JOURNAL OF BACTERIOLOGYVol. 88, No. 5, p. 1380-1387 November, 1964Copyright © 1964 American Society for Microbiology

Printed in U.S.A.

ATTACHMENT AND GROWTH OF BACTERIA ON SURFACES OFCONTINUOUS-CULTURE VESSELS

DON H. LARSEN' AND R. L. DIMMICKNaval Biological Laboratory, School of Public Health, University of California, Berkeley, California

Received for publication 16 July 1964

ABSTRACT

DON H. LARSEN (Brigham Young University,Provo, Utah), AND R. L. DIMMICK. Attachmentand growth of bacteria on surfaces of continuous-culture vessels. J. Bacteriol. 88:1380-1387. 1964.-Initial attempts to induce synchrony in a con-tinuous culture of Serratia marcescens by alter-nating growth temperatures produced fluctuationsin the population of a magnitude and at a densityhigher than predicted by theory. Without temper-ature change, the density in the 14-ml volumechanged with dilution rate, but the total outputof cells per hour remained constant, even at dilu-tion rates greater than critical. When glass woolwas added to the culture vessel, the total outputper hour increased 30-fold. Nonlethal ultrasonicagitation applied to the vessel reduced the popu-lation density in continuous culture under both astatic and a cyclic temperature program. Thedecrease in population density, when the washoutrate was momentarily increased about tenfold,was less than theoretically predicted, and thesubsequent rapid rise, when flow was terminated,indicated the presence of a reservoir of cells on thewalls of the vessel continually discharging theirprogeny into the medium. Several genera wereexamined in the latter manner; it is estimatedthat in some cases as many as 90% (S. marcescens,Escherichia coli), and in others (Bacillus spp.)possibly none, of the cells in suspension arose fromwall inhabitants. Growth of bacteria on the wallsof continuous-culture vessels can significantlyinfluence the population density and, hence, thekinetics of continuous growth.

Single or multicyclic changes of temperatureapplied to bacterial cultures during exponentialgrowth may induce synchronous cell division in aportion of the cells for two or three generations(Hotchkiss, 1954; Szybalski and Hunter-Szy-balska, 1955; Kjeldgaard, Maal0e, and Schaecter,1958). One instance wherein cyclic temperaturechanges were combined with continuous-culture

1 Present address: Department of Bacteriology,Brigham Young University, Provo, Utah.

methodology for the purpose of attaining syn-chronous growth in a continuous culture was re-ported (Johnson and Larkin, 1963). Proof thatsynchrony had occurred would depend, in part,upon continuous-culture kinetics.

Several investigators observed that data fromcontinuous-culture experiments deviate fromtheoretical predictions (Herbert, Elsworth, andTelling, 1956; Holme, 1957; Contois, 1959). Theexistence of steady states, at dilution rates greaterthan the critical rate, was attributed by Herbertet al. (1956) primarily to incomplete mixing orinflowing medium with the culture. They furthersuggested that apparatus effects, such as detach-ment of growth from the walls, could have con-tributed to the observed anomalies. Contois(1959) presented evidence that fluctuation ofgrowth rate with population density was responsi-ble, in part, for the deviations.Our attempts to induce synchrony in a con-

tinuous culture of Serratia marcescens grownunder conditions of alternating temperature indi-cated that the population fluctuated at a levelgreatly exceeding the expected value at criticaldilution rates. Experiments showed that growth,of a nature not readily visible, on the walls of aculture vessel, can be a highly significant factor inthe interpretation of continuous-culture kinetics.

MATERIALS AND METHODS

Description of the apparatus. The culture vessel(Fig. 1) consisted of an all-glass unit having acommon air and medium inlet to facilitate rapidmixing and to minimize contamination by aero-solized particles. Medium drained into the cell bygravity and out by a syphon tube that projectedthrough the bottom of a small water bath. Thevessel was immersed in the bath to a point belowthe level of the contained medium. The moataround the inlet (Fig. 1), which was half-filledwith 1 M KOH, was effective in preventing con-tamination of media in the supply vessel. Asample port, closed with a vaccine stopper, per-

1380

GROWTH ON CONTINUOUS-CULTURE VESSELS

Me d iuminlet

vaccinestopper

Continuous Culture Vesse

FIG. 1. Continuous-culture vessel.

mitted inoculation or removal of sarna 3-in. (7.62 cm) hypodermic needlePumps, controlled by an auto

mechanism, circulated water alternatstorage reservoirs through the smareservoir was maintained at 32 Cthe experiments, unless otherwise stathe temperature of the second reservcas required over the range of 0 to 24of the medium was regulated by meaclamp attached to the inlet tubirmeasured by collecting the effluent incylinder.

Operation of the apparatus. Operalnology conforms to that of Herbert (

The organism, S. marcescens (8 UKin half-strength Bunting's (1940) l

dium that supported a maximal population ofapproximately 2 X 1010 cells per ml. At the startof an experiment, we adjusted the flow rate andtemperature to the desired value, and seeded the

3Air inlet medium with, usually, a 24-hr-old suspension of3 Air inlet cells. Samples were withdrawn by means of a)Ht syringe. The number of colony-forming units

(CFU) per milliliter was then determined by thecommonly employed procedure of decimal dilu-tion and surface colony enumeration.

Additional genera were tested (Table 3) inmedia appropriate to their satisfactory growth,and a vessel that could be immersed and operatedin a temperature-controlled ultrasonic cleaningbath (DiSONtegrator, model 40; Ultrasonic In-dustries, Inc., Plainview, N.Y.) was utilized incertain experiments. This second model differedfrom the one previously described, in that theeffluent tube extended within the vessel in avertical position, and overflow was removed by

Liquid level reduced pressure.

RESULTSA cyclic pattern of increase and decrease in cell

numbers, concurrent with temperature change,developed within 10 hr after the medium was in-oculated. Typically, the number increased two-to sixfold during the interval at 32 C, and thendeclined a corresponding amount during the coldinterval. The regularity of this pattern, and therelative stability of the average populationdensity over a 13-day period of continuous opera-tion, are shown in Fig. 2. Each segment of thecurve shows the results of a 1-hr sampling periodon the day indicated. The rise and fall of CFU

iples through coincident with temperature shift was essentially(no. 20). constant when several cycles were sampled se-

Imatic-timingtely from two tA11 1 INTERVALS AT 4 C (40 MlINUTES)11 bath. One g 8.Oxt7 INTERVALS AT 32 C (20 MINUTES)

throughout 44 3rd DAYT 4th DAY 6th DATS 7th DAY 110th DAY \13th DAY

ited, whereas .007 x I)irwas varied A0\ I IC. Flow rate 2.0-0 I|tnsof ascrew ItIng, and was I.cub1 a craduated zA a, rA"c&%A"VVuA

tional termi-et al. (1956).:), was grownbroth, a me-

,,,, ... .... .. . ... W.0 204060 0 60 0 60 0 60 0 60 0 60

Time in minutes

FIG. 2. Typical cyclic growth of Serratia mar-cescens with change of temperature during a 2-weekperiod of continuous culture.

VOL. 88, 1964 1381

LARSEN AND DIMMICK

quentially on the same day. There was, however,a tendency for the maximal population level toincrease gradually, day to day.The influence of the temperature of the cold

interval on the resulting growth patterns is shownin Fig. 3. The cold interval was varied by 4 Cincrements over the range of 0 to 20 C, whereasthe warm interval was maintained at 32 C. Themore the temperature was lowered, the greaterwas the difference between the calculated washoutrate and the rate of decrease of CFU. Not shownin the figure is our finding that 24 C was themaximal temperature of the cold interval thatconsistently produced an abrupt rise and fall ofthe numbers of cells.

Other parameters were examined; for example,the influence of dilution rate during a 40-min cold,20-min warm sequence on the population and onthe fluctuation of numbers is shown in Fig. 4.Dilution rate was varied from 0.2 volume per hrto approximately 3 volumes per hr. Increased

A., ^ wIINTERVAS AT LOWERTEM PERATURE$ INTERVALS AT32a

WASH-OUT RATE

1S 3045 O 45 0 450 -45 45 45

Time in minutes

FIG. 3. Influence of temperature change on thecyclic growth of Serratia marcescens in a continuous-culture vessel.

109

PeS

0)

eo

-A

EC 100

a07Sz

Time in minutes

FIG. 4. Influence of rate of flow on the growth ofSerratia marcescens in a continuous-culture vessel.

TABLE 1. Comparison of direct microscopic cellcounts with the number of viable cells in samples

from a Cyclostat

No. of Direct cellTime of sample viable Icount percells perml ml (X 107)

(X 107)

End of cold interval ......... 0.9 1.2End of warm interval........ 5.6 5.0Middle of cold interval. 1.8 1.2End of cold interval ......... 1.4 2.0End of warm interval........ 5.5 6.4Middle of cold interval. 1.8 2.5End of cold interval ......... 0.9 1.7

dilution rates decreased the average population;however, the percentage change in numbers dur-ing the warm interval was greatest at the highestdilution rates.

In all instances, results were not in accord withthe relationships of cell numbers that would beexpected if the population were in synchrony; thegeneration times calculated from established dilu-tion rates yielded highly improbable values.Clumping was not a significant factor in the cyclicfluctuation caused by alternating temperature,because direct counts correlated satisfactorilywith the number of CFU (Table 1), and no clumpswere observed.When the dilution rate was temporarily in-

creased to 7 volumes per hr, the average popula-tion density was reduced to approximately Y50of the initial value. Only a few minutes, however,were required for the population to return to theoriginal level after flow was stopped and the tem-perature maintained at 32 C. When a portion of aculture at the end of a cold interval was trans-ferred to an identical, but clean, culture vessel at32 C, the concentration of cells in the clean vesseldid not increase for 40 min, whereas that in theoriginal vessel evinced the typical abrupt rise.The walls of the continuous-culture vessel ap-

peared to be clean while it was being used. How-ever, when we positioned a used vessel in a beamof light, we observed a thin opalescent film on thewalls below the internal liquid level. We rinsedthe vessel twice with sterile medium, and thenadded a measured volume of sterile saline andscrubbed the walls vigorously with a sterile brush.Colonies obtained from the saline wash indicatedthat the total population on the inner surface ofof the vessel was 2.8 X 109 CFU. (The inlet tube

1382 J. BACTERIOL.

GROWTH ON CONTINUOUS-CULTURE VESSELS

was not examined.) Similar results were obtainedwhen we examined a vessel employed for con-tinuous culture at constant temperature.The population density and output per hour of

S. marcescens grown in continuous culture at 32 Cis shown in Table 2. It is evident that, for thedilution rates tested, the total output remainedconstant, indicating again the presence of a reser-voir of cells that were not being diluted at theexpected rates. When glass wool was added to thevessel, the output per hour increased about 30-fold; apparently the output and, hence, the meanpopulation density were a function of availablesurface area. As before, there was no evidence ofgrowth on the walls unless the vessel was ob-served by oblique lighting, but the glass woolreadily became pink in hue, indicating, beyonddoubt, that the fibers supported growth on theirsurfaces. Conversely, plating the inner surfaceswith silicone delayed the build-up, but did notreduce the numbers of apparent wall inhabitants.

Preliminary experiments with the second vesselindicated that intermittent ultrasonic agitationreduced the extent of attachment of bacteria tothe inner surfaces. The bath was subjected to ul-trasonic vibration at 90 kc for 30 sec at intervalsof 7.5 min. Neither the growth rate of S. marces-cens in tubes suspended in the bath, nor theviability of cultures grown either continuously orin batch culture, was altered significantly byintermittent ultrasonic excitation.The influence of ultrasound on continuous cul-

ture of S. marcescens in Bunting's (1940) mediumat 32 C is shown in Fig. 5. The abrupt rise in thenumber of viable cells immediately after the startof ultrasonic agitation was a constant charac-teristic of such experiments with S. marcescens.We attribute this increase to detachment of cells

TABLE 2. Influence of dilution rate and glass woolon population density and yield of cells in a

continuous culture of Serratia marcescens

Dilution rate* Population Total celldensity (per ml) yield/hr

0.35 3.2 X 108 1.6 X 1090.92 1.1 X 108 1.4 X 1092.0 4.7 X 107 1.3 X 1093.9 3.5 X 107 1.9 X 109

3.8 (withglass wool 1.3 X 109 6.6 X 1010added)

* Critical dilution rate = 0.83 volumes per hr.

cn

E

c00

t

E

08Dilution rate =1.5 vols/ Hr

AlK._

00

107 Snic excitation started~a_[30 seconds each 7 i minutes) -

94 96 98 t00 \116 118Time in hours

FIG. 5. Influence of intermittent ultrasonic agita-tion on the population density of a continuous cul-ture of Serratia marcescens.

from the walls of the vessel, rather than to deag-gregation, because sonic excitation did not in-crease the number of CFU of a continuous culturetransferred to a fresh second vessel.

After the initial increase, the number graduallydecreased until a new steady-stage population wasestablished at a level about one-eighth thatpresent prior to the start of excitation. Becausethe growth rate of batch cultures of S. marcescenswas not reduced by ultrasonic agitation, we inter-pret this decrease in steady-state population to a

reduction in the number of organisms growingand continuously detaching from the walls.Further, the minimal generation time of S. mar-

cescens in one-half strength Bunting's mediumwas 49 min. The calculated critical dilution ratewas 0.8 volume per hr (Herbert et al., 1956).Maintenance of a steady-state population of 5 X107 CFU per ml, at a dilution rate double thecritical dilution rate during ultrasonic agitation,indicated a reduction but not elimination ofgrowth on the walls of the culture vessel.The influence of sonic energy on a continuous

culture subjected to alternating temperature isshown in Fig. 6. The average peak populationlevel was decreased from 8.0 X 107 to 9.7 X 106CFU per ml. The cyclic increase and decrease incounts concomitant with temperature changepersisted, but the magnitude of the shift was

decreased. As before, a steady-state populationwas maintained at a dilution rate greater thanthe calculated critical dilution rate.The results of a brief drastic increase in the

dilution rate of a previously stabilized continuousculture, followed by complete stoppage of flow(Fig. 7), showed that an increase in the dilutionrate of a 43-hr continuous culture of S. marcescens

VOL. 88, 1964 1383

LARSEN AND DIMMICK

from 1.9 to 24.0 volumes per hr for 38 min de-creased the count from 9.9 X 107 to 1.1 X 107CFU per ml. If there had been no reservoir fromwhich cells could arise, then this rate of dilutionwould have reduced the numbers to less than 100per milliliter. Within 1 hr after the flow was com-pletely stopped, the count had increased to1.2 X 109 cells per ml, whereas maximal rate ofgrowth could have increased the number to only2.8 X 108 cells per ml; i.e., over 98% of the cellsin the liquid culture originated from the walk ofthe vessel during this 1-hr interval.

01)

,a._

c

0)r-

-o

.E

D1

>1

*-*32 C intervals*--..16 C intervals*-*Wash out rate: lW

Without sonic excitationAfter

18 hr intermittent excitation

/03~~ ~ ~

/ *

I)114 115 '138 140

Time in hours

FIG. 6. Influence of intermittent ultrasonic agi-tation on the population density of a continuousculture of Serratia marcescens under a cyclic temper-ature program.

109

Q Experimental

*0

0)

inbahDincreasedtfrom1.9vs/Hrto

24.0 vols/Hr

C 10F-stoppeIo4osHrLe/\ Maximum :rowth rate

Diuto rat at\ in batch culture

42 43 44 45 46Time in hours

FIG. 7. Influence of a change of dilution rate onthe population density of a continuous culture ofSerratia marcescens.

E0

00

66 76

%106 Flow stopped

0-0.0 1 -

D 0

a -- D

66 67 68Age of culture in hours

FIG. 8. Influence of a change of dilution rate onthe population density of a continuous culture ofBacillus cereus. Refer to Table S for definition ofA, B, C, D, and E.

The results of a similar experiment with a 66-hrcontinuous culture of Bacillus cereus are shown inFig. 8. When the dilution rate was increased from0.4 to 32 volumes per hr for 13 min, the numbersdropped from 4.2 X 108 to 1.7 X 105 per ml.Dilution during this interval should have reducedthe count to 4.2 X 104 per ml. By the samereasoning as above, we conclude that B. cereusdid not grow on, and detach from, the culturevessel as readily as did S. marcescens. After theflow was stopped, the increase of numbers in theculture was less than that predicted from theminimal doubling time of B. cereus in the mediumemployed. The rapid dilution of the culture prob-ably induced a temporary lowering of the growthrate.

Results of similar experiments with severalspecies of bacteria are summarized in Table 3.The tendency for growth on, and detachmentfrom, the vessel surfaces seemed to differ greatlyamong various species. No correlation betweenthe tendency to grow on the wall and other char-acteristics such as motility, morphology, and

1384 J. BACTERIOL.

GROWTH ON CONTINUOUS-CULTURE VESSELS

TABLE 3. Influence of one cycle of alternate high flow and zero flow rate on population densityin continuous cultures

Colony-forming units per ml X 106*Organism F

A B C D E

Serratia marcescens (24 hr)........... 48 0.048 22 55 930 97S. marcescens (43 hr) ................ 86 0.000086 11 28 120 93Escherichia coli ...................... 27 0.026 3.7 13 170 94Aerobacter aerogenes.................. 520 0.135 37 120 280 67Bacillus cereus.420 0.042 0.17 0.57 0.30 0B. subtilis.430 0.43 0.68 26 2.0 0Sarcina lutea ........................ O.81 0.00081 0.0057 0.011 0.024 72Staphylococcus epidermidis.190 0.40 2.4 6.7 68 93S. aureus.50 0.10 1.1 4.4 35 90

* A, steady state; B, theoretical number after interval at high flow rate, calculated from the volumeof medium that flowed through the vessel during the interval at high dilution rate, and assuming nodetachment of cells from the walls; C, experimental count after the high dilution rate interval; D,theoretical number 1 hr after flow was stopped; calculation based on minimal generation time of organ-ism in medium employed, and assuming that no cells detached from the walls; starting point was experi-mental count made 1 min after flow stopped; E, experimental count 1 hr after flow stopped; F, percentageof cells originating from walls after flow stopped. F = 1 - (D -C)/(E - C) X 100. See Fig. 8.

reaction to Gram stain is indicated by theselimited observations. We do not know whetherthe observed minimal surface growth and subse-quent detachment of the two bacilli are charac-teristics of the genus as a whole.

DISCUSSIONIt is evident that if a population not subject to

washout (captive population) discharges itsprogeny within a culture vessel, then the liquid-borne cells will be more numerous than predictedby theory, especially at flow rates that should re-duce the population to near zero. There are manyreports that bacterial cells tend to become at-tached or adsorbed onto surfaces (ZoBell, 1937,1943; Gunnison and Marshall, 1937; Conn andConn, 1940; Heukelekian and Heller, 1940;Zvyagintsev, 1959; Helmstetter and Cummings,1963).

ZoBell (1937, 1943) observed that surfaces in-creased the bacterial population, and enhancedthe physiological activity of bacteria in seawater.The beneficial effect was more pronounced whenthe concentration of nutrients was low. He sug-gested that the point of contact of the bacterialcell and the solid surface may serve as concentra-tion foci for nutrients, and thereby aid in theproduction of optimal oxidation-reduction orother physicochemical conditions. A similar rela-

tionship between nutrient concentration and sur-face growth was reported by Heukelekian andHeller (1940).An essential feature of most continuous-culture

theories is the consideration of the specific growthrate as a function of the concentration of thelimiting nutrient (Monod, 1942, 1950; Novickand Szilard, 1950; Spicer, 1955; Herbert et al.,1956; Moser, 1958). If the limiting nutrientshould adsorb and concentrate on the walls of acontinuous-culture vessel, the population on thewalls would have access to a higher concentrationof limiting nutrient than would the cells in theliquid phase; consequently, the "two populations"would grow at different rates.The amount of growth on surfaces of a con-

tinuous-culture device, compared with growth inthe liquid phase, is dependent on populationdensity and surface-to-volume ratio. A tenfoldincrease in the volume of a sphere decreases thesurface-to-volume ratio by a factor of approxi-mately one-half. Progeny from surface inhabi-tants could be significant if vessels of a capacityof several liters were operated at populationdensities in the range of 107 to 101 cells per ml.Extrapolation of our results, especially thoseshowing a stable population over a wide range ofdilution rates with S. marcescens, from the 14-mlvessel employed to a vessel of a capacity of 20

VOL. 88, 1964 1385

LARSEN AND DIMMICK

liters, indicates that over 50% of the total cellspresent at a level of 108 per ml, could originatefrom growth on the walls.The observed variability in surface activity of

different species of bacteria is in agreement withthe data of Zvyagintsev (1959), who reportedthat adsorption of a variety of species on glassslides varied from none to very strong. He ob-served no clear-cut dependence of adsorption onthe taxonomic characteristics of the microor-ganism or on its reaction to Gram stain.The relatively constant rate of cell production

from vessel surfaces, over a wide range of dilutionrates, indicates that sites or spaces for firm at-tachment are limited. It was reported that at-tachment of HeLa cells anid monkey kidney cellsin synthetic medium requires a critical number ofnegative attachment sites on the surface of theglass (Rappaport, Poole, and Rappaport, 1960;Rappaport, 1960). These investigators noted thatthe rate of attachment and outgrowth, and thetime the monolayers of mammalian cells could bemaintained before retraction occurred, were func-tions of the total negative charge and the proton-exchange capacity of the glass.The possibility that bacterial cells might also

grow as a monolayer under these specialized con-ditions is shown in Fig. 9, where the observedpopulation densities at several dilution rates are

comlpared with equivalent theoretical densities.

O---'O Observed

*- * Calculated

109

X 10

1 2 3 4 5 6 7 8

Dilution Rate,Vol/Hr

FIG. 9. Relationship of population density atthe end of a warm cycle to the dilution rate whenSerratia marcescens was grown continuously undera cyclic temperature program.

The following assumptions or approximationswere made to obtain the calculated values: (i) thateach ,2 of the total surface area was occupied byone bacterium; i.e., 3 X 109 ,2 for the vessel, plus2 X 108 ,A2 for the inlet tube, or 3.2 X 109 A2 total;(ii) that the mean generation time (49 min) ofthe captive bacteria was equivalent to that inbatch culture; (iii) that generation occurred onlyduring the warm interval; i.e., one-third of thehourly cycle; and (iv) that the volume into whichall progeny were discharged was that of the vessel(14 ml), plus one-third of the volume exchangedat a given flow rate.There is good agreement between the two sets

of data. It is evident that at rapid dilution ratesthe captive population supplies most of the cells,whereas at slow flow rates most cells arise fromliquid-borne bacteria. These data further implythat one cannot calculate the true generationtime of bacteria in continuous culture by com-monly employed formulas, especially at low celldensities, without considering the influence of thecaptive population.Although we did not extensively study the

mechanisms of attachment and detachment, weshowed their importance to kinetics of growth incontinuous culture, and we wonder whether asimilar phenomenon could operate in many of theroutine activities of microbiology. We showed thatit is not necessary to postulate unusual growthrates to account for the maintenance of anomal-ous population densities at high dilution rates incontinuous culture.

ACKNOWLEDGMENT

The technical assistance of James B. Gonzalesand Stephen A. Dunn is gratefully acknowledged.

This work was sponsored by the Office of NavalResearch under a contract with the Regents ofthe University of California.

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CONTOIS, D. E. 1959. Kinetics of bacterial growth:relationship between population density andspecific growth rate of continuous cultures.J. Gen. Microbiol. 21:603-613.

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1386 J. BACTERIOL.

GROWTH ON CONTINUOUS-CULTURE VESSELS

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