separation of viable and non-viable yeast using dielectrophoresis

E L S E V I E R Journal of Biotechnology 32 (1994) 29-37

journal of blotechnology

Separation of viable and non-viable yeast using dielectrophoresis

Gerard H. Markx *, Mark S. Talary, Ronald Pethig Institute of Molecular and Biomolecular Electronics, University of Wales, Dean Street, Bangor, Gwynedd LL57 1UT, UK

(Received 16 February 1993; revision accepted 17 April 1993)

Abstract

Dielectrophoresis, the movement of particles in non-uniform AC electric fields, was used to rapidly separate viable and non-viable yeast cells with good efficiency. Known mixtures of viable and heat-treated cells of Saccharomyces cerevisiae were separated and selectively isolated using positive and negative dielectrophoretic forces generated by microelectrodes in a small chamber. Good correlations with the initial known relative compositions were obtained by direct microscopic counting of cells at the electrodes after initial dielectrophoretic separation (r = 0.995), from methylene blue staining (r = 0.992) and by optical absorption measurements (r = 0.980) of the effluent after selectively flushing out the viable and non-viable cells from the chamber. Through measurement of cell viability by staining with methylene blue and plate counts, for an initial suspension of approx. 1.4 x 10 7 cells per ml containing 60% non-viable cells, the dielectrophoretically separated non-viable fraction contained 3% viable cells and the viable fraction 8% dead cells. The separation efficiency is increased by dilution of the initial suspension or by repeat operation(s). Cell viability was not affected by the separation procedure.

Key words: Dielectrophoresis; Yeast; Viability; Cell separation

I. I n t r o d u c t i o n

The determination of cell viability is not straightforward and results are often very dependent on the technique employed. However, such determination is of considerable practical and theoretical importance (Jones, 1987; Higgins, 1992; Kaprelyants and Kell, 1992) and the devel- opment of new techniques for the study of cell death, as well as for the physical separation of viable and non-viable cells in a mixed population, would be very useful. We demonstrate here that

* Corresponding author. Abbreviation: DEP, dielectrophoresis.

the phenomenon of dielectrophoresis is capable of providing the basis for such techniques.

Dielectrophoresis (DEP) is the movement of particles in non-uniform AC electric fields, the theory and practice of which is well documented (Pohl, 1978a,b; Pethig, 1979, 1991). As a result of an externally imposed electric field a dipole mo- ment is induced in the particle (cell), and if the field is non-uniform the particle experiences a net translational force which may direct it either towards or away from high field regions. This induced motion constitutes the DEP effect, and for ceils is comprised of several frequency-dependent components (Burt et al., 1990; Pethig, 1991; Pethig et al., 1992). Below around 1 kHz it is largely controlled by polarisations associated with

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30 G.H. Markx et al. /Journal of Biotechnology 32 (1994) 29-37

surface charge effects, whilst between 1 kHz and 1 MHz surface conduction, dipolar relaxations at membrane or cell wall surfaces, membrane fluid- ity, as well as transmembrane ion transport pro- cesses, are dominant influences. Above 1 MHz the controlling influences on the DEP response are membrane capacitance and interfacial polarisations associated with surface and internal cell structure. The main variables under the experi- menter's control are the conductivity and permit- tivity of the suspending medium and the frequency of the applied field. Thus, it is possible to choose the variables such that a mixture of particles with different DEP properties can be separated, and this is greatly facilitated using microelectrodes of an interdigitated, castellated, design (Price et al., 1988; Burt et al., 1989, 1990; Pethig et al., 1992).

It has already been shown (Pohl, 1978a,b; Huang et al., 1992) that the DEP properties of viable and non-viable yeast cells are significantly different, and differences have also been re- ported using the closely related techniques of dielectric spectroscopy (Boulton et al., 1989; Stoi- cheva et al., 1989; Markx et al., 1991) and electro-rotation (H61zel and Lamprecht, 1992; Huang et al., 1992). In the DEP methods employed by Pohl (Pohl and Hawk, 1966; Crane and Pohl, 1968; Pohl, 1978a,b) and by Mason and Townsley (1971) cell separation was achieved using the effect of positive dielectrophoresis cre- ated by means of a simple two-electrode system, and only poor efficiency in cell separation was obtained. The high efficiency of separation obtained by us has resulted from two new features, namely the use of interdigitated microelectrode arrays and the controlled application of both positive and negative dielectrophoretic forces. Also, the method is in principle generic since the dielectrophoretic properties can vary considerably between cells of different organisms, and indeed is also dependent on physiological states other than the viability (Mason and Townsley, 1971; Pohl, 1978a,b; Pethig, 1991; Gascoyne et al., 1992).

The dielectrophoretic separation method described here operates on the basis, as described elsewhere (Huang et al., 1992), that frequency

ranges can be found where: (a) both viable and non-viable yeast cells exhibit positive DEP; and (b) viable cells exhibit positive DEP and non-viable cells negative DEP. The other phenomenon exploited is associated with the fact that when using interdigitated, castellated microelectrodes, cells collected under positive DEP are held in deep and steep-sided potential energy wells at the electrode edges, whereas under the influence of a negative dielectrophoretic force the cells are retained as triangular-shaped aggregations in shallow potential energy wells (Gascoyne et al., 1992; Pethig et ai., 1992). Thus, cells attracted to the electrodes by positive DEP are not easily dislodged by flushing fluid over the electrodes, whereas those cells retained by negative DEP are readily and selectively removed by such action.

2. Materials and Methods

Yeast The yeast used was baker's yeast (Sac-

charomyces cere~'isiae, strain RXII, obtained from the Institute of Biophysics, Free University of Berlin) grown at 30°C in a medium of pH 5 consisting of 5 g 1 - i yeast extract (Oxoid), 5 g I - 1 bacterial peptone (Oxoid) and 50 g 1-1 sucrose. The yeast was grown overnight, harvested and washed four times in 280 mM mannitol. The cells were rendered non-viable by heating to 90°C in a waterbath for 20 min, after which they were washed as before. Suspensions with different relative amounts of viable and non-viable cells were made by mixing.

Dielectrophoretic spectrometer The DEP spectra of suspensions of viable and

of non-viable yeast cells were measured so as to ascertain the frequency ranges where the viable and non-viable ceils exhibited either positive or negative DEP. Suspensions of viable and non-viable (heat-treated) yeast cells were prepared hav- ing an absorption of 0.6 at 655 nm in a cuvette of 1-cm path length (corresponding to 1.4 x 10 7 cells per ml), and their DEP spectra were obtained using a split-beam spectrometric system, based on a previous design from this laboratory (Price et

G.H. Markx et al. /Journal of Biotechnology 32 (1994) 29-37 31

al., 1988; Burt et al., 1989, 1990). One component of the split laser beam monitored the optical density of the cell suspension located between two interdigitated electrode arrays, of the same geometry as those used in the cell separation chamber. The other component of the split-beam corrected for random fluctuations of the beam intensity and also provided a reference signal to give increased sensitivity of measurement. Posi- tive DEP manifested itself as a reduction in optical density of the cell suspension, whilst the effect of negative DEP was to increase the optical density as a result of cells being repelled away from the electrodes into the bulk suspending solution. As described elsewhere (Price et al., 1988; Burt et al., 1989) the initial rate of change of the optical absorbance, on application of the AC voltage signal to the electrodes, is proportional to the DEP collection rate of the cells.

Dielectrophoretic separation The cell separation chamber incorporated in-

terdigitated, castellated microelectrodes of the same basic design and construction as those used in DEP studies of colloidal particles, bacteria, yeast and mammalian cells (Burt et al., 1989, 1990; Price et al., 1988; Pethig et al., 1992). The electrodes were fabricated onto a microscope slide and the characteristic dimension defining the castellated geometry was 80 ~tm. The chamber, of volume 50/~1, was constructed by placing a poly- acetate spacer and a microscope cover slip on top of the electrodes, and sealing the system with epoxy resin. The cells and suspending fluid could be injected into and flushed out of the chamber through two small diameter tubes.

The first stage of the separation process con- sisted of applying to the electrodes a sinusoidal voltage of such a frequency that both the viable and non-viable ceils collected at the electrode tips as a result of a positive dielectrophoretic force. With this voltage signal still applied, the chamber was then flushed through with clean suspending fluid so as to remove cellular debris and cells not captured by the electrodes. The frequency of the applied voltage was then ad- justed so that the non-viable cells redistributed themselves so as to collect in triangular aggrega-

tions at the electrode bay regions under the influence of a negative dielectrophoretic force, whilst the viable cells remained at the electrode tips under a positive force. With this voltage signal still applied, the chamber was then flushed through to selectively remove the non-viable cells from the chamber. The final stage involved switching off the applied voltage to the electrodes and flushing the chamber in order to remove the viable cells.

Measurement of the separation of ceils of different viability was accomplished in two ways. In the first method the cells were brought into the chamber by injection, a 5 V (pk-pk) 10 MHz voltage was applied to the electrodes and the number of cells occurring in triangular aggregations and on top of the electrodes, and of those collected at the electrode edges, were counted by direct microscopic observation and from pho- tographs of areas representative for the electrode arrays. To compensate for the fact that some cells were present in the chamber from previous experiments, cell counts were also made before intro- ducing the new sample.

In the second method ceils were brought into the cell by injection and collected at the electrode edges by applying a 10 V (pk-pk) 10 kHz signal. Non-captured ceils and any cellular debris were flushed out with 280 mM mannitol. The signal was then changed to 10 V (pk-pk) 10 MHz, which had the effect of causing non-viable ceils to migrate into triangular aggregations and on top of the electrodes, whilst leaving the viable ones located at the electrode edges. By passing a gen- tle stream of fluid medium through the DEP chamber with the 10 MHz signal applied, the non-viable cells were selectively removed from the chamber. The passage of these cells was monitored as an increase of optical absorbance at 500 nm, using a 1-cm flow-through cell and a Pye-Unicam SP6-400 spectrophotometer (Fig. 1). On removal of the non-viable ceils, the voltage was switched off and the subsequent flushing out of the viable cells from the electrode edges was also recorded as an increase in optical absorbance. The absorbance signal was followed in time and the area under the two absorption peaks was measured. The flow rate through the cham-


lrl Cell UV/Vis photo- collection spectrometer

/ ~ Dielectrophorefic ~----~feed seporotion

amber ~ I

• L T ~ . J ~ Syringe / \

Microscope Frequency generotor

Fig. 1. Schematic outline of experimental system. Cells were syringed into the DEP separation chamber containing the microelectrodes, and after DEP separation their flushing-out was monitored by optical absorbtion.

ber was 30 ml h - 1, and suspensions of viable and non-viable yeast cells of the same concentration exhibited the same absorbance at 500 nm.

Estimation of viability To estimate the viability of cells, they were

stained with methylene blue (Stoicheva et al., 1989), and they were plated out on plates containing growth medium with 1.2% agar.

3. Results and Discussion

shaped aggregations in the electrode 'bay' regions. Non-viable cells also collect as diamond- shaped aggregations onto the surface of the electrodes away from the electrode edges under the influence of a negative dielectrophoretic effect (Pethig et ai., 1992). This rearrangement of the cells is completed within 30-60 s. The two types of cell were thus easily recognisable and physi- cally separated on a local scale by application of the 10 MHz signal. Observations using methylene blue-treated cell suspensions confirmed that the stained cells collected in the triangular formations and on top of the electrodes, whereas the unstained (hence viable) cells collected at the electrode edges and in pearl chains.

The relative numbers of viable and non-viable cells were obtained by direct microscopic inspec- tion, as well as from photographic records, of cell collection at the electrodes (e.g., Fig. 4). Cell viability was also determined using methylene blue staining. Fig. 6 shows the measured cell viability vs. the viability expected from the known composition of the cell mixtures. Good correlations can be seen (correlation coefficient r = 0.992 and 0.995 for methylene blue staining and dielectrophoresis, respectively).

The DEP spectra of suspensions of viable and non-viable yeast cells, measured using the split- beam spectrometer, are shown in Fig. 2. These spectra provided the information required to en- able the conditions for cell separation to be es- tablished, namely that both the viable and non-viable cells exhibit a positive DEP of similar magnitude at 10 kHz, whilst above 2 MHz the non-viable cells exhibit a negative DEP effect and the viable ones a positive effect.

The result of applying a 5 V (pk-pk) 10 kHz voltage signal to the electrodes for a suspension containing both viable and non-viable cells is shown in Fig. 3. Both cell types collect (within 10 s) at the electrodes. In Fig. 4 the result of chang- ing the frequency of the applied voltage to 10 MHz is shown. The viable cells remain collected at the electrode edges and in 'pearl chains' between the electrodes, whilst the non-viable cells have rearranged themselves into triangular-

2

1.

"~" t Vlable cells

i " .~ -

0

Non-vlable cells _ g

Log frequency (Hz)

Fig. 2. The dielectrophoretic spectra of viable and non-viable yeast suspensions as measured with the split-beam dielectrophoretic spectrometer. The relative change in the absorbance of the yeast suspension is measured after the application of AC voltages to the electrodes, and provides an indication of the magnitude and polarity of the DEP cell collection rate.


The cells were also separated by flushing the DEP chamber as described in Materials and Methods, so as to first selectively remove the non-viable cells (Fig. 5) and then the viable ones. The relative numbers of negative DEP collected (non-viable) and positive DEP collected (viable) ceils were determined by optical absorbance measurements. Previous studies (Burt et al., 1989) have shown for yeast concentrations up to around 1.4 x 107 cells per ml that the optical absorbance in 1-cm path length cuvettes varies linearly with concentration (i.e., Beer's law is obeyed). Apart from the linear relationship between cell concentration (checked for viable and non-viable cell suspensions) the advantage of operating within Beer's law is that errors associated with multiple light scattering are avoided. In this work we did not use cell concentrations above 1.4 x 107 ml- i. The results obtained are shown in Fig. 7, and a reasonable correlation is seen (r = 0.980) with the

initial known relative compositions of the suspensions.

After DEP separation of a suspension prepared using 40% viable and 60% non-viable (heat-treated) yeast ceils, the two separated components were stained with methylene blue and plated-out on growth medium with 1.2% agar. Viable cells (3%) were still present in the fraction supposed to contain non-viable cells, whilst the fraction containing mainly viable ceils also contained dead cells (8%). This shows that at the relatively high cell concentrations used in these experiments (approx. 107 ml - t ) the separation was not 100% successful. At these concentrations non-viable (stained) cells were sometimes trapped or sterically hindered by the viable cells at the electrode edges. This effect was reduced if suspensions with a lower concentration of cells were used. On plating out good growth (cell recovery 100% to within experimental error) was obtained

Fig. 3. Viable and non-viable (methylene blue stained) yeast cells collected at the electrodes after applying a 5 V (pk-pk) 10 kHz signal.


from fractions with viable cells, whilst only very few (3%) colonies were obtained from fractions containing non-viable ceils. Our finding that the yeast viability was not affected by the applied electric field is in accord with the earlier work of F6rster and Emeis (1985). These workers demon- strated that the viability of yeast protoplasts, which are more fragile than the intact yeast cells used in our work, was unaffected by dielectrophoresis.

4. Conclusions

From analyses of the dielectrophoretic and electrorotational behaviour of yeast cells, Huang et al. (1992) showed that the cytoplasmic mem-

brahe conductivity of the cells increased on heat treatment from 2.5 x 10 -7 S m - l to 1.6 X 10 -4 S m-m, in parallel with a decrease of the internal cell conductivity from 0.2 S m - t to 7 x 10 -3 S m-1. These changes in cellular electric properties give rise to the differences in dielectrophoretic behaviour described here and form the basis of the separation technique.

The process of injecting cells into the separation chamber, trapping the ceils using a 10 kHz signal and locally separating the viable from non- viable cells at the electrodes using a 10 MHz signal, can be achieved within 2 min. The measurements in which the numbers of viable and non-viable cells were counted at this stage of dielectrophoretic separation were made here by simple counting procedures, but this can be auto-

Fig. 4. Dielectrophoretic separation of viable and non-viable yeast cells using interdigitated, castellated electrodes and a 5 V (pk-pk) 10 MHz signal. The viable yeast cells collect on the edges of and in pearl chains between the electrodes, whilst the non-viable cells collect in triangular aggregations between the electrodes and in diamond-shaped formations on top of the electrodes.

G.H. Markx et al. / Journal of Biotechnology 32 (1994) 29-37 35

Fig. 5. The viable cells remain in the chamber after flushing out the non-viable cells with the 10 MHz signal applied to the electrodes.

IOO

9o-

80-

70-

60-

50- >

40-

30"

~ 20-

I0-

• , , , , . . . . . ,

20 40 60 80 100

Expected viability (%)

J - ~ - Methylene blue --0- Theoretical ~ Dielectrophores's J

Fig. 6. Percentage viability o f mixed cell suspensions determined by methylene blue staining and dielectropborctic behaviour, versus the expected viability from the mixtures made. A good correlation is obtained for both methods (correlation coefficient r = 0.992 and 0.995 for methylene blue and DEP, respectively).

too

90-

80.

70-

so- >.

~o 50-

-~ 40"

~, 3O-

20-

10-

( ) . . . . . . . . . .

20 40 60 80 100 Expected viability (7.)

Fig. 7. Viability obtained from absorbance measurements of the outflow of the chamber on selective flushing-out of first the non-viable and then the viable yeast cells, versus the viability expected from the mixtures made (r = 0.980).


m a t e d using image analysis t echniques (Gascoyne et at., 1992). This p r o c e d u r e can the re fo re pro- vide a r ap id m e t h o d for ascer ta in ing cell viabili ty, wi thout the need for chemica l t r e a t m e n t of the cells, and for select ively col lect ing the cells af ter - wards.

Fo r 1.4 × 10 7 cells p e r ml of 40% viability, a s ignif icant n u m b e r (8%) of d e a d cells a p p e a r e d in what should have been the fract ion conta in ing the select ively f lushed-out v iable cells a lone. F r o m direct microscopic observa t ions of the D E P effect on me thy lene b lue - t r e a t ed suspensions, this ' con- t amina t ion ' was found to occur because non-viable cells were s ter ical ly h i n d e r e d and even t r a p p e d by the v iable ceils. This effect was r e d u c e d significantly on 10-fold d i lu t ion of the initial suspension. Improved eff iciency of s epa ra t ion can also be achieved by passing the cells th rough two or more s tages of d i e l ec t rophore t i c separa t ion . W e are also explor ing the advan tages to be ga ined from o the r mic roe l ec t rode s t ruc tures and geome- tries. Final ly, p re l iminary da t a with s ta t ionary cu l tu res (da ta not shown) indica te tha t cells at d i f fe ren t physiological s ta tes can be ident i f ied th rough the i r d i e l ec t rophore t i c behaviour , and the behav iour of mor ibund cells may be d i f fe ren t f rom tha t of bo th viable and non-viable cells. A p a r t f rom the po ten t i a l for select ive cell s epa ra - t ion technologies , a compar i son of the die lec- t r opho re t i c t echn ique with s ta ining me thods for de t e rmin ing cell viabi l i ty and physiological s ta te could thus prove scientif ical ly rewarding.

Acknowledgements

This work has been s u p p o r t e d by the Basic Science P r o g r a m m e of the Na t iona l F o u n d a t i o n for Cance r R e s e a r c h (USA) , by a S E R C stu- den t sh ip to M.S.T. and a pos tdoc to ra l r e sea rch ass is tantship to G . H . M unde r a p u m p - p r i m i n g S E R C G r a n t ( G R / H 5 4 3 7 9 ) . W e thank Y. H u a n g and J .P.H. Burt for va luab le prac t ica l advice, J. T a m e for the e l ec t rode pho to l i thography , R. H/Slzel for the yeas t s train, and A.S. Kapre lyan ts , X-B. W a n g and X-F. Z h o u for va luable discus- sions.

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separation of viable and non-viable yeast using dielectrophoresis

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