PUMPING AS A MEANS FOR THE MECHANICAL RUPTURE OFMICROORGANISMS MIXED WITH GLASS BEADS'

CARL LAMANNA, M. A. CHATIGNY, AND E. H. COLLEDGE

The Naval Biological Laboratory, School of Public Health, University of California, Berkeley, CaliforniaReceived for publication July 28, 1958

Agitation with small glass beads causes amechanically induced rupture of microorganisms.A number of means for agitating mixtures ofbeads and suspensions of organisms have beendescribed (Curran and Evans, 1942; Mickle,1948; Lamanna and Mallette, 1954). However,since no reports seem to exist on the possibleeffectiveness of pumping mechanisms for im-parting motion to suspensions of glass beads andmicroorganisms, it is our purpose to report ourefforts. The term pumping is used herein in thesense of transmission of energy to a fluid orslurry by pressure (compression or suction). Aninherent theoretical advantage of a pumpingdevice is the ability to move fluids within acompletely filled closed system without foaminigand the danger of creating an aerosol of potenti-ally dangerous organisms.

METHODS AND DESIGN OF EQUIPMENT

Test organisms. It has been a general experi-ence that yeasts are among the most difficultorganisms to rupture. Therefore, commerciallyavailable yeast has been employed routinely inour studies with the thought that successfulrupture of yeasts would insure successful ruptureof bacteria. This has actually been our experi-ence. Bacterial spores and Serratia marcescenshave been successfully ruptured when yeastshave been ruptured.

Glass beads. Mlinnesota Mining and Manufac-turing Company 3 M "Superbrite" glass beadshave been used for all studies. The effective beads

'This work was sponsored by the Office ofNaval Research, U. S. Navy, and the Bureau ofMedicine and Surgery, U. S. Navy, under a con-tract between the Office of Naval Research andthe Regents of the University of California.

Opinions expressed in this report are not to beconstrued as reflecting the view of the NavyDepartment or of the naval service at large(Article 1252, U. S. Navy Regulations, 1948). Re-production in whole or in part is permitted for anypurpose of the United States Government.

are catalogue no. 070 (0.47 mm average diameter)and 110 (0.15 mm average diameter), the 070size beads being somewhat better than the 110beads.

Commerctally available putmps. A number ofcommercially available pumps have been studied:centrifugal type laboratory circulating pumps, aRobbins and Myers Company "Moyno" (2L3)pump, which operates on the progressing cavityprinciple, and an Aminco peristaltic pump. Noneof these have proved satisfactory. Rupture ofmicroorganisms in the circulating slurry of glassbeads and organisms does occur but at an un-satisfactory rate. In addition, considerable risesin temperature of the circulating slurry areobserved. In the case of the peristaltic pump, thethickness of the slurry (viscosity) that could bemoved was limited. In sum, none of the commer-cial pumps tried worked satisfactorily. While asearch for a satisfactory pump will go on in thehope of finding a suitable commercially avail-able one, experience indicated it would be worth-while to design our own equipment.

Principle of design of equipment. The design ofequipment should permit the impartation ofsufficient motion to a slurry of beads and micro-organisms completely enclosed in tubing to resultin the mechanical rupture of 90 per cent or moreof the organisms within 1 hr at ambient and lowertemperatures.

Tubing for holding the slurry of glass beads andmicroorganisms. For use with the equipment wedesigned and which is described, we desired ameans for joining together the open ends of apiece of tubing to yield a continuous completelyclosed tube, a hollow hoop, containing slurry.Such a tube should have a flexible wall so thatmotion could be imparted through the wall to theslurry from an outside source. Such a tube wouldbe safe to handle since the organisms within thetube would have no way of access out of the tubebarring accidental breakage in the wall of thetube itself.

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Various means were employed unsuccessfullyfor tying the two open ends of a rubber tubetogether into a single circular unit of hollowtubing. No simple method for routine use in thelaboratory was found for vulcanizing the twoopen ends of a rubber tube together. Nor wereany tested industrial tapes and adhesives satis-factory. Under tension, the joint tore apart,spilling the internal contents of the tube. Studiesof rubber tubing were therefore abandoned andattention turned to plastic tubing.

"Tygon-B," a trade name for a polyvinylchloride plastic that can be autoclaved, provedsatisfactory. It can be welded by the simpleapplication of heat. Pressing the heated ends ofTygon-B tubing together yields a welded jointwith a tensile strength of the order of the un-treated tubing itself. One-sixteenth in wallthickness tubing of 14 and j! in internal diameterhas been employed.

Filling the plastic tube. The required quantity ofbeads is poured into an open end of a tube througha funnel. Two methods have been used to add thesuspension of organisms: (a) Using a syringeequipped with a long needle, the suspension oforganisms is injected into the mass of beads tofill the tube. The tube's open ends are thenjoined together. (b) The open ends of thetube containing the beads are joined together.Then a syringe with needle is used to puncturethe tube and air removed from the tube whilesimultaneously injecting a suspension of organ-isms into the tube with another syringe andneedle. After addition of the suspension, theneedles are withdrawn and the needle holes in thetubing heatsealed.With practice, either of these techniques can

be mastered to permit safe loading of the plastictubing with virulent microorganisms. After theopen ends of the tube have been sealed together,and organisms added, the outside of the tube canbe freed of any living organisms accidentallyspilled on the outside of the tubing by washingthe surface with a proper disinfectant.

Joining the open ends of the plastic tube by heator cementing. Pieces of Tygon-B plastic are dis-solved in cyclohexanone to give a thick, viscoussolution. This solution can be used to glue theopen ends of tubing together. The result is atight seal with tensile strength equivalent to thetubing itself. A disadvantage of this method ofjoining the open ends of tubing together is that

the glue takes a long time to dry. It may takeovernight or longer for complete drying.A satisfactory technique for joining the ends of

the tubing is to use a bunsen burner to heat oneend of a strip of aluminum metal. At the oppositeend of the hot metal strip the ends of the opentubing are pressed, the ends of the tubing beingplaced on the opposite faces of the hot strip.When the ends of the tubing become soft, theyare moved along the strip and slowly moved offthe edge of the strip, pressed where they makecontact, and permitted to fuse by cooling. Whenfully cooled, the hollow hoop of tubing is foundto be completely sealed against the escape of theinternally contained slurry, and can be pulledwith great strength without rupture of the joint.

Fool-proof sealing of pin holes has provedmore difficult, and cannot be said to have beenachieved in an absolute sense. In sealing pinholes or holes made in the tubing by injection ofsuspensions or for removal of small samples, ahot metal spatula is passed over the hole. Inaddition, small bits of thin strips of plastic areplaced over the hole and sealed into place with ahot metal rod or spatula.A degree of skill is required for the sealing

operations. With sufficient practice, anyone withnormal manual dexterity can master the tech-nique.When the tube with slurry has been prepared,

it can then be placed on either of two machinesfor the purpose of inducing mechanical ruptureof microorganisms. The two machines whichhave been designed at the Naval BiologicalLaboratory are now described.

Centrifuge accessory for the mechanical rupture ofmicroorganisms. This machine consists of a pulleyfitted to the drive shaft of a refrigerated centri-fuge (International PR1), and a plate, clampedto the drive housing, bearing two fixed sheavesand a third adjustable sheaf. The latter is usedto control the tension of the tubing which isdraped around the rotors (figure 1). The centri-fuge is used to move the tube as a continuousbelt, and to control the temperature. As thetubing moves over the rotors, heat is generatedby friction, thus the desirability of using a refrig-erated centrifuge (it is also desirable to lubricatethe tubing). Apparently, as the tubing movesover the faces of the rotors the tubing is squeezedand a pumping action results. Sufficient move-ment is imparted to the beads to result in mechan-

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LAMANNA, CHATIGNY, AND COLLEDGE

Figure 1. Centrifuge device for mechanical rupture of microorganisms with a 10.5 ft length of tubingin position. The coils of the tubing are placed so that they all move in the same direction.

Figure 2. The compression roller device for the mechanical rupture of microorganisms. Figure A shows atube properly placed for use. Figure B is a rear view showing the spring used to hold the variable positionrotor (top roller in figure A) with pressure against the fixed roller attached to the motor for direct drive.

ical rupture of microorganisms. Variable lengthsof tubing of different diameter can be used. Fortubing of 14 in diameter, the maximal length oftubing employed has been 10.5 feet which holds400 ml of slurry.

Compression roller device for the mechanicalrupture of microorganisms. This device (figure 2)consists basically of a 1725 rpm, split phase, 18hp motor to which is attached a rubber surfacedcompression roller and an apposed idler roller of

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similar design. The latter is mounted to provideadjustable spring loading (figure 2B) of the pres-

sure between the two rollers. A guide sheafsliding freely on a splined vertical shaft attachedto the motor base positions the tubing betweenthe rollers and maintains gentle tension on theloop. As the tubing moves between the surfacesof the rollers, a pumping action is exerted on itwhich results in mechanical rupture of the con-

tained organisms contacting the glass beads.The tubing can be placed so as to pass through

ice water on passage to and from the compressionrollers. This is a desirable feature since it serves

to maintain low temperatures during the ruptureof the organisms. A chamber has been builtaround the compression rollers to preventsplashing of water during vigorous rotation of thetubing placed in ice water.

Engineering drawings of the device have beenprepared and are available to those who requestthem.

Recovery of fluid and ruptured microorganismsfrom slurry. The tube is cut and the slurry forcedout by any suitable means. In the case of virulentorganisms it may be desirable to remove the fluidby means of a sterile syringe and needle throughpuncture of the unopened tube.The slurry of bead-organisms can be filtered

through a stainless steel screen of fine enoughmesh to retain the beads and to let only thefluid pass. A 120 by 120 fine mesh with openingsof 0.1168 mm and wire diameter of 0.094 mm hasbeen built into a screen for insertion into a

Seitz type filter and has proved to be satisfactory.Efficient operation of devices. As the plastic

tube passes over the faces of the rotors of thedevices, sufficient pressure must be exerted tocause changes in shape of the tube. It is thissqueezing or pumping action that results in therupture of microorganisms. With the centrifugeaccessory this effect of pressure is illustrated bythe following experience. Using a ratio of 3.5 to5 in the volumes of beads to suspension of yeastand with the centrifuge rotating at 3000 rpm,

50 per cent of the yeast cells were ruptured in a

belt "loosely" fitted whereas 99.99 per cent ofthe yeast cells were ruptured in a belt fittedtightly on the rotors.The centrifuge device operates with less genera-

tion of heat and with almost no tendency for the

plastic tubing to jump off the rotors if a thin

layer of Vaseline is used to grease the faces of therotors.

It is not necessary for rupture of the micro-organisms that the slurry within the tube circu-late freely. This has been tested in both devicesby placing a plug in the tubing which preventsfree circulation. Tests with plugged tubes showedsatisfactory rupturing rates of yeast but with amuch greater generation of heat.The slurry can be so thick with, beads that

circulation of the fluid phase with microorganismsdoes not take place. In spite of this conditionrupture does take place. But there has been ob-served a tendency for increased heterogeneityin the rates of rupture in different local areaswithin the tubing. This has been found by takingafter a run a number of samples from a singletube at different places along the tube.Any concentration of yeast suspension was

ruptured that had enough fluidity to flow freely.Rupture also occurred in mixtures of dried yeastcake and beads, but the rate was low and heatgeneration great.

EXPERIMENTAL FINDINGS

Yeasts prove to be more difficult to rupture thanbacteria. No failures with bacteria and bacterialendospores are encountered when yeast can beruptured with the devices developed. To demon-strate that endospores can be ruptured, the fol-lowing experience with spores of Bacillus cereusis cited. When 9 ml of a spore suspension with 1X 109 spores per ml were added to 12 ml ofbeads in a length of 4 in plastic tubing and thetube placed on the centrifuge device, more than

TABLE 1Trial of rupture of Serratia marcescens employing

the centrifuge device

Time Plate Count

min no. organisms/ml

0 2.11 X 101015 1.35 X 10'°30 5.07 X 10945 2.77 X 10960 1.42 X 10990 6.1 X 108120 2.4 X 108

Conditions of trial: Tube used, /1 in internaldiameter; beads used, 070, 10 ml; Serratia marces-cens, 2.11 X 1010 per ml, 25 ml; centrifuge run at2500 rpm and 0 C.

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LAMANNA, CHATIGNY, AND COLLEDGE

TABLE 2

Influence of the relative proportions of beads toyeast suspension on the rate of rupture of yeasts

in the Naval Biological Laboratory devices

Apparatus

Centrifuge de-vice operatedat 2000 rpm

Compressionroller device

Expt

1

2

3

Ratio,Beads (ml):

YeastSuspension

(ml)

1.5:1.02:33:5

1:11:21:41:8

1:11:21:41:8

Relative Count* per min:

0

128140102

100100100100

100100100100

15

18

5242

30

1

23

40582882

25395878

60

211

* Microscopic determination: the number ofunruptured cells in a 1:100 dilution of the yeastsuspension in 16 of the smallest squares of aclinical hemocytometer.

TABLE 3

Influence of rpm on the rate of rupture of yeastorganisms in the Naval Biological Laboratory

centrifuge device

Relative Countt per min-

Run* Speed i 0 6(rpm) 15 30 600

A B C A B C AI B C

1 1000 137 72 79 8173 7677 4145 272000 137 10 11 14 16 22 14 11 12 304000 137 18 9 13 10 17 8 11 13 16

2 1000 137 60 58 62 54 34 474000 137 16 18 18 12 19 20

Run 1- Internal tube diameter, 14 in; tuLelength, 27Y4 in; ratio of beads (ml) to yeast sus-pension (ml), 1:2; temperature, 0 C.Run 2: Internal tube diameter, }f in; tube

length, 27% in; ratio of beads (ml) to yeast sus-pension (ml), 1:2; temperature, 0 C.

t Microscopic determination: the number ofunruptured cells in a 1:100 dilution of the yeastsuspension in 16 of the smallest squares of aclinical hemocytometer. A, B, and C stand forthree separate experiments.

99 per cent of the spores were ruptured within 15min of operation of the centrifuge at 1000 rpm.

In table 1 an experiment with a suspension ofSerratia marcescens is recorded which shows a93 per cent drop in viable numbers after 1 hr ofoperation of the centrifuge device. The rate ofrupture could be increased by increasing the ratioof beads to cell suspension or the rpm of thecentrifuge run. A graphical plot of these datawould show a characteristic feature of the processof rupture of microorganisms, namely, that thedecrease in count of unruptured organisms in timeis exponential for a limited time and then tendsto plateau (see also table 3). This finding indicatesthat sterility of the suspension of microorganismscannot be achieved unless the runs were to becontinued for inordinate periods of time.The rate of rupture of organisms is related to

the ratio of the volumes of beads to suspensionof organisms (table 2). The greater the relativeamount of beads to suspension the more rapid isthe rate of rupture. This finding is similar to thatreported for the use of the Waring Blendor toagitate a bead-organism slurry (Lamanna andMallette, 1954). It would seem that the maximalrate would be attained only if enough suspensionwere used to fill the spaces between the beads.As common sense suggests, it was found that

the higher the speed at which the centrifugedevice is operated the more rapid is the rate atwhich yeasts are ruptured. Some results ob-tained are recorded in table 3. The data alsoillustrate another finding, namely, that with agiven slurry and the same length of tubing, theefficiency of rupture is not influenced by theinternal diameter (volume) of the tubing. Weinterpret this observation to mean that it is onlythe surface area of the tubing moving across theface of the rotors which affects the rate of ruptureof organisms.

SUMMARY

Pumping can be employed to cause glassbeads to rupture microorganisms. Since to dateno commercially available pump tested hasproved satisfactory in operation, two deviceshave been designed at the Naval BiologicalLaboratory which successfully utilize the princi-ple of pumping for achieving rupture of micro-organisms in a closed system. A slurry of micro-organisms and glass beads is placed in atransparent polyvinyl tube (Tygon-B) and the

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tube moved rapidly over rotating pulleys. Themachines devised rotate the tube; one is a centri-fuge accessory and the other a compression rollerdevice. These devices transmit kinetic energy tothe slurry within the tube by pressure exertedon the flexible walls of the tube as the tube movesalong the faces of rotors. Data are presentedwhich show these devices to be practical instru-ments for laboratory rupture of microorganisms.Variables affecting the rates of rupture have beeninvestigated.

REFERENCES

CURRAN, H. R. AND EVANS, F. R. 1942 Thekilling of bacterial spores in fluids by agita-tion with small inert particles. J. Bacteriol.,43, 125-138.

LAMANNA, C. AND MALLETTE, M. F. 1954 Use ofglass beads for the mechanical rupture ofmicroorganisms in concentrated suspensions.J. Bacteriol., 67, 503-504.

MICKLE, H. 1948 A tissue disintegrator. J.Roy. Microscop. Soc., 68, 10-12.

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