Variability of the Turgor Pressure of Individual Cells of the Gram- Negative Heterotroph Ancylobacter aquaticus

Transcription

1 JOURNAL OF BACTEROLOGY, OCt. 1987, p /87/ $02.00/0 Copyright 1987, American Society for Microbioogy Vo. 169, No. 10 Variabiity of the Turgor Pressure of ndividua Ces of the Gram- Negative Heterotroph Ancyobacter aquaticus M. F. SUZANNE PNETTE AND ARTHUR L. KOCH* Bioogy Department, ndiana University, Boomington, ndiana Received 16 March 1987/Accepted 23 Juy 1987 Ces of Ancyobacter aquaticus were observed under phase microscopy in a chamber to which a measured pressure coud be appied. The initia coapse pressure (Ca), i.e., the owest pressure needed to coapse the most pressure-sensitive gas vesices, was measured for 69 ces. The ces were taken from cutures in ow-density baanced exponentia growth, and the experiments were performed quicky so that the bacteria were in a uniform physioogica state at the time of measurement. The turgor pressure, Pt, is the difference between the pressure, C, that woud cause coapse of vesices when removed from the ce and Ca. n this paper we focus on the variabiity of Pt from ce to ce. Part of the observed variabiity of Ca was due to the variabiity of the coapse pressure of individua vesices (standard deviation [SD = 90 kpa), but because there were about 100 vesices per ce and because a change in refracted ight after the fifth vesice (approximatey) -coapsed probaby coud be detected by the human eye, the pressure woud ony have an SD of 18.6 kpa due to this type of samping error. The observed SD of Pt was 42 kpa, indicating that turgor pressure did vary consideraby from ce to ce. However, the turgor pressure was independent of ce size. Statistica anaysis showed that Pt woud decrease 6.9 kpa over a ce cyce, but with too arge an SD (19.9 kpa) to be significant. This impies that the observed change in Pt over the ce cyce is not statisticay significant. The accumuation of materias by a growing bacteria ce produces an osmotic pressure difference across the ce enveope. Thus, water moves in and out of the ce unti the osmotic pressure is baanced by the tension which deveops in the stretched eastic fabric of the ce wa. The hydrostatic pressure on the ce wa under these conditions is caed the turgor pressure. The turgor pressure in individua bacteria ces has never been precisey measured. We have devised a method to measure turgor pressure in singe bacteria ces containing gas vesices. The method is based on the earier studies of Kebahn (4), who observed iving organisms in a pressurized chamber, and of Wasby (19), who carried out turbidometric studies of cyanobacteria with gas vesices. Gas vesices are hoow gas-fied cyinders with conica end pieces. The vesices are composed amost excusivey of a particuar hydrophobic protein (8). The most hydrophobic part faces inwards, aowing the accumuation of gases during vesice assemby (20). Wasby (18) demonstrated that the gases inside the vesices are in equiibrium with dissoved gases in the environment of the ce. Most vesices coapse instantaneousy when a critica pressure is appied, providing a means of measuring turgor pressure in intact bacteria ces (19, 23, 24). n a turgid ce, there are three components of the pressure impinging on the vesice. Foowing Wasby (23), we designate C as the mean pressure differentia just needed to coapse a vesice. Then C = Ca' + P, - Pv, where C,' is the critica absoute appied pressure, P, is the turgor pressure of the ce, and P, is the partia pressure of gases inside the vesice. Because the cutures are aerated by gente bubbing, measurements are conducted so that P, is equa to the atmospheric pressure. Because the gauge reads the pressure above atmospheric pressure, we can substitute C, + P, for Ca ' and obtain C = Ca + P,. The vaue of Ca is the appied pressure required to cause coapse in a turgid ce and wi be referred to as the coapse pressure. On the ikey assump- * Corresponding author tion that the physica properties of the vesices are constant and independent of the phase of the ce cyce, C can be taken to be constant, and thus Ca varies in a compementary fashion with P,. Since our method aows us to measure Ca, we are abe to assess the variabiity of P, from ce to ce. This method cannot distinguish between the coapse of a singe vesice and the simutaneous coapse of more than one vesice among the hundreds present within the ce. We therefore focused ony on the pressure causing the first noticeabe decrease in refractiity due to vesice coapse in a particuar ce rather than on the pressure at which the observabe ight scattered by a popuation of ces was decreased by 50%, as was done in the nepheometric studies of Wasby (19, 21) and ourseves (7; M. F. S. Pinette and A. L. Koch, in M. Saton, G. D. Shockman, L. Daneo- Moore, and M. Higgins, ed., Antibiotic nhibition of Bacteria Surface Assemby and Function, in press). t was found that the turgor pressure is variabe from ce to ce, but does not vary systematicay with ce size. MATERALS AND METHODS Strain and cuture conditions. Ancyobacter aquaticus (formery Microcycus aquaticus [13]) is a gas-vacuoated, gramnegative heterotroph (11, 12, 17). The term vacuoe has come to refer to aggregations of individua gas vesices, We chose to work with A. aquaticus despite the morphoogica compications discussed beow because it is the ony organism with gas vesices which has the required characteristics for a study of pressure reations in a gram-negative heterotroph. Our strain, M158, was obtained from A. E. Konopka. t produces vesices in virtuay a ces and at a stages of the ce cyce (Konopka, persona communication). n eectron micrographs of A. aquaticus, the vesices do not appear to be grouped but seem to be dispersed throughout the ce (8). We wi therefore use the term vesice throughout this paper. A the cutures used in this study were grown in Casamino Acids (Difco Laboratories)-gucose-vitamin medium (CAGV) (8).

2 4738 PNETTE AND KOCH Cutures were grown with forced aeration at 30 C in CAGV; the average doubing time was 4.1 h. To ensure baanced growth (5), cutures were kept in eary exponentia growth for approximatey 20 generations, maintaining ce density beow 30 plg (dry weight) of ces per m by periodic subcuture. Optica densities were measured with a Cary mode 16 spectrophotometer at 660 nm immediatey after a the vesices in a sampe were coapsed with 550 kpa of nitrogen gas for 1 min. When a compete growth curve was obtained, the dry weight rose exponentiay up to 50 pug/m (0.120 OD600). Apparatus. The experiments were performed with a Zeiss Photomicroscope at a magnification of 1,000x with a 2 x Optovar ens incuded in the optica train. The ces were viewed in rectanguar capiaries (Vitro Dynamics, Rockaway, N.J.) made of borosiicate gass, about 10,um thick with a path ength of 100,um. The inside surfaces of each capiary were coated by fiing the capiaries with 0.1% poy-l-ysine (poy-l-ysine HBr; Mr, 240,000; Sigma Chemica Co.) in phosphate-buffered saine (ph 7) (3) before the bacteria were mounted. This pretreatment caused the bacteria to adhere to the gass (16) so that they remained in the fied of view and in focus as pressure was appied. The capiaries had been previousy fitted with a seeve made of shrinkabe Tefon tubing (Smna Parts, nc., Miami, Fa.) and cemented with epoxy so that they coud be connected to a stainess stee cannua, which was in turn connected to a gas reguator and nitrogen tank. Measurement of coapse pressure and bacteria size. The bacteria suspension was drawn into the capiary immediatey after the poy-l-ysine soution was removed, and the dista end was dipped in partiay hardened 5-min epoxy (Devcon, Danvers, Mass.). The capiary was connected to the cannua and paced on a temperature-controed (30'C) microscope stage as quicky as possibe. A pressure of 204 kpa, which is just beow the minima vesice coapse pressure, was immediatey appied. The pressure was raised in 17-kPa increments at 5-s intervas unti the first noticeabe decrease in ight scattered from the refractie bodies of a particuar ce was observed. This pressure was designated the initia coapse pressure. The pressure was then raised, usuay in the same increments, to 550 kpa to coapse a remaining vesices. The ce size was estimated after the vesices had been coapsed with an ocuar micrometer by recording the product of the ength and breadth of the ce, which we wi ca the projected area. Both measurements are approximate, since the ces are vibrioid to toroida in shape. Severa additiona measurements were taken when an unusua ce shape required it, and appropriate adjustments of the projected area were made. Finay, the pane of focus was raised and owered and the ce was checked carefuy for constrictions or evidence of ce division and to confirm that a vesices had been coapsed. One to three ces coud be viewed during a singe experimenta tria. Ces with overapping portions or which overapped other ces were eft out of the fina resuts. An important aspect of our technique was that the ces were in a steady state of baanced growth at the time that the coapse pressure was actuay measured. This was achieved by taking care to use sampes from diute exponentiay growing batch cutures. The critica coapse pressure determination was competed between 5 and 7 min after the sampe was removed from the aerated cuture in the water bath and ess than 2 min after the first increment of pressure was appied. During this time the partia pressure of gas in the vesices shoud not have changed significanty because J. BACTEROL. the ce density was kept ow. Furthermore, the consumption of 02 is partiay compensated for by the reease of CO2 into the medium. Significant oss of gases by diffusion woud be unikey due to the design of the apparatus. The temperature was maintained within a few tenths of a degree of the growth temperature throughout the necessary manipuations. Coapse pressure in the absence of turgor. Three methods were tried to measure coapse pressure in the absence of turgor. First, the above techniques were used to measure initia coapse pressures in ces immediatey after high concentrations of sucrose or NaC were added to the medium. Second, sampes from growing cutures were treated with 500 p,g of ampiciin per m for 20 min before being oaded into the capiary. Third, initia coapse pressures were obtained after treating sampes of growing cuture with a ysozyme protoco which produced spheropasts containing intact vesices as foows. A 10-m amount of an exponentiay growing cuture was diuted with 200 mm Tris hydrochoride buffer containing 1 M sucrose (ph 8.0), and 3 m of 100 mm EDTA (ph 7.6) (Sigma) was added, foowed by gente stirring for 5 min. Then, 3 m of 10-mg/m hen egg white ysozyme (Sigma) was added. The sampe was incubated at 37 C with occasiona gente stirring, diuted 50:50 with distied water after 30 min, and then subjected to coapse pressure measurement. Pressure measurements. Pressures are reported in kiopascas, which were converted from the pounds per square inch with which standard pressure reguators are marked. Severa different units of pressure are currenty in use. We ist the foowing conversion factors for the reader's convenience: 1 PS = atm = bar = knm-2 = kpa. A 1 osmoa soution has an osmotic pressure of 2,520.5 kpa. Errors given are standard deviations. RESULTS The growth morphoogy of A. aquaticus is quite irreguar. Not ony are the organisms sometimes curved or spira, but aso the thickness of the ce varies from one end to the other. Because our measurements of coapse pressure require that the ces be immobiized on the gass surface, we can estimate size in two, but not three, dimensions. Other studies facing this kind of probem have frequenty assumed that the ces were cyindricay symmetrica. To see whether this was appropriate here, we observed ces in wet mounts with a phase microscope and a Zeiss Neofuor 63 x objective with oi. The movement of the ces by Brownian motion aowed us to subjectivey ascertain that the organisms had roughy cyindrica symmetry around their curved axes. Though they appear to have a circuar cross-section, the area is not constant. The cross-sectiona area does not appear to be systematicay arger or smaer as ces grow and increase their contour ength. Therefore, we decided to assume that the projected area was proportiona to the voume of the ce in question; i.e., it was assumed that the projected area is a rough measure of the ce's progress through the ce cyce. Strain M158 appears to have a quite variabe ce cyce. This incudes both asymmetric division and variabiity in size at division. The coefficient of variation of ce size (45%) in the measured sampe was broader than that expected (23%) if the arge majority of ces divided precisey into equa-sized daughters (6). Pressure variation with ce size. Observations of the initia coapse pressure of vesices in 69 individua ces of A. aquaticus under externay appied pressure are shown as grouped data in Fig. 1. The coapse pressure varied widey,

3 VOL. 169, 1987 ANCYLOBACTER AQUATCUS TURGOR PRESSURE X ca X 1 2 cn 300 e.. so n cr (1) 0.25 c-j 0. J z Z to g + Tta t1o 12 PROJECTED AREA (Mpm2) FG. 1. nitia coapse pressure, Ca, as a function of ce size. C,, is the appied pressure at which the first detectabe decrease in ight refracted by the vesices in a ce was observed. The soid ine is the inear east-squares regression ine of measurements of the 69 individua ces. The space between vertica ines at 4 and 8 p.m2 indicates ces within the size range that most ces woud have in traversing the norma ce cyce. Projected area is an estimate of ce size (see the text). The data have been grouped in ce size casses, for which the means (with bars indicating 1 standard error) are indicated. Numbers indicate the number of ces observed in each size cass. from 207 to 414 kpa; the initia coapse pressure had a mean vaue of 289 ± 42 kpa. The ce size varied in projected area from 2 to 18 p.m2, with a mean projected area of 7.9 ± 3.5 p.m2. Statistica anaysis of a the data indicated no significant correation between ce size and the pressure at the first noticeabe change in refractiity (product-moment correation coefficient, [P << 0.01]). Because the ce cyce of A. aquaticus is variabe, the resuts were anayzed so as to excude the very arge and very sma ces and thereby estimate the variation over a typica ce cyce. Based on the measured sizes of ces, we made the assumption that a typica ce divides in haf at a critica projected size of 8 p.m2. Anaysis of the subset of data for ony those ces whose projected area ranged between 4 and 8 p.m2 ed to a sope of the inear regression of 1.72 ± 4.97 kpa/p.m2, which is not significanty different from zero. This sope corresponds to a 6.9 ± 19.9 kpa increase in initia coapse pressure over a nomina ce cyce. The turgor pressure woud decrease by the same amount. Neither change was statisticay significant. Dividing ces, that is, ces with constrictions observabe with the phase microscope, were eft out of the above cacuations. The size of a dividing ce can be assigned in two ways, either as the size of the whoe ce or as the separate sizes of the prospective daughter ces. Neither choice woud have changed our resuts had the constricting ces been pooed with the other ces. Dividing ces, considered by themseves, did not differ significanty from the other ces in our study. Statistica anaysis of dividing ces gave an average initia vesice coapse pressure of kpa (n = 10). Measurement of turgor pressure. On account of the reationship C = Ca + P,, we can estimate turgor pressure if C is accuratey known. Wasby's method depends on the assumption that P, is in fact reduced to zero in the presence of high concentrations of osmoytes (19). nitia coapse pressures were measured as soon as possibe after diuting a sampe of growing cuture with sucrose or NaC soutions prepared in the growth medium. There was no systematic trend with concentration or type of osmoyte (Tabe 1). The pooed data obtained with fina concentrations of added osmoyte greater than 0.3 M NaC or 0.4 M sucrose was 432 ± 9.4 kpa (n = 32). The pooed data for growing untreated ces in these experiments was 325 ± 12 kpa (n = 17). This corresponds to a turgor pressure of = 107 kpa. These data do not ead to an accurate estimate of P, because TABLE 1. nitia coapse pressures of gas vesices in ces suspended in media of different osmotic pressures Osmoyte added Mean individua and fina concn Ntr. f initia coapse (osmoaity)" pressure (kpa) Sucrose None NaC None a Contro treatments with no added osmoyte were interspersed with hyperosmotic treatments.

4 4740 PNETTE AND KOCH we beieve that during the 7-min exposure to the osmoyte (sucrose or NaC), the ces were abe to accommodate and thus partiay regain their turgor. As an aternative, the coapse pressure of the vesices in the absence of turgor pressure was measured by treating the ces with 500,ug of ampiciin per m. As shown previousy (7; Pinette and Koch, in press) by nepheometric studies, when the synthesis of the ce wa is stopped, P, increases in some of the ces and decreases in another fraction. t is assumed that this atter fraction of ces ost their turgor pressure competey. After 20 min of treatment, about 84% of the vesices appeared to be in this turgor-free state. Experiments on individua ces with the microscope equivaent to those conducted nepheometricay gave two distinct popuations of ces. One group, component, had a coapse pressure (176 ± 55 kpa [n = 9]) ower than that of the untreated contro (292 ± 80 kpa [n = 15]). The significance of component is discussed esewhere (7; Pinette and Koch, in press). The other group had a higher coapse pressure ( kpa [n = 15]). f these atter ces had competey ost their turgor pressure, then the turgor pressure in the growing ces was = 122 kpa. A third aternative procedure was tried in which the ces were treated with EDTA and ysozyme in the presence of sucrose. Such ces had ost the rigid ayer, as shown by the fact that they burst when diuted twofod with water. The observed coapse pressure of eight individua ces was kpa. This corresponds to a turgor pressure of = 132 kpa. DSCUSSON This study focused on avoiding experimenta error in coapse pressure measurements due to accommodation of the ce to the appied hydrostatic and osmotic pressure and to the effect of the coapse of the most susceptibe vesices in changing ceuar voume and turgor pressure. The first source of error was reduced by minimizing the time (1 to 2 min) after the ces were exposed to pressure unti the coapse coud be detected. Accommodation of the gases inside the vesices to the increased pressure was unikey because the ces under observation were 2 to 3 cm from the gas phase and itte or no additiona gas coud diffuse over this distance during the time of the experiment. The second source of error was minimized by measuring the first observabe coapse of vesices. For discussion purposes, imagine that the voume of the ce is decreased 6% by the coapse of vesices. The interna voume of the ce woud decrease instantaneousy by neary 6%. But in seconds, water woud enter the ce, since the ce enveope is quite permeabe to water (14, 23). Amost a of the 6% oss in ce voume woud be recovered when water activity became equa on both sides of the ce wa. However, the turgor pressure woud remain 6% ower unti the ce coud restore the origina hydrostatic pressure through the active transport of osmoytes or by forming new gas vesices. Therefore, this systematic error in the estimation of Ca woud be smaer if fewer coapsed vesices were detected. This creates a major probem. Athough an individua ce may have hundreds of vesices, we ony measured the coapse of the few most fragie ones. This operationa constraint causes the measurements of coapse pressure, Ca, to be more variabe than if we had measured the pressure that coapsed 50% of the vesices. The coapse pressure for individua vesices in the absence of turgor pressure varies; i.e., the standard deviation is 80 kpa in Anabaenafos-aquae J. BACTEROL. (1) and 90 kpa in A. aquaticus (7). Athough this is a arge variation, it affects the measurement of Ca and P, to a much esser degree because there are many vesices per ce. The standard deviation due to this cause was estimated by a Monte Caro cacuation. t was assumed that (i) each ce had 100 vesices, (ii) the experimenta coapse was recorded when the fifth most sensitive vesice coapsed, (iii) the sensitivity to coapse was distributed in a norma, Gaussian manner, and (iv) the standard deviation of vesice coapse was 90 kpa, as estimated by our nepheometric (7) studies. The computer program chose 100 random vaues from a norma distribution with unit standard deviation, arranged them in increasing order, and seected the fifth from the smaest. The standard deviation of the fifth-smaest vaues of 600 such repetitions of sets of 100 was Therefore the standard deviation of either Ca or P, woud be x 90 kpa = 18.6 kpa, if this samping error were the ony source of error. This vaue is smaer than the observed standard deviation for normay growing ces (42 kpa), so it is not ikey that vesice samping statistics are the dominant source of error in measurements. For comparison, if the pressure causing coapse of 50% of the vesices were used as the criterion and if there are 100 vesices per ce, the error contribution is 90/V10 = 9 kpa. Variabiity of turgor pressure. t is concuded that the initia coapse pressure does vary from ce to ce even in cutures undergoing strict baanced growth. Athough the observation of the initia coapse is subjective to some degree, it coud not cause fuctuations of 42 kpa. Severa quaitative observations support the concusion. One is that the intensity of scattered ight appeared to dim uniformy throughout the ength of singe ces as pressure was increased. This is consistent with the assumption that the turgor pressure is the same at any ocation within a procaryotic ce, couped with the assumption that the vesices are numerous and randomy distributed within these ces. Sister ces that are sti attached to each other but have separate ce compartments have simiar initia coapse pressures. However, separated ces within the same fied often have quite different coapse pressures. This impies that the variabiity of Ca observed is not due to inaccuracy of pressure measurement at the time of initia coapse of vesices. t further impies that cosey reated ces have more simiar turgor pressures than distanty reated ces, without impying whether this resuts from physioogica or quasigenetic phenomena. The second concusion is that this variation is not correated with ce size. Because conditions were not atered during the few minutes required for oading the capiary, etting the epoxy dry, and increasing the pressure, the range of initia coapse pressure for 69 measurements of intact ces which varied from 207 to 414 kpa refects bioogica variation of the turgor pressure in the growing popuation. This concusion is consistent with the coapse pressure range of A. fos-aquae, which was 440 to 605 kpa (19). Since coapse pressure in turgid ces is the sum of turgor pressure and C, which is assumed to be constant, we concude that turgor pressure varies from ce to ce. Additiona evidence that the turgor pressure is not constant from ce to ce is our repeated observation of distincty different vesice coapse pressures for two ces suspended in growth medium under simutaneous observation in a singe microscope fied. That is, even though the ces are indistinguishabe morphoogicay, they are ikey to have disparate vesice coapse pressures. The resuts shown in Fig. 1 demonstrate that neither Ca or P, varied systematicay with ce size. These measurements

5 VOL. 169, 1987 were made to observe the variation in turgor pressure through the ce cyce. However, the variabiity in ce morphoogy and in the ce size at division prevented a strong concusion as to whether change in P, occurred during the division cyce. t is possibe that a negigibe fraction of the variance was associated with the regression of Ca on ce size. t is concuded that any cycica change in turgor pressure as an integra part of the ce cyce is smaer than our techniques can now measure. A more precise study of pressure reations during the ce cyce in a gram-negative heterotroph requires an organism with a more reguar ce cyce and a more constant turgor pressure from ce to ce. Absoute vaue of the turgor pressure. The average turgor pressure of a popuation of growing gram-negative heterotrophic bacteria (Prosthemicrobium pneumaticum) has been measured (22); the turgor pressure was 300 kpa. The few estimates in the iterature indicate a range of 300 to 500 kpa for Escherichia coi and Samonea typhimurium (10, 15). Wasby has measured the turgor pressures of severa gas-vacuoate procaryotic ces by his sucrose method. He found mean turgor pressures of 312 kpa for A. fos-aquae and 76 kpa for Rhodotheca conspicua (19). He has aso provided us with data indicating the A. aquaticus has a mean turgor pressure of 190 kpa (persona communication). This vaue and our own (7) estimate of popuations of ces by 90 ight-scattering methods (210 kpa) are greater than any of the three estimates presented here (110, 122, and 132 kpa). This discrepancy is probaby not the resut of a systematic error in the measurement of Ca of the growing cuture, but in the estimation of the coapse pressure of turgor-free vesices, C. The microscope method is unsuitabe for the measurement of the coapse pressure of individua vesices after they are iberated from the ce. The vesices are too sma, too mobie, and too unstabe to be observed. Consequenty, three ways were tried to reieve the ceuar turgor pressure whie retaining the vesices within the ceuar structure. None of these methods proved totay satisfactory. The sucrose method required that the ces be exposed to a higher osmotic environment for at east 7 min, during which time the ce amost certainy accommodates to some degree and reestabishes some of its turgor pressure. The ampiciin method aows observation of the vesices of ces unti the moment they burst, but the judgment that turgor has been ost competey is probematica. Thus, this procedure is not as usefu or as reiabe as the same technique appied to popuations with nepheometric methods (7). With the nepheometric technique, vesices that have been iberated from the burst ces and not yet degraded contribute to the measurements. Thus, the nepheometric coapse pressure measurements refect the properties of these turgor-free vesices as we as of those retained inside ces with reduced or no turgor. Consequenty, the turgor pressure is underestimated with the microscope version of the ampiciin procedure because an increased proportion of vesices are under some pressure from the ce enveope. The ysozyme technique was successfu in aowing the measurement of Ca of osmoticay unstabe vesices. The vaues were higher than the contros, but this coud be in part due to the sucrose treatment and in part due to the partia persistence of the ce wa. Consequenty, we presenty beieve that our 480-kPa estimate for C made with nepheometric measurement (7) of ampiciin-treated ces is the most reiabe vaue now avaiabe and that the turgor pressure, P,, is about 210 kpa. With further refinement, the microscope technique not ony shoud be capabe of precise measurements of the ANCYLOBACTER AQUATCUS TURGOR PRESSURE 4741 variation of turgor pressure throughout the ce cyce, but shoud provide the absoute vaue of the turgor pressure of individua ces. This wi require, however, not ony instrumenta improvements, but the study of a bacterium that divides more reguary than does A. aquaticus. The point that we wish to make here is that since there is no systematic decrease in the initia coapse pressure, there is no gradua increase or decrease in turgor pressure associated with the ce cyce eading to ce division. Any change must be very sma compared with average ce turgor pressure. Aso, if there is any discrete (stepwise) change in turgor pressure at any point in the ce cyce, we were unabe to detect it. n particuar, the turgor pressure of constricting ces was simiar to that of nonconstricting ces. ACKNOWLEDGMENTS This work was supported by Pubic Heath Service grant GM from the Nationa nstitute of Genera Medica Sciences. We thank W. W. Badwin, A. E. Konopka, H. E. Kubitschek, and A. E. Wasby for hepfu comments and suggestions. LTERATURE CTED 1. Buckand, B., and A. E. Wasby A study of the strength and stabiity of gas vesices isoated from a bue-green aga. Arch. Microbio. 79: Cohen-Bazire, G., R. Kunisawa, and N. Pfennig Comparative study of the structure of gas vacuoes. J. Bacterio. 100: Dubecco, R., and M. Vogt Paque formation and isoation of pure ines with poiomyeitis viruses. J. Exp. Med. 99: Kebahn, H Neue Untersuchungen uber die Gasvakuoen. Jahrb. Wiss. Bot. 61: Koch, A. L Growth measurement, p n P. Gerhardt, R. G. E. Murray, R. N. Costiow, E. W. Nester, W. A. Wood, N. R. Krieg, and G. B. Phiips (ed.), Manua of methods for genera bacterioogy. American Society for Microbioogy, Washington, D.C. 6. Koch, A. L., and M. L. 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