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Brandon W. Peterson, Prashant K. Sharma, Henny C. van der Mei and Henk J. Busscher Appl. Environ. Microbiol. 2012, 78(1):120. DOI: 10.1128/AEM.06780-11. Published Ahead of Print 28 October 2011.
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A centrifugation coefﬁcient.63 Vbacterium nbacteria Vpellet (1) in which the inﬂuence of the centrifugation speed is expressed by the ratio between the total bacterial volume occupied (i. Consequently. bacterial cell surface damage can be expected to affect surface-sensitive phenomena.000 g) of bacterial cultures was found to reduce the surface charge of organisms not protected by a dense layer of extracellular polymeric substances (9).000 g was demonstrated to cause signiﬁcant reductions in Escherichia coli viability compared to centrifugation at 5. In general. Moreover. which depends on the orientation of the bacteria with respect to each other and the direction of the centrifugal force. 2013 by guest entrifugation is a common laboratory practice used for harvesting planktonic bacteria. including DNA.org/ on January 28. The factor 0. the volume of a single bacterium [Vbacterium]) multiplied by the number of bacteria in the pellet (nbacteria) and the volume of the pellet (Vpellet). using Staphylococcus aureus ATCC 12600 as an example strain. bacteria in a suspension are compacted into a pellet. Moreover. Kolff Institute.asm. Initial deposition rates of Staphylococcus aureus ATCC 12600 to a glass surface decayed exponentially from 4.. which is partly because there is no simple method available to predict or evaluate bacterial cell surface damage due to centrifugation. University Medical Center Groningen and University of Groningen. a wide variety of forces (roughly ranging from 1.org 0099-2240/12/12. American Society for Microbiology. above which centrifugation was considered to impact the outcome of surface-sensitive experiments due to cell surface damage. 10–12) is used without mentioning a reason for a particular choice. the aim of this study is to demonstrate how centrif- C ugal compaction affects the outcome of surface-sensitive experiments due to cell surface damage and to propose a predictive method from which possible cell surface damage can be inferred. doi:10. a signiﬁcant change in initial deposition rate or zeta potential distribution. the resulting bacterial pellet volume. Here. Busscher Department of Biomedical Engineering. 10).Bacterial Cell Surface Damage Due to Centrifugal Compaction Brandon W. and the number of cells within the pellet. harvesting by the high-speed centrifugation (15. van der Mei. W. Very few studies exist on bacterial damage caused by centrifugation because of the difﬁculty in relating centrifugation speed and container geometry to the damage caused. Groningen. for instance. strain SW8 or Staphylococcus epidermidis (9). Peterson. MATERIALS AND METHODS Theoretical background: coefﬁcient of centrifugation. and further investigation revealed a 10-fold drop in virulence. which may easily lead to cell surface damage with a potential effect on the outcome of surface-sensitive experiments. The coefﬁcient C is suggested to be predictive for cell surface damage to occur or not during centrifugation. Centrifugation in essence involves compacting bacteria into a pellet.1128/AEM. The large range of applied centrifugation speeds likely yields unknown bacterial cell surface damage. Sharma.000 g) (1. while the proportion of staphylococci with a zeta potential of around 15 mV decreased from 97 to firstname.lastname@example.org to 1.nl. Sharma.00 Applied and Environmental Microbiology p. versatile method of analysis for describing the compaction of bacteria during centrifugation based on a proposed centrifugation coefﬁcient. For harvesting. Possible effects of centrifugation often are assumed to be absent or nonexistent. such as their adhesion to surfaces. whereas the centrifugation speed could predict damage in only 58% of all cases. Therefore. 120 –125 . This creates a shear force that is active on the contact points between bacteria during compaction. Prashant K. suggesting that interior structures also were modiﬁed (1). Many experimental protocols choose high centrifugation speeds to collect as many bacteria as possible while assuming that it does not cause any bacterial cell damage or cell death (4. These surface-sensitive parameters were used independently to derive a critical centrifugation coefﬁcient (0. However. we provide a simple.580 g. Kinetic experiments demonstrated differences in surface characteristics in Chlamydia psittaci after centrifugation at 1.478 cm 2 s 1 with increasing C.asm. During centrifugation. The Netherlands Centrifugal damage has been known to alter bacterial cell surface properties and interior structures.. Copyright © 2012.000 g. describing the compaction within a pellet can be deﬁned according to the equation C 0. in 84% of all cases included here. Downloaded from http://aem. a coefﬁcient of centrifugation. i. controlling the centrifugation coefﬁcient within narrow limits over a series of experiments yielded 43% smaller standard deviations in initial staphylococcal deposition rates than with centrifugation at ﬁxed speeds for replicate experiments. is proposed that can be easily calculated from the measured volume of a single bacterium.63 Received 2 September 2011 Accepted 19 October 2011 Published ahead of print 28 October 2011 Address correspondence to Prashant K. and its predictive nature will be validated by studying initial deposition to glass and zeta potentials of staphylococci centrifuged at different speeds.000 to 12. J.e. causing collisions against each other that result in shear forces on the bacterial cell surface.k. causing variations between reported results. The critical centrifugation coefﬁcient could successfully predict staphylococcal cell surface damage. while little effect was detected on the viability of Psychrobacter sp. All Rights Reserved. p. Chemical and physical surface damage to Pseudomonas aeruginosa. Henny C. Values of C can be related to different bacterial cell surface properties. Changing the geometry of the centrifugation container or centrifugation speeds changed the value of C signiﬁcantly. C.06780-11 120 aem.040). yielded lower initial deposition rates to substratum surfaces than were observed for undamaged bacteria (2). C. cell surface damage during bacterial centrifugation at 15. C. and Henk J.e.
with volumes given by Vpellet Vpellet cone R2d' 3 a2 3R2 b d'2 R (2) (3) (4) wedge h 2 3b a 3R Vpellet cap d' 2 6 3a where R is the radius of the container. S. or by more indirect techniques. (b) Pellet shapes and dimensions used in the calculation of the pellet volume (Vpellet) (equations 2 to 4). d= is the depth of the pellet of a spherical cone or cap. Pellet geometry strictly coincides with the shape of the container used for centrifugation. the number of bacteria in a pellet. For spherically shaped bacteria. and the pellet volume. represents the random angle orientation of the bacteria with respect to each other and the direction of the centrifugal force (Fig. and rotor JA18. Sixty-ml aliquots were taken from the culture and centrifuged (rotor JA14. a is the measured radius across the straight edge of the cylinder. (a) The contact point between two bacteria during centrifugation. Measured dimensions are 2a. Beckman J2-MC. 70 ml at 23° incline. England) for 24 h and subsequently subcultured into 200 ml fresh TSB under static conditions at 37°C for 18 h. atomic force. The determination of the coefﬁcient of centrifugation according to equation 1 requires the measurement of the volume of a single bacterium. 2013 by guest FIG 1 Schematic of applied forces and geometries of bacterial pellets.org/ on January 28. In a pellet. such as dynamic light scattering. followed by direct counting under a phase-contrast microscope.asm. Bacterial strain. 1a) (8). Oxoid Ltd. Beckman Coulter. 200 ml at a 25° incline. the angle takes random values between 0 and 90 degrees. or spherical cap (equation 4) shape.org 121 . With increasing centrifugation speed. h is p cos ( being the ﬁxed angle of the rotor and p the measured diameter across the vertical plane). CA. and harvesting by centrifugation. The measured radius then can be propagated into the bacterial volume (Vbacterium). The shear force acting on the two cells due to compaction during centrifugation can be expressed as the sinus component ( ) of the centrifugal force. b is the radius across the curved edge of the cylinder (equal to p sin ).Centrifugal Cell Surface Damage Downloaded from http://aem. Beckman January 2012 Volume 78 Number 1 aem.. pellets form according to three speciﬁc geometric shapes at the base/side of the container and possess a cone (equation 2). other measures and formulas should be applied.asm. Typically. aureus ATCC 12600 was precultured from a blood agar plate in 10 ml of tryptone soya broth (TSB. and is the angle of incline of the pellet segment. or phase-contrast microscopy. p. culturing. while increased compaction leads to a smaller pellet. more bacterial cells leave the suspension and enter into the pellet. such as scanning electron. and d= for both the cylindrical wedge and spherical cap geometries. The cone geometry was not used in the manuscript. which is set equal to cos 1[1 (b/R)] (Fig. Palo Alto. cylindrical wedge (equation 3). the radius of a single bacterium can be determined by microscopic techniques. 1b). which yields a higher coefﬁcient of centrifugation. For rod-shaped bacteria. Basingstoke. The enumeration of the total number of bacteria in a pellet can be done after the dispersal of the pellet into suspension.
the bacterial suspension ﬂowed through the ﬂow chamber at a wall shear rate of 15 s 1 for 60 min at 21°C.300 and 14. Subsequently. the reported P values were multiplied by the number of tests performed: two for comparison of container geometry (at 6. aureus ATCC 12600. Brieﬂy. and 14.05) or double asterisks (P 0.330. 2). 150 mM NaCl. Experimental determination of centrifugation coefﬁcients. In all cases. 2013 by guest FIG 2 Effects of centrifugation speed and container geometry on (a) the number of S. aureus ATCC 12600 appeared as single cells during enumeration. the pellet dimensions 2a. Zeta potential distributions were measured with a Lazer Zee meter 501 (PenKem. aureus of 480 and 500 nm. or 4 was chosen to calculate the pellet volume depending on the geometry of the container. (b) the pellet volume after centrifugation. Galway. Fig.330. combined with numbers of staphylococci in the pellets (4. Number of bacteria harvested.00) (Fig. S. respectively. (i) Bacterial deposition in a parallel plate ﬂow chamber.800 g in a JA18 rotor).0). Statistically signiﬁcant differences for centrifugal force are indicated with an asterisk (P 0. staphylococcal deposition experiments were carried out in a parallel plate ﬂow chamber. and those for rotor geometry are indicated as ### (P 0. staphylococci were resuspended in PBS at a density of 3 108 per ml after the different centrifugations. Shaded quartiles indicate those determined using the JA14 rotor. 5. To determine the volume of a single bacterium.800. pH 7. aureus ATCC 12600 in PBS was found to be 480 86 nm. 3. a 60-ml aliquot was centrifuged at medium speed (6.org/ on January 28. Each zeta potential distribution presented contains a minimum of 100 individual bacteria.0) and was resuspended by ﬂushing PBS against the side wall of the container using a pipette. 6.5 1010 to 5. and the number of bacteria adhering per cm2 was enumerated using in-housedeveloped software based on MATLAB. 2b). Surface-sensitive experiments. Pellet volume.asm. the initial deposition rate (j0. and the supernatant was discarded. pH 7. 6. 122 aem. Therefore. The pellet was washed twice with 10 ml phosphate-buffered saline (PBS. the microelectrophoresis chamber was ﬁlled with a bacterial suspension at a density of 107 to 108 bacteria per ml. From a plot of the number of adhering bacteria versus time. United Kingdom). Fig. Pellets then were resuspended in 10 ml PBS and diluted. container volumes. aureus ATCC 12600. Zeta potentials were determined six times from six different bacterial cultures. however. and degrees of ﬁlling.300 g in a JA14 rotor and 1. bacteria were suspended after the different centrifugations in low-ionic-strength PBS (5 mM K2HPO4. 5 mM KH2PO4. The hydrodynamic radius of S.250 g and at 15. First. pellet volume.01). Images were taken every minute. with whiskers representing minimum and maximum values (GraphPad Prism v. 3 and 4). Data analysis.org Applied and Environmental Microbiology .05) from multiple Student t tests. which. Bedford Hills. RESULTS Centrifugation coefﬁcients. 2a) and the pellet volumes (378 to 405 mm3 in the JA14 rotor and 182 to 268 mm3 in the JA18 rotor. 5 mM KH2PO4. 5 mM K2HPO4.300 g for 5 min at 10°C. Bacterial deposition to surfaces is highly sensitive to changes in bacterial cell and substratum surface properties. as derived from initial deposition rates and zeta potential distributions (Fig. Malvern Instruments. The ﬁnal pellet was resuspended in 10 ml PBS to a density of 108 per ml using a Bürker-Türk counting chamber for enumeration. and d= were measured using a caliper.480 and 6.800 g) and three for speed comparisons (6. Changing the centrifugation speed or geometry of the container did not signiﬁcantly alter the number of bacteria harvested in a pellet. 14. Pellet volumes were measured after the different centrifugations in JA14 and JA18 rotor containers. or 15. To reveal possible cell surface damage from differences in zeta potentials.480 g) for 5 min at 10°C (JA14 rotor).001). The horizontal line in panel c indicates the critical C value. 2c as a function of the centrifugation speed. The velocity of each individual bacterium was determined by image sequence analysis and expressed as a zeta potential.0002) with the geometry of the centrifugation containers. Worcestershire. yielded the centrifugation coefﬁcients presented in Fig. in cm 2 s 1) was calculated by linear regression analysis. and calculated centrifugation coefﬁcients as a function of centrifugation force then were compared using unpaired twotailed Student t tests with Welch’s correction for unequal variances.075 cm. NY) equipped with image analysis options for zeta sizing.250. Incidentally. Three measurements were combined from three subcultures to obtain the average radius for S. atomic force and phase-contrast microscopy yielded an average radius of S. Downloaded from http://aem. 6. p. Coulter. assuming that the Helmholtz-Smoluchowski equation holds. 30 mM NaCl. and a voltage difference of 150 V was applied to the chamber. Whiskers indicate the minimum and maximum values for ﬁve measurements.8 1010 staphylococci. After discarding the supernatant. Initial bacterial deposition rates (j0) were determined on the bottom glass plate of the ﬂow chamber with dimensions (3) of 17. while unﬁlled quartiles designate those determined using the JA18 rotor.250. Ireland) at 1. All experiments were carried out ﬁve times with different bacterial cultures. aureus ATCC 12600 cells harvested in a pellet. The hydrodynamic radius of the staphylococci was measured using dynamic light scattering (Zetasizer nano series nano-ZS.Peterson et al.6 by 0. (ii) Zeta potential distribution. To eliminate increased alpha errors (0. Centrifugation coefﬁcients were grouped according to the centrifugal force applied and compiled into box-plot format with median and quartiles. Note that the two rotors differed in rotor axis angles. equation 2. decreased with increasing speed and changed signiﬁcantly (P 0. and (c) the centrifugation coefﬁcient (C) for S. and the number of bacteria in the pellet was determined using a Bürker-Türk counting chamber.480.480 and 15. Brieﬂy.asm.5 by 1.
4).037) coincides well with the one derived from initial deposition rates (0. The dotted horizontal line indicates the median deposition rate from which a critical centrifugation coefﬁcient of 0. (a) The mean number of S. Downloaded from http://aem. 3a) and resulted in an exponential decay (R2 0. aureus ATCC 12600 to glass as a function of the centrifugation coefﬁcient applied during the harvesting of the staphylococci. The dotted horizontal line indicates the median percentage size of this subpopulation.037 is derived. (b) The initial deposition rate of S. ranging from 32 to 4 mV. 3). from which a critical centrifugation coefﬁcient of 0.040).Centrifugal Cell Surface Damage FIG 3 Bacterial deposition in a parallel plate ﬂow chamber. we determined the percentage of experiments fulﬁlling this condition. The numbers of staphylococci adhering to the glass substratum increased linearly with time for at least 15 min under the present experimental conditions (Fig. then the outcome of any surface-sensitive experiment at a centrifugation coefﬁcient higher than the critical one would yield data below the median outcome of that experimental set and vice versa. The slope of the linear deposition during the ﬁrst 15 min varied with the value of C (Fig.org/ on January 28. 2013 by guest Effects on the outcome of surface-sensitive experiments. Predictive capability of the critical centrifugation coefﬁcient.org 123 . The median of the initial deposition rates observed amounted to 2. Note that the critical centrifugation coefﬁcient from zeta potential distributions above which centrifugation is considered to severely impact the cell surface properties (0. The curves obey an exponential decay with a correlation coefﬁcient (R2) equal to 0. This yielded a predictive capability with respect to the initial staphylococcal deposition rate and zeta potential distribution of 95 and 74%.040).75) with increasing centrifugation coefﬁcients (Fig.703 cm 2 s 1 and was adapted to derive a critical centrifugation coefﬁcient (0. To quantify the predictive nature of the critical coefﬁcient of centrifugation. Each distribution is based on the measurement of the zeta potentials of between 100 and 125 bacteria. which was concurrent with the development of other subpopulations of staphylococci with less negative zeta potentials. (b) The percentage of staphylococci contained within the subpopulation with a zeta potential of around 15 mV as a function of the centrifugation coefﬁcient.75. deﬁned similarly to the method used for the initial deposition rates (Fig. 3b) but based on the median percentage size of the 15-mV subpopulation. aureus ATCC 12600 cells deposited on glass as a function of time for different ranges of the centrifugation coefﬁcient.asm. 4b and also can be used to determine a critical centrifugation coefﬁcient. The zeta potential distribution of S. The percentage of the staphylococcal subpopulation with a zeta potential of around 15 mV is given as a function of the centrifugation coefﬁcient in Fig. albeit in different proportions of the total population. respectively. The occurrence of bacteria with a zeta potential of around 15 mV decreased from 97 to 58% with increasing centrifugation coefﬁcients. above which centrifugation was considered to severely affect the initial staphylococcal deposition rate. If the predictive nature of the critical centrifugation coefﬁcient was 100%.040 is derived. The major subpopulation had a zeta potential of around 15 mV (Fig.asm. 3b). aureus ATCC 12600 for two different centrifugal forces. (a) Example of the effect of centrifugation on the zeta potential distribution in a low-ionic-strength suspension of S. January 2012 Volume 78 Number 1 aem. 4a) regardless of the centrifugation coefﬁcient. which is higher than that from a similar analysis on the basis of centrifugation speed that yielded a predic- FIG 4 Bacterial velocities and derivation of zeta potentials. the average predictive capability of the critical centrifugation coefﬁcient amounts to 84%. Therefore. aureus ATCC 12600 showed multiple subpopulations with different zeta potentials and full widths at half maximum (Fig.
a critical value of 0.040 for C was found.org Applied and Environmental Microbiology . based on a so-called centrifugation coefﬁcient. one can use equation 2. 3. The horizontal line indicates the critical centrifugation coefﬁcient value for C established here. aureus ATCC 12600.Peterson et al. Furthermore.05. The centrifugation of S. As an example.5 nN for anchored organic bonds therefore was used to estimate whether centrifugal forces could cause cell surface damage.asm. or 4 depending on the container geometry to calculate the volume affected. C.org/ on January 28. and it is likely that cell surface material is stripped off the cell surface by centrifugation (5). The centrifugation coefﬁcient describes the average compaction observed throughout the pellet rather than the compaction at one point due to differential compaction observed at different depths within a pellet. and even when centrifugation is performed below the critical centrifugation coefﬁcient a sizeable fraction of the bacteria can be expected to experience a shear force of 4. (6) have demonstrated that most anchored organic bonds rupture at 4. The measurement of the centrifugation coefﬁcient is simple and solely requires 124 aem.5 nN at two centrifugation speeds.000 g can be as high as 10 atm.5 nN and inserting the known centrifugal acceleration f and the measured coefﬁcient of centrifugation C. We believe this assumption to be valid. and Grandbois et al. Importantly.asm.02 units over a series of experiments yielded 43% smaller standard deviations in initial staphylococcal deposition rates than when the centrifugation speed was ﬁxed. Cell surface molecules are anchored to the membrane. and the volume of a single bacterium. and it is indicative of the degree of compaction of bacteria in a pellet and thus of the shear forces acting on the cell surface during compaction. suggesting that other experiments involving the bacterial cell surface also could have been used to arrive at this critical value. The proposed centrifugation coefﬁcient. aureus ATCC 12600 at coefﬁcients above 0. they did not severely affect the surface-sensitive properties measured here. C. yielding a coefﬁcient C value of 0. since it may help to improve the reproducibility within one and among different laboratories when performing cell surfacesensitive experiments with centrifuged bacteria. aureus ATCC 12600 centrifuged at 6. For S. tive capability of only 58%. The centrifugation coefﬁcient should be kept low by using a wide. and the potential damage arising from centrifugal compaction usually is ignored due to a lack of an easy assessment method. can be calculated from equation 5. 5a. since we observed no difference in the zeta potential distribution of staphylococci resuspended by spontaneous dissolution over time and by pipetting up to six times (data not shown). The upper threshold of 4. In summary. including their deposition to surfaces and surface charge. controlling the centrifugation coefﬁcient within narrow limits of 0. Hydrostatic pressures within a 15-ml centrifuge container at 10. The centrifugation coefﬁcient can be easily calculated from the pellet volume after centrifugation. The mass at the threshold height m(hthres). However. we assumed that possible effects of bacterial resuspension during harvesting could be neglected. Figure 5b shows that the volume fraction of bacteria affected by centrifugation increases with the coefﬁcient of centrifugation. Downloaded from http://aem. controlling the centrifugation coefﬁcient within narrow limits decreased the standard deviations between replicates more strongly than controlling the centrifugation speed. assuming the forces experienced [F(h)] to be equal to the molecular bond rupture force of 4.5 nN or higher. It is interesting that the critical centrifugation coefﬁcient appeared independent of whether it was derived from the deposition behavior of the strain or its negative cell surface charge density (zeta potential distribution). It can be seen that only a fraction of all bacteria in a pellet actually experience a shear force above 4. f is the centrifugal acceleration. This is an important conclusion of general applicability. 2013 by guest FIG 5 Volume affected within a bacterial pellet. and C is the centrifugation coefﬁcient. ﬂat-bottom container to minimize the pellet height and the shear forces during compaction. The forces that bacteria in a pellet experience depend not only on their position in the pellet but also on the centrifugation speed and the degree of compaction according to the equation F(h) m(h)fC (5) in which m(h) is the total mass of the bacteria above height h. In the proposed analysis of cell surface damage. we have introduced a new method for predicting cell surface damage from effects on the outcome of two surface-sensitive experiments. as a function of fractional volume affected by shear force larger than 4. Volume affected is indicated by the green shaded region. we have plotted the pellet volume as a function of its height in Fig. the number of bacteria in the pellet.5 nN. Data represent a 5-mmthick pellet of S. Here. shear forces active during the centrifugal compaction cause surface damage and affect bacterial cell surfacesensitive properties. below which bacteria are affected by centrifugation.480 g. (b) The centrifugation coefﬁcient.5 nN. resulting in an average success rate of 84% in predicting cell surface damage affecting initial staphylococcal deposition rates and zeta potentials. since these shear forces occur below the critical coefﬁcient value. From the calculated mass affected. (a) Example of the relation between the height in a bacterial pellet with the volume of the pellet (left axis) and the shear force exerted on the bacteria (right axis).040 resulted in a signiﬁcant reduction in initial staphylococcal deposition rates to glass and a change in their zeta potential distributions. can be used to predict cell surface damage with an impact on the cell surface-sensitive properties selected. DISCUSSION Centrifugation is a necessary evil in harvesting microorganisms from liquid cultures.
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