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The concentration and separation of bacteria and cells by ferrography
159
Wear, 90 (1983) 159 - 165
THE CONCENTRATION AND SEPARATION CELLS BY FERROGRAPHY*
OF BACTERIA
AND
ANTHONY P. RUSSELL and VERNON C. WESTCOTT The Foxboro
Company, Foxboro,
MA 02035
ALFRED DEMARIA and MARGARET
Maxwell Finland Laboratory for Infectious Street, Boston, MA 02118 (U.S.A.) (Received November 17,1982;
(U.S.A.)
JOHNS Disease, Boston City Hospital, 774 Albany
in revised form December 29,1982)
Summary When micro-organisms are found in nature they are frequently accompanied by other matter including organic and inorganic particles. Also, the organisms are extremely dilute so that to examine them it is necessary to grow them or to concentrate them by some means. The disadvantage of growing them is that a suitable nutrient must be known and time is needed. With the introduction of ferrography it has become possible to separate micro-organisms from other particulate material by chemically magnetizing the organisms and recovering them with the Ferrograph. The chemistry of organism magnetization is discussed and examples given. The recovery of five species of bacteria is described. The separations of eukaryotic from prokaryotic cells as well as the separation of white from red blood cells are also demonstrated.
1. Introduction Techniques for the isolation of biological material and particles with a size of one to several hundred micrometers lack the specificity of separation which is obtained with dissolved compounds by chromatography. Particles are commonly separated by filtration or centrifugation. Filtration is a simple technique, but separation is incomplete; the filter retains all particles greater than the filter pore size, presenting them in a jumbled distribution that obscures the distinction between populations. Centrifugation requires manipulation to select and display the fraction of interest. The use of ferrography as a means of investigating particles of cartilage and bone in synovial fluid *Paper presented at the First International Conference on Advances in Ferrography, University College, Swansea, Gt. Britain, September 22 - 24, 1982. 0043-1648/83/$3.00
@ Elsevier Sequoia/Printed in The Netherlands
160
[ 1, 21 raises the possibility of recovering micro-organisms and other biological particles. The ability to extract selectively from solution particles according to size and chemical composition offers the possibility of sefectively isolating bacteria and separating them from other material, We report here the successful precipitation of bacteria and mammalian cells and the separation of bacteria from mammalian cells by ferrography. Initial work on the separation of bacteria, cells and clusters of bacteria based on size and chemical composition is also reported.
2. Ferrography In the Ferrograph the liquid sample is pumped along the length of a special glass substrate called a Ferrogram and is drained into a waste bottle. Particles in the liquid are pulled down onto the surface of the Fenogram by a high gradient magnetic field [3]. In order to precipitate small particles of low magnetic susceptibility a high field B, and field gradient dB,/dy are essential. A maximum field intensity of approximately 18 000 G and a maximum gradient of the order of 200 000 G cm-l in the vertical plane (pe~endicular to the substrate) are employed. A considerable amount of literature about ferrography for particles from mechanical systems exists [4]. The force on a particle in the magnetic field of a Ferrograph is given in SI units by the expression 15, 61:
F, =
f&&%(XP -
XLIVP
Where p,, = 47~X 10V7 is the permeability of space, B, (T) is the horizontal field intensity, l.lB,/lily (T m-‘) is the vertical field gradient, VP is the particle volume and xp and xL are the volumetric susceptibilities of the particle and the liquid respectively (it should be noted that, although x is non-dimensional, it is larger by the factor 4w in SI units than in c.g.s. units), The quantity (l//.QB, aB,/ay has a maximum value of approximately 250 X lo7 N rnp3 at the surface of a Ferrogram placed directly on the magnet of the Ferrograph employed. The net magnetic attraction experienced by a particle is proportional to the difference in susceptibility between the particle and the fluid in which it is suspended. If the fluid has a higher susceptibility than the particle, the particle will be repelled. In order to be attracted, the particle must have a higher magnetic susceptibility than the solution.
3. Biological materials Biological materials are generally bacteria, of such materials are repelled
diamagnetic. Particles, including by a magnetic field and do not
161
precipitate on a Ferrogram. Solutions of magnetic salts have been prepared that cause suspended particles of biological origin to become paramagnetic. Magnetic cations bind to the negative sites in the biological material to increase the magnetic susceptibility. Transitional metal ions of the lanthanide family have been particularly useful because they carry high magnetic moments not easily modified by chemical reaction, have a small hydration sphere and have equilibrium constants favoring concentration in the biological material. Several solutions employing erbium chloride (ErCls) have been developed. These solutions have been employed to precipitate various species of bacteria by ferrography. Figure 1 is a diagram showing the region of the Ferrogram on which bacteria are deposited.
I
60
50
40 MM
30 ALONG
20
10
0
FERROGRAM
Fig. 1. Diagram of a Ferrogram. The larger and the most magnetic particles precipitate near the entry and the smaller and less magnetic particles precipitate toward the exit. The position of the precipitated particles on the Ferrogram is measured according to the scale.
The results obtained from various suspensions of bacteria and of mixtures of bacteria and cells are reported here. These Ferrograms were prepared by adding 0.4 ml of 0.01 M ErCl, to 0.1 ml of suspension and the mixture was pumped over the Ferrogram at a flow rate of 0.028 ml min-‘; 1 ml of distilled water was then pumped over the Ferrogram at the same rate to remove residual ErCl,. Figure 2 illustrates Escherichia coli deposited at the 40 mm position on a Ferrogram. Figure 3 is a graph illustrating the separation of mammalian cells (mouse lymphocytes) from E. coli. The experimental details were similar to those given above, with the exception that the ErCls was dissolved in isotonic saline. In the figure the abscissa represents the distance along the Ferrogram and the ordinate represents the number of particles observed in a given area. Since the lymphocytes have varying sizes there is a variation in the maximum range that depends on the size of the cells. As a result the concentration of lymphocytes varies along the Ferrogram. They are completely separated from the bacteria within the first half of the Ferrogram. In order to investigate a separation according to size with particles that are more homogeneous chemically than the mixture of E. coli and mammalian cells, a Ferrogram of clusters of Staphylococcus aureus was prepared. S. aureus clusters containing different numbers of cells were counted at the
162
on a Ferrogram at the 40 mm position Fig. 2. Photomicrograph of E. Coli precipitated under the conditions described in the text. The three-dimensional appearance was obtained with a dark field condenser and an objective with a numerical aperture slightly larger than the occluded angle of the opaque disc. (Magnification 610x.)
Q
.--._\ _ -.---__
..
‘\
‘.
-...__---
_.
0 60
t
50
40
30
20
ENTRY MM
ALONG
FERROGRAM
10
END
t
OF
0
MAGNET
Fig. 3. The separation of E. Coli (0) from mouse lymphocytes (0) by ferrography. ordinate represents the number of bacteria or cells counted along the Ferrogram. position along the Ferrogram where the counts were taken is given by the abscissa.
The The
50 and 25 mm positions on a Ferrogram (data not shown). At the 50 mm position the average cluster contained six cells, while at the 25 mm position the average cluster contained three cells. However, large and small clusters could be observed at both positions, possibly because of variations in the morphologies of the clusters or variations in the flow pattern on the Ferrogram. The separation of particles of different sizes may be enhanced by injecting the suspension of particles into a carrier liquid as it flows over the substrate. With this method a particle-free solution of 0.01 M ErCl, is pumped over the Ferrogram at a flow rate of 0.2 ml min-‘. The particle suspension is injected through a syringe needle with an inside diameter of 0.3 mm as a thin stream on the center-line at the 45 mm position. The Ferrogram substrate is elevated 1 mm above the magnet at the end near the
163
ENTRY
TUBE
SUBSTRATE
0
1 50
40
30
POSITION
20
ON
10
0
FERROGRAM
Fig. 4. The separation of cell clusters of different sizes from an aspergillus suspension: (a) the experimental arrangement showing that the Ferrogram was tilted at an angle of 2” to the magnet so that the particles experienced an increasing magnetic field as they traveled down the Ferrogram; (b) the relationship between the average particle size (micrometers) and the position on the Ferrogram (the bars indicate the standard deviations).
entry so that particles experience an increasing magnetic field as they progress down the Ferrogram (Fig. 4(a)). This method was used to separate clusters of yeast-like cells from an Aspergillus sp. bread mold. The aspergillus was suspended in 0.01 M ErCl, and injected, at a flow rate of 0.01 ml min-‘, into the carrier fluid. The results of the separations are shown in Fig. 4(b). The particles are graded in size from 35 + 5 pm near the entry to 5.5 f 2.5 I.crnnear the exit. The combination of the increasing magnetic field and the restriction of particles in the stream to a region over the poles results in a separation in which there is little overlap in the size distribution of particles between the beginning and the end of the Ferrogram. The other bacterial species studied include strains of Klebsiellu pneumoniae, type 2, Caroli strain, Proteus vulgaris and Salmonella typhi. Several Ferrograms are compared in Table 1, from which it will be observed that K.
164 TABLE 1 The rate of deposition
of bacteria -
Organism
Comparative of deposition
~-IE. coli, Kl K. pneumoniae P. vuZgaris S. typhi S. aweus
rate a
0.07 0.18 0.05 0.06 0.06
*The rate of deposition is defined as the reciprocal of the distance along the axis of the Ferrogram from the point of maximum concentration to the point where the concentration is one-half as much. All Ferrograms were run in duplicate except for that for S. aureus, and at a flow rate of 0.028 ml min-‘.
~~e~mon~e is deposited much faster than the other strains tested. This is consistent with the fact that the surface is surrounded by an acidic polysaccharide capsule [7f which appears to increase the amount of erbium absorbed and, therefore, the strength of the magnetic attraction. A more dramatic example of the effects of differential binding of the magnetizing ions is seen with human blood cells. White cells become more magnetic than the solution and precipitate above the magnet poles. Red cells have a lower susceptibility than the solution and are therefore repelled so that the majority run off the Ferrogram while a few are pushed out to the outer edges of the flow path where they remain. Figure 5 is a graph of the 1000
100
. K
800
80 k s
t: :
600
60
2
ii
-I
5
ii! ”
z
40
400
:: 0 d
8 d 200
20
s
: i z
0
0 0
1 DISTANCE
2
3
ACROSS
FERROGRAM
4
5
Fig. 5. The concentrations of human red blood celk (*) and white blood cells (0) at right angles to the axis of the Ferrogram at the 40 mm position. The count was made at a magnification of 400x over an area of 0.06 mm*. The difference between the two ordinate scales should be noted. The flow rate for the Ferrogram was 0.5 ml min-‘.
165
distribution path.
of human
red and white blood cells at right angles to the flow
4. Conclusions Separation of micro-organisms and cells by ferrographic techniques has been demonstrated. Further work will be directed toward increasing the number of organisms studied and toward determining the most suitable operating conditions for separation, Chemical studies have been initiated to devise methods of generating differential magnetic susceptibilities on the basis of the chemical composition of the organisms. Many histological stains that react with specific tissues contain paramagnetic salts. These stains may be used to magnetize tissues differentially.
References 1 C. H. Evans, E. R. Bowen, J. Bowen, W. P. Tew and V. C. Westcott, J. Biochem. Biophys. Methods, 2 (1980) 11 - 18. 2 D. C. Mears, E. N. Hanley, Jr., and R. Rutkowski, J. Biomed. Mater. Res., 12 (1978) 867 - 875. 3 D. Scott, W. W. Seifert and V. C. Westcott, Sci. Am., 230 (5) (1974) 88 - 97. 4 Ferrograph bibliography, 1978 (Foxboro Analytical, P.O. Box 435, Burlington, MA 01803). 5 Wear in fluid power systems, Rep. ONR CRl69-004-2, 1979 (Fluid Power Research Center, Oklahoma State University, Stillwater, OK 74074) (Office of Naval Research Contract N00014-75-C-1157). 6 W. W. Seifert and V. C. Westcott, Wear, 21 (1972) 27. 7 P. R. Edwards and W. H. Ewing, Identification of the Enterobacteriaceae, Burgess, Minneapolis, MN, 3rd edn., 1972, pp. 57 - 59.
Wear, 90 (1983) 159 - 165
THE CONCENTRATION AND SEPARATION CELLS BY FERROGRAPHY*
OF BACTERIA
AND
ANTHONY P. RUSSELL and VERNON C. WESTCOTT The Foxboro
Company, Foxboro,
MA 02035
ALFRED DEMARIA and MARGARET
Maxwell Finland Laboratory for Infectious Street, Boston, MA 02118 (U.S.A.) (Received November 17,1982;
(U.S.A.)
JOHNS Disease, Boston City Hospital, 774 Albany
in revised form December 29,1982)
Summary When micro-organisms are found in nature they are frequently accompanied by other matter including organic and inorganic particles. Also, the organisms are extremely dilute so that to examine them it is necessary to grow them or to concentrate them by some means. The disadvantage of growing them is that a suitable nutrient must be known and time is needed. With the introduction of ferrography it has become possible to separate micro-organisms from other particulate material by chemically magnetizing the organisms and recovering them with the Ferrograph. The chemistry of organism magnetization is discussed and examples given. The recovery of five species of bacteria is described. The separations of eukaryotic from prokaryotic cells as well as the separation of white from red blood cells are also demonstrated.
1. Introduction Techniques for the isolation of biological material and particles with a size of one to several hundred micrometers lack the specificity of separation which is obtained with dissolved compounds by chromatography. Particles are commonly separated by filtration or centrifugation. Filtration is a simple technique, but separation is incomplete; the filter retains all particles greater than the filter pore size, presenting them in a jumbled distribution that obscures the distinction between populations. Centrifugation requires manipulation to select and display the fraction of interest. The use of ferrography as a means of investigating particles of cartilage and bone in synovial fluid *Paper presented at the First International Conference on Advances in Ferrography, University College, Swansea, Gt. Britain, September 22 - 24, 1982. 0043-1648/83/$3.00
@ Elsevier Sequoia/Printed in The Netherlands
160
[ 1, 21 raises the possibility of recovering micro-organisms and other biological particles. The ability to extract selectively from solution particles according to size and chemical composition offers the possibility of sefectively isolating bacteria and separating them from other material, We report here the successful precipitation of bacteria and mammalian cells and the separation of bacteria from mammalian cells by ferrography. Initial work on the separation of bacteria, cells and clusters of bacteria based on size and chemical composition is also reported.
2. Ferrography In the Ferrograph the liquid sample is pumped along the length of a special glass substrate called a Ferrogram and is drained into a waste bottle. Particles in the liquid are pulled down onto the surface of the Fenogram by a high gradient magnetic field [3]. In order to precipitate small particles of low magnetic susceptibility a high field B, and field gradient dB,/dy are essential. A maximum field intensity of approximately 18 000 G and a maximum gradient of the order of 200 000 G cm-l in the vertical plane (pe~endicular to the substrate) are employed. A considerable amount of literature about ferrography for particles from mechanical systems exists [4]. The force on a particle in the magnetic field of a Ferrograph is given in SI units by the expression 15, 61:
F, =
f&&%(XP -
XLIVP
Where p,, = 47~X 10V7 is the permeability of space, B, (T) is the horizontal field intensity, l.lB,/lily (T m-‘) is the vertical field gradient, VP is the particle volume and xp and xL are the volumetric susceptibilities of the particle and the liquid respectively (it should be noted that, although x is non-dimensional, it is larger by the factor 4w in SI units than in c.g.s. units), The quantity (l//.QB, aB,/ay has a maximum value of approximately 250 X lo7 N rnp3 at the surface of a Ferrogram placed directly on the magnet of the Ferrograph employed. The net magnetic attraction experienced by a particle is proportional to the difference in susceptibility between the particle and the fluid in which it is suspended. If the fluid has a higher susceptibility than the particle, the particle will be repelled. In order to be attracted, the particle must have a higher magnetic susceptibility than the solution.
3. Biological materials Biological materials are generally bacteria, of such materials are repelled
diamagnetic. Particles, including by a magnetic field and do not
161
precipitate on a Ferrogram. Solutions of magnetic salts have been prepared that cause suspended particles of biological origin to become paramagnetic. Magnetic cations bind to the negative sites in the biological material to increase the magnetic susceptibility. Transitional metal ions of the lanthanide family have been particularly useful because they carry high magnetic moments not easily modified by chemical reaction, have a small hydration sphere and have equilibrium constants favoring concentration in the biological material. Several solutions employing erbium chloride (ErCls) have been developed. These solutions have been employed to precipitate various species of bacteria by ferrography. Figure 1 is a diagram showing the region of the Ferrogram on which bacteria are deposited.
I
60
50
40 MM
30 ALONG
20
10
0
FERROGRAM
Fig. 1. Diagram of a Ferrogram. The larger and the most magnetic particles precipitate near the entry and the smaller and less magnetic particles precipitate toward the exit. The position of the precipitated particles on the Ferrogram is measured according to the scale.
The results obtained from various suspensions of bacteria and of mixtures of bacteria and cells are reported here. These Ferrograms were prepared by adding 0.4 ml of 0.01 M ErCl, to 0.1 ml of suspension and the mixture was pumped over the Ferrogram at a flow rate of 0.028 ml min-‘; 1 ml of distilled water was then pumped over the Ferrogram at the same rate to remove residual ErCl,. Figure 2 illustrates Escherichia coli deposited at the 40 mm position on a Ferrogram. Figure 3 is a graph illustrating the separation of mammalian cells (mouse lymphocytes) from E. coli. The experimental details were similar to those given above, with the exception that the ErCls was dissolved in isotonic saline. In the figure the abscissa represents the distance along the Ferrogram and the ordinate represents the number of particles observed in a given area. Since the lymphocytes have varying sizes there is a variation in the maximum range that depends on the size of the cells. As a result the concentration of lymphocytes varies along the Ferrogram. They are completely separated from the bacteria within the first half of the Ferrogram. In order to investigate a separation according to size with particles that are more homogeneous chemically than the mixture of E. coli and mammalian cells, a Ferrogram of clusters of Staphylococcus aureus was prepared. S. aureus clusters containing different numbers of cells were counted at the
162
on a Ferrogram at the 40 mm position Fig. 2. Photomicrograph of E. Coli precipitated under the conditions described in the text. The three-dimensional appearance was obtained with a dark field condenser and an objective with a numerical aperture slightly larger than the occluded angle of the opaque disc. (Magnification 610x.)
Q
.--._\ _ -.---__
..
‘\
‘.
-...__---
_.
0 60
t
50
40
30
20
ENTRY MM
ALONG
FERROGRAM
10
END
t
OF
0
MAGNET
Fig. 3. The separation of E. Coli (0) from mouse lymphocytes (0) by ferrography. ordinate represents the number of bacteria or cells counted along the Ferrogram. position along the Ferrogram where the counts were taken is given by the abscissa.
The The
50 and 25 mm positions on a Ferrogram (data not shown). At the 50 mm position the average cluster contained six cells, while at the 25 mm position the average cluster contained three cells. However, large and small clusters could be observed at both positions, possibly because of variations in the morphologies of the clusters or variations in the flow pattern on the Ferrogram. The separation of particles of different sizes may be enhanced by injecting the suspension of particles into a carrier liquid as it flows over the substrate. With this method a particle-free solution of 0.01 M ErCl, is pumped over the Ferrogram at a flow rate of 0.2 ml min-‘. The particle suspension is injected through a syringe needle with an inside diameter of 0.3 mm as a thin stream on the center-line at the 45 mm position. The Ferrogram substrate is elevated 1 mm above the magnet at the end near the
163
ENTRY
TUBE
SUBSTRATE
0
1 50
40
30
POSITION
20
ON
10
0
FERROGRAM
Fig. 4. The separation of cell clusters of different sizes from an aspergillus suspension: (a) the experimental arrangement showing that the Ferrogram was tilted at an angle of 2” to the magnet so that the particles experienced an increasing magnetic field as they traveled down the Ferrogram; (b) the relationship between the average particle size (micrometers) and the position on the Ferrogram (the bars indicate the standard deviations).
entry so that particles experience an increasing magnetic field as they progress down the Ferrogram (Fig. 4(a)). This method was used to separate clusters of yeast-like cells from an Aspergillus sp. bread mold. The aspergillus was suspended in 0.01 M ErCl, and injected, at a flow rate of 0.01 ml min-‘, into the carrier fluid. The results of the separations are shown in Fig. 4(b). The particles are graded in size from 35 + 5 pm near the entry to 5.5 f 2.5 I.crnnear the exit. The combination of the increasing magnetic field and the restriction of particles in the stream to a region over the poles results in a separation in which there is little overlap in the size distribution of particles between the beginning and the end of the Ferrogram. The other bacterial species studied include strains of Klebsiellu pneumoniae, type 2, Caroli strain, Proteus vulgaris and Salmonella typhi. Several Ferrograms are compared in Table 1, from which it will be observed that K.
164 TABLE 1 The rate of deposition
of bacteria -
Organism
Comparative of deposition
~-IE. coli, Kl K. pneumoniae P. vuZgaris S. typhi S. aweus
rate a
0.07 0.18 0.05 0.06 0.06
*The rate of deposition is defined as the reciprocal of the distance along the axis of the Ferrogram from the point of maximum concentration to the point where the concentration is one-half as much. All Ferrograms were run in duplicate except for that for S. aureus, and at a flow rate of 0.028 ml min-‘.
~~e~mon~e is deposited much faster than the other strains tested. This is consistent with the fact that the surface is surrounded by an acidic polysaccharide capsule [7f which appears to increase the amount of erbium absorbed and, therefore, the strength of the magnetic attraction. A more dramatic example of the effects of differential binding of the magnetizing ions is seen with human blood cells. White cells become more magnetic than the solution and precipitate above the magnet poles. Red cells have a lower susceptibility than the solution and are therefore repelled so that the majority run off the Ferrogram while a few are pushed out to the outer edges of the flow path where they remain. Figure 5 is a graph of the 1000
100
. K
800
80 k s
t: :
600
60
2
ii
-I
5
ii! ”
z
40
400
:: 0 d
8 d 200
20
s
: i z
0
0 0
1 DISTANCE
2
3
ACROSS
FERROGRAM
4
5
Fig. 5. The concentrations of human red blood celk (*) and white blood cells (0) at right angles to the axis of the Ferrogram at the 40 mm position. The count was made at a magnification of 400x over an area of 0.06 mm*. The difference between the two ordinate scales should be noted. The flow rate for the Ferrogram was 0.5 ml min-‘.
165
distribution path.
of human
red and white blood cells at right angles to the flow
4. Conclusions Separation of micro-organisms and cells by ferrographic techniques has been demonstrated. Further work will be directed toward increasing the number of organisms studied and toward determining the most suitable operating conditions for separation, Chemical studies have been initiated to devise methods of generating differential magnetic susceptibilities on the basis of the chemical composition of the organisms. Many histological stains that react with specific tissues contain paramagnetic salts. These stains may be used to magnetize tissues differentially.
References 1 C. H. Evans, E. R. Bowen, J. Bowen, W. P. Tew and V. C. Westcott, J. Biochem. Biophys. Methods, 2 (1980) 11 - 18. 2 D. C. Mears, E. N. Hanley, Jr., and R. Rutkowski, J. Biomed. Mater. Res., 12 (1978) 867 - 875. 3 D. Scott, W. W. Seifert and V. C. Westcott, Sci. Am., 230 (5) (1974) 88 - 97. 4 Ferrograph bibliography, 1978 (Foxboro Analytical, P.O. Box 435, Burlington, MA 01803). 5 Wear in fluid power systems, Rep. ONR CRl69-004-2, 1979 (Fluid Power Research Center, Oklahoma State University, Stillwater, OK 74074) (Office of Naval Research Contract N00014-75-C-1157). 6 W. W. Seifert and V. C. Westcott, Wear, 21 (1972) 27. 7 P. R. Edwards and W. H. Ewing, Identification of the Enterobacteriaceae, Burgess, Minneapolis, MN, 3rd edn., 1972, pp. 57 - 59.