MICROORGANISMS MIXED WITH GLASS BEADS'

Transcription

1 PUMPING AS A MEANS FOR THE MECHANICAL RUPTURE OF MICROORGANISMS 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, California Received for publication July 28, 958 Agitation with small glass beads causes a mechanically induced rupture of microorganisms. A number of means for agitating mixtures of beads and suspensions of organisms have been described (Curran and Evans, 942; Mickle, 948; Lamanna and Mallette, 954). However, since no reports seem to exist on the possible effectiveness of pumping mechanisms for imparting motion to suspensions of glass beads and microorganisms, it is our purpose to report our efforts. The term pumping is used herein in the sense of transmission of energy to a fluid or slurry by pressure (compression or suction). An inherent theoretical advantage of a pumping device is the ability to move fluids within a completely filled closed system without foaminig and the danger of creating an aerosol of potentially dangerous organisms. METHODS AND DESIGN OF EQUIPMENT Test organisms. It has been a general experience that yeasts are among the most difficult organisms to rupture. Therefore, commercially available yeast has been employed routinely in our studies with the thought that successful rupture of yeasts would insure successful rupture of bacteria. This has actually been our experience. Bacterial spores and Serratia marcescens have been successfully ruptured when yeasts have been ruptured. Glass beads. Mlinnesota Mining and Manufacturing Company 3 M "Superbrite" glass beads have been used for all studies. The effective beads 'This work was sponsored by the Office of Naval Research, U. S. Navy, and the Bureau of Medicine and Surgery, U. S. Navy, under a contract between the Office of Naval Research and the Regents of the University of California. Opinions expressed in this report are not to be construed as reflecting the view of the Navy Department or of the naval service at large (Article 252, U. S. Navy Regulations, 948). Reproduction in whole or in part is permitted for any purpose of the United States Government. are catalogue no. 070 (0.47 mm average diameter) and 0 (0.5 mm average diameter), the 070 size beads being somewhat better than the 0 beads. Commerctally available putmps. A number of commercially available pumps have been studied: centrifugal type laboratory circulating pumps, a Robbins and Myers Company "Moyno" (2L3) pump, which operates on the progressing cavity principle, and an Aminco peristaltic pump. None of these have proved satisfactory. Rupture of microorganisms in the circulating slurry of glass beads and organisms does occur but at an unsatisfactory rate. In addition, considerable rises in temperature of the circulating slurry are observed. In the case of the peristaltic pump, the thickness of the slurry (viscosity) that could be moved was limited. In sum, none of the commercial pumps tried worked satisfactorily. While a search for a satisfactory pump will go on in the hope of finding a suitable commercially available one, experience indicated it would be worthwhile to design our own equipment. Principle of design of equipment. The design of equipment should permit the impartation of sufficient motion to a slurry of beads and microorganisms completely enclosed in tubing to result in the mechanical rupture of 90 per cent or more of the organisms within hr at ambient and lower temperatures. Tubing for holding the slurry of glass beads and microorganisms. For use with the equipment we designed and which is described, we desired a means for joining together the open ends of a piece of tubing to yield a continuous completely closed tube, a hollow hoop, containing slurry. Such a tube should have a flexible wall so that motion could be imparted through the wall to the slurry from an outside source. Such a tube would be safe to handle since the organisms within the tube would have no way of access out of the tube barring accidental breakage in the wall of the tube itself. 04

2 959 PUMPING DEVICE FOR RUPTURE OF MICROORGANISMS 05 Various means were employed unsuccessfully for tying the two open ends of a rubber tube together into a single circular unit of hollow tubing. No simple method for routine use in the laboratory was found for vulcanizing the two open ends of a rubber tube together. Nor were any tested industrial tapes and adhesives satisfactory. Under tension, the joint tore apart, spilling the internal contents of the tube. Studies of rubber tubing were therefore abandoned and attention turned to plastic tubing. "Tygon-B," a trade name for a polyvinyl chloride plastic that can be autoclaved, proved satisfactory. It can be welded by the simple application of heat. Pressing the heated ends of Tygon-B tubing together yields a welded joint with a tensile strength of the order of the untreated tubing itself. One-sixteenth in wall thickness tubing of 4 and j! in internal diameter has been employed. Filling the plastic tube. The required quantity of beads is poured into an open end of a tube through a funnel. Two methods have been used to add the suspension of organisms: (a) Using a syringe equipped with a long needle, the suspension of organisms is injected into the mass of beads to fill the tube. The tube's open ends are then joined together. (b) The open ends of the tube containing the beads are joined together. Then a syringe with needle is used to puncture the tube and air removed from the tube while simultaneously injecting a suspension of organisms into the tube with another syringe and needle. After addition of the suspension, the needles are withdrawn and the needle holes in the tubing heatsealed. With practice, either of these techniques can be mastered to permit safe loading of the plastic tubing with virulent microorganisms. After the open ends of the tube have been sealed together, and organisms added, the outside of the tube can be freed of any living organisms accidentally spilled on the outside of the tubing by washing the surface with a proper disinfectant. Joining the open ends of the plastic tube by heat or cementing. Pieces of Tygon-B plastic are dissolved in cyclohexanone to give a thick, viscous solution. This solution can be used to glue the open ends of tubing together. The result is a tight seal with tensile strength equivalent to the tubing itself. A disadvantage of this method of joining the open ends of tubing together is that the glue takes a long time to dry. It may take overnight or longer for complete drying. A satisfactory technique for joining the ends of the tubing is to use a bunsen burner to heat one end of a strip of aluminum metal. At the opposite end of the hot metal strip the ends of the open tubing are pressed, the ends of the tubing being placed on the opposite faces of the hot strip. When the ends of the tubing become soft, they are moved along the strip and slowly moved off the edge of the strip, pressed where they make contact, and permitted to fuse by cooling. When fully cooled, the hollow hoop of tubing is found to be completely sealed against the escape of the internally contained slurry, and can be pulled with great strength without rupture of the joint. Fool-proof sealing of pin holes has proved more difficult, and cannot be said to have been achieved in an absolute sense. In sealing pin holes or holes made in the tubing by injection of suspensions or for removal of small samples, a hot metal spatula is passed over the hole. In addition, small bits of thin strips of plastic are placed over the hole and sealed into place with a hot metal rod or spatula. A degree of skill is required for the sealing operations. With sufficient practice, anyone with normal manual dexterity can master the technique. When the tube with slurry has been prepared, it can then be placed on either of two machines for the purpose of inducing mechanical rupture of microorganisms. The two machines which have been designed at the Naval Biological Laboratory are now described. Centrifuge accessory for the mechanical rupture of microorganisms. This machine consists of a pulley fitted to the drive shaft of a refrigerated centrifuge (International PR), and a plate, clamped to the drive housing, bearing two fixed sheaves and a third adjustable sheaf. The latter is used to control the tension of the tubing which is draped around the rotors (figure ). The centrifuge is used to move the tube as a continuous belt, and to control the temperature. As the tubing moves over the rotors, heat is generated by friction, thus the desirability of using a refrigerated centrifuge (it is also desirable to lubricate the tubing). Apparently, as the tubing moves over the faces of the rotors the tubing is squeezed and a pumping action results. Sufficient movement is imparted to the beads to result in mechan-

3 06 LAMANNA, CHATIGNY, AND COLLEDGE [VOL. 77 Figure. Centrifuge device for mechanical rupture of microorganisms with a 0.5 ft length of tubing in position. The coils of the tubing are placed so that they all move in the same direction. Downloaded from on April 6, 209 by guest Figure 2. The compression roller device for the mechanical rupture of microorganisms. Figure A shows a tube properly placed for use. Figure B is a rear view showing the spring used to hold the variable position rotor (top roller in figure A) with pressure against the fixed roller attached to the motor for direct drive. ical rupture of microorganisms. Variable lengths of tubing of different diameter can be used. For tubing of 4 in diameter, the maximal length of tubing employed has been 0.5 feet which holds 400 ml of slurry. Compression roller device for the mechanical rupture of microorganisms. This device (figure 2) consists basically of a 725 rpm, split phase, 8 hp motor to which is attached a rubber surfaced compression roller and an apposed idler roller of

4 959] PUMPING DEVICE FOR RUPTURE OF MICROORGANISMS 07 similar design. The latter is mounted to provide adjustable spring loading (figure 2B) of the pressure between the two rollers. A guide sheaf sliding freely on a splined vertical shaft attached to the motor base positions the tubing between the rollers and maintains gentle tension on the loop. As the tubing moves between the surfaces of the rollers, a pumping action is exerted on it which results in mechanical rupture of the contained organisms contacting the glass beads. The tubing can be placed so as to pass through ice water on passage to and from the compression rollers. This is a desirable feature since it serves to maintain low temperatures during the rupture of the organisms. A chamber has been built around the compression rollers to prevent splashing of water during vigorous rotation of the tubing placed in ice water. Engineering drawings of the device have been prepared and are available to those who request them. Recovery of fluid and ruptured microorganisms from slurry. The tube is cut and the slurry forced out by any suitable means. In the case of virulent organisms it may be desirable to remove the fluid by means of a sterile syringe and needle through puncture of the unopened tube. The slurry of bead-organisms can be filtered through a stainless steel screen of fine enough mesh to retain the beads and to let only the fluid pass. A 20 by 20 fine mesh with openings of 0.68 mm and wire diameter of mm has been 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 the devices, sufficient pressure must be exerted to cause changes in shape of the tube. It is this squeezing or pumping action that results in the rupture of microorganisms. With the centrifuge accessory this effect of pressure is illustrated by the following experience. Using a ratio of 3.5 to 5 in the volumes of beads to suspension of yeast and with the centrifuge rotating at 3000 rpm, 50 per cent of the yeast cells were ruptured in a belt "loosely" fitted whereas per cent of the yeast cells were ruptured in a belt fitted tightly on the rotors. The centrifuge device operates with less generation 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 the rotors. It is not necessary for rupture of the microorganisms that the slurry within the tube circulate freely. This has been tested in both devices by placing a plug in the tubing which prevents free circulation. Tests with plugged tubes showed satisfactory rupturing rates of yeast but with a much greater generation of heat. The slurry can be so thick with, beads that circulation of the fluid phase with microorganisms does not take place. In spite of this condition rupture does take place. But there has been observed a tendency for increased heterogeneity in the rates of rupture in different local areas within the tubing. This has been found by taking after a run a number of samples from a single tube 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 yeast cake and beads, but the rate was low and heat generation great. EXPERIMENTAL FINDINGS Yeasts prove to be more difficult to rupture than bacteria. No failures with bacteria and bacterial endospores are encountered when yeast can be ruptured with the devices developed. To demonstrate that endospores can be ruptured, the following experience with spores of Bacillus cereus is cited. When 9 ml of a spore suspension with X 09 spores per ml were added to 2 ml of beads in a length of 4 in plastic tubing and the tube placed on the centrifuge device, more than TABLE Trial of rupture of Serratia marcescens employing the centrifuge device Time Plate Count min no. organisms/ml 0 2. X X 0' X X X X X 08 Conditions of trial: Tube used, / in internal diameter; beads used, 070, 0 ml; Serratia marcescens, 2. X 00 per ml, 25 ml; centrifuge run at 2500 rpm and 0 C.

5 08 LAMANNA, CHATIGNY, AND COLLEDGE [VOL. 77 TABLE 2 Influence of the relative proportions of beads to yeast suspension on the rate of rupture of yeasts in the Naval Biological Laboratory devices Apparatus Centrifuge device operated at 2000 rpm Compression roller device Expt 2 3 Ratio, Beads (ml): Yeast Suspension (ml).5:.0 2:3 3:5 : :2 :4 :8 : :2 :4 :8 Relative Count* per min: * Microscopic determination: the number of unruptured cells in a :00 dilution of the yeast suspension in 6 of the smallest squares of a clinical hemocytometer. TABLE 3 Influence of rpm on the rate of rupture of yeast organisms in the Naval Biological Laboratory centrifuge device Relative Countt per min- Run* Speed (rpm) i A B C A B C AI B C Run - Internal tube diameter, 4 in; tule length, 27Y4 in; ratio of beads (ml) to yeast suspension (ml), :2; temperature, 0 C. Run 2: Internal tube diameter, }f in; tube length, 27% in; ratio of beads (ml) to yeast suspension (ml), :2; temperature, 0 C. t Microscopic determination: the number of unruptured cells in a :00 dilution of the yeast suspension in 6 of the smallest squares of a clinical hemocytometer. A, B, and C stand for three separate experiments per cent of the spores were ruptured within 5 min of operation of the centrifuge at 000 rpm. In table an experiment with a suspension of Serratia marcescens is recorded which shows a 93 per cent drop in viable numbers after hr of operation of the centrifuge device. The rate of rupture could be increased by increasing the ratio of beads to cell suspension or the rpm of the centrifuge run. A graphical plot of these data would show a characteristic feature of the process of rupture of microorganisms, namely, that the decrease in count of unruptured organisms in time is exponential for a limited time and then tends to plateau (see also table 3). This finding indicates that sterility of the suspension of microorganisms cannot be achieved unless the runs were to be continued for inordinate periods of time. The rate of rupture of organisms is related to the ratio of the volumes of beads to suspension of organisms (table 2). The greater the relative amount of beads to suspension the more rapid is the rate of rupture. This finding is similar to that reported for the use of the Waring Blendor to agitate a bead-organism slurry (Lamanna and Mallette, 954). It would seem that the maximal rate would be attained only if enough suspension were used to fill the spaces between the beads. As common sense suggests, it was found that the higher the speed at which the centrifuge device is operated the more rapid is the rate at which yeasts are ruptured. Some results obtained are recorded in table 3. The data also illustrate another finding, namely, that with a given slurry and the same length of tubing, the efficiency of rupture is not influenced by the internal diameter (volume) of the tubing. We interpret this observation to mean that it is only the surface area of the tubing moving across the face of the rotors which affects the rate of rupture of organisms. SUMMARY Pumping can be employed to cause glass beads to rupture microorganisms. Since to date no commercially available pump tested has proved satisfactory in operation, two devices have been designed at the Naval Biological Laboratory which successfully utilize the principle of pumping for achieving rupture of microorganisms in a closed system. A slurry of microorganisms and glass beads is placed in a transparent polyvinyl tube (Tygon-B) and the

6 959] PUMPING DEVICE FOR RUPTURE OF MICROORGANISMS 09 tube moved rapidly over rotating pulleys. The machines devised rotate the tube; one is a centrifuge accessory and the other a compression roller device. These devices transmit kinetic energy to the slurry within the tube by pressure exerted on the flexible walls of the tube as the tube moves along the faces of rotors. Data are presented which show these devices to be practical instruments for laboratory rupture of microorganisms. Variables affecting the rates of rupture have been investigated. REFERENCES CURRAN, H. R. AND EVANS, F. R. 942 The killing of bacterial spores in fluids by agitation with small inert particles. J. Bacteriol., 43, LAMANNA, C. AND MALLETTE, M. F. 954 Use of glass beads for the mechanical rupture of microorganisms in concentrated suspensions. J. Bacteriol., 67, MICKLE, H. 948 A tissue disintegrator. J. Roy. Microscop. Soc., 68, 0-2.