tuttle

A THESISSubmitted to the faculty of Michigan State

College in partial fulfillment of the requirements for the degree

cfDoctor of Philosophy

Departments of Chemistry and Bacteriology East Lansing, Michigan

1933

ProQuest Number: 10008230

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(4- IS (6 f 7JO

Fig. 6. - A composite curve of triplicate electrometric titrations on acidified 0.1W FeSO^ withKMnO^t using a graphite electrode*

44Results of study (continued)

Figures 1, 2, and 3 are electrometric titrations on 20 cc. of FaOH with 0.1H KC1. These three titrations were carried out in the same manner except for a differ­ence in the electrodes that were used in following this reaction. A study of Figures 1 and 2 showed that plati­nized platinum and platinized graphite agree. The change in voltage at the inflection point was a,bout 0.5 cf a volt. The curve in Figure 3 shows that graphite electrodes do not agree in alkaline solutions and that the voltage change at the inflection point was only about 0,2 of a volt. This observation would indicate that graphite electrodes do not follow a pH change as well as platinised electrodes.

Figures,4, 5, and 6 are electrometric titrations of 20 cc. of acidified 0.1N FeS04(hH4)S04 with EMn0 4 . The three titrations were carried out in the same manner except for a difference in electrodes. The curves in Figures 4, 5, and 6 show that platinized platinum, platinized graphite, and graphite are in agreement. The only difference appeared in the upper knee of the electrometric tit rat ?_ on curve when graphite was used as an electrode. The bend in the curve is not so sharp. The graphite electrodes required a little more time for equilibrium between readings than was requir­ed for the platinized electrodes. All three of the eleo« • trodes followed this oxidation-reduction change. It is evi­dent from the titration curves that platinized platinum and platinized graphite agree. Graphite electrodes do not follow a change in pH# but do follow oxidation-reduction changes.

DiscussionIn preliminary experiments it was difficult to produce

platinized platinum and platinized graphite hydrogen revers­ible electrodes that would agree with each other closer than0.0002 of a volt in either acid or alkaline solutions. With­out any change in procedure, the agreement was increased from ten to a hundred fold by the addition of an electrical shield (2) surrounding the cell. Then, with the elimination of all errors due to contact potentials and heat effects and the development of better technic, individual readings were made accurately to one millionth of a voltcPlatinized graphite electrodes were found to be a little slower in reaching their equilibrium with the hydrogen ions than the platinized platinum. This was especially tT»e in alkaline solutions.

Four hours were required to reach equilibrium after starting the flow of hydrogen. The length of time depended somewhat upon the size of the cell and the rate of flow of hydrogen.

The results of these experiments show that platinized graphite may be substituted for platinized platinum in elec­trometric titrations. In acid solutions and weak alkaline solutions up to one normal, their agreement was within 50

45millionths of a volt. In more alkaline solutions than one normal, their agreement could not he relied upon within0.2.of a millivolt. Without using the proper shielding, the agreement was not greater than three or four tenths of a millivolt in all solutions studied. The graphite elec­trodes do not agree with these electrodes when used in ti­trating an alkali with an acid. They do not even agree with each other in alkaline solutions. The change in voltage at the inflection point of an alkali-acid titration was small compared with that of the platinized platinum and plati­nized graphite electrodes. Graphite electrodes agree with the platinized electrodes in oxidation-reduction titrations if sufficient time is allowed for equilibrium. It was found that graphite electrodes when treated as described, were slightly more sluggish when compared with the plati­nized electrodes. In view of these results, a platinum- graphite electrode couple for electrometric titrations would be very satisfactory for alkali-acid titrations, but unsuitable for oxidation-reduction titrations. Thus, it would appear that the graphite electrode is very suitable for following an oxidation-reduction change in a solution where the pH change was slight as in buffered liquid cul­ture medium inoculated with bacteria.

Summary1. Platinized graphite electrodes may be substituted

for platinized platinum electrodes. They may be used in acid solutions, weak alkaline solutions, and often in stronger alkaline solutions.

2. -Graphite electrodes do not register a change in pH.3. Graphite electrodes register oxidation-reduction

changes if sufficient time is allowed for equilibrium.References

1. Ralzton, Robert R.Potentiometric titration of acidity in oils.University of Michigan, Ann Arbor. Ana.lytic Industrial and Engineering Chemistry, 4, 109, 1932.

2. White, Walter P.Leakage prevention by shielding especially in poten­tiometer systems.Journal of American Chemical Society, 36, 2011, 1914.

3. Clark, William MansfieldThe determination of hydrogen ions.

4. Poulk, C.W* and Marion HollingsworthA precision measurement of the composition of the constant boiling mixture of hydrogen chloride and water.Journal of the American Chemical Society, 45, 1220, 1923.

THE BEHAVIOR OF THE GRAPHITE ELECTRODE INELECTRO-CHEMICAL CELLS

I The Determination of the Oxidation- Reduction Potential of Sterile Culture Media by Means of the Graphite Electrode

II Oxidat1 on-Reduction Studies in Relation to the Growth and Differenbiation of the Species of the Genus Brucella

III The Preparation and Use of the Platinized- Graphite Electrode

byC* D. Tuttle

A THESISSubmitted to the faculty of Michigan State

College in partial fulfillment of the requirements for the degree

ofDoctor of Philosophy

Departments of Chemistry and Bacteriology East Lansing, Michigan

1933

r m BEHAVIOR OP TEE GRAPHITE EIECTRODE INEIECTRO-CMEMICAL CEjITiS

I The Determination of the Oxidation- Reduction Potential of Sterile Culture Media "by Means of the Graphite Electrode

II Oxidation-Reduction Studies in Relation to the Growth and Differentiation of the Species of the Genus Bruce1la

p.l

p. 11

III The Preparation and Use of the Platinized-Graphite Electrode p.31

X - T h e Determination of the Oxidation- Reduction Potential of Sterile Culture Media

by Means of the Graphite Electrode

It has already been demonstrated that the potential of inorganic chemical oxidation-reduction systems and a few organic ones may be measured at the surface of bright no­ble metal electrode with the conventional potentiometer without difficulty. On the other hand in many organic oxi- dation-reduction systems, it is often difficult to make ox­idation- reduction potential measurements with bright noble metal electrodes by using the conventional potentiometer due to the ease with which the electrodes are polarized.It has been shown that this difficulty is increased in or­ganic systems that are poorly poised (l). Bacteriological culture media are examples of extremely poorly poised or­ganic systems. Pour general methods have been employed to overcome or to avoid polarization and its effects. They are: the use of oxidation-reduction dyes to indicate the extent of oxidation or reduction through their color changes(2), the use of a vacuum tube voltmeter, or a potentiometer with a vacuum tube arrangement or electrometer as the null instrument (3), by making use of poising effects such as poising substances, the passage of highly purified nitrogen or the exclusion of air (4), and the use of an electrode that is not easily polarized and permits of the use of the conventional potentiometer (5), (6).

In the light of previous studies of this nature it ap­pears desireable to find a suitable electrode which would materially reduce the laborious technic -required in meas­uring the oxidation-reduction potentials in systems of a- biological nature. With this view in mi-nd, a study of elec­trodes for use in measuring the oxidation-reduction poten­tials of liquid culture media used for bacteriological pur­poses was undertaken.

ExperimentalApparatus and Materials. The apparatus used in these

experiments wasthat ordinarily employed in making elec­tromotive force measurements in physical chemistry. Two such sets of apparatus were used in connection with this work; one at 25c C. and the other at 37DC. A small thermo­stat was built to operate at 37°C £O.01°C. The thermostat for 25° G was larger and could easily be maintained within .005°C at 25°C for a long period of time.

A Leeds and Worthrup student type potentiometer with accessories was used in connection with the work at 37°C.The galvanometer was a high grade Leeds and Northrup pointer type. The type K potentiometer and accessories was used for the work done at 25°C. This included a pre-

2cision type of- gadveenomeLter which was very sensitive o smallr changes in current. Both potentiometers were shxeld- ed'as described by 'White (7), The metal parts of the ther­mostats were also connected to the main shield.

The cells used in these experiments were of simple design, A IE KC1 calomel half cell with a long side arm was used as the reference electrode for the studies at 37°C. The side arm dipped into a large intermediate ves­sel which also received one side of the agar bridges from several half cells containing the electrodes to be tested,A similar arrangement was used for the work at 25°C. except that a IP hydrochloric acid hydrogen half cell was used in place of the calomel half cell as the reference electrode* This cell was connected with the intermediate vessel by use of a salt bridge containing a stopcock. The measure­ments were made through the closed stopcock. The electrodes were tested in half cells made from pyrex test tubes 25 mm, diameter and 100 mm. long. These tubes were fitted with a rubber stopper-containing holes to carry the elec­trode, an agar bridge, and a glass tube plugged with cot­ton. A second type of half cell was also used. It was a special flat bottom freezing point tube fitted with a rub­ber stopper having holes for three electrodes, an agar bridge, and a glass tube plugged with cotton,'

All chemicals used were Baker’s analyzed. The calo­mel was prepared as described by Lewis, Brighton and Se­bastian (8) from Baker1s analyzed chemicals without fur­ther purification, The mercury was cleaned by running it through a fine capillary several times. It was-dried in vacuum over phosphorus pentoxide, The 1M KC1 solution was made up by weighing the KC1 and dissolving it in the required amount of distilled water. 13? H'Cl was prepared as described by Foulk and Hollingsworth (9). The KC1 agar, for agar bridges, was a 2 per cent agar in IE potassium chloride solution.

The electrodes used in this study were made from one- fourth inch sticks of special Acheson graphite cut to a ' length of four inches. After considerable study of methods of preparing the electrode for satisfactory use the fol­lowing procedure was adopted; The electrodes were first cleaned with a fine grade of steel wool and polished with a clean cheese cloth.They were then placed in distilled water and boiled slowly from four to six hours. The boil­ing under distilled water must be of such length as to remove all traces of material left on the electrodes from a previous experiment. Frequent changes of distilled wa­ter hastened this process. They were cooled under distilled water. Acheson graphite electrodes when prepared in this manner may be used in measuring potentials with an ordi­nary student type of potentiometer.

All of the experiments with electrodes were made on two kinds of bacteriological culture media. A liquid

3synthetic medium and liver infusion medium were used.The synthetic medium was prepared by Hershey in this lab­oratory and was composed of the following ingredients:

Water (distilled) 100 cc.Yeast extract 0.3 gramsAmmonium citrate 0.5Sodium chloride 0.5Potassium dihydrogen phosphate 0.2Ammonium sulphate 0.2Ferric ammonium citrate 0.001Magnesium sulphate 0-.05Potassium nitrate .0.05The pH was adjusted to 6.6 with sodium bicarbonate.The beef liver broth was made by extracting one lb,

of finely ground beef liver with 500 cc. of tap water in flowing steam for two hours. To 500 cc* of the extract was added 500 cc. of tap water, 10 grams of Bacto-peptone and 5 grams of c. p. sodium chloride*. The mixture was heated in flowing steam for 30 min, f filtered and steri­lized in the autoclave at 15 lb. pressure for 30 min-. The pH was adjusted to 6.6 after sterilization.

Method of filling agar bridges and half cell. The agar bridges were made by filling a H tube- made of soft glass 2 mm. in diameter to within an inch- of the top of the limbs with 2% agar in molar KC1. They were then steamed for 20 min. and -cooled. The ends of the glass tubing, were sealed off and steamed for 20 m-in.- to test- the- seals for leakage. A mark was made- with, a file jUs-t below- the level of the- agar on each arm of the bridges. The bridges we-re wrapped in paper and sterilized in- the au-todave- at 15' -lb. pressure f-or- 30 minutes-. The other- glassware was washed and sterilized in h-ot-air. The assembled half c-ells minus the agar bridges were placed in a large suitable vessel, wrapped in paper and-sterilized- in -the'autoclave*- Follow­ing. sterilization and cooling, the half cells were partly filled with the sterile liquid medium and one limb- of the agar bridges inserted through the holes in the rubber stoppers.'Only the tip on the side of the agar bridge which passed through the rubber stopper was broken off at its file mark when each half cell was assembled. The half cells were connected to the calomel -intermediate cell by breaking off the tip of the other limb of the agar bridges at' its file mark and inserting the arm in the hole of the stopper on the intermediate vessel.

Standardizing the calomel half cell. The hydrogen for the hydrogen half cell was produced by electrolyzing a 10 per cent solution of HaOE. The hydrogen generator wa.s a modification of that described by Clark (10). The hydrogen was purified by'passing it -over an electrically heated platinum spiral. A piece of heavy capillary- glass tubing about 1 meter long was placed between the generator and the hot platinum wire as a safety device. Since the chem­icals used in the calomel half cell were not repurified

Table

Iof

different

electrodes

in synthetic

medi

um

4

02 to o in rH totD z> H4 H4 in inCO 03 O o o ocO to H4 H1 Kjf* • 0 e • •o o o O O o4* 4 Hr + H*-

CO Ct] O rH CO 03to to n to in ino o o O o oH4 H4 H4 H4 H40 • • • • •o o o O O o* 4 4- 4 4 4

to

03 o 03 c\? o O o- 02 CO CO 03CO CO o CO H 4 o to £> to CO E**c- 00 03 03 03 03 03 03 03 03 03cO CO to CO CO CO to CO to CO CO

• • 0 • • •* • • 0 • oo o o o o o o o o o o4 4 4 4 4 4 4~ 4 4 4 4>

ino-o>to•o■4

00toa>toaO

H4CDrOou+3o0)

rHm

O<D

CO

C\2

OOto*o

H4r-00to3o

03rH<J>tooo

CO020>CO

toc\2a>to•o

H4(MO'.CO

inc\203too

to0203CO•o

00o03CO•o

to0203CO*o

03 I—I03CO

00r~i03CO

.>o

rHrH03CO

4 4 4 4 4 4 4 + 4 4 4 4 4-

H 4 CO to H 4 co CO co o CO in to to CO03 o 03 CO to C3 o H o rH rH rH oz> 03 03 03 03 03 o O o O O O oCO CO CO CO co CO «t?4 H* H4 H4 H4• A * • • 0 • * • • •o o o o o o o O o O O O o4 4 4 + 4 4 *+ 4 + 4 4 4 4

03. in CO in rH H 4 H 4 to H 4 03 to O 03Z> co o o O O O O 03 H H ,-H H03 o rH rH rH rH rH rH O m rH fw-j rHCO H 4 H 4 H 4 H 4 H 4 H 4 H 4 H 4 H 4 H 4 H 4<» ♦ • • • V » • * • • • • *o O O O O O O O O O O O O4 4 4 + 4 H 4 4 4 4 4 4 4

&

03 m co CO 03 00 03 CO 03 rH 03 CN2 00CO C\2 to to to to H 4 02 C" rH CO 03 oCO H 4 H 4 H4 H 4 H 4 H 4 H 4 C2 02 rH rH 02H 4 H 4 H 4 H 4 H 4 H 4 H 4 H 4 H* H 4 H 4 H 4 H 4• 0 • • • • • • • o • • •o O O O O O O O O o O O O4 '4- 4

6•H W0) Ct3 O rH CO H 4 LO t O O C O O H C\2 CO H 4 l O

rH rH rH rH rH rH•H

0.4208

+ 0.4126

+ 0.4009

t 0.3921

4 0.3978

+ 0.

4063

Table

IIof

different

electrodes in

liver

infusion medium

0

o CM CO O to lO £> CO m z> CflVO to rH o CO in rH r- co c- .in Kt« toi* CO co CO CM 02 02 02to , CO CO CO CO CO cO CO CO CO to CO• • • • ■ • to - • to • to ' • •o o O o o o o o o o o o

o in o o rH ' <0 CO in o o 02r- c- H O VO CO o 00 c& £- oo in. CO to CO 02 02 02 COCO CO CO co CO CO to to CO co CO to• • • • * • • • • • • •o o o o o o o o o o o o

r- 02 CO o xj* cr> CO CM m02 02 02 VO cO in lO CO o rH rH o>CO in LO in in CO CO CO CO CO CMto CO CO CO co CO to CO co CO CO CO0 • • « • ■ to to to to to • • «Xt o o o o o o o O o o o oOfH * *♦ to- •to to •to + to to to to +--PO0rH rH 00 rH in in VO CM in CO CM r-W O in in CO 02 rH o O CO CO coCO z> 2> t- o r- £> O- vo VO VO vo<H CO CO co to co CO CO CO cO CO to cO COO to to to • to to to • 0 • • tofj o O o o o o o o o o o oM0 4 + to* + ■to +■ . Hr -to ■to­ to to to­

Jj 00 o o in o- CO CM o rn cr> E'­ olCT> CO CO 02 o rH C- vO CM en vorH o cr> CO £> ID CO to02 CO CO to CO CO co CO c0 CO CO• to • • to to • to to • • •O o o o o o o o o o o o

CM o O o rH O co o o CM o>00 in rH in CM O vo in CO rHo CO GO C- o- VO VO VO vo VO VOCO CO CO to co co CO co CO CO coto • to • • • to to • • • •o O o o O o o o o o o o-to to to to- to ♦ to­ to- to to- to- to-

G•H CO0 d o CO in vo t- o rH CM CO inrH rH H rH rH rH•Htr»

6

fHHH0t—I .0 ClJ§H

erapiHO>

Ft*rH(DOC0Jh0CmC*H•HtJ

§♦rHXd0bDC•H*5O

CO

CO 0 <d o Ft P o 0 I—I 0pfl0Ft0«RCH•H'd

ChoP3

00oFJ0Ft0c«Ch*H

IP 0 «HP 0rH O

*C(

Io 0 0 rH 0 O 0 *d

> Ft o 0 fcl0 F C P F! -PH Ch H d P

to m o> CO into CM CO CM 00CM CO to CM too o o o o• • • • •o o o o o

0Ch 0 O 'd O 0 Ft Ft P 0 p P p O *H 0 0 0 3 rH Ofe 0

•r-t !>

to CO CM

■H m CO co toH o> to rH to0 to in co CO cr>o O o o o oc • • • • *0 o o o o o

00 rHCM H

<0rH 00rH

C\J in

?and the concentration in the agar bridges varied slightly, it was necessary to standardize the calomel half cell. Standardization was accomplished by substituting a hydro­gen reversible half cell for one of the half cells con­taining an electrode to be tested and measuring this cell. The resulting E is corrected for temperature and pressure. All values can now be calculated into values as though the readings were made with a hydrogen reversible elec­trode (EjJ by a simple algebraic addition.

Results of StudyIn table I are tabulated the corrected readings for

six graphite electrodes in the synthetic medium measured against the hydrogen reversible electrode over a period of fifteen days. The cell measured is represented as fol­lows: graphite electrode, synthetic medium, KC1 agar bridge, IF HC1, 1%, Pt at 25°C. These readings are repre­sentative of many similar experiments*

In table II are tabulated the corrected readings for six graphite electrodes in the liver infusion medium measured against the hydrogen reversible electrode over a period of fifteen days. The cell measured is represented as follows: Graphite electrode, liver infusion medium,KC1 agar bridge, IF HC1, ET̂ , Ft at 25°C. These readings are representative of many similar experiments.

The values given in the table I show that the elec­trodes in the synthetic medium changed slightly from their original values after a period of fifteen days incubation. As the period of incubation increases, there was a tenden­cy for the electrodes to drift more positive. The elec­trodes in liver infusion medium moved from their original values more than those in the synthetic medium. The change in potential of the electrodes, in this case, was in the negative direction. But even at that the negative drift was small in comparison to that which obtains for metal electrodes.

The tabulated E^ readings in the two tables indicate that there is a much closer agreement between electrodes as the period of incubation increases. The electrodes ap­proach a close agreement after a period of seven to ten days incubation in either media. A longer period of incu­bation does not bring about any closer agreement.

In continuing this study it was decided to hold the complete half-cell in an incubator for seven days at 37°C before mounting in the thermostat. The E^ readings of several electrodes in liver infusion broth held in this manner are tabulated in table III.

Table III contains condensed data from five experi­ments using 87 electrodes. It shows the number of elec­trodes used in each experiment, the maximum variation of voltage between all the electrodes and the maximum differ­ence in voltage between all electrodes after removing the most erratic ones for each of the five experiments. The

8data in the table reveal that only 18,4 per cent of the electrodes deviate to a considerable degree from the aver­age potential of the graphite electrode in liver infusion medium. If large noble metal electrodes had been used in the liver infusion medium from one-fourth to one-half of the electrodes would have been even more irregular than the graphite electrodes, ^If a small amount of IF acid or base is added to the medium in one of the half cells, the value of the oxida­tion-reduction potential is not affected as much as one would expect.

DiscussionThe results of these experiments indicate that Ache­

son graphite electrode is a suitable electrode for oxida­tion-reduction potential measurements in liquid culture media used for growing bacteria. In view of the fa,ct that it was not necessary to exclude the air from the half cell or to make use of a vacuum tube arrangement or electrom­eter in making the measurements, it is quite evident that the electrode was very well poised. The measurements were easily made with the ordinary student type potentiometer using a high grade pointer galvanometer. The use of this type of electrode has a distinct advantage over electrodes made of noble metals in that it is often necessary to ex­clude the air and to use a vacuum tube arrangement or electrometer in making the measurements. It was necessary to use a potentiometer shield and to connect all the met­al parts of the thermostat to this shield in order to ob­tain very close agreement between several electrodes in the same cell. The agreement was within 35 millivolts, A slight change in pH had only a negligible effect upon the oxidation-reduction potential.

The possibility of using a common metal as an oxida­tion-reduction electrode in the synthetic media was exam­ined. Several of the metals, after standing in the medium a few days, were discovered to be dissolving slowly. As a result of this finding the more common metals were exam­ined for solubility in the synthetic medium.

It was found that silver, nickel, mercury, lead, magnesium, manganese, aluminum, antimony, cobalt, cadmi­um, tin, zinc, and copper were slightly soluble. Chromium was the only metal of the less noble ones which was not soluble. The solubility of the metal was increased by autoclaving in the synthetic medium at fifteen pounds steam pressure for 45 minutes, it was possible to detect the metals in solution by qualitative analysis.

SummaryAn electrode made of special Acheson graphite is

satisfactory for oxidation-reduction studies of linuid me, dia used in growing bacteria.

9The electrode in liquid media is stable enough to

peTmit the use of a potentiometer.Graphite electrodes when treated as described above

can be made to agree with each other within 35 milli­volts .

The oxidation-reduction potential of graphite elec­trodes is not affected by small changes in pH of the medium.

References1. Michaelis, L.

Oxidation-reduction potentials.Translated by Louis B. Elexner.J.B.Lippincott Co., 1930.

2. Lubos, ReneObservations on the oxidation-reduction properties of sterile bacteriological media.The Journal of Experimental Medicine, vol. XLIX,Ho.3, p. 507-523, 1929.

3. Allyn, W. P. and I. L. Baldwin Oxidation-reduction potentials in relation to the growth of an aerobic form of ba.cteria.Journal of Bacteriology, vol. XXIII, Ho. 5, 1932.

4. Knight, B.C.J.G.Oxidation-reduction studies in relation to bacterial growth. IX. A method of poising the oxidation-reduc­tion potential of bacteriological culture media.The Biochemical Journal, vol. XXIV, Ho.4 ,p.1075,1930.

5. Coulter, Calvin B.Oxida.ti on-reduct ion equilibria in biological systems.1. Reduction potentials of sterile culture bouillon.The Journal of General Physiology, vol. XII, Ho.l, p. 139-146, 1928.

6. Elexner, Louis B, , and E. S, Guzman Barron Oxidation-reduction potentials at carbon and tungsten electrodes.Journal of American Chemical Society, vol. 52,P. 2773-76, 1930.

7. 7/hite, Walter P.Leakage prevention of shielding especially in potentiometer systems.Journal of American Chemical Society, vol. 36, p. 2011, 1914.

8. Lewis, Gilbert H., Thomas Brighton and Ruben L. Se­bastian, A study of hydrogen and ca.lomel electrodes. Jour, of American Chemical Society, vol. 39 ,p. 2245 , lfl"7.

109. Foulk, C, W., and Marion Hollingsworth

A precision measurement of the composition of the constant hoiling mixture of hydrogen* chloride and water*Journal of American Chemical Society, vol. 45, p. 1220, 1923.

10. Clark, W. M.The determination of hydrogen ions, Baltimore,, 2nd edition, 1922.

II - Oxidation-Reduction Studies in Relation to the Growth and Rifferentiation of the Species of

the Genus BrucellaThe object of this investigation has been to deter­

mine the oxidation-reduction time potential associated with the growth of strains of Bruce 11a melitensis, Bru­cella abortus t/ and Brucella suds in a suitable liquid-cul- ture medium Tn the piesence of and without the addition of certain growth inhibiting dyes. It was thought that the results of such a study might furnish further informa­tion on the biological relationship of the three species and enlightenment as to the mechanism of the selective action of the dyes toward the growth of Bruce 11a (l). It is obvious that in making a comparative study of this na­ture of closely related aerobic bacteria, the growth con­ditions that are to be maintained should closely approx­imate those normally used in growing the organism. In oth­er words, if there are metabolic differences in intensity and capacity in the three species of Brucella when grown on culture medium under aerobic conditions, the differences should be measurable in an oxidation-reduction system if the same growth conditions are maintained. The measure­ment of electrode potentials of culture media under aero­bic conditions requires the employment of a system in which the E^ of the cell will remain at or near the same level for a period before inoculation. The maintenance of electrode potentials at a constant level in liquids con­taining organic materials has been a difficult problem without the introduction of some dynamic method to pre­vent the drift of potential and polarization of the elec­trode, These troublesome factors may be overcome by deaer- ating the liquid in the electrode vessel and measuring the E.1T.F, changes with a vacuum-tube potentiometer or an- electrometer.

By making use of an electrode made of Acheson special graphite.as in this study, the troublesome drift of poten­tial and the polarizing tendency so characteristic of metal electrodes was avoided. Further, one does not en­counter marked potential differences in different graphite electrodes that are commonly found when different platinum or gold electrodes are used in the same cell.

Another important advantage of using the graphite electrode for measuring oxidation-reduction potentials is, that a large number of cells may be studied simultane­ously with a high degree of accuracy.

The experimental procedure and results of this study followsExperimental Procedure

Apparatus. A thermostat was used to maintain the

12

V'V'ai

/- L/veh infusion medium.Aaar* hrM#* UN.KCt).

3- /M KCI.4 ~ fffzClz puste.

S-'Mer c u>~y. 6~-Gir*phiie e/eettode 7- InecaiGt-jng tube, 8~*$t opcoct\«

Figure A - Schematic Arrangement of Complete Cell

13cells at a constant temperature* An insulated woodenbox lined with galvanized sheet iron was used as a container. The thexmoregulator was of the mercury in glass expansion type and was made in our laboratory. A Bernell polarized telegraphic relay was used in conjunction with the thermo­regulator for controlling the current in a 250 watt elec­tric heater. The stirring device was constructed from an ordinary laboratory stirring pulley and was driven by a small electric motor. The temperature was maintained at 37 C + .01 C.A Leeds and Forthrup student type potentiometer and accessories were used throughout these experiments. A high grade Leeds and Forthrup pointer galvanometer was found satisfactory for this work.

The potentiometer assembly and working battery were placed on sheets of metal and all were connected to the metal case of the thermostat, thus forming a shield against stray currents (2). Care was taken to see that each post was well insulated from the shield. The shielding helps to prevent drifting of the potential, due to stray currents, and to stabilize the galvanometer during damp or stormy weather.

The cell used in these experiments is shown in FigureA. A calomel cell with a long side tube leading into the intermediate vessel was used as a reference electrode for ten oxidation-reduction half cells. The intermediate ves­sel was a small jar fitted with a cork stopper containing ten holes for receiving the agar bridges from the oxida­tion-reduction half cells. A center hole received the cal­omel side tube* The oxidation-reduction half cells were pyrex test tubes 25 mm, by 100 mm. fitted with rubber stoppers containing holes for the electrode, agar bridge, and an inoculation tube. The' inoculation tube was a short piece of small bore glass tubing passing through the rub­ber stopper and projecting above the stopper. It was cov­ered with a short piece of larger bore glass tubing plugged at one end with cotton.

Inoculating pipettes were made by drawing out pieces of soft glass tubing 5 mm, in diameter to a capillary end of a sufficient diameter to pass through the inoculation tube in the cell*. The large end of the pipettes was plugged with cotton. They were sterilized in a dry oven.

Materials. All the chemicals used in filling the calomel half cell were prepared from "Baker*s Analyzed" chemicals. The mercury was cleaned by running it through a fine capillary several times. It was dried in vacuum over phosphorus pent oxide. The calomel was prepared as described by Lewis, Brighton, and Sebastian (3j without further purification of the chemicals. 1M potassium chlor­ide solution was made by weighing the potassium chloride and dissolving it in the required amount of distilled wat e r.

The agar bridges contained two per cent washed agar

14in 3JT potassium chloride solution# ^The electrode used in an oxidation-reduction hall cell is of great importance. After repeated trials with many metal electrodes, it was found that they could no e used in liver infusion broth under aerobic conditions the measurements were made with a potentiometer system# T e current required by the galvanometer can easily polarize poorly poised electrodes and make measurements un­certain. Many investigators studying oxidation-reduction po tentials in bacteriological culture media have overcome this difficulty in a limited way by using some form of an electrometer or vacuum tube amplifier in place of the gal­vanometer. Oftentimes a special form of vacuum tube volta­meter is used. The electrometer, vacuum tube amplifier, and vacuum tube voltmeter require elaborate shielding, skill in making the measurements and oftentimes the control of the humidity of the air if accurate readings are to be ob­tained for each individual potential reading* klexner and Barron (4) make the following statement in their conclu­sions on carbon and tungsten electrodes when used as oxida­tion-reduction electrodes, "Graphite gives the same results as platinum in a dyestuff system, be it well or poorly poised, whereas tungsten gives reliable results only when the system is well poised." Flexner and Barron recommended the use of lead pencil graphite for electrodes. While this type of electrode was found more satisfactory than metal ones in liver infusion medium, it was inferior to those made of Acheson special graphite. Large electrodes 1/4 inch by 4 inches of this material gave surprisingly good re­sults, This type of electrode does not become polarized easily and when used in the potentiometer system described herein, each individual reading can be made with an accur-. acy of .3 or .4 of a millivolt.

The Oxidation-Reduction Half Cell; The pyrex glass containers are thoroughly cleansed, rinsed in distilled wa­ter and sterilized in the dry oven, hew electrodes are cleaned with 00 steel wool and polished with a clean cotton cloth. Fext, they are rinsed in distilled water and insert­ed into rubber stoppers. One should avoid touching that part of the surface of the electrode which projects below the bottom of the rubber stopper after it is cleaned and rinsed in distilled water. The glass cell is fitted with the rubber stopper containing the electrode and glass tube through which the medium is inoculated, covered with paper and sterilized in the autoclave at 15 pounds pressure for 30 minutes. The sterile cells are stored until ready for use,

The agar bridges are made by filling 2 mm. diameter soft glass tubes bent in a IT shape to within one inch of the openings with two per cent agar containing m KC1 Thev are heated in flowing steam for 20 minutes and cooled’ The ends of the glass tubing are sealed off, heated again’in flowing steam for 20 minutes to test the seals for leakage.

15A mark is made with a file just below the agar level so that the sealed ends may be broken off easily. They are wrapped in paper and sterilized in the autoclave.

A half-cell is assembled for study by using every possible aseptic precaution. First, 25 cc. of sterile liv­er infusion broth pH 6.6 are added to the- sterile glass cell by removing the stopper. Next, the end of one arm of the agar bridge is broken off and inserted through a hole in the stopper until it is well below the surface of the broth. The other sealed end of the bridge is not broken off until the cell is placed in the water bath and connect­ed to the calomel intermediate cell. The assembled half­cell is now placed in an incubator at 37°C for four to seven days to permit the electrode potential to become sta­ble. It is now placed in the constant temperature water bath and connected to the calomel intermediate cell by means of the agar bridge. After a period of 24 hours the initial potential of the half-cell is taken and recorded-.

Inoculation of the Malf-cell. The inoculum is pre- pared by suspending the growth of a 48 hour agar slant in sterile physiological salt solution a few minutes before use. Approximately 100,000 living cells are introduced into the medium by means of a sterile capillary pipette through the glass inoculating tube in the rubber stopper.

Preparation of Solutions of Dyes. The dyes used in this study were thionin (certified) and basic fuchsin (certified) manufactured by the national Aniline and Chem­ical Co. They were made up in one per cent suspensions in sterile distilled water. The suspensions are heated in flowing steam for 20 minutes before use to obtain uniform suspension of the dyes. They must be heated each time they are used as the dyes do not remain in suspension in this concentration. The hot dye suspensions are added to the broth just' before the inoculum is added. The amount of thionin solution that was found satisfactory was 0.2 cc., of basic fuchsin 0.3 cc.

Procedure of Cleaning Electrode at the Completion of an Experiment. The cells were ^aken^apart and the elec­trode placed in a container of distilled water. The con­tainer was then placed in flowing steam for 24 hours. The water was then poured off, fresh distilled water added/ and the container placed in flowing steam again. The water is changed about every two hours until no more of the dye is present. The electrode is cleaned with 00 steel wool and polished with a clean cotton cloth. It is placed in a clean glass tube or beaker and stored until ready for use.

Method of Standardizing the Calomel Kalf-cell and Calculating values. Since the chemicals used in i’hecalomel half-cell were not repurified and the concentra­tions in the agar bridge vary slightly, it was necessary to standardize the calomel half-cell. This can be accom­plished by measuring the following cell and applying the proper corrections for temperature and pressure:

16(1) (-) Pt, Ha (1 atm.), W HC1; 1ST KC1 2% agar

bridge; 1STKC1, Hg.Cla, Hg ( + ). at 37 C.The E-.K.E. of the following cell was measurea:(2) C, liver infusion medium; Iff KG1 2% agar bridge;

1M KC1, Hg«Ola, Hg at 37°C. Our results are reported in terms of E*̂ values which are the same as though the 1o±-lowing celT had been measured: . *(3) (+) C, liver infusion medium; Iff HC1, Hgll atm.j,Pt (-} at 37°C•If we place cell (l) in series and in opposition tocell (2 ) we have cell (3). .(4 ) ( + ) C, liver infusion medium; Iff KC1 2% agarbridge; Iff KOI, Hg^Cl *, Hg - Hg, Hg^Cla , Iff KC15 Iff KC12% agar bridge; Iff HCl, (l atm.), Pt (-) at 37 C.

In order to calculate the values of the cells meas­ured in terms of Eh cell (3) we simply add the values of cell (1) and cell (2) algebraically. The sign on the elec­trode in these experiments has always been taken as that onthe potentiometer. This is a convenient convention to fol­low since a + E^ value represents a degree of oxidation.

ResultsThe Eh values of cells containing sterile liver infu-

sion broth of a pH of 6.6 without and with dyes are setforth in table I on the following page. The time potential curves of the broth without and with dyes are illustrated in figure 1. The E^ values and curves obtained on these cells are examples taken from determinations made on a large number of cells with different batches of the medium. It is interesting to note that the addition of the dyes to the medium in the cells causes no appreciable differ­ences between the E^ readings of the cells.

In table II is set forth a list of the values of cells containing medium, without and with dyes, after inoc­ulating one set with Br, melitensis strain 344, and another set with strain 608. Both strains are of human origin. The E^ values of cells inoculated with strain 344 plotted a- gainst time are compared in figure 2. There is very little difference between the speed and degree of reduction ofthe medium in each of the three cells as a. result of thegrowth of the organism.

The time Eh values obtained after inoculating liver infusion broth without and with dyes with Br. abortus strain 109 and strain 93 are present in taFIe TTl. The time potential curves for strain 109 are shown in figure 3. Strain 109 is of human origin and strain 93 is of bovine origin.

There is only a slight difference in the direction of the curve and final E^ value of the inoculated medium with­out dye and the one containing basic fuchsin. The time potential curve of the inoculated medium containing thionin takes an entirely different direction from that of the oth­er two curves. The change in the Eh value of the medium

17containing thionin would indicate that a slight reduction of the medium was in progress during the period of incu­bation.

Two sets of time E^ values of the medium inoculated with two strains of Br, suis are presented in table IV.Figure 4 shows the ETme potential curves of the medium

without and with dyes after inoculating with strain 533, Strain 533 is of porcine origin a.nd strain 570 is of human origin. The successive E^ values in the table and position of the curve plainly show that the organism causes little if any reduction in the medium in the presence of basic fuchsin. The reduction of the medium in the presence of thionin progresses just as rapidly af­ter inoculation with Br. suis as does the medium without the dye.

Table I, - Time E^ of Sterile Liver Infusion Brothwith and without dyes .

Broth without dye

Broth with Thionin

Broth with B. Fuchsin

TimeHours Eh Time

Hours Eh TimeHours Eh

0 0.3225 0 0.3179 0 ♦ 0.315620 ♦ 0,3189 30 + 0.3117 20 0.316554 ♦ 0.3162 46 0.3131 54 + 0.322468 + 0.3128 70 0,3137 68 + 0.327592 + 0,3098 94 0.3136 92 + 0.3296

116 + 0.3090 118 + 0.3165 116 ■f 0.3248140 + 0.3102 142 t 0.3162 140 0.3186164 ♦ 0.3108 166 + 0.3166 164 + 0.3176188 + 0.3116 188 0.3229

18Table II. - Time Eh of Liver Infusion Broth ^jjoculated

with Br. melitensis Strain 3445 Br. melitensis StrainInoculated wit

Broth without Broth widye Thionin

TimeHours

0

Eh 4 0.3153

TimeHours

0 t

24 + 0.2530 22

48 + 0.2062 46 ♦72 f 0.1772 70 f96 4- 0.1667 94 f

120 ♦ 0.1596 118 *

156 f 0.1537 154 t

168 + 0.1400192 4- 0.1335216 t 0.1330

Inoculated with0 + 0.2778 0 t

24 t 0.2431 24 ■f48 ♦ 0.2014 4872 + 0.1817 72 +96 ♦ 0.1752 96 ♦

120 + 0.1751 120 +156 * 0.1617 156 *168 t 0.1511 168 4192 ♦ 0.1522 192 t216 t 0.1556 216 +

strain 344 Broth withB. Puchsin

Eh0.3073

TimeHours

0

Bh + 0.3026

0.2459 24 ♦ 0.29790.2139 48 4 0.25590.1862 74 ♦ 0.22610.1692 96 + 0.20850.1599 120 4 0.18900.1566 146 ♦ 0.1761

170 4 0.1651192 4 0•1538216 4 0.1397

strain 6080.2798 0 4 0.26910.2305 24 4 0.26280.2173 48 4 0.24450.2052 72 f 0.21680.1951 96 4 0.20340.1942 120 4 0.18630.1920 156 4 0.16000.1816 168 4 0.16020.1746 192 4 0.16660.1624 216 4- 0.1489

19Table III. - Time Eh of Liver Infusion Broth Inoculated

with Br. abortus Strain 109; Br, abortus Strain 93

Inoculated with Strain 109Broth without Broth with Broth with

dye Thionin B* FuchsinFime Time *®h Time EhTours n Kours Hours

0 4 0.3130 0 0.2957 0 4 0.321820 4 0.2634 24 0,2897 20 4 0.316454 4 0,1995 48 0.2758 54 4 0.253192 4 0.1731 74 ' 0.2568 68 4 0.2248

116 4 0.1648 96 0.2520 92 4 0,2006140 + 0.1596 120 0.2423 116 4 0.1855164 4 0.1541 146 0.2225 140 + 0.1747188 4 0.1509 170 0.2291 164 4 0.1676

192 0.2199 188 t 0.1638216 0 .2 2 0 1

Inoculated with Strain 930 4 0.3099 0 0.2893 0 4 0.2810

30 4 0.2349 30 0.2696 24 4 0.272146 4 0.2305 46 0,2732 48 4 0.227770 f 0.2048 70 0.2703 72 4 0.206794 4 0.1833 94 0.2635 96 4 0.1923118 + 0,1694 118 0.2570 120 4 0.1661142 4 0.1558 142 0.2532 170 4 0.1518166 4. 0.1438 166 0.2488 192 4 0,1534

20Table XV. - Time of liver Infusion Broth Inoculated

with Br. suls Strain 533;.Br. suis Strain 570Inoculated with Strain 533

Broth without Broth with Broth withdye Thionin B, Fuchs in

Time Eh Time Eh Time EhHours Hours Hours0 0 o 3132 0 4.0.2497 0 4 0.2715

. 24 0.2632 24, 4 0.1481 24 4 0.258548 0.2035 48 4 0.1285 48 4 0.248272 0.1662 72 + 0.1207 72 4 0.250096 0.1465 96 4 0^1084 96 + 0.2514

120 0.1264 120 4 0.0897 120 4 0.2517146 0.1320 146 4 0.0982 170 4 0.2437168 0.1215 168 + 0.0897 192 4 0,2510192 0.1148 192 4 0.0866216 0.0939 216 4 0.0857

Inoculated with Strain 5700 0.3300 0 f 0.2940 0 4 0.307630 0.2509 30 4 0.1864 24 4 0.294646 0.2345 46 4 0.2062 48 4 0.279470 0 .2 1 2 0 70 4 0.1768 74 4 0,269494 0.1975 94 4 0.1539 96 4 0.2699

118 0.1795 118 4 0.1525 120 4 0,2698142 0.1719 142 4 0.1362 146 4 0.2689166 0 *1 6 1 6 166 4 0.1340 170 4 0.2704

192 4 0,2722216 4 0.2748

21

..— &

Fig. 1. - Time potential curves of sterile liver infusionbroth; ■*-*- without dye, -SMt- with thionin, -e— e-witli basic fuchsin.

22

2 + 4 a 72 36 12,0 /44Time in hours,

Pig, 2. - Comparison of time potential curves of liverinfusion broth inoculated with Br, melitensis strain 344; without dye, witJTthionin, -e— e— withbasic fuchsin.

23

4

t«

/

O 2 4 4 3 72 96 /SO /44 t&S /$ 2 240Time in hours.

Fig* 3* - Comparison of time potential curves of liver infusion "broth inoculated with Br. abortus strain 109;

without dye 9 -x--x- with thionin, -Q~ with basic fuchsin*

24

2.+ 48 72 86 /SO MTime in hours<

Fig. - Comparison of time potential curves of liver infusion broth inoculated with Br. suis strain 533;

without dye, with thionin, -o- -o with basicfuchsin*

25

24 48 72 96 tzo }4Time in hours.

Fig,5. - Comparison of time potential curves of liver infusion broth without dye; -++. inoculated with Br, abortus strain 109, X—x inoculated with Br. suis strain 533, inoculated with Br. melitensis strain"S44.

26

4

3

. <• ^.2

/9

0 £4 46 /£ $e> /ao 14 + i£& !$£. QlI 34-0Time ih heats*

Fig. 6. - Comparsion of time potential curves of liver infusion "broth containing thionin; ■■ «■■-» inoculated with Br* abortus strain 109,*rjh inoculated with Br* suia strain 533, -o ...q- inoculated with Br. melitensis strain 344*

21

Fig. 7. - Comparison of time potential curves of liver infusion broth containing basic fuchsin; '■ t—+ ■ inoculated with Br. abortus strain 109, inoculated with Br.suis strain 533, inoculated with Br. melitensisstrain 344.

28A comparison of the reducing capacity of the three spe­

cies of Brucella in liver infusion "broth without and with the dyes is illustrated more clearly in figures 5, 6, and 7. A comparison of the time potential curves resulting^ from the growth of each of the three species in liver in­fusion broth is seen in figure 5. There is little variation between the direction of the three curves or individual

values. The final E, value for each of the three spe­cies varies to only a slight degree. The addition of thio- nin to the medium considerably retards the growth of Br* abortus and as a consequence checks the reduction of the medium as illustrated in figure 6* While Br, melitensis does not reduce the medium in the presence of this dye as rapidly or to the same degree as does Br. suds, there is on the other hand, a noticeable difference in its reduc­ing capacity over that of Br, abortus.

The differences in the reducing"”capacities of the three species in the presence of basic fuchsin is well il­lustrated in figure 7. The reducing capacity of Br. suis is held almost completely in check while that of the other two species is not affected by the dye.

DiscussionThe foregoing data indicate that sticks of special

graphite (Acheson) when properly prepared are suitable as electrodes for measuring the oxidation-reduction intensity of bacteria when grown in a sterile liquid culture medium maintained under aerobic conditions. The potential of sterile beef liver broth after a preliminary incubation period of 4 to 7 days, lies between - 0.31 and - 0.34 volt.

The time potential curves of the medium during the growth of the three species of Bruce 11a do not vary to a great degree. During the first 48 hour growing period there is a, decline in potential to + 0,2 volt. At this point the curves separate and as the time of incubation increases the separation becomes more ma.rked, Br. suis grows more rapidly than the other two species of Brucella and as the curve indicates, has much greater reducing ca­pacity than the other two species. The rate of reduction due to the growth of Brucella proceeds at a much slower pace than that which has been observed for many bacteria when measured in oxidation-reduction systems under aerobic conditi ons,

Tn view of the fact that there has not been found a marked variation in the reducing capacity of the three species of Bruce 11a after seven days of incubation in liver infusion broth, one would presume that there is not a marked difference in the metabolism of the organisms in the same medium.

The differences in metabolism of the organisms become apparent when bacteriostatic dyes are added to the liquid medium. The addition of a suitable amount of thionin to the

29medium retards or prevents its reduction when inoculated with Br, abortus . The presence of basic fuchsin holds the reduction of the medium in check when inoculated with Br. suis, Neither dye, to any extent, prevents the medium from Toeing reduced by the growth’" of Br. melitensis.

Each of the cells were examined at the end of the growth period for viable organisms, but no attempt was made to determine the number on a quantitative basis. It was rare to find Br. abortus alive at the end of the in­cubation growth period in the cells to which thionin had been added. Br. suis was always recovered from the cells containing basic fuchsin although the potentials of the cells indicated the absence of growth activity.

In those cells in which growth occurred, the pH shift­ed from 6.6 to 7. There would be very little difference in the time potential curves were they corrected for the change in pH of the medium which occurred during the growth of the bacteria.

The differences recorded in the metabolism of the spe­cies of Brucella in a liquid medium in the presence of bacteriostatic dyes by means of oxidation-reduction poten­tial measurements, serve to confirm that which is already known of the differences in their growth behavior on a sol­id medium in the presence of the same dyes (l).

Summary1, Electrodes made of graphite (Acheson special) are

suitable for measuring the oxidation-reduction reactions of aerobic bacteria in liquid media under aerobic condi­tions. ‘

2. The reduction potentials of Brucella in beef liv­er infusion broth under aerobic conditions show a negative drift that attains the value: E^ + 0.15 to 0.1 volt at the end of an incubation period of eight days, Br. suis shows a slightly more negative drift than the other two species.

3* abortus in the presence of thionin is unableto reduce the potential of the medium,

4* Br* suis in the presence of basic fuchsin is un­able to reduce the potential of the medium.

5. The presence of neither thionin or basic fuchsin retards the negative potential drift of the medium caused by the growth of Br. melitensis.

30References

1* Huddleson, I. ForestThe differentiation of the species of the Genus Brucella.Technical Bulletin No. 100, Michigan State College, East Lansing, Michigan.

2» White, Walter P.Leakage prevention by shielding especially in poten­tiometer systems.Journal of American Chemical Society, p. 2011, vol. 36, 1914.

3. Lewis, Gilbert F., Thomas Brighton and Reuben L. Se-■ bastian. A study of hydrogen and calomel electrodes.* * Journal of American Chemical Society, p. 2245, vol.39, 1917.

4. Flexner, Louis B,, and E. S. Guzman Barron Oxidation-reduction potentials at carbon and tungsten electrodes. • ‘ ■ ■ **Journal of American Chemical Society, p. 2773, vol.52, 1930.

Ill - The Preparation and Use of the Platinized-Graphite Electrode

In a search for a more suitable electrode for meas­uring the oxidation-reduction potential of liquid culture media for the growth of bacteria, considerable time was devoted to the study of the behavior of graphite electrodes. In the course of this work it was decided to determine the suitability of this type of electrode, platinized as a re­versible electrode. In addition to this study a more exten­sive comparative study was formulated of platinized plati­num, platinized graphite, and graphite as electrodes for electrometric titrations (l).

Apparatus. The apparatus that was employed in connec­tion with the work on hydrogen reversible cells was of precision type and quality. A water thermostat'was used that maintained the temperature within 0.005 C. of the working temperature, 25 C.

A type K 9 Leeds and Northrup potentiometer was used. This instrument and the galvanometer were very carefully shielded according to the method described by White (2).The metal tank of the thermostat and the copper shields which surrounded the cells were connected to the main shield. All alternating current circuits were removed as far as possible from the immediate vicinity of the poten­tiometer system. The galvanometer was a Leeds and Forthrup precision type. The distance between it and the reading telescope and scale was about 5 ft. The sensitivity of the potentiometer system was such that one millionth of a volt difference in potential would cause a deflection in the galvanometer system of 1 mm. This sensitivity was obtained on the 16 millivolt scale of the potentiometer and in read­ing the cell of highest resistance.

The cell finally used in these experiments was of very simple design. A special flat bottom freezing point tube which was closed with a number 8 rubber stopper was found satisfactory, holes were made in the stopper for gas inlet and exit tubes and also for four electrodes. The four electrodes, two platinized platinum and two platinized graphite, were conveniently spaced around the gas inlet tube which was placed in the center of the rubber stopper.A hole was also provided for the exit tube which led into a trap.

The hydrogen was produced by electrolyzing a 10% so­lution of FaOF in a generator similar to that described by Clark (3). The hydrogen was purified by passing it over an electrically heated platinum spiral. A piece of heavy cap­illary glass tubing about 1 meter long was placed between the generator and the hot platinum wire as a safety device. The hydrogen was brought into the cells from the generator and purifier through glass tubing coupled together with short rubber connections.

32The apparatus used in connection with the electro­

metric titrations was that commonly employed for this Kina of work. It consisted of a heeds and Uorthrup student^po­tentiometer, motor driven stirrer, and a burette. A sil­ver, silver chloride electrode in 1 molar potassium chlor­ide solution served as the reference electrode*

Materials. The hydrochloric acid solutions were made from Baker’s chemical pure acid as described by Foulk and Hollingworth (4). The solutions were IF, 0.1F, and 0.01F in hydrogen chloride. The sodium hydroxide solutions were made up in the usual way and titrated with the above acids using phenolphthalein as the endpoint indicator. The plat­inum electrodes were made of platinum foil# The graphite electrodes were Acheson special graphite rods 1/4 inch in diameter and 6 inches long. This grade of graphite is free from interfering impurities and is more dense than other grades.

The electrodes were platinized by electrolyzing in a 7>% chloroplatinic acid solution containing a trace of lead acetate (about 0.02^), This solution was prepared as follows: Scraps or pieces of platinum were dissolved in concentrated chemically pure hydrochloric acid by vigor­ously electrolyzing the platinum as the anode. Once the platinum begins to dissolve it will continue slowly with­out further electrelyzing if kept tightly stoppered. The platinum black on platinized electrodes was rapidly re­moved by electrolyzing in concentrated hydrochloric acid. In one case the platinum was dissolved in the usual way in aqua regia. The solution of platinum in concentrated hydrochloric acid was placed in a casserole and boiled down to a syrupy consistency over a Bunsen burner. From time to time small amounts of concentrated hydrochloric acid were added to prevent precipitation. If precipita­tion did occur during this process, sufficient concen­trated hydrochloric acid was added to the hot solution to redissolve the precipitate. The rich orange solution was reduced to dryness over a steam bath. The resulting mass had a very deep orange color. The residue was taken up in the required amount of distilled water containing a trace of lead acetate and brought near the boiling point over a flame. The precipitate thus formed was redissolved by adding concentrated hydrochloric acid ( a drop at a time from a capillary pipette) to the solution until nearly all the platinum was back in solution. The addition of acid was stopped just before the lead precipitated as the chloride. The cool solution was filtered, decanting as much as possible. The small amount of precipitate which was not redissolved was dissolved in concentrated hydro­chloric acid and used as an electrolytic strip for plati­nized electrodes. Cleanliness was found to be of great importance throughout the above procedure. At first de­posits from this solution were of a d.ark gray color in­stead of black. However, it was found that such deposits

33were as satisfactory as the black*

Procedure; The platinum black was removed from the electrodes by electrolyzing in chemically pure hydrochlor­ic acid. The platinum electrodes were heated to a white heat in the reducing cone of a Bunsen flame preparatory to platinizing. The platinum electrodes were platinized by making them the cathode and electrolyzing for 3 or 4 min­utes in the platinizing solution. The graphite electrodes were heated to a dull red and immediately placed in cold distilled water. They were further cleaned with a fine grade of steel wool and polished with a new cheese cloth. They were placed in distilled water and slowly boiled from four to six hours. At the end of this time they were allowed to cool under distilled water, wrapped in clean paper, and dried over night at 110°C. The graphite elec­trodes were platinized from 5 to 10 minutes. The anode was a spiral of platinum wire around the graphite elec­trode and one-half inch distance from its surface. The current passed was sufficient to produce a vigorous evo­lution of hydrogen. Since platinized electrodes should never be allowed to dry before using, they were immedi­ately rinsed with and placed under distilled water. The electrodes and gas inlet tube were very carefully placed in the stopper used in closing the top of the cell.. The electrodes were rinsed several times with the solution to be used in the cell. The cell was filled to the proper level and the stopper carrying the electrodes and inlet tube was carefully fitted to the cell which was placed in its shield in the thermostat. The trap assembly was placed on the gas exit of the cell and the flow of hydrogen started. Pure hydrogen was continuously passed during the life of the cell. No measurements were made until the gas had passed for 6 hours. Daily readings were made on all the combinations between the four electrodes (two plati­nized platinum and two platinized graphite) until check readings were obtained.

The electrometric titrations were conducted in the usual manner. No special precautions were taken outside of that ordinarily employed in such work. The cell was open to atmospheric air. A 20 ml, sample of the reagent to be titrated was pipetted into a 250 ml. beaker and di­luted to 50 ml. for all titrations reported.

Results of StudyThe tabulated results of the experiments on the

hydrogen reversible electrode are given in Tables I, II,III, IV, V, and VI which follow. The readings from one cell for four days are given for each concentration of hydrochloric acid and sodium hydroxide studied..

Theoretically, the potential difference between the electrodes in all cases represented in Tables I, II, III,IV, V, and VI is exactly zero. That this is true comes from the following consideration. The electrodes of these

34cells are hydrogen gas electrodes* The reversible^reaction at the surface of a hydrogen electrode is 2e.The finely divided platinum, covering the surface of the electrode, adsorbs the gaseous hydrogen and acts^as a cat­alyst for this reaction,, The only differences which re­main between platinized platinum and platinized graphite, when used as a gas electrode, are the differences in the adsorptive properties of platinum and graphite* Tables I, IX, rxr, V, and VI show that very little adsorption of either a positive or negative ion had taken place because all the values are very close to the theoretical value. Table IV shows that in all probability some adsorption of the sodium ion on the graphite had taken place because there is a tendency for the platinized graphite to become more positive than platinized platinum. This adsorption was aided by the removal of minute particles of platinum black due to the soapy action generated by the bubbling of the gas through the alkali*

Key to symbols used in table. Pt-Pt difference in volts between the two platinized platinum electrodes*

Ptp-CPtjj difference in volts between the right plat­inized platinum electrode and the right platinized graph­ite electrode.

Pt^-CPt^ difference in volts between the right plat­inized platinum electrode and the left platinized graphite electrode,

CPt-CPt difference in volts between the two plat­inized graphite electrodes.

Pt̂ --CFtpj difference in volts between the left plati­nized platinum electrode and the right platinized graph­ite electrode.

Ptr-CFtj^ difference in volts between the left plati­nized platinum electrode and the left platinized graphite electrode.

R-*' right electrode connected to the positive terminal of the potentiometer,L* left electrode connected to the positive terminal of the potentiometer.C+ platinized graphite electrode connected to the

positive terminal of the potentiometer.C- platinized graphite electrode connected to the

negative terminal of the potentiometer.The results of the electrometric titrations are shown

graphically in Figures 1 to 6 inclusive which follow.These curves are composites of triplicate electrometric titrations. A silver, silver chloride electrode in one mo­lar potassium chloride solution served as the reference electrode. The same standardized reagents were used in making all the electrometric titrations represented in Figures 1, 2, and 3. The experiments represented in Fig­ures 4, 5, and 6 were conducted in a similar manner.

Hours in Thermostat

*896120144

35Table I

Hydrogen Cell of IFP t - P tVolts JU 0*0000020.000000 0.000000 0.000000

CPt - CPtvolts

PtR-CPtRvolts

C+ 0.000015C* 0.000021C+ 0.000030C+ 0.000039PtL- CPtRvolts

HC1-PtR-CPti

volts 0+ 0.000006C + 0.000004C+ 0.000004G+ 0.000006

« r CPtivolts

72 KC + 0 <,000021 C + 0.000019 C- 0.00000796 HC + 0.000021 c+ 0.000024 C + 0.000004

120 RCt 0.000034 c+ 0.000030 Cf O • o o o o o

144 RC + 0.000032 c+ 0.000039 c+ 0.000006

Hours in Thermostat

Table II Hydrogen Cell of 0.1F

P t - P t

24487296

voltsPtR-CPtRvolts

HC1PtR -CPtt

volts24 It* 0.000010 c- 0.000010 c+ 0.00003048 L+ 0.000004 0.000000 c+ 0.00000472 I>+ 0.000003 c+ 0.000002 c+ 0.00000596 L+ 0.000004 0.000000 c+ 0.000004

CPt - CPt PtL- CPtR PtL- CPtLvolts

LC+ 0.000039LC+ 0.000004I.C+ 0.000002LC+ 0.000004

volts C- 0.000020C- 0.000004

0.000000C- 0.000004

volts C+ 0.000018

0.000000C+ 0.000002

0.000000

36Table III

Hydrogen cell of 0.01P MCIHours in Pt -Pt Pt^-CPtpj PtR-CPtj^Thermostat volts volts volts

24 0.000000 C- 0.000002 C+ 0.00000248 R+ 0.000001 C+ 0.000004 C* 0.00000472 0.000000 C+ 0.000004 C+ 0.00000496 0.000000 C+ 0.000004 C+ 0.000004

CPt - CPt PtL-CPtR PtL-CPtL

24volts

LC+ 0.000004volts

C- 0.000002volts

C+ 0.00000248 0.000000 Cf 0.000005 C+ 0.00000572 0.000000 C* 0.000004 C+ 0.00000496 0.000000 C+ 0.000004 C* 0.000004

Table IVHydrogen cell of 1.004P MaOH

Hours in Pt - Pt Pt^-CPt^ Pt -CPtTThermostat R

volts volts volts24 R* 0,000002 C- 0.000005 C- 0.00001548 R+ 0.000003 C+ 0.000017 04 0.00007372 R+ 0.000005 C + 0.000040 C ■+ 0.00012196 R4 0.000005 c+ 0.000054 c+ 0.000174

CPt - CPt Pt’I-CPtK Pt,£-CPt-jrvolts volts volts24 RC + 0.000010 C- 0.000007 C- 0.000017

48 LC+ 0.000052 c* 0.000020 c+ 0.00007872 LC + 0.000081 c+. 0.000045 C4 0.00012696 I.C4 0.000120 C4 0.000051 CU 0.000170

Hours in Thermostat

24487296

37 Table V

Hydrogen cell of 0.0998F HaOH Pt -Ptvolts0.0000000.0QQ0000.000000 0.000000

CPt - CPtvolts

PtR-CPtRvolts

C* 0.000035C* 0.000021

0.000000C+ 0.000010PtT~CPt„L Rvolts

PtR-CPtLvolts

C+ 0.000102C4 0.000045C+ 0.000005C4 0.000010Pt̂ -CPtj,

volts24 LC + 0.000066 C4 0.000035 C4 0.00010248 LC+ 0.000024 C4 0.000021 C4 0.00004572 XC+ 0.000005 0.000000 C+ 0.00000596 0.000000 C4 0.000010 C4- 0.000010

Table VI Hydrogen cell of 0.00992F HaOH

Hours in Thermostat

24487296

24487296

Pt - Ptvolts

R* 0.0000040.000000 0.0000000.000000

CPt - CPtvolts

RCt 0.000130RC4 0.000003LC+ 0.000005LC4 0.000010

PtR-CPtR PtR-CPtLvolts

C*f 0.000094C- 0.000006C- 0.000010C- 0.000020PI^-CPtRvolts

C4 0.000100C- 0.000006C- 0.000010C- 0.000020

volts C- 0.000036C- 0.000009C- 0.000005C- 0.000010Ptj.-CPtj.

volts C- 0.000030

0.000009C- 0.000005C- 0.000010

38

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Pig. 5. - A composite curve of triplicate electrometrictitrations on acidified O.ltf FeSO^KH^JSO^ with KE&1O4 ,using a platinized graphite electrode.