J. Cell Sci. 35, 43i-44i (i979) 431Printed in Great Britain © Company of Biologists Limited 19J9

CONTROLLED CELL DISRUPTION: A

COMPARISON OF THE FORCES REQUIRED TO

DISRUPT DIFFERENT MICRO-ORGANISMS

M. V. KELEMEN

Microbiology Section, Department of Pharmaceutics,The School of Pharmacy, University of London, England

AND J. E. E. SHARPE

Department of Meclumical Engineering, Engineering Faculty,Queen Mary College, University of London, England

SUMMARY

A cell disrupter has been developed which can measure the forces required to disrupt botheukaryotic and prokaryotic cells. It operates a continuous process and will disrupt both largeand small volumes. Shear forces are set up when a suspension under laminar flow conditions isreleased under high pressure through a short orifice. If the applied pressure is altered, the shearforces are simultaneously changed so that the amount of cell disruption can be compared underdifferent known and repeatable conditions. The disrupter is now manufactured and supplied byStansted Fluid Power Limited, Stansted, England.

Phase-contrast microscopy has shown that the disrupter will break a variety of organismsincluding Chlorella, Aspergillus fumigatis, Fusarium sp., Saccharomyces cerevisiae, Eschericluacoli, Lactobacillus casei, Bacillus subtilis, Clostridium perfringens, Streptococcus faecalis, Strepto-coccus zooepidermicus and Staphylococcus aureus. The cells are not all broken at one pressure buta certain pressure must be applied before disruption starts which will then increase rapidly asthe applied pressure is increased. The applied pressure required to disrupt half the populationin a culture is different from one species to another, rods being disrupted more easily thanspheres.

The ease of disruption seems to be related to the shape and chemical composition of the cellwall. Furthermore, the disrupting process, in an unsynchronized culture is not random andmay be related to the statistical size distribution of the cells.

INTRODUCTION

Cell disruption has been achieved using different types of equipment. Originally,

yeast cells were broken by grinding with Kieselguhr and sand (Buchner, 1897) and,

since then, different techniques have been developed to obtain both cell envelopes and

intracellular material. The methods have included subjecting a suspension of cells to a

source of ultrasonic vibration, shaking with glass beads, in addition to subjecting cell

suspensions to a shear stress gradient achieved by a large reduction in pressure

through a very short orifice (Rogers & Perkins, 1968; Wright, Edwards & Jones, 1974).

Milner, Lawrence & French (1950) have described an apparatus which disrupted

chloroplasts, yeast and Escherichia coli by means of a shear stress gradient and

measured the pressure required to obtain different amounts of chloroplast disruption.

Although this disrupter can be used for the quantitative measurement of the forces

432 M. V. Kelemen andj. E. E. Sharpe

required to break cells, it cannot conveniently handle large volumes as it is designedto process small single samples.

A cell disrupter has been developed which can measure the forces required to dis-rupt eukaryotic and prokaryotic cells. In addition, it operates a continuous process andcan, therefore, disrupt large volumes of cells (Sharpe, 1975). This apparatus cansubject cells to extremely high shear forces causing disruption. The shear forces areset up when the suspension is released under controlled high pressure through a shortorifice. If the applied pressure is altered, the shear forces are simultaneously changedand the degree of cell disruption can be compared under different known and repeat-able conditions.

Two properties of the cell disrupter have been demonstrated. Firstly, it has beenused to compare the conditions required to disrupt several species of micro-organismsand, secondly, the apparatus has been used to disrupt cells by a continuous process ofb̂ oth large and small volumes.

MATERIALS AND METHODS

The apparatus

The apparatus for disrupting cells in suspension consists of an orifice formed from a conicalseating with a hardened ball loaded into it which allows a flow of fluid only after a given pressurehas been exceeded (Sharpe, 1975). The liquid passing through the disrupter obeys laminar flowconditions (Patel & Head, 1968). The disrupter is now manufactured and supplied by StanstedFluid Power Limited, Stansted, England.

Temperature Tc

Temperature rise A7"

Force F

Temperature T

Pressure />,

Zone of high shear

Flow

Pressure Po

Fig. 1. Diagrammatic section through disruption valve.

Fig. 1 shows a diagrammatic section through the valve. The ball may be loaded either by thecompression of a stiff spring or by an air-loaded piston, allowing the loading on the ball andhence the pressure drop to be continuously adjusted.

Fig. 2 shows the inlet funnel (a), high-pressure cylinder assembly (b) and disrupter valve (c)which are made in high-grade stainless steel and may be autoclaved if required. The componentsof the disrupting valve are designed to be disposable and may be replaced at will. The fluidcontaining the cells in suspension is pumped through the orifice against the loaded ball valve bya suitable electrically or pneumatically operated high-pressure pump. For the i-s-mm ball

Motorand

gearbox1

Pressurerelief

mechanism1

Speed regulator

Loadregulator

(disruption fcrce)

Self-actingpneumaticcylinder

Loadregulator

(disruption force)High

pressure cylinder

Fig. 2. Cell disruption apparatus, A, the electric disrupter, B, the pneumatic disrupter.

434 M. V. Kelemen andj. E. E. Sharpe

valve used for these experiments, pressures up to 27 x io8 Pa (4 x io4 lb/in.1, 2-76 x io! kNm"1) were investigated. Either a i-hp (7^6 x io! W) electrical pump made by Stansted FluidPower Limited, England capable of processing 10 l./h or a smaller bench mounted pneumaticpump (also made by Stansted) which can handle up to 4 l./h were used for the experimentsdescribed.

The work done in shearing the suspension fluid and hence, the cells may be calculated fromthe applied pressure and the instantaneous flow or measured as the temperature rise of the fluidacross the apparatus using a thermocouple in the inlet and outlet streams. For a given valve sizeand fluid there is a linear relationship between the applied pressure or the shear stress gradient,and the fluid temperature rise. The relationship is not affected by the flow rate, allowing thetemperature rise to be used as a continuous monitor of the shear stress gradient and hence, theforces acting on the cells. The fluid temperature rise was used to calibrate and standardizethe two sets of apparatus. For the valve used for these tests 6-8 x 10* Pa (1 x 10' lb/in.1,6-9 x io3 kN m~a) pressure drop produced 1 °C rise in the temperature of water.

Bacterial strains

The organisms used were Escherichia coli 114, Staphylococcus aureus, Oxford, Lactobacilluscasei MTX/r, Bacillus subtilis 168, Streptococcus faecalis A.T.C.C. 9790, Clostridium perfringensN.C.T.C. 8237, Streptococcus zooepidermicus, Saccharomyces cerevisiae N.C.Y.C. 239, Aspergil-lus fumigatis, Fusarium sp. and CMorella.

Growth conditions

E. coli and 5. aureus were maintained on nutrient agar purchased from Oxoid Limited,London, England, and grown on Oxoid no. 2 nutrient broth. L. casei was maintained on HedleyWright agar (Wright, 1933) and grown on medium described by Dunlap, Harding & Huen-nekens (1971). B. subtilis was maintained on a tryptone yeast extract agar derived from L-medium (Karamata & Gross, 1970) and grown on casein hydrolysate-yeast extract medium(CHY) (Hughes, 1968). S. faecalis was maintained on Hedley Wright agar containing 5 %horse blood and grown on CHY medium. S. cerevisiae was maintained on a yeast plate medium(Mortimer & Hawthorne, 1969) and grown on the same medium with agar omitted. Cultures ofCl. perfringens, S. zooepidermicus, A. fumigatis, Fusarium sp. and Chlorella were provided.

Assay for cell disruption

Cell disruption was demonstrated qualitatively by phase-contrast microscopy. The organ-isms were photograhed before and after being processed through the disrupter. The detailedmorphological damage caused by disruption was observed by electron microscopy, usingpositive staining with 1 % uranyl acetate or negative staining with 2 % sodium phosphotung-state, a service provided by Dr Wyrick and Dr Burdett.

The extinction at 260 and 280 nm of soluble material released from the cells was also used asan estimate of cell disruption. The overnight cultures (15 1.) were centrifuged at 6000 g for20 min at 5 °C and resuspended in 0-05 M sodium phosphate buffer, pH 6-8 (i-o 1.). Samples(10 ml) of this suspension taken before and after passing through the disrupter at increasingapplied pressure o to 27 x io8 Pa (o to 4 x io1 lb/in.1, 2-76 x io5 kN m"1), were centrifuged at3000 g for 30 min and the supernatant solutions were diluted 10-fold with 005 M sodiumphosphate buffer, pH 6'8, for measurement. A sample of the soluble material from B. subtiliswas prepared by adding lysozyme (1 mg) to the bacterial suspension (10 ml). After incubationat 37 °C for 18 h, centrifugation and dilution, this sample was compared with those which hadpassed through the disrupter.

A quantitative estimate of cell disruption was made by viable counts. A viable count wasperformed on each culture before disruption, after passing through the apparatus with noapplied pressure and also at increasing applied pressures. Serial 10-fold dilutions were pre-pared for each sample and the viability in 2 successive dilutions tested. Samples of E. coli andS. auretis were diluted with nutrient broth and the colony-forming units were counted onnutrient agar. The samples of L. casei, S. faecalis and B. subtilis were diluted in a minimal saltmedium with glucose omitted (Davis & Mingioli, 1950) and the colony-forming units were

Controlled cell disruption 435

counted on Hedley Wright agar. 5. cerevisiae was also diluted in a minimal salt medium and thecolony-forming units were counted on yeast-plate medium. Counts for any given dilution weremade on duplicate plates. Two dilutions were routinely sampled giving effectively four countswhich were averaged.

RESULTS AND DISCUSSION

Microscopic observation

Clear evidence was obtained by phase-contrast and electron microscopy that avariety of micro-organisms can be disrupted after subjection to a high shear stressgradient in the disrupter. Fig. 3 shows a selection of organisms which have beendisrupted at different pressures. Chlorella, which has a thick cellulose wall, wasdisrupted at 4 '8xio7Pa (7 x io3 lb/in.2, 4-8 x io4 kN m~2). Filamentous fungisuch as A.fumigatis and Fusarium could be disrupted at 6-8 x io7 Pa (1 x io4 lb/in.2,6-9 x io4kN m~2), and unicellular fungus, S. cerevisiae was disrupted at 1-5 x io8 Pa(2-26 x 1 o4 lb/in.2, 1-56 x io5kN m~2). Smaller micro-organisms such as E. coli, L.casei, B. subtilis, Cl. perfringens, S. faecalis, S. zooepidermicus and S. aureus may alsobe disrupted. Electron micrographs of B. subtilis and S. aureus (Fig. 4) showed thatthe cells were broken by shearing the outer membrane. In the case of the Bacillus thiswas not restricted to a specific region of the rod.

Cell disruption estimated by the measurement of u.v. absorption

The u.v. absorption in the supernatant fluids increased considerably after passingcultures through the cell disrupter. Fig. 5 A shows the results for B. subtilis. In thisinstance it was possible to compare the absorption of the different samples which hadpassed through the disrupter with the supernatant fluid remaining after a culture wastreated with lysozyme. The results demonstrate that disruption is almost completeat 9-5 x ior Pa (1-4 x io4 lb/in.2, 9-65 x io4 kN m~2), when the absorption of the celllysate is compared with the supernatant derived from the bacterial suspensions passingthrough the cell disrupter. This comparison is not possible with the other species used,as they are not susceptible to lysozyme, but as shown with S. aureus (Fig. 5 B), it wasalways possible to demonstrate an increase in absorption with a concomitant increasein the applied pressure.

Cell disruption estimated by viable counting

The percentage viability remaining in a cell suspension was determined with respectto increasing applied pressure. It is assumed that any viable cells are intact andundamaged while those that are not viable have been disrupted. It was observed thatthe cells were not all broken at any one setting of the disrupter. The pattern found forall the micro-organisms tested was that little or no disruption occurred until a certainlevel of applied pressure was reached, and that subsequently the proportion of cellsdisrupted increased rapidly with increasing disrupter pressure until almost all thecells were disrupted. Fig. 6 shows the proportion of cells disrupted at different appliedpressures for each organism tested. Disruption followed a sigmoid curve in each case.

28-3

436 M. V. Kelemen andj. E. E. Sharpe

Fig. 3. Phase-contrast photographs of disrupted organisms, viewed originally at x 600.A, Chlorella; B, A.fumigatis; C, Fusarium sp.; D, S. cerevisiae.

Controlled cell disruption 437

4A B

Fig. 4. Electron micrographs of A, B. subtilis ( x 21000) and B, 5 . aureus (x 50000).

Distinctly different applied pressures were required to disrupt half the population ineach culture; E. colt (Gram-negative rods, 2-4 x 0-5 /tm) required i-5xio7Pa(2-2 x io3 lb/in.2, 1-52 x io* kN m~2), B. subtilis (Gram-positive rods 1-5-3 x

0-5-0-8 /im) required 2-4 x io7 Pa (3-6 x io3 lb/in.2, 2-4 x io4 kN m~2), L. casei (pleo-morphic Gram-positive rods, varying in length up to 4 x 0-4-0-7 /tm) required3-1 x io7 Pa (4-6 x io3 lb/in.2, 3-1 x 10* kN m~2), S. faecalis (Gram-positive ovoidcocci 1 -02 /im in diameter) required r j x io8Pa(2-26x io4 lb/in.2, 1-56 x io5kNm~2),S. aureus (Gram-positive cocci, 1 /im in diameter) required 19 x io8 Pa (2-86 x io4 lb/in.2, i ^x io ' kNm" 2 ) , and S. cerevisiae (oval yeast cells 7-12 x 5-8/tm) required1-5 x io8 Pa (2-21 x io4 lb/in.2, 1-56 x io6 kN m~2). The applied pressure required toachieve complete disruption in all cultures tested was beyond the capability of theapparatus, although greater than 95 % disruption of all cultures was achieved at2-7 x io8 Pa (4-0 x io4 lb/in.2, 2-76 x ioB kN m~2).

From the few species tested it seems probable that the pressure required for dis-ruption is related to the shape of the organism, the rods being more easily disruptedthan the cocci (Fig. 6). In addition to the shape, the composition of the cell wall alsodetermines the ease with which an organism is disrupted as may be seen from the fact

438 M. V. Kelemen andj. E. E. Sharpe

that E. coli, a Gram-negative rod, although similar in size to B. subtilis (a Gram-positive rod) is disrupted more easily. The force required to disrupt 50 % of the cellsin a culture seems dependent both on the shape of the organism and on the structureof the outer membranes.

0-3 -260 nm + lysozyme

• 260 nm

280 nm + lysozyme

10 15 20 (X 103) Ib/in2

0-5 1-0Disrupting pressure

1-5 (X 108) Pa

260 nm

30 (X 103) Ib/in2

I i i

0-5 1-0 1-5 2 0Disrupting pressure

2-5 (X 10s) Pa

Fig. s A,B. Effect of applied pressure on the release of u.v.-absorbing material fromA, B. subtilis, and B, 5. aureus. io3 Ib/in.3 = 6-9 x io3 kN m"1.

In addition to the species specificity which might be anticipated on the basis ofshape and cell wall composition, all the cells in an unsynchronized culture are notdisrupted at one given pressure. When the percentage of the individual cells disruptedis plotted against applied pressure, a characteristic sigmoid curve is obtained for eachmicro-organism.

To determine whether cell disruption is a random or non-random process, a cellsuspension was passed through the disrupter at a relatively low applied pressure whichdisrupted 20 % of the cell population. This suspension was then passed once morethrough the disrupter, beginning again at zero pressure. It was observed that passing

Controlled cell disruption 439

the cells through the disrupter a second time at a relatively low pressure did notincrease the number of disrupted cells. The number of viable cells decreased onlywhen the applied pressure was increased beyond the initial maximum (Fig. 7). From

40 (X 103) Ib/in2

I I0-5 10 1-5 20

Disrupting pressure2-5 30 (X 108) Pa

Fig. 6. Effect of applied pressure on the cell viability of different microorganisms.io'lb/in.a = 6-oxio'kNm-». A, E. coli; • , S. faecalh; U, B. subtilis; Q, S.cerevisiae; O, L. casei; and A, S. aureus.

10Disrupting pressure

1-5(X 10s) Pa

Fig. 7. Effect of applied pressure on a partially disrupted suspension of L. casei.10' Ib/in. • = 69 x 103 kN m~*. O, initially undisrupted cells; # , initially partiallydisrupted cells.

this it seems most probable that the process of cell disruption is not random but isdetermined by some physical property of the cell population.

The theory of thin shells (Goldenveizer, 1961) suggests that the cells of a givenculture are disrupted on the basis of their size, the large cells being disrupted first, theamount of disruption at a given applied pressure being related to the number of cells

440 M. V. Kelemen andj. E. E. Sharpe

10

- 5

o^ 10

0-5

I I I I0-01 0-1 O5 2 5 10 20 40 60 80 90 95 98 99-8 99-99

Cell population, %

Fig. 8. Number of cells with a given volume versus Log volume. Data obtained fromKubitschek (1969).

Ib/in- ,

1 X 105 -

Pa

5X 10"

2 X 108

1 X 10"

8 5X 107= 1 X 104

'5 X 103

Q- 2 X 107

1 X 10'

5 X 106

2 X 106

1 X 1 0 * -

5 X 104

'2 X 104

2 X 103

- 1 X 103

1 X 103 L I I I I I I I I I I I I I II I I I I99-8 99 95 90 80 60 40 20 10 5 2 1 0-5 0-1

% viable cells

Fig. 9. The cell viability versus Log applied pressure for different micro-organisms.io3lb/in.2 = 69 x io'kNm"1. A, E. coli; • , 5. faecalis; • , B. subtilis; D, S.cerevisiae; O, L. casei; and A, S. aureus.

having a given size or volume. If the forces required to disrupt a cell are inverselyrelated to the physical dimension of the cells, then it should follow that the statisticaldistribution of disrupted cells reflects the statistical size distribution.

Kubitschek (1969) measured the individual cell volumes of E. coli in an unsyn-chronized growing culture and found a large distribution of cell volumes at any given

Controlled cell disruption 441

time. If the number of cells of a given volume is plotted against the logarithm of thatvolume, it can be shown that the cultures of E. coli have a Log Normal volume distri-bution (Fig. 8). Similarly, it can be shown that cell viability when plotted against theLog of the applied pressure also follows a Log Normal distribution (Fig. 9). Theo-retically, this suggests that the statistical distribution of disrupted cells reflects theirstatistical volume distribution.

It is evident that there are several factors which affect the ease with which a cell canbe disrupted. Among these are cell shape, the composition of the outer membraneand, possibly, the cell volume. The advantage of the apparatus described is that dif-ferent pressures can be accurately reproduced, making it possible to determine therelative forces required to disrupt each type of cell.

We wish to thank colleagues at the School of Pharmacy, University of London and at theNational Institute for Medical Research, London, for the valuable help in this work.

REFERENCES

BUCHNER, E. (1897). Alkoholisch Gahrung ohne Hefezellen. Ber. dt. cheni. Ges. 30, 1, 117-124.DAVIS, B. D. & MINGIOLI, E. S. (1950). Mutants of Escherichia coli requiring methionine or

vitamin B12. J. Bad. 60, 17-28.DUNLAP, R. B., HARDING, N. G. L. & HUENNEKENS, F. M. (1971). Thymidylate synthetase

from a methopterin-resistant Lactobacillus casei. Biochemistry, N.Y. 10, 88-97.GOLDENVEIZER, C. (1961). Theory of Thin Shells. London: Pergamon.HUGHES, R. C. (1968). The cell wall of Bacillus liclieniformii NCTC 6346. Biochem. J. 106,

49-59-KARAMATA, D. & GROSS, J. D. (1970). Isolation and genetic analysis of temperature sensitive

mutants of B. subtilis defective in DNA synthesis. Molec. gen. Genet. 108, 277-287.KUBITSCHEK, H. E. (1969). Growth during the bacterial cell cycle. Analysis of cell size distri-

bution. Biophys.J. 9, 792-809.MILNER, H. W., LAWRENCE, N. S. & FRENCH, C. S. (1950). Colloidal dispersion of chloroplast

material. Science, N.Y. i n , 633-634.MORTIMER, R. K. & HAWTHORNE, D. C. (1969). Yeast genetics. In Tlie Yeasts, vol. 1 (ed. A. H.

Rose & J. S. Harrison), pp. 391-392. London and New York: Academic Press.PATEL, V. C. & HEAD, M. R. (1968). Reversion of Turbulent to Laminar Flow. Aeronautical

Research Council Report No. 29859.ROGERS, H. J. & PERKINS, H. R. (1968). The disruption of bacteria and the preparation of cell

walls. In Cell Walls and Membranes (ed. C. Long), pp. 196-205. London: Spon.SHARPE, J. E. E. (1975). A disrupter for bacteriological and mammalian Cells. Lab. Pract. 25,

28-29.WRIGHT, B. M., EDWARDS, A. J. & JONES, V. E. (1974). Use of a cell rupturing pump for the

preparation of thymocyte subcellular fractions. J. Immun. Meth. 4, 281-296.WRIGHT, H. D. (1933). The importance of adequate reduction of peptone in the preparation of

media for the pneumococcus and other organisms. J. Path. Bad. 37, 257-282.

[Received 22 May 1978)