the effects of certain chemical compounds upon the

THE EFFECTS OF CERTAIN CHEMICAL COMPOUNDSUPON THE COURSE OF GAS PRODUCTION BY

BAKER'S YEAST'

SARA E. BRANHAMDepartment of Bacteriology, School of Medicine and Dentistry, The University of

Rochester, Rochester, New York

Received for publication April 1, 1929

INTRODUCTION

During a recent investigation of a method for standardizingantiseptics on the basis of their ability to inhibit fermentation byyeast, observations were made on the effects of chemical com-pounds upon the course of gas production by these organisms.The method used was a modification of the technic employedby Pilcher and Sollmann (1922-1923) and by Peterson (1926)whose work was based upon Dreser's (1917) suggestion that theinhibition of gas production by certain compounds acting uponyeast could be used as a measure of the antiseptic efficiency ofthese compounds. As the apparatus usedby these authors permit-ted large and undetermined amounts of the gas to escape aroundthe outside of the fermentation tubes, it was necessary to changetheir procedure by employing a vessel which would collect all ofthe gas. The introduction of a simple gasometer at once en-larged the scope of the method, making it a more accurate meansof determining the inhibiting effect of antiseptics, and at thesame time converting it into a means for obtaining informationin regard to some phases of the biology of yeast. A comparisonof antiseptics on the basis of the results obtained through thecollection of the total gas evolved has been published elsewhere(Branham, 1929). The chief purpose of this paper is to report

1 This investigation was aided by a fellowship granted by the FleischmannCompany.

247

on February 17, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

SARA E. BRANHAM

observations of more general physiological interest in connectionwith the action of chemicals upon the rate and volume of gasproduction by Saceharomyces cerevisiae in solutions of sucrose.

HISTORICAL SKETCH

A group of workers at Greifswald in 1884-1888 were among thefirst, after Pasteur, to study the effects of chemicals upon yeastfermentation. They observed the rate and amount of carbondioxide production in mixtures of yeast cells and sugar solutionsto which these substances had been added. Hoffmann (1884),Thol (1885), Gottbrecht (1886), and Schulz (1887, 1888) pre-sented extensive observations made with thallium tartrate, mer-curic chloride, iodine, bromine, arsenic acid, chromic acid, formicacid, and salicylic acid, showing that high dilutions caused astriking increase in fermentative activity, whereas carbon dioiddeformation was markedly inhibited by greater concentrations.More recently Peterson (1926) found a stimulation of carbondioxide production in high dilutions of a few of the mercury com-pounds with which he worked. Harden and Young (1911) foundthe same phenomenon in the action of arsenates and arsenitesupon yeast juice. The enzymes in the juice are unaffected bymany substances that are very toxic for the living cells, so thatresults obtained with this type of material are not consistentlycomparable in this respect with those obtained with whole yeast.Joachimoglu (1922) was unable to confirm the observations

of the Greifswald investigators in that he found that smallamounts of mercuric chloride, phenol, and quinine did not stimu-late fermentation in the mixtures of sugar and yeast which heused. He agreed that some substances apparently cause an in-creased activity in high dilutions, but did not consider that theseobservations could be generally applied.Many other investigators have studied the toxicity of certain

compounds for yeasts. Lindner and Grouven (1913) found mer-curic chloride, fluorides, formalin, and antiformin to be regularlytoxic for yeasts in all dilutions studied. Dreser (1917), Zernerand Hamburger (1921), and Pilcher and Sollmann (1922-1923)have used the inhibition of carbon dioxide production by yeasts

248


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

COURSE OF GAS PRODUCTION BY BAKER'S YEAST

as a criterion for comparison in their studies of silver compounds.Euler and Emberg (1919) noted appreciable changes in the rateof growth and the cell composition of yeasts under the influenceof various hydrogen ion concentrations. Somogyi (1921) foundmany organic and inorganic acids to have a harmful effect uponyeasts. Euler and Nilsson (1925) and Brown and Wikoff (1927a)have found hydrogen peroxide to prevent fermentation to amarked degree, and these latter workers also found (1927b)hexyl resorcinol powerfully toxic for yeasts. Myers (1927) foundthat certain volatile oils, particularly thymol, exerted a markedfungicidal action. Dann and Quastel (1928) have made asystematic investigation of the effects of a number of glucosidesand polyhydric phenols on yeast fermentation. They foundthat allyl alcohol, acrylic acid, and phloroglucinol inhibited fer-mentation, both by the living cells and by zymin, whereas phlor-izin inhibited zymin, but not living microorganisms.The methods used to estimate the effect of the compounds

upon the activity of yeasts have varied widely. Schulz (1887,1888) measured the pressure of the evolved carbon dioxide bymeans of a mercury manometer attached to the apparatus whichhe used. Slator (1906, 1908) employed a method similar to thatof Schulz. Euler and Lindner (1915) used a Meissl ventilationvalve which allowed the carbon dioxide to escape, but retainedthe water vapor by causing the gas to bubble through sulphuricacid. The losses in weight were calculated as carbon dioxideevolved. Alwood (1908) adopted a similar apparatus which heused in the same way. Joachimoglu (1922), described an appara-tus in which the amount of carbon dioxide produced was deter-mined by weight. Dreser (1917) and Pilcher and Sollmann(1922-1923) collected the carbon dioxide in an inverted tubeand then measured the column of gas in linear centimeters.Dann and Quastel (1928) measured the carbon dioxide in cubiccentimeters by means of an apparatus originally devised by Try-horn and Jessop, which, while accurate, seems too elaboratefor general or large scale use. Lindner and Grouven (1913) usedthe killing power of a compound as a criterion. Zerner andHamburger (1921) also used killing power, but determined it in

299


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

SARA E. BRANHAM

a different way. Euler and Nilsson (1925) measured the totalyeast and total sugar present in samples withdrawn from theirtest mixtures at definite intervals. Similarly Balls and Brown(1925) and Brown and Wikoff (1927a, 1927b) made regularlytimed estimations of total solid, total sugar, weight of yeast crop,and rate of inversion of sugar. Somogyi (1921) determined theamount of sugar in the fermenting yeast-glucose-acid mixtureswhich he was investigating. Quite different from these methodswas that of Gutstein (1927) who made a microscopical study ofthe morphology of yeast cells treated with the dyes and salts ofthe heavy metals which he studied.

Interesting studies have been made of the chief factors whichcontrol the rate and amount of fermentation by yeasts. Byusing a bottle connected with a manometer for measurement ofgas, and making readings every five minutes, Slator (1906, 1908)found the velocity of fermentation to be directly proportional tothe concentration of the yeast, and independent of the concen-tration of the sugar between 1 and 10 per cent. Sucrose, glucose,and levulose were fermented at the same rate by the breweryyeasts which he used. Slator (1913) stated that fermentation,as well as growth of cells, is logarithmic. Tammann (1889)found that the course of fermentation, when observed from be-ginning to end, did not fall on a logarithmic curve, but morenearly approached a straight line. Balls and Brown (1925)reported that yeast growth is logarithmic only when the weightof the total yeast is considered instead of the number of cells.Kohler (1920) considered the sugar concentration to be a con-trolling factor in growth velocity, and claimed that the processes,both of carbon dioxide production and growth, occurring inalcoholic fermentation follow a rhythm; i.e., an increased ratefollows a decreased rate according to the variation in sugar andalcohol content of the medium. The studies of Lindner andGrouven (1913) and of Zerner and Hamburger (1921) indicatethat various yeasts show great differences in activity, as well as inresistance to toxic substances, and that there is a direct rela-tion between the amount of each yeast used and the concentra-tion of such substances required to kill or inhibit its action.

250r


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


Joachimoglu (1922) called attention to the fact, doubtless noticedbymany, thatfermentation in aseries of flasks, containing thesameamount of the same mixture, may not be equal in degree or rate.

EXPERIMENTAL WORK

The apparatus used in the studies reported in this paper isillustrated in figure 1. The larger tube (a), graduated for 30 cc.,

FiG. 1

is fitted with a two-hole rubber stopper carrying two pieces ofglass tubing; one extending to the bottom of the test tube, andone, bent at right angles, which barely reaches the inner surfaceof the stopper. The larger graduated tube (a) is filled with water,inverted, and fitted by means of a second rubber stopper intothe smaller tube (c) containing the yeast-sugar solution. Thenthe completed apparatus is placed in a water bath deep enoughto cover the opening of (b). The amount of gas can be read in

I

c

251

a

rb

c


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

SARA E. BRANHAM

cubic centimeters by means of the graduations on (a). Specialcopper racks were made for these simple gasometers so that num-bers of them could be handled at one time.

Before attempting to observe the effects of chemicals uponfermentation it was necessary to determine the most suitableconcentrations of yeast and sugar for regular use. With someproportions more gas was evolved than could be convenientlycollected; with others, fermentation proceeded so slowly that itwas difficult to determine the end-point and to keep other factorsconstant.

In order to find suitable proportions of yeast and sugar forsatisfactory experimentation, various sets of 12 tubes each wereset up with different combinations of yeast and sugar: Two, 4, 5,and 10 per cent suspensions of yeasts were used with 1, 2, 5, and10 per cent solutions of cane sugar. Fermentation did not pro-ceed at a uniform rate in all tubes containing the same amount ofthe same yeast and sugar mixture; i.e., the apparatus containingthe greatest amount of gas after 15 minutes would not regularlybe the one containing most after thirty or forty-five minutes, orone hour. But when fermentation was allowed to proceed tocompletion the total amount of carbon dioxide formed in eachapparatus was found to be approximately equal. With 10 percent yeast and 1 per cent sugar the reaction proceeded quicklyand was practically finished within 1 hour. The total gas formedin these tubes was relatively uniform, and the volume was easilycontained in gasometers of the size described. These proportionswere used in all of the following studies.The experiments were repeated with many samples of Fleisch-

mann's yeast, including both one-pound bricks, containing 1 percent starch as a binder, and the usual small cakes, with 2 percent corn starch. Both glucose and sucrose were used; therewas no appreciable difference in the results obtained, so thatMerck's "Highest Purity-C.P." sucrose was used throughoutthe rest of this study.

Individual samples of yeast, through shipping and storage,necessarily vary in activity. Consequently, if the effect ofdifferent chemicals upon the course of gas production was to be

252


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


studied, it was obviously important to observe the normal courseof carbon dioxide evolution with every sample of yeast used.As a matter of fact, not only with every sample of yeast, butevery time that a test was made, a series of tubes containingonly the 10 per cent yeast and 1 per cent sugar mixture wasincluded. Besides serving as a check on the variability of theyeast used in each experiment, this obviated the necessity formaking corrections for daily changes in barometric pressure whichwould be theoretically necessary for absolute accuracy.

There was always some residual air in the small tubes (c)containing the 10 cc. of yeast-sugar mixture. The temperatureof the water bath caused this to expand and also caused much ofthe dissolved gases to separate out so that readings of 0.5 to 1.0cc. were always obtained in the large tubes (a) whether fermenta-tion had occurred or not. By taking prelimin,%ry readings afterthe first five minutes of incubatio'n these amounts could be readdirectly and corrections made by subtracting them from thetotal amounts of gas formed. A "lag" of about eight minutes,which occurred before carbon dioxide began to be collected intube (a), gave ample time for these preliminary readings to bemade.No corrections were made for dissolved carbon dioxide or for

carbon dioxide pressure within the tubes. Since the gasometerswere of approximately the same size, and uniform quantities ofyeast and sugar were put into them, the dissolved carbon dioxideand carbon dioxide pressure may be considered as practically aconstant in any given series of tubes. However this would notbe true of other series, particularly if they contained varyingdilutions of chemicals that would alter the pH of the medium,thereby changing the solubility and rate of liberation of carbondioxide, since both of these factors are affected by the hydrogenion concentration. The influence of hydrogen ion concentrationshould be taken into consideration when absolute accuracy isdesired in comparing the action of different dilutions of such com-pounds.An investigation of the effect of certain chemicals upon yeast

fermentation was now begun. The materials studied were of

253


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

SARA E. BRANHAM

widely different composition, and were chosen with reference tothe development of a satisfactory method for standardizing an-tiseptics reported in another paper (Branham, 1929). Thesecompounds were as follows: 3 samples of mercuric chloride, 3 ofmercurochrome, 3 of silver nitrate, 2 of phenol, lysol, tricresol, 2creosote oil preparations, hexyl resorcinol, metaphen (4-nitro-3,5-bisacetoxymercuri-2 cresol), formaldehyde, copper sulphate,tincture of iodine, chloramine-T (sodium toluene-p-sulphonchlor-amine), sodium hypochlorite, ethyl alcohol, sodium chloride, andhydrochloric acid.The stock solutions of the chemicals studied were made with

care. The solid compounds were weighed on accurate balancesto within 0.1 mgm. of the theoretical amount; solutions weremade in standard volumetric flasks; and calibrated pipettes wereused in making the desired dilutions. Final concentrations of10 per cent yeast and 1 per cent sugar were used throughout.The yeast suspensions and the sugar solutions were freshly madein distilled water each day, and were kept at 3 to 6°C. To setup the tests, equal parts of 20 per cent yeast and 2 per cent sugar,with the desired amount of test chemical were placed in a smallflask, and the mixture distributed in 10-cc. amounts into thesmall tubes (c). Six tubes of each dilution of every chemicalwere routinely used in all experiments. These tubes, and alsothe control series referred to above, which contained only yeastand sugar, were all placed in a water bath at 38°C. The timeelapsing between the mixing of the yeast, sugar, and chemical,and the immersion of the series of tubes in the water bath wasapproxmately two minutes. Readings were made after fifteenminutes, thirty minutes, one hour, and every hour thereafteruntil fermentation ceased. Then curves were plotted with thereadings obtained, each curve representing the average of 6 tubescontaining the same material. It is impossible to present herecharts which illustrate the action of all of the chemicals includedin this study. Much of the information gleaned from the curveson these charts is presented in a condensed form in the accom-panying table. (See table 1.)The effect of mercuric chloride upon yeast fermentation is

254A


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

TABLE I

The effects of varying dilutions of sixteen chemical compounds upon the course of CO2production by yeastAMOUNT CO2 PRODUCED

C A U PEB UNIT OFTIME_N Time inhoursR

Stimulation of fermentationduring first 30 minutes in1:30,000 to 1:80,000

Excess of total gas over thatin control in 1:15,000 to1:80,000; greatest in1:20,000

Excess of total gas over thatin control in 1:1,000 to1:4,000

Practically no increase infermentation over thecontrol in any dilutions

Excess of gas over that incontrol in 1:250 to 1:600.Greatest total amount ofgas in 1:350. Fermenta-tion much delayed in 1:200to 1:300

Excess of gas over that incontrol in 1:400 and 1:500.Greatest amount in 1:500

1

2

HgCl2

Mercuro-chrome

cc.

08181918151414

3 1 AgNO,

cc.

09181918151414

047121116171612

05111212141212

04171618181512

5,00010,00015,00020,00030,00050,00080,000Control

5080100200500

1,0002,0004,000

Control

6,0008,00010,00015,00020,00030,00060,000Control

100200250300350400600

Control

200300350

cc.0291313131314

03791010111411

013711121212

0134.5691112

00.54.0

cc.

06151917141414

047101013141511

02101212131212

0281016161512

01.09.0

cc.0

10181918151414

047131116171612

08

111212141212

07

21182119

-1512

0

047111116171612

03111212141212

031215.517171512

029.0

4 Phenol

5 Lysol a

255


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

TABLE 1-Continued

Po AMOUNT C00 PRODUCEDa PUR UIT OF TIME

e P CIRNCEICAL DIUTION Time in hours RAMRAPS

K _ _ 2 | 3 | 4 1

Late increase in fermenta-tion in 1:500 to 1 :1,000

0 Increased fermentation in4 1:5,OOO to 1:10,000

Increased fermentation. in1:5,000 to 1:10,000, great-est in 1: 8,000, but quickestin 1:10,000

Very marked late increasein fermentation in 1:600to 1:1,000

Some increased fermenta-tion in 1:10,000 to 1:20,000

Very slight, but definite fer-mentation, regardless ofdilution, between 1:80and 1 :1,000

cc.

o

cc.

a

c4

181612

0

5

15

5

6

7

8

9

10

Lysol

Tricresol

Hexylre-sorcinol

Metaphen

Formal-dehyde

Coppersulphate

0

400500

Control

300400500

1,000Control

2,0002,5003,0008,00010,000Control

2,0003,0003,5004,0005,0006,0008,00010,000Control

200400600800

1,000Control

801,ooo2,0006,0008,00010,00020,000Control

cc.

8.017.0L5.0

0281311

025141412

0012.558111510

0132

1112

22348111213

cc.

16.0l22.0l16.0

05161713

038161612

00.5491316201810

031291912

224612131313

cc.

18.022.016.0

0

0411171612

01.04.510.01517211810

0516191912

224

151413

101 10

06

20201912

2

2

4

16

13

0

20211912

224

16

13

256I


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

_ AMOUNT C00 PRODUCED0 PER UNIT OF TIME

M; CHEMCAL DIUTON Time in hours ARK

54Z

cc.

0717171314

022

189

cc.

01020171314

0

1

131 13

0

0371111

091225319

0

047

1112

091226319

Delayed fermentation inlower dilutions. Late in-crease in total gas formedin 1:500 to 1:1,000

Vigorous fermentation af-ter long latent period in1:S,000

Active fermentation afterlong latent period in lowerdilutions

Slightly increased fermenta-tion in 1:10

Pronounced stimulation offermentation by all dilu-tions higher than 1:10

* The amount of free available chlorine in different samples of sodium hypo-chlorite varies widely, and the results obtained here would by no means be con-stant with other samples. With the sample represented in this table a dilution of1:100 represented only 50 parts per million of free available chlorine.

257

11

12

13

14

15

Tinctureof iodine

Chlora-mine-T

Sodiumhypo-chlorite*

Ethylalcohol

HCI

250300500

1,00010,000Control

2,0005,0007,0008,00010,00020,000Control

5075100200500

1,0005,000

Control

( 2%) 5(10%) 10Control

N/10N/20N/50N/100Control

5102050100

Control

CC.

00481113

016891312

000910111313

01212

00446

021224299

cc.

0012161314

071012111313

0351011111413

01513

01677

081225309

cc.

0415161314

016

121413

069

13

01713

027109

091225319

16 1 NaCl

1 ADJLJV 1--tOoT&c4uv


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

SARA E. BRANHAM

shown in chart 1, which records observations made over a periodof five hours. Carbon dioxide production was inhibited com-pletely in dilutions up to 1:5000. Although a tremendous lagoccurred in dilutions of 1: 5000 to 1: 10,000, an appreciableamount of gas was ultimately evolved. Fermentation was re-tarded in dilutions up to 1: 80,000, but inhibition in some of thesehigher dilutions of mercuric chloride was only temporary for thetotal amount of carbon dioxide ultimately formed was oftenmuch greater than that in the control tubes containing only yeast

20I,,

Hrs.

'4

CHART 1. EFFECT OF VARYING DILUTIONS OF MERCURIC CHLORIDE UPON CARBONDIOXIDE PRODUCTION BY 10 PER CENT YEAST AND 1 PER CENT SUCROSE

and sugar. It is evident from a study of these curves that thisincrease in the total gas, above the amount formed in the tubeswithout mercuric chloride, occurred after the normal reaction inthe control series was practically finished. Readings made justat the completion of fermentation in the control tubes wouldshow a definite inhibition by dilutions of mercuric chloride up to1:80,000, the degree depending directly upon the concentrationof the mercuric chloride. But if the total amount of gas pro-duced, regardless of time, be taken as an end-point, readingsmight be interpreted to indicate stimulation of fermentativeactivity in certain of these same dilutions. Frequent readings

258


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


show the degree and duration of the inhibitory effect of differentdilutions of mercuric chloride as well as the amount and rate ofcarbon dioxide production. The maximum amount of gas wasby no means always produced in tubes containing the minimumquantity of mercuric chloride which produced an effect. Chart1 shows that the greatest total carbon dioxide production was indilutions of 1: 15,000 to 1: 30,000, the amount formed being 25to 35 per cent greater than that produced in the tubes withoutmercuric chloride. This excess of gas was evolved rather slowly,reaching the maximum at the end of about three hours, whereasfermentation in the tubes without mercuric chloride had ceasedat the end of one hour.Another effect of this compound may be seen in the curves

representing dilutions of 1:30,000 to 1:80,000. Here a genu-ine stimulation of fermentation seems to have occurred duringthe first fifteen minutes; after that the rate of carbon dioxideproduction fell off; at one hour the total amount was less thanthe control, and when fermentation was finally complete theexcess was very little. If the final observations had been madeat some arbitrarily chosen time interval, the interpretation of theexperiment would have differed greatly according to whetherfifteen minutes, one hour, or three hours had been selected as theend-point. With the simple apparatus described above it ispossible to observe the whole process.Thus we find, in the case of mercuric chloride, that low dilu-

tions exert a marked inhibiting action upon fermentation; higherdilutions cause definite inhibition temporarily, but the totalamount of carbon dioxide formed may be far in excess of thatproduced by the control tubes without mercuric chloride; andin some of the highest dilutions which effect the process at allthere is an actual initial stimulation, followed by a decreasedrate of fermentation.The course of fermentation in the presence of mercuric chloride

has been discussed here in detail because some of its phases areshown by other chemicals. An excess of carbon dioxide abovethe amount in the control tubes was produced regularly in somedilutions of a number of compounds other than mercuric chloride,

259


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

SARA E. BRANHAM

when fermentation was allowed to proceed to completion. Al-most invariably this phenomenon occurred after the cessation offermentation in the control series of tubes, and usually in dilu-tions that caused a definite inhibition of fermentation duringthe first hour. Charts representing the action of phenol (seechart 2), lysol, tricresol, mercurochrome, formaldehyde, tinctureof iodine, chloramine-T (see chart 3), copper sulphate, hexyl re-sorcinol, metaphen, ethyl alcohol, hydrochloric acid, and sodiuimchloride all indicate such an effect. Table 1 shows that in someof these cases the excess of gas produced beyond that in the

/20 - / 000-

CHART 2 EFPEcT OF VARYING DILUTIONS OF PHENOL UPON CARBON DIOXIDEPRODUC ON BY 10 PER CENT YEAST AND I PER CENT SUCROSE

controls was considerable; in formaldehyde there was 60 per centmore than the control in dilutions of 1: 600 to 1:1000; in lysol, 25per cent in 1:400 to 1:500; in phenol, 60 per cent in 1:350; inhexylresorcinol, 50 per cent in 1:8000; in sodium chloride, morethan 200 per cent with a one per cent solution. With some otherchemicals the excess was less. In most cases this excess g'as wasfound in the highest dilutions of the chemicals that had causedany iniitial inhibition. But in others (phenol, chloramine-T, andtincture of iodine) the greatest final amount of gas was formed inconcentrations that had caused marked inhibition for a consider-

260


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


able period of time. The most extreme case of that type waschloramine-T (chart 3) where tubes containing a concentrationthat completely prevented gas formation for one hour, ultimately,at the end of four hours, contained nearly twice as much carbondioxide as the control, a ratio of 22 to 13 cc.

This increased fermentation in the presence of certain dilutionsdid not occur with all of the chemicals studied. None of thecurves plotted for any sample of silver nitrate (chart 4) showedan appreciably increased amount of carbon dioxide beyond that24

22 _ 0oo

20

14

:L1000~ ~~~~~~~~~~~010/4

Ct_ 1 &~~~~~~~~~~2,000

0

Mrts. Q 1 2 -3 S

CHART 3. EFCT OF VARYING DILUTIONS OF CHLORAMaNE-T UPON CARBONDIOXIDE PRODUCTION BY 10 PER CENT YEAST AND 1 PER CENT SUCROSE

in the control tubes, nor did sodium hypochlorite show it duringthe period of observation.

Initial stimulation of fermentation by high dilutions, as wasnoted in the chart representing the action of mercuric chloride,can also be seen strikingly with sodium chloride. Here thestimulation is enormous, the total gas formed in' 1 per cent sodiumchloride being more than 3 times as much as in the controls (31to 9 cc.) within the same period of time. The action of sodiumchloride, and its effect upon the activity of other chemicals havebeen discussed by many. Pilcher and Sollmann (1923-1924) refer

261


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

SARA E. BRANHAM

to its action in preventing the inhibition of yeast fermentation bysilver salts. Explanations of its action have been offered byKronig and Paul (1896), by Rideal and Rideal (1921), by Halvor-son and Cade (1928) and by Speakman, Gee and Luck (1928).The action of copper sulphate was unique. With this salt

there was some fermentation during the first hour, even in con-centrations as great as 1:80, but only in dilutions greater than1: 5000 did any great degree of gas formation occur.A similarity in the general behavior of yeast in the presence of

related chemicals was apparent, even though the concentrations

/+~~~~~~~~~~~~~~~~J0000

200000

/4

0Y.S 0 5

CHART 4. EFrEcT OF VARYING DILUTIONS OF SILVER NITRATE UPON CARBONDIOXIDE PRODUCTION BY 10 PER CENT YEAST AND 1 PER CENT SUCROSu

necessary to produce the same effects varied widely in individualmembers of these groups. For example, the curves representingthe effect of mercurochrome definitely resemble those of mercuricchloride, showing the same phases, although a concentration of1: 50 of mercurochrome is necessary to produce the same degreeof inhibition of fermentation that is caused by 1: 5000 mercuricchloride. The charts showing the effect of phenol (chart 2),lysol, and tricresol are very similar to each other in generalappearance, as are also those for two creosote oil preparationsexamined. Within a wide range of dilutions (see chart 2) the

262


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


curves are the same for the first 15 minutes, after which they be-come very divergent. Charts representing the curves plottedfor those compounds which liberate free halogen; viz., sodiumhypochlorite, chloramine-T (chart 3), and tincture of iodine,show an especially striking resemblance to each other. The mostremarkable feature of these is a sudden abundant gas formation inconcentrations tbat completely inhibited fermentation for oneor two hours.

SUMMARY

A simple gasometer has been devised in which carbon dioxideproduction by yeasts can be measured with a fair degree of accur-acy, and the course of the fermentation observed. This appara-tus offers a means of studying some interesting phases of theprocess of fermentation under the influence of many agents.The concentration at which fermentation is completely in-

hibited has been readily determined for every chemical studied.An apparent stimulation of carbon dioxide production by highdilutions of compounds that inhibited this activity in greaterconcentrations often occurred, the total amount of gas producedfrequently being far in excess of that evolved by an equal amountof the yeast-sugar mixture to which no chemical had been added.

Curves plotted with readings made at frequent intervals showthat this increased fermentation commonly occurred in dilutionsthat had definitely inhibited activity for a greater or less periodof time. This phenomenon was manifested by the majority ofcompounds studied, though their behavior was by no meansidentical. Sometimes the greatest amount of gas was formed indilutions which caused relatively slight initial inhibition; i.e.,mercuric chloride, mercurochrome, metaphen, and hexylresorcinol.With other chemicals a sudden outburst of activity occurred indilutions which had caused complete inhibition for one or twohours; i.e., those which liberated free halogen such as chloramine-T, tincture of iodine, and sodium hypochlorite.

In some instances there was a transient initial stimulation indilutions too high to exert any inhibitory action; i.e., mercuricchloride and sodium chloride.

263


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

264 SARA E. BRANHAM

A few compounds showed no phases of increased fermentationin any dilutions; i.e., silver nitrate..The data presented indicate that the effect of individual chemi-

cals is characteristic, and that related compourds act upon theyeasts in a similar way.

REFERENCES

ALWOOD, W. B. 1908 U. S. Dept. Agric. Bureau Chem. Bull. 111. .BALLS, A. K., AND BROWN, J. B. 1925 Jour. Biol. Chem., 62, 789; ibid., 823.BRANHAM, S. E. 1929 Jour. Inf. Dis., 44, 142.BROWN, J. B., AND WIKOFF, H. L. 1927a Ann. Applied Biol., 14, 428.BROWN, J. B., AND WIKOFF, H. L. 1927b Ann. Applied Biol., 14, 436.DANN, W. J., AND QUAESTEL, J. H. 1928 Biochem. Jour., 22, 245.DRESER, H. 1917 Ztschr. f. exper. Path. u. Therap., 19, 285.EULER, H., AND EMBERG, F. 1919 Zeit. Biol., 69, 349.EULER, H., AND LINDNER, P. 1915 Chemie der Hefe unter der alkoholischen

Giirung, Leipzig.EULER, H. V., AND NILSSON, R. 1925 Chem. Zelle u. Gewebe, 12, 238.GOTTBRECHT, C. 1886 Inaug. Diss., Greifswald (Quoted by Schulz).GUTSTEIN, M. 1927 Centralbl. f. Bakt., I, Orig., 104, 410.HALVORSON, H. O., AND CADE, A. R. 1928 Proc. Soc. Exper. Biol., and Med.,

25,506.HARDEN, A., AND YOUNG, W. J. 1911 Proc. Roy. Soc., Series B, 83, 451.HOFMAN, G. 1884 Inaug. Diss., Griefswald (Quoted by Schulz).JOACHIMOGLU, G. 1922 Biochem. Zeit., 130, 239.KOHLER, E. 1920 Biochem. Zeit., 106, 194.KR6NIG, T., AND PAIUL, B. 1896 Zeit. Physikal. Ohem., 21, 414.LINDNER, P., AND GROUVEN, 0. 1913 Wchschr. Brau., 30, 133.MYERS, H. B. 1927 Jour. Amer. Med. Assoc., 89,1834.PETERSON, J. B. 1926 Jour. Amer. Med. Assoc., 87, 223.PILCHER, J. D., AND SOLLMANN, T. 1922-1923 Jour. Lab. and Clin. Med., 8, 301.PILCHER, J. D., AND SOLLMANN, T. 1923-1924 Jour. Lab. and Clin. Med., 9, 256.RIDEAL, S., AND RIDEAL, E. 1921 Chemical Disinfection and Sterilization,

p. 182.SCHULZ, H 1887 Virchow's Arch. f. path. Anat. u. Physiol., 108, 423.SCHULZ, H. 1888 Pfluger's Arch. f. d. ges. Physiol., 42, 517.SLATOR, A 1906 Jour. Chem. Soc., 89, 128.SLATOR, A. 1908 Jour. Soc. Chem. Ind., 27, 653.SLATOR, A. 1913 Biochem. Jour., 7,197.SoMoGYo, R. 1921 Biochem. Zeit., 120, 100.SPEAKMAN, H. B., GEE, A. H., AND LUCK, J. M. 1928 Jour. Bact., 15, 319.TAMMANN, G. 1889 Zeit. f. physik., Chem., 3, 25.THOL, W. 1885 Inaug. Diss., Greifswald (Quoted by Schulz).ZERNER, E., AND HAMBURGER, R. 1921 BiochemL Zeit., 122, 315.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

the effects of certain chemical compounds upon the

Documents