Quantitative Measurement of the Growth ofPleuropneumonia-like Organisms1

PAUL F. SMITH

Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

Received for publication April 23, 1956

Accurate and reliable methods for the quantitativemeasurement of the growth of pleuropneumonia-likeorganisms have never been developed. Studies onmembers of this group of organisms have advanced inrecent years to the stage at which further develop-ments will be impeded by the lack of such methods.Generally techniques employed for rapid quantita-tion of bacterial growth, such as turbidity, packedcell volume, and dry weight, are unsuitable becauseof the small size and low cellular protein yields ofthese organisms.

Estimations of the relative growth on solid mediarecorded as 4+, 3+, and so forth, or comparison ofthe relative amount of growth on a test plate withthat on a control plate in which "optimal" growthoccurred have been employed most frequently in thepast. Holmes and Pirie (1932) attempted to relate theamount of growth with reduction time of methyleneblue in the presence of lactate, and thereby constructa growth curve for the bovine pleuropneumonia or-ganisms. Keller and Morton (1954) depicted thefutility of turbidimetric measurement of whole culturein constructing growth curves for human strains. Theseworkers successfully estimated growth by the methodof least probable numbers and by calculation of thenumbers of organisms from average colony counts of100 microscopic fields. These two methods are timeconsuming and are not adaptable to routine use.Liebermeister (1954) assessed the suitability of mediafor cultivation of pleuropneumonia-like organisms bycounting the number of colonies developing from 0.01ml of liquid culture spread over the surface of an agarplate. Mean colony diameters of randomly selectedcolonies were employed as a measure of growth andgrowth inhibition by Smith and Morton (1951, 1953).Measurement of cellular nitrogen as related to

optical density of resting cell suspensions have beenused to standardize preparations for metabolic studies(Lecce and Morton, 1954; Rodwell and Rodwell,1954).Although these methods are of value, none have

been analyzed in regard to their accuracy and relia-bility. The purpose of this study was to develop and

1 This work was supported in part by a contract (Nonr551(04)) between the Office of Naval Research and the Uni-versity of Pennsylvania.

assess all conceivable methods to ascertain which aremost applicable for routine use.

MATERIALS AND METHODSStrains 07 and 48 of human origin were employed

as test organisms. These were selected because oftheir typical nature, and because they represent twodifferent types in regard to their nutritional require-ments (Smith, 1955). The media employed were thebroth described by Morton et al. (1951) supplementedwith Difco PPLO serum fraction (Smith and Morton,1951) and the synthetic medium reported by Smith(1955). Measurement of dose response in the crudemedium was performed with the PPLO serum frac-tion as the required component; in the syntheticmedium arginine was the required factor. Inocula weredeveloped by seeding 10-ml amounts of the crudemedium with agar blocks containing colonies and in-cubating 48 hours at 37 C. Standardized inocula wereprepared by inoculation of 200-ml amounts of thecrude medium with 10-ml broth cultures prepared asabove followed by incubation for 48 hours. The brothculture was centrifuged under sterile conditions at12,000 rpm in the Servall angle centrifuge for 5 min-utes, the supernatant decanted, and the sedimentedcells resuspended in 5 ml of physiologic saline. Theturbidity of the suspension was determined in a Klett-Summerson photoelectric colorimeter and the numberof viable cells determined from the curve shown infigure 5. The suspension was diluted to contain 1010organisms per ml. Additional description of prepara-tion of inoculum is given under results of each method.

Acid production was assessed by titration withstandard alkali or by measurement of pH change witha Beckman pH meter. Zones of growth were determinedby the cup plate method of antibiotic assay, except thatthe essential nutrient was added to the cups and thediameters of the resulting areas of growth measured.Turbidimetric determinations were performed with aKlett-Summerson photoelectric colorimeter using a420-m, filter. Colony diameters were measured with anocular micrometer at 100>X magnification. Colonycounts were performed at 24X magnification. Assess-ment of growth on solid media always was aided byflooding the plate culture with a 1: 20 dilution of Dienes'stain for a few seconds.

254

GROWTH OF PLEUROPNEUMONIA-LIKE ORGANISMS

RESULTSAcid production or change of pH. Ten-ml aliquots of

broth containing concentrations of PPLO serum frac-tion varying from 0 to 2.0 per cent vere inoculated with0.05 ml of a 48-hour-old broth culture and incubated48 hours. No titratable acidity was demonstrable. Thecultures exhibited an alkaline reaction to phenol red.Titration with standard acid yielded results which wereproportional to the level of protein present in themedium. No change of pH was noted during the courseof growth.Zone of growth by the cup plate method. The entire

surfaces of agar plates containing the unsupplementedcrude medium were inoculated with agar blocks of 48-hour-old cultures. Varying levels of PPLO serum frac-tion were added to the metal cylinders employed forantibiotic testing. After 4 days' incubation, the zone ofgrowth was measured. A gradual increase of diameterof the zone of growth from 17 mm for the lowest concen-tration to 31 mm for the highest concentration wasnoted. The differences between varying levels of re-quired nutrient were not great enough to permit con-struction of accurate dose response curves. In addition,great difficulty was experienced in detecting the outeredge of the zones due to the increasingly smaller size ofthe colonies.

Turbidimetric methods. Ten-ml cultures with varyinglevels of PPLO serum fraction, prepared as describedfor determination of change of pH, were employed tomeasure turbidity in both the Klett-Summersonphotometer and the Beckman DU spectrophotometer.Photometric readings of tubes giving optimal growthdid not exceed 5 or 10, which are insignificant. Difficultywas experienced blanking the uninoculated medium inthe spectrophotometer at 280 m,u. Therefore no readingswere obtained.

It was surmised that turbidimetric measurementswould be of value if cultures wvere concentrated, therebyincreasing the density of cell suspensions. Cultures of30- and 50-ml volumes were grown in the presence ofvarying concentrations of the PPLO serum fraction.Tubes were inoculated either with a standardized salinesuspension of 48-hour-old cells or with an aliquot ofwhole 48-hour broth culture. Following an incubationperiod usually of 4 days, the cultures were centrifugedat 13,000 rpm in a Servall angle centrifuge. The super-natant was decanted and the sedimented cells resus-pended in physiologic saline. Density of the suspensionswas determined in both the colorimeter and the spectro-photometer. The latter offered no advantage overthe colorimeter.

Preliminary examination of the method gave noticeof two possible sources of error. One is the loss of cellswhile decanting the supernatant. This can be minimizedby taking care not to disturb the sediment whichadheres to the wrall of the centrifuge tube. The other

RPAWx/03

FIG. 1. Effect of centrifugal speed on sedimentation of cells.Strain 07.

source of error is the fact that all of the cells are notsedimented by centrifugation.An experiment was designed to determine the per

cent removal of cells at various centrifugal speeds. A10-ml sample of a pooled 600-ml culture was centri-fuged at various speeds in a Model L Spinco centrifuge.Following 5 minutes at a given speed, a 0.1-ml aliquotof the supernatant was removed for viable cell counting.The 5-minute intervals do not account for the time re-quired for acceleration and deceleration. Figure 1 showsthe effect of the speed of centrifugation on the numbersof viable organisms remaining in the supernatant. Veryfew cells are removed at 5,000 rpm. A logarithmic de-crease of cells in the supernatant occurs between 5,000and 20,000 rpm. Increasing the speed to 40,000 rpmhas little effect. Apparently a fraction of the cells(about 0.01 per cent) are small enough to resist sedi-mentation at this speed or unavoidable movementsresuspend some organisms. In general, sedimentationin the Servall at 13,000 rpm for 5 minutes permittedrecoveries of about 90 per cent of the cells. Resuspensionand resedimentation resulted in 95 to 99 per cent re-coveries, dependant upon the turbidity of the suspen-sion. Usually 108 cells per ml remained in thesupernatant.

Figure 2 shows the typical dose response curve toPPLO serum fraction by measurement of turbidities ofconcentrated cultures. The use of whole culture or re-suspended cells as inoculum had little effect on the

1956] 255

PAUL F. SMITH

~404

2 30 ___

20

to~ ~ ~ o .

os r.o 2D 3.0% PPLO SERUM FRACTION

FIG. 2. Dose response to PPLO serum fraction measured byturbidity of concentrated cultures. Strain 07. Four days' in-cubation.

resulting turbidities when the number of organismsadded was relatively constant. Thus, a 5 per centinoculum of a 48-hour-old whole culture (equivalentto 1.5 X 109 cells) or 109 cells suspended in 0.1-ml salineinoculated into 30-ml medium gave the same result.Increase in size of inoculum tended to flatten the doseresponse curve unless the incubation time was short-ened. The use of 50-ml cultures offered no significantadvantage over 30-ml cultures. Variation betweenturbidity readings of duplicate or triplicate tubes wasnot significant. Replicate curves prepared on differentoccasions were equivalent. Variation between turbidityreadings can be extensive if great care is not taken toprevent loss of sedimented cells.Measurement of mean colony diameters. Agar plates

containing PPLO serum fraction at levels varyingfrom 0 to 3.0 per cent were inoculated with 0.05 ml of a104 dilution of a 48-hour-old broth culture. The inocu-lum was spread over the agar surface with a bent glassrod and the plates incubated for 4 days at 37 C. Thediameters of 10 and 25 random colonies were measuredon each plate and were recorded as units on the ocularmicrometer scale. The conversion factor is equal to13.2 ut per unit.No significant difference was found between the

average diameters of 10 and 25 colonies. Analysis ofvariance in both cases yielded a probability of 0.001that the variation of the diameter of colonies measuredfor a given concentration of essential nutrient was equalto the variation of the colony diameters among thedifferent doses of nutrient. Dilution of the inoculum inbroth, saline, or water had no effect on the shape or

magnitude of the dose response curve. Increased incuba-tion time up to 6 days increased the magnitude of thecurve. However, the shape of the curve became atypicalof dose response curves after 4 days' incubation. Ageof the inoculum up to 6 days was without effect, pro-vided the dilution of the cultures was adequate toprevent crowding of the colonies on the plates. A 102dilution of a 24-hour-old broth culture was adequate,while a 104 dilution of cultures over 24 hours old wasrequired. Maintenance of a 1.3 per cent agar concentra-tion and addition of equal volumes of the essentialnutrient eliminated the limiting effect of agar oncolony size.Measurement of mean colony diameters on plates

inoculated with 104 dilution of a 48-hour-old broth cul-ture and incubated 4 days yielded comparable resultson repetition. The straight line regression coefficientsfor seven replicate curves approached 1.0 in all in-stances. This is indicative of a positive correlationbetween dose of nutrient and colony size. Employingthe t-test (Snedecor, 1940) the probability that thedeviation between any two replicate curves is significantis less than 10 per cent. A typical dose response curveemploying mean diameters as a measure of growth isshown in figure 3.

Viable cell count. Viable cell counts were made bycounting the number of colonies appearing on plates ofcrude solid medium following inoculation with aliquotsof appropriate broth dilutions of cultures or saline sus-pensions of the organisms. Exactly 0.01-ml aliquotswere plated in duplicate without spreading. This volumecovered an area on the plate which was almost com-

Z PPLO SERUM FRACTIONFIG. 3. Dose response to PPLO serum fraction measured

by increase in mean colony diameters on solid medium. Strain07. Four days' incubation.

256 [VOL. 4

GROWTH OF PLEUROPNEUMONIA-LIKE ORGANISMS

pletely visible in one microscopic field at 24X magni-fication. Since the colonies are distinctly visible onlyupon magnification, counting over this small areareduces the error which occurs when it is necessary toobserve more than one field. Furthermore, the possi-bility that some organisms would be removed byadhering to a glass spreader was avoided. Followingincubation in upright position for 4 days at 37 C,plates were flooded with dilute Dienes' stain andcounts performed.A preliminary analysis was made of the variability

of the counts between aliquots of the same dilution andof the average total viable cell counts between differentdilutions. Variation was found to be small. For ex-ample, on plates on which the mean colony count was32.1, the 95 per cent confidence limits were 27.0 and37.2; on plates with a mean colony count of 2.4, theconfidence limits were 1.4 and 3.4. There was no statis-tically significant difference between replicate testsperformed on different days.Measurement of dose response by the viable cell

count method was carried out by inoculating 10-mlaliquots of broth, supplemented with varying levels ofPPLO serum fraction, with 0.1 ml of a standard inocu-lum containing 109 organisms per ml. Following anincubation period of 4 days, appropriate dilutions(104, 105, or 106) were plated as above. Figure 4 showsthe typical dose response curve. Replication of thecurves employing the same lot of serum fraction waspossible. Incubation of the broth tubes for 3 and 4 daysdid not affect the magnitude of the curve. Extension ofthe incubation period to 6 days resulted in a 1- to 2-logdrop in viable cell counts. Decrease of the inoculum

% SERUI FRACTIONFIG. 4. Dose response to PPLO serum fraction measured

by viable cell counts. Strain 07. Four days' incubation.

I0~~~~~~~~~~~~~Lo~~~~~~~ 00 )069l l l

C.z

7

FIG. 5. RelationshipStrain 07.

200 300 400KLETT READA9of turbidity to viable cell count.

size below 101 organisms per 10 ml of medium neces-sitated lengthening the incubation time. Increase ofinoculum size above 109 organisms tended to flattenthe dose response curve. Since 48-hour-old broth cul-tures contain about 109 organisms per ml, it is possibleto employ a 5 per cent inoculum of whole 48-hourculture instead of cells resuspended in saline for studiesin the crude medium. No difference was noted in thedose response to PPLO serum fraction when eitherinoculum was used.

Rela.tionship between turbidity and numbers of viablecells. Turbidity is the usual measurement employedwhen resting cell suspensions are prepared. Cellularnitrogen has been related to turbidity of suspensions ofpleuropneumonia-like organisms by Lecce and Morton(1954). It would be advantageous to correlate turbiditymeasurements to numbers of viable cells in order togain some knowledge of the activities of individualcells.Employing the method outlined for determination of

dose response by colony counts with the exception thathigher dilutions were made for plating, viable cellcounts were performed on suspensions of cells withdifferent turbidity measurements. Cell suspensionswere prepared by sedimentation in the Servall anglecentrifuge at 13,000 rpm and resuspension in physiologicsaline. Figure 5 shows the resulting straight line rela-tionship when the log of the numbers of organisms isplotted against turbidity measurements. The uppercurve shows the relationship for 4-day-old cultures whilethe lower curve is representative of 6-day-old cultures.Although turbidity of cultures increases with incubationtime, viability decreases after 4 days at 37 C. It isevident from the slope of the curve that a great increasein turbidity is not attended by a great increase innumbers of viable cells. In addition, readings of 30to 50 in the photometer represent the usual yields of

2 -719561

PAUL F. SMITH

0~~ ~ ~~~00

o c0 200 300 400 500 600KLETT READING

FIG. 6. Relationship of turbidity to dry cell weight. Strain07. Four-day-old cells.

viable cells when optimal growth is obtained. Thisfact alone can account for the uselessness of turbidityof unconcentrated cultures as a measure of growth.

Relationship between turbidity and dry weight. Analyti-cal determinations on microorganisms usually arerecorded in per cent of dry weight. Therefore, it seemeddesirable to relate the usual turbidimetric measure-ments to dry weight. Saline-washed cell suspensions ofdifferent turbidimetric readings were centrifuged at13,000 rpm, the supernatant decanted, and the sedi-mented cells in celluloid tubes placed over calciumchloride under vacuum until constant weights wereobtained. The dried cells were removed and the tubestared. Figure 6 shows the resulting relationship betweenturbidity and dry weight of cells per ml of suspension.

Experiments with the synthetic medium. Application ofthe methods found suitable for measurement of doseresponse to PPLO serum fraction in the crude mediumto the growth response to some other required nutrientin the synthetic medium was desirable. Arginine wasselected as the assay material because it is an absoluterequirement for growth.

Turbidity measurements of concentrated cultureswere found not to be applicable to the measurement ofdose response to arginine since growth is suboptimal.However, measurement of mean colony diameters andof viable cells yielded a quantitative measure of thegrowth response to arginine. The latter method wasmore reliable since colony diameters are small due tothe suboptimal nutritional environment of the syn-thetic medium. All 3 methods were found to be ap-plicable to measurement of the factor or factors inBacto-peptone which are required for the growth ofcertain strains.

DIscUSSIONThree methods for assessment of growth of pleuro-

pneumonia-like organisms have been demonstrated tobe applicable for measuring dose response. Thesemethods are measurements of turbidities of concen-trated cultures, of colony diameters, and of the numbersof viable cells by colony counts.

Quantitation of the numbers of viable cells by colonycounts is applicable to studies on growth, growthinhibition, and stability of the organisms under variousconditions. The use of these methods for assessment ofgrowth should permit a more quantitative approach tothe study of this group of microorganisms.Extreme variation in growth response to different

lots of Bacto PPLO serum fraction was noted. Theuse of lots of the fraction comparable to the preparationoriginally described by Smith and Morton (1951) per-mitted optimal yields of about 109 organisms per mlwhen added at the level of 1 per cent. Increasinglylower optimal yields were obtained with other lots.In most instances this variation in ability to promotegrowth was related to the protein concentration of theserum fraction.The use of the pleuropneumonia group of organisms

for the detection and assay of new growth factors hasnot been attempted widely because of the lack of suit-able methods for quantitation of growth. The demon-strated methods should alleviate this condition to someextent. Although the methods are more cumbersome toemploy than the routine methods for assay of bacterialgrowth, the potential of discovering new growth factorsby studies on the nutrition of this group of organismsshould not be overlooked. The vast majority of strainsare unable to multiply on presently developed syntheticmedia unless supplemented with crude components.

SUMMARY

Three methods useful for the quantitation of growthof pleuropneumonia-like organisms are described.These are measurement of turbidities of concentratedcultures, of colony diameters, and of number of viablecells by colony counting. The accuracy and statisticalsignificance of these methods are demonstrated. Themethods are applicable to quantitation of dose response.Measurement of acid production, of turbidities of un-concentrated cultures, and of zone growth by the cupplate method are of no value for assessment of growth.The relationship between turbidities of cell suspensionsand viable count or dry cell weight is discussed.

REFERENCES

HOLMES, B. E., AND PIRIE, A. 1932 Growth and metabolismof the bovine pleuropneumonia virus. Brit. J. Exptl.Pathol., 13, 364-370.

KELLER, R., AND MORTON, H. E. 1954 The growth of pleuro-pneumonia-like organisms of human origin: Cultivationin the developing chick embryo and an in vitro growthcycle. J. Bacteriol., 67, 129-134.

LECCE, J. G., AND MORTON, H. E. 1954 Metabolic studies onthree strains of pleuropneumonia-like organisms isolatedfrom man. J. Bacteriol., 67, 62-68.

LIEBERMEISTER, K. 1954 Ein Niahrsubstrat zur Ziichtungvon Organismen der Pleuropneumonia- (PPLO) -Gruppe.Z. Hyg. Infektionskrankh., 140, 132-141.

258 [VOL. 4

BACTERIAL FLORA OF LOW SALT CUCUMBER BRINES

MORTON, H. E., SMITH, P. F., AND LEBERMAN, P. R. 1951Investigation of the cultivation of pleuropneumonia-likeorganisms from humans. Am. J. Syphilis, Gonorrhea,Venereal Diseases, 35, 361-369.

RODWELL, A. W., AND RODWELL, E. S. 1954 The breakdownof carbohydrates by Asterococcus mycoides, the organismof bovine pleuropneumonia. Australian J. Exptl. Biol.Sci., 7, 18-30.

SMITH, P. F., AND MORTON, H. E. 1951 The separation andcharacterization of the growth factor in serum and ascitic

fluid which is required by certain pleuropneumonia-likeorganisms. J. Bacteriol., 61, 395-405.

SMITH, P. F., AND MORTON, H. E. 1953 Nature of the growthinhibitor in some mammalian sera for pleuropneumonia-like organisms of human origin. Proc. Soc. Exptl. Biol.Med., 83, 65-69.

SMITH, P. F. 1955 Synthetic media for pleuropneumonia-likeorganisms. Proc. Soc. Exptl. Biol. Med., 88, 628-631.

SNEDECOR, G. W. 1940 Statistical Methods Applied to Experi-ments in Agriculture and Biology. The Iowa State CollegePress, Ames, Iowa.

Variations in Bacterial Flora of Low Salt Cucumber Brines'CARL S. PEDERSON AND MARGARET N. ALBURY

New York State Agricultural Experiment Station, Cornell University, Geneva, New York

Received for publication April 25, 1956

In commercial pickling, the cucumbers are packedin salt brines to be held until later when they are re-

freshed and converted to pickles of the desired type. Inthe brine, an active lactic acid fermentation occurs.

Greater knowledge of the bacterial and chemicalchanges occurring during fermentation and curing mayoffer information to aid in preventing undesirablechanges.

Until recently little attention has been given to thesystematic study of changes in flora occurring duringthe active fermentation. Numerous studies indicatedthat several species of bacteria were involved; Fabian,et al. (1932), Henneberg (1926), Wuistenfeld and Kreipe(1933), Vahlteich et al. (1935), Fabian and Wickerham(1935), Etchells and Jones (1946), Pederson and Ward(1949), Pederson and Albury (1950, 1953), andCostilow et al. (1956). Fabian and Wickerham (1935)have indicated and Pederson et al. (1949, 1950, 1953)have demonstrated that there occurs, under certainconditions, a sequence of growth of the several bac-terial species similar to that of the kraut fermentation.These laboratory-scale studies on pickles were con-

ducted in small containers. Since losses due to softening,bloating, and discoloration are so serious at times, itseemed advisable to conduct the present study in

larger or semi-commercial containers.

MATERIALS AND METHODS

The methods used were based upon those used inprevious studies of cucumber fermentations. Themethod of determining the course of fermentation, theisolation and identification of cultures, and chemicalanalyses were similar to those used in! previous studies

1 Accepted by the Director of the New York State Agri-cultural Experiment Station as Journal Paper No. 1007.

(Pederson et al.; 1949, 1950, 1953). At intervals duringfermentation, brine samples were withdrawn for bac-teriologic analysis and determination of acidity, hydro-gen ion concentration, and salt content. These sampleswere obtained at more frequent intervals during theearly active stages of fermentation than during thelater stages. The brine samples were plated at ap-propriate dilutions. After incubation and counting ofplates, 20 representative colonies per plating wereselected for isolation and identification. From theidentification of the 20 isolates from each plating andthe total bacterial count for that plating, the totalbrine count of the individual species for each intervalwas estimated. A quantitative estimation of the ef-fectivity of the various species was obtained from theseresults and chemical analysis.Although this study primarily involved normal

fermentation in low salt concentration brines, somepure culture inoculation studies were made to demon-strate the comparative effects of individual species. Forpure culture inoculation studies, 1 per cent by volumeof a 24- to 48-hour glucose broth culture of the desiredorganism was added to the brine. An equivalent amountof sterile broth was added to the uninoculated control.The cucumbers were packed in 45-gallon barrels with

sufficient brine so that the cover was immersed. The con-centration of salt was calculated to give the desired per-centage on the basis of the combined weights of cucum-bers and brine. In three series, barrel no. 1 to 4, 5 to 8,and 9 to 12, 3.5 to 3.75 per cent salt, and in a fourthseries, barrel no. 15 to 19, 2.5 to 2.7 per cent salt wasused. The salt concentration was increased at weeklyintervals beginning about 2 to 3 weeks after packinguntil 14 to 16 per cent salt was present.

In previous studies medium-sized cucumbers were

1956] 259