scholarly journals Surface charge characteristics of Bacillus subtilis NRS-762 cells

2018 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Sodium Chloride ◽  
Ionic Strength ◽  
Zeta Potential ◽  
Cell Surface ◽  
Surface Charge ◽  
Sodium Nitrate ◽  
Actual Surface ◽  
Wash Buffer ◽  
Ph Range ◽  
Ph Profiles

Bacterial cell surface carries an electrical charge due to the myriad functional groups present, as well as assortment of ions and molecules nonspecifically adsorbed to the cell surface. Thus, solution in contact with the bacterial cell surface play a critical role in influencing the overall surface charge characteristics through conferring nonspecifically adsorbed ions and molecules. Various wash buffers are commonly used in removing nonspecifically adsorbed ions and molecules for revealing the real surface charge of the bacterium. Using electrophoretic mobility measurement of zeta potential, this study attempted to understand the surface charge characteristics of Bacillus subtilis NRS-762 (ATCC 8473) with the help of three wash buffers: deionized (DI) water, 0.1M sodium nitrate, and 9 g/L sodium chloride. Experiment results revealed that B. subtilis NRS-762 was negatively charged over the entire pH range from 1.5 to 12. Specifically, with deionized water as wash buffer, the point-of-zero-charge (pHzpc) was at pH 1.5, which indicated that large amount of negatively charged functional groups were present on the cell surface. Comparison between the zeta potential-pH profiles of B. subtilis NRS-762 cultivated at 30 oC and 37 oC revealed that the profile for growth at 37 oC was more negatively charged over the entire pH range compared to that for growth at 30 oC. This highlighted that physiological adaptation might had occurred on the cell surface for coping with growth at a higher temperature. Zeta potential-pH profiles obtained revealed that DI water could not remove significant quantities of the nonspecifically adsorbed ions and molecules. On the other hand, the zeta potential-pH profiles of cells washed with 0.1M sodium nitrate and 9 g/L sodium chloride overlapped each other substantially, and were more negatively charged over the pH range from 2 to 11, compared to that of cells washed with DI water. This revealed substantial removal of nonspecifically adsorbed ions and molecules with the use of 0.1M sodium nitrate (0.1M ionic strength) and 9 g/L sodium chloride (0.15M ionic strength), which helped reveal the actual surface charge of B. subtilis NRS-762 cells. Collectively, actual surface charge of B. subtilis NRS-762 was masked by nonspecifically adsorbed ions and molecules, which could be removed by 0.1M sodium nitrate and 9 g/L sodium chloride wash buffer. Thus, in the case of B. subtilis NRS-762, 0.1M ionic strength wash buffer was the threshold at which there was complete removal of nonspecifically adsorbed ions and molecules from the cell surface.

scholarly journals Surface charge characteristics of Bacillus subtilis NRS-762 cells

2018 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Sodium Chloride ◽  
Ionic Strength ◽  
Zeta Potential ◽  
Cell Surface ◽  
Surface Charge ◽  
Sodium Nitrate ◽  
Actual Surface ◽  
Wash Buffer ◽  
Ph Range ◽  
Ph Profiles

Bacterial cell surface carries an electrical charge due to the myriad functional groups present, as well as assortment of ions and molecules nonspecifically adsorbed to the cell surface. Thus, solution in contact with the bacterial cell surface play a critical role in influencing the overall surface charge characteristics through conferring nonspecifically adsorbed ions and molecules. Various wash buffers are commonly used in removing nonspecifically adsorbed ions and molecules for revealing the real surface charge of the bacterium. Using electrophoretic mobility measurement of zeta potential, this study attempted to understand the surface charge characteristics of Bacillus subtilis NRS-762 (ATCC 8473) with the help of three wash buffers: deionized (DI) water, 0.1M sodium nitrate, and 9 g/L sodium chloride. Experiment results revealed that B. subtilis NRS-762 was negatively charged over the entire pH range from 1.5 to 12. Specifically, with deionized water as wash buffer, the point-of-zero-charge (pHzpc) was at pH 1.5, which indicated that large amount of negatively charged functional groups were present on the cell surface. Comparison between the zeta potential-pH profiles of B. subtilis NRS-762 cultivated at 30 oC and 37 oC revealed that the profile for growth at 37 oC was more negatively charged over the entire pH range compared to that for growth at 30 oC. This highlighted that physiological adaptation might had occurred on the cell surface for coping with growth at a higher temperature. Zeta potential-pH profiles obtained revealed that DI water could not remove significant quantities of the nonspecifically adsorbed ions and molecules. On the other hand, the zeta potential-pH profiles of cells washed with 0.1M sodium nitrate and 9 g/L sodium chloride overlapped each other substantially, and were more negatively charged over the pH range from 2 to 11, compared to that of cells washed with DI water. This revealed substantial removal of nonspecifically adsorbed ions and molecules with the use of 0.1M sodium nitrate (0.1M ionic strength) and 9 g/L sodium chloride (0.15M ionic strength), which helped reveal the actual surface charge of B. subtilis NRS-762 cells. Collectively, actual surface charge of B. subtilis NRS-762 was masked by nonspecifically adsorbed ions and molecules, which could be removed by 0.1M sodium nitrate and 9 g/L sodium chloride wash buffer. Thus, in the case of B. subtilis NRS-762, 0.1M ionic strength wash buffer was the threshold at which there was complete removal of nonspecifically adsorbed ions and molecules from the cell surface.

scholarly journals Zeta potential of bacterial cells: Effect of wash buffers

2013 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Sodium Chloride ◽  
Ionic Strength ◽  
Zeta Potential ◽  
Cell Surface ◽  
Phosphate Buffer ◽  
Bacterial Cells ◽  
Ph Profile ◽  
Wash Buffer ◽  
Ph Range ◽  
Zeta Potential Analysis

Zeta potential - defined as the electrical charge at the shear plane - is widely used as a proxy for cell surface charge. Consequent of its definition, nonspecific adsorption of ions on the cell surface may alter the value - and polarity - of the measured zeta potential, thereby, leading to erroneous results. Multiple wash and centrifugation steps are commonly used in preparing cells for zeta potential analysis – where various wash buffers (such as 9 g/L sodium chloride and 0.1M sodium nitrate) help remove ions and charged molecules nonspecifically bound to the cell surface. Nevertheless, little information on the wash buffers’ relative efficacies in removing nonspecifically bound ions hamper the comparison of zeta potential results across laboratories even for the same bacterial strain cultured under identical conditions. Thus, the present study sought to evaluate the effect of various wash buffers on zeta potential of bacterial cells grown in two culture media differing in salt content – thereby, allowing potential differential efficacy of buffers in removing nonspecifically adsorbed ions and metabolites to be discerned. Preliminary data revealed that for Escherichia coli DH5α (ATCC 53868) grown in LB Lennox (supplemented with 2 g/L glucose), the zeta potential-pH profile was not significantly different over the pH range from 2 to 12 for deionized water, 9 g/L sodium chloride, and phosphate buffer saline (PBS) wash buffers. As the glucose supplemented LB medium was a low salt medium without a phosphate buffer, it was unlikely that nonspecific adsorption of ions on the cell surface was extensive – thus, supporting the observation that the various wash buffers used did not have differential effect on zeta potential measurement. On the other hand, the zeta potential-pH profile of E. coli grown in a semi-defined medium with a high capacity phosphate buffer system, was significantly different over the pH range from 1 to 12 for deionized water, 9 g/L sodium chloride, 0.1M sodium nitrate, 0.1M sodium acetate, and 0.1M sodium citrate with the extent of difference positively correlated with wash buffers’ ionic strength. A similar relationship was also observed between the measured point of zero charge (pHzpc) and ionic strength of wash buffer, which, taken together, suggested that charge screening might be an important mechanism for removing the adsorbed ions. Collectively, although the experimental data suggests possible use of high ionic strength wash buffer in removing nonspecifically adsorbed ions from bacterial cell surface prior to zeta potential analysis, possible structural damage to the surface from removing intrinsic ions - necessary for stabilizing the bacterial cell wall - could not be discounted.

scholarly journals Zeta potential of bacterial cells: Effect of wash buffers

2015 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Sodium Chloride ◽  
Ionic Strength ◽  
Zeta Potential ◽  
Cell Surface ◽  
Phosphate Buffer ◽  
Bacterial Cells ◽  
Ph Profile ◽  
Wash Buffer ◽  
Ph Range ◽  
Zeta Potential Analysis

Zeta potential - defined as the electrical charge at the shear plane - is widely used as a proxy for cell surface charge. Consequent of its definition, nonspecific adsorption of ions on the cell surface may alter the value - and polarity - of the measured zeta potential, thereby, leading to erroneous results. Multiple wash and centrifugation steps are commonly used in preparing cells for zeta potential analysis – where various wash buffers (such as 9 g/L sodium chloride and 0.1M sodium nitrate) help remove ions and charged molecules nonspecifically bound to the cell surface. Nevertheless, little information on the wash buffers’ relative efficacies in removing nonspecifically bound ions hamper the comparison of zeta potential results across laboratories even for the same bacterial strain cultured under identical conditions. Thus, the present study sought to evaluate the effect of various wash buffers on zeta potential of bacterial cells grown in two culture media differing in salt content – thereby, allowing potential differential efficacy of buffers in removing nonspecifically adsorbed ions and metabolites to be discerned. Preliminary data revealed that for Escherichia coli DH5α (ATCC 53868) grown in LB Lennox (supplemented with 2 g/L glucose), the zeta potential-pH profile was not significantly different over the pH range from 2 to 12 for deionized water, 9 g/L sodium chloride, and phosphate buffer saline (PBS) wash buffers. As the glucose supplemented LB medium was a low salt medium without a phosphate buffer, it was unlikely that nonspecific adsorption of ions on the cell surface was extensive – thus, supporting the observation that the various wash buffers used did not have differential effect on zeta potential measurement. On the other hand, the zeta potential-pH profile of E. coli grown in a semi-defined medium with a high capacity phosphate buffer system, was significantly different over the pH range from 1 to 12 for deionized water, 9 g/L sodium chloride, 0.1M sodium nitrate, 0.1M sodium acetate, and 0.1M sodium citrate with the extent of difference positively correlated with wash buffers’ ionic strength. A similar relationship was also observed between the measured point of zero charge (pHzpc) and ionic strength of wash buffer, which, taken together, suggested that charge screening might be an important mechanism for removing the adsorbed ions. Collectively, although the experimental data suggests possible use of high ionic strength wash buffer in removing nonspecifically adsorbed ions from bacterial cell surface prior to zeta potential analysis, possible structural damage to the surface from removing intrinsic ions - necessary for stabilizing the bacterial cell wall - could not be discounted.

scholarly journals Bacteria surface charge in “layers”: revealed by wash buffers of different ionic strength

2016 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Ionic Strength ◽  
Zeta Potential ◽  
Cell Surface ◽  
Surface Charge ◽  
Shake Flask ◽  
Cell Envelope ◽  
Cell Mass ◽  
Deionized Water ◽  
Growth Environment ◽  
Wash Buffer

Bacteria surface charge derives its meaning from the cell’s environment; thus, there is no one specific surface charge. Determining the surface charge of bacteria in its native environment requires measuring the proxy variable, zeta potential, using cells obtained from field studies. However, lack of adequate cell mass and concerns over measurement of a mixed species consortia rather than a specific species meant that bacteria surface charge measurement require biomass obtained from laboratory shake flask pure culture. Often grown in rich medium where myriad proteins and ions nonspecifically adsorbed on the cell envelope or peptidoglycan layer, standard procedures for preparing the cell mass require repeated steps of washing and centrifugation with various wash buffers, the efficacies of which are poorly understood. This report describes a systematic study on how wash buffers of different composition and salinity affect the efficiency of removing nonspecifically adsorbed biomolecules and ions from Escherichia coli DH5a (ATCC 53868) cultured aerobically (shake flask, 37 oC and 230 rpm) in LB Lennox medium. Using zeta potential-pH profiles over pH 1 to 12 as readout, proxy measurement of wash buffers’ efficacies showed that efficiency of removing nonspecifically adsorbed ions and metabolites positively correlates with wash buffer ionic strength. More importantly, 0.15M ionic strength (i.e., 9 g/L NaCl and phosphate buffer saline) seems to be the minimum below which there appeared to be little removal of nonspecifically adsorbed biomolecules (deionized water wash as control). On the other hand, high ionic strength of 0.6M (0.1M sodium citrate) significantly changed the point of zero charge (pHzpc), a reference marker for the removal of ions intrinsic to the cell envelope; thus, indicating significant cell surface damage. Collectively, results obtained provided important pointers for wash buffer choice concerning preservation of cell envelope integrity. Finally, is there a true cell surface charge? Yes, but how to define it? How many “layers” of adsorbed biomolecules? Philosophically, cells in culture broth are coated with layers of metabolites, proteins and ions; hence, desire to reveal the true surface charge is essentially a decoating process, where wash buffers of increasing ionic strength could remove each layer through charge screening. Where is the endpoint? This research suggests that ionic strength of wash buffers chosen should be similar to that of the environment the research is seeking to address. Imagine a single bacterium suspended in LB medium, what is its relevant surface charge? It is the one where the loosely associated ions and metabolites can be removed, similar to the constant adsorption and desorption processes that the cell experiences in its growth environment. Thus, deionized water wash provides a good estimate of the bacteria surface charge as grown in specific medium.

scholarly journals Bacteria surface charge in layers: revealed by wash buffers of different ionic strength

2016 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Ionic Strength ◽  
Zeta Potential ◽  
Cell Surface ◽  
Surface Charge ◽  
Cell Envelope ◽  
Cell Mass ◽  
Surface Damage ◽  
Culture Broth ◽  
Point Of Zero Charge ◽  
Wash Buffer

Bacteria surface charge derives its meaning from the cell’s environment; thus, there is no specific surface charge. Determining the surface charge of bacteria in its native environment requires measuring the proxy variable, zeta potential, using cells obtained from field studies. However, lack of adequate cell mass and concerns over measurement of a mixed species consortia rather than a specific species meant that bacteria surface charge measurement require biomass obtained from pure culture. Often grown in rich medium where myriad proteins and ions nonspecifically adsorbed onto the cell envelope or peptidoglycan layer, standard procedures for preparing the cell mass incorporate repeated steps of washing and centrifugation with various wash buffers, the efficacies of which are poorly understood. This report describes a systematic study on how wash buffers of different composition and salinity affect the efficiency of removing nonspecifically adsorbed biomolecules and ions from Escherichia coli DH5α (ATCC 53868) cultured aerobically (shake flask, 37 oC and 230 rpm) in LB Lennox medium. Using zeta potential-pH profiles over pH 1 to 12 as readout, proxy measurement of wash buffers’ efficacies showed that efficiency of removing nonspecifically adsorbed ions and metabolites positively correlates with wash buffer ionic strength. More importantly, 0.15M ionic strength (i.e., 9 g/L NaCl and phosphate buffer saline) seems to be the minimum below which there appeared to be little removal of nonspecifically adsorbed biomolecules. On the other hand, high ionic strength of 0.6M (e.g., 0.1M sodium citrate) significantly changed the point of zero charge (pHzpc), a reference marker for removing ions intrinsic to the cell envelope; thus, indicating significant cell surface damage. Collectively, results obtained inform wash buffer choice with regards to preserving cell envelope integrity. But, is there a true cell surface charge? Yes, but how do we define it in number of “layers” of adsorbed biomolecules? Philosophically, cells in culture broth are coated with layers of metabolites, proteins and ions; hence, desire to reveal the true surface charge is essentially a decoating process, where wash buffers of increasing ionic strength remove each layer via charge screening. However, where is the endpoint? This research offers a different perspective and answer: i.e., ionic strength of wash buffers chosen should be similar to that of the environment the research is seeking to address. Imagine a single bacterium suspended in LB medium, where there is constant adsorption and desorption of biomolecules as the cell grows: what is its relevant surface charge? It is the one where the loosely associated ions and metabolites is removed. Thus, deionized water wash provides a good estimate of the bacteria surface charge as grown in specific medium.

scholarly journals Zeta potential of bacterial cells: Effect of wash buffers

2017 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Ionic Strength ◽  
Zeta Potential ◽  
Cell Surface ◽  
Bacterial Cell ◽  
Phosphate Buffer ◽  
Defined Medium ◽  
Deionized Water ◽  
Nonspecific Adsorption ◽  
E Coli ◽  
Ph Range

Zeta potential, defined as the electric charge at the shear plane, is widely used as a proxy parameter for bacterial cell surface charge. Nonspecific adsorption of ions or polyelectrolytes onto the cell surface, however, alters the value and polarity of the measured zeta potential, leading to erroneous results. Multiple wash and centrifugation steps are commonly used in preparing cells for zeta potential analysis, where various wash buffers (such as 9 g/L NaCl, 0.001M KCl, and 0.1M NaNO3) are routinely used for removing (by charge screening) ions and charged molecules that bind nonspecifically to the cell surface. Using Escherichia coli DH5α grown in LB Lennox (with 2 g/L glucose), experiment data showed that the zeta potential-pH profile was not significantly different over the pH range from 2 to 12 for deionized water, 9 g/L NaCl, and phosphate buffer saline (PBS) wash buffers. As LB Lennox is a low salt medium without a phosphate buffer, it was likely that the extent of nonspecific adsorption of ions on the cell surface was not severe, and the different wash buffers would correspondingly not exert much effect on measured zeta potential. Zeta potential-pH profiles for E. coli grown in a semi-defined medium (with a high capacity phosphate buffer system), on the other hand, was significantly different over the pH range from 1 to 12 for deionized water, 9 g/L NaCl, 0.1M NaNO3, 0.1M sodium acetate, and 0.1M sodium citrate wash buffers with the deviation positively correlated with wash buffer’s ionic strength. Furthermore, the point of zero charge (pHzpc) for E. coli grown in the semi-defined medium varies between 1.5 and 3, in an ionic strength dependent manner, for the various wash buffers tested. Collectively, this preliminary study highlights the importance of wash buffer ionic strength in affecting removal efficiency of non-specifically absorbed ions on bacterial cell surface, where a threshold exists (0.15M) for charge screening to be effective. At the upper bound, 0.6M ionic strength might remove cations intrinsic to the cell envelope, leading to possible cell surface damage and erroneous measurements.

scholarly journals Bacterial surface charge in “layers”: revealed by wash buffers of different ionic strength

2018 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Ionic Strength ◽  
Zeta Potential ◽  
Surface Charge ◽  
Cell Envelope ◽  
Cell Mass ◽  
Culture Broth ◽  
Point Of Zero Charge ◽  
Bacterial Surface ◽  
Incomplete Removal ◽  
Wash Buffer

Bacterial surface charge derives its meaning from the cell’s environment such as the solution in contact with the cell. Determining the surface charge of bacteria in its native environment requires measuring the proxy variable, zeta potential, using cells obtained from field studies. However, lack of adequate cell mass and concerns over measurement of a mixed species consortia rather than a specific species meant that bacterial surface charge measurement require biomass obtained from pure culture. Often grown in rich medium where myriad proteins and ions nonspecifically adsorbed onto the cell envelope or peptidoglycan layer, standard procedures for preparing the cell mass incorporated repeated steps of washing and centrifugation with various wash buffers, the efficacies of which are poorly understood. This report describes the results of a systematic study on how wash buffers of different composition and ionic strength affect the efficiency of removing nonspecifically adsorbed biomolecules and ions from Escherichia coli DH5α (ATCC 53868) cultured aerobically (shake flask, 37 oC and 230 rpm) in LB Lennox medium with 2 g/L glucose and a formulated medium. Using zeta potential-pH profiles over pH 1 to 12 as readout, the results showed that efficiency of removing nonspecifically adsorbed ions and metabolites positively correlated with wash buffer ionic strength. More importantly, 0.15M ionic strength (i.e., 9 g/L NaCl) seemed to be the minimum below which there was incomplete removal of nonspecifically adsorbed biomolecules. On the other hand, high ionic strength of 0.6M (e.g., 0.1M sodium citrate) significantly changed the point of zero charge (pHzpc), a reference marker for removal of ions intrinsic to the cell envelope. Collectively, results obtained inform wash buffer choice with regards to preserving cell envelope integrity, and avoidance of adsorption of buffer ions such as citrate. But, is there a true cell surface charge? Yes, but how do we define it in number of “layers” of adsorbed biomolecules? Philosophically, cells in culture broth are coated with layers of metabolites, proteins and ions. Hence, desire to reveal the true surface charge is essentially a decoating process, where wash buffers of increasing ionic strength remove each layer via charge screening. However, where is the endpoint? This research offers a different perspective and answer. Imagine a single bacterium suspended in LB medium, where there is constant adsorption and desorption of biomolecules as the cell grows: what is its relevant surface charge? It is the one where the loosely associated ions and metabolites are removed while retaining the nonspecifically adsorbed ions and biomolecules. Thus, deionized water wash provides a good estimate of the bacterial surface charge as grown in specific medium.

scholarly journals Zeta potential of bacteria cells: Effect of wash buffers

2016 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Ionic Strength ◽  
Zeta Potential ◽  
Cell Surface ◽  
Phosphate Buffer ◽  
Defined Medium ◽  
Deionized Water ◽  
Ph Profile ◽  
Nonspecific Adsorption ◽  
E Coli ◽  
Ph Range

Zeta potential, defined as the electric charge at the shear plane, is widely used as a proxy for bacteria cell surface charge. Nonspecific adsorption of ions or polyelectrolytes onto the cell surface, however, alters the value and polarity of the measured zeta potential, leading to erroneous results. Multiple wash and centrifugation steps are commonly used in preparing cells for zeta potential analysis, where various wash buffers (such as 9 g/L NaCl, 0.001M KCl, and 0.1M NaNO3) are routinely used for removing (by charge screening) ions and charged molecules that bind nonspecifically to the cell surface. Preliminary data showed that, for Escherichia coli DH5α (ATCC 53868) grown in LB Lennox (with 2 g/L glucose, or LBG), the zeta potential-pH profile was not significantly different over the pH range from 2 to 12 for deionized water, 9 g/L NaCl, and phosphate buffer saline (PBS) wash buffers. As LBG is a low salt medium without a phosphate buffer, it was likely that the extent of nonspecific adsorption of ions on the cell surface was not substantial, and the different wash buffers would correspondingly not affect measured zeta potential much. For E. coli grown in a semi-defined medium (with a high capacity phosphate buffer system), the zeta potential-pH profile was significantly different over the pH range from 1 to 12 for deionized water, 9 g/L NaCl, 0.1M NaNO3, 0.1M sodium acetate, and 0.1M sodium citrate wash buffers with the extent of difference positively correlated with wash buffer’s ionic strength. Furthermore, the point of zero charge (pHzpc) for E. coli grown in the semi-defined medium varies between 1.5 and 3, in an ionic strength-dependent manner, for the various wash buffers tested. Collectively, this preliminary study suggests that wash buffers' ionic strength strongly affect removal efficiency of nonspecifically absorbed ions on bacteria surfaces, where a threshold exists (0.15M) for charge screening to be effective. At the upper bound, 0.6M ionic strength might result in removal of cations that stabilizes the cell envelope; thus, leading to possible cell surface damage and erroneous measurements.

scholarly journals Bacterial surface charge in “layers”: revealed by wash buffers of different ionic strength

2018 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Ionic Strength ◽  
Zeta Potential ◽  
Surface Charge ◽  
Cell Envelope ◽  
Cell Mass ◽  
Culture Broth ◽  
Point Of Zero Charge ◽  
Bacterial Surface ◽  
Incomplete Removal ◽  
Wash Buffer

Bacterial surface charge derives its meaning from the cell’s environment such as the solution in contact with the cell. Determining the surface charge of bacteria in its native environment requires measuring the proxy variable, zeta potential, using cells obtained from field studies. However, lack of adequate cell mass and concerns over measurement of a mixed species consortia rather than a specific species meant that bacterial surface charge measurement require biomass obtained from pure culture. Often grown in rich medium where myriad proteins and ions nonspecifically adsorbed onto the cell envelope or peptidoglycan layer, standard procedures for preparing the cell mass incorporated repeated steps of washing and centrifugation with various wash buffers, the efficacies of which are poorly understood. This report describes the results of a systematic study on how wash buffers of different composition and ionic strength affect the efficiency of removing nonspecifically adsorbed biomolecules and ions from Escherichia coli DH5α (ATCC 53868) cultured aerobically (shake flask, 37 oC and 230 rpm) in LB Lennox medium with 2 g/L glucose and a formulated medium. Using zeta potential-pH profiles over pH 1 to 12 as readout, the results showed that efficiency of removing nonspecifically adsorbed ions and metabolites positively correlated with wash buffer ionic strength. More importantly, 0.15M ionic strength (i.e., 9 g/L NaCl) seemed to be the minimum below which there was incomplete removal of nonspecifically adsorbed biomolecules. On the other hand, high ionic strength of 0.6M (e.g., 0.1M sodium citrate) significantly changed the point of zero charge (pHzpc), a reference marker for removal of ions intrinsic to the cell envelope. Collectively, results obtained inform wash buffer choice with regards to preserving cell envelope integrity, and avoidance of adsorption of buffer ions such as citrate. But, is there a true cell surface charge? Yes, but how do we define it in number of “layers” of adsorbed biomolecules? Philosophically, cells in culture broth are coated with layers of metabolites, proteins and ions. Hence, desire to reveal the true surface charge is essentially a decoating process, where wash buffers of increasing ionic strength remove each layer via charge screening. However, where is the endpoint? This research offers a different perspective and answer. Imagine a single bacterium suspended in LB medium, where there is constant adsorption and desorption of biomolecules as the cell grows: what is its relevant surface charge? It is the one where the loosely associated ions and metabolites are removed while retaining the nonspecifically adsorbed ions and biomolecules. Thus, deionized water wash provides a good estimate of the bacterial surface charge as grown in specific medium.

scholarly journals Zeta potential of bacterial cells: Effect of wash buffers

2015 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Ionic Strength ◽  
Zeta Potential ◽  
Cell Surface ◽  
Phosphate Buffer ◽  
Defined Medium ◽  
Deionized Water ◽  
Ph Profile ◽  
Nonspecific Adsorption ◽  
E Coli ◽  
Ph Range

Zeta potential, defined as the electric charge at the shear plane, is widely used as a proxy for bacterial cell surface charge. Nonspecific adsorption of ions or polyelectrolytes onto the cell surface, however, alters the value and polarity of the measured zeta potential, leading to erroneous results. Multiple wash and centrifugation steps are commonly used in preparing cells for zeta potential analysis, where various wash buffers (such as 9 g/L NaCl, 0.001M KCl, and 0.1M NaNO3) are routinely used for removing (via charge screening) ions and charged molecules that bind nonspecifically to the cell surface. Preliminary data revealed that, for Escherichia coli DH5α grown in LB Lennox (with 2 g/L glucose, LBG hereafter), the zeta potential-pH profile was not significantly different over the pH range from 2 to 12 for deionized water, 9 g/L NaCl, and phosphate buffer saline (PBS) wash buffers. As LBG is a low salt medium without a phosphate buffer, it was likely that the extent of nonspecific adsorption of ions on the cell surface was not severe, and the different wash buffers would, correspondingly, not exert much effect on measured zeta potential. For E. coli grown in a semi-defined medium (with a high capacity phosphate buffer system), the zeta potential-pH profile was significantly different over the pH range from 1 to 12 for deionized water, 9 g/L NaCl, 0.1M NaNO3, 0.1M sodium acetate, and 0.1M sodium citrate wash buffers with the extent of difference positively correlated with wash buffer’s ionic strength. Furthermore, the point of zero charge (pHzpc) for E. coli grown in the semi-defined medium varies between 1.5 and 3, in an ionic strength-dependent manner, for the various wash buffers tested. Collectively, this preliminary study highlights that wash buffer ionic strength plays important roles in affecting removal efficiency of nonspecifically absorbed ions on bacterial surfaces, where a threshold exists (0.15M) for charge screening to be effective, while 0.6M ionic strength might result in intrinsic cations removal, leading to possible cell surface damage and erroneous measurements.

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