bacterial body. supported a flat-topped mushroom-shaped electrode upon which 361 Crystalline quartz has a certain characteristic which is fundamental

Transcription

1 SONIC ENERGY AS A LETHAL AGENT FOR YEAST AND BACTERIA T. D. BECKWITH Department of Bacteriology, University of California, Los Angeles AND C. E. WEAVER Department of Water and Power, Los Angeles Received for publication July 8, 1936 Crystalline quartz has a certain characteristic which is fundamental to the experiments which we have performed. It is piezo active. Wood and Loomis have described an apparatus which we have utilized with certain modifications and which gives rise to the piezo effect. With this apparatus we have conducted certain experiments dealing with the killing effect of this source of energy upon bacteria and yeasts.' Wood and Loomis (1927) have demonstrated that ultrasonic radiation can disrupt certain higher forms of life. Williams and Gaines (1930) have shown germicidal effect of this wave motion but they used much lower frequency than that which we have applied. Harvey and Loomis (1929) demonstrated the death of luminous bacteria within 1 and 11 hours when treated by this energy. Chambers and Flosdorf (1936) have utilized a low frequency for extraction of certain fractions of the bacterial body. The reader is referred to the description of Wood and Loomis for an understanding of the principles involved and of the circuit utilized. We departed from their type in certain respects, as follows. The generator consisted of a circular cast-bronze housing of approximately 20 cm. diameter at the bottom and curved inward to 10 cm. in diameter at the top. One side was grounded. The bottom of this container was of plate glass and upon it was supported a flat-topped mushroom-shaped electrode upon which 361 JOU=AL 01 RAC Y, vo 32, x0. 4

2 362 T. D. BECKWITH AND C. E. WEAVER rested two quartz crystals, each 3 mm. thick by 5.3 cm. diameter. The dielectric was circulated by means of a pump and was cooled in order to obviate temperature effects upon the microorganisms under test. The power applied to the quartz crystals was high but corona effects were reduced to a minimum by careful selection of a dielectric and by use of the cooling system. Material to be treated may be placed in a container either entirely of glass, or better still, in a glass tube of 1.25 to 5 cm. in diameter and covered over one end with cellophane. By use of the thin tissue carefully cemented over the glass tube, less loss of energy occurs and the effect of the vibration upon exposed materials is greater in intensity. EXPERIMENTS WITH YEAST The yeast which has been used in most instances in this portion of our work has been a strain of Saccharomyces ellipsoideu. It has been propagated as needed in grape juice, previously sterilized by the intermittent method. In this medium, luxuriant growth takes place within a few hours and the organism is found to be somewhat larger than the cells constituting the more usual strains utilized by bakers. Our initial experiments were concerned with the effects of ultrasonic vibration upon cell morphology. It was determined that the cells of rapidly -growing yeast were ruptured, following exposure to this form of energy. If a suspension of this yeast in grape juice be treated thus, subsequent microscopic examination shows that most of the field shows merely fragments of cells. Occasionally there remains an intact unit, but if this treated culture now be put back into the incubator, growth is not likely to continue. Evidently, therefore, such cells, although unruptured, must be dead. Further experiments dealing with this yeast were now carried out with the idea of determining the rate of death when treated with ultrasonic vibration. To make dilution preparations to determine by plating the exact numbers of organisms per unit volume wad impracticable; therefore the following technique was adopted. By means of a 6 mm. loop, transfers of somewhat

3 SONIC ENERGY AS LETHAL AGENT FOR YEAST constant volumes of inoculum may be made repeatedly and a count of resulting colonies following plating upon Sabouraud -agar will yield valid indication of a death curve should there be reduction of the numbers of viable organisms. It is realized, however, that this method is not sufficiently refined to yield the most exact data. By use of this technique, however, the following experiment has been performed repeatedly. The proliferating culture of yeast was placed to a depth of approximately 2.5 cm. in a heavy glass tube with cellophane bottom and this in turn was lowered into the dielectric bath containing the quartz crystal. When in proper position, the cellophane membrane was directly over the disc of the crystal so that the maximum intensity of vibration might be obtained. TABLE 1 Effectiveness of ultrasonic vibration in killing actively-growing yeast (Numbers in the volume of a 6 mm. loop) 363 Before treatment ,000 28,000 54,000 50,000 36,500 I minute... 17,000 39,000 2,100 1,800 4,200 3 minutes , S minutes minutes minutes Treatment of the culture was interrupted at definite time intervals in order to remove a loopful of the yeast suspension for plating, and, following these momentary stops, treatment was resumed until the experiment was terminated. The results obtained in this portion of the work proved to be variable. Frequently, treatment for a short interval is followed by an increase of the apparent numbers of viable cells and one is led to conclude therefore that the violent agitation first breaks up clumps of cells. In turn, this seeming increase is succeeded by definite lowering of counts as the experiment progresses. In some instances counts dropped precipitously while in other tests the curve of death was more gradual. At random, however, there have been selected five sets of figures which together give a just idea of the effectiveness of this mode of treatment when

4 364 T. D. BECKWITH AND C. E. WEAVER applied to yeasts under the conditions outlined. They are presented in table 1. From these results, it appears that sterilization was complete in 15 minutes in some instances while in others it was partial. All treated materials following the close of the experiments were incubated. In some instances there was no indication that life persisted following irradiation, while in others delayed growth appeared after 4 or 5 days showing that a small number of viable organisms must have persisted, although weakened considerably. In all of these experiments, however, a microscopic examination revealed many disrupted organisms. The conclusion which may be drawn here is that ultrasonic irradiation of this yeast results in death of the cells. The rate of death, however, was variable under the conditions of our work and the causes of such variation were not revealed. It is probable that certain forms may have been sheltered in minute crevices. It is equally possible that spattering on the walls of the culture container deposited some cells temporarily out of the suspending fluid. Either or both probabilities would account for the discrepancies. Resultant heat as determined by thermometer was of no importance in this outcome since in all instances the final temperature was never higher than 39C.. Wave length may be changed by substituting another crystal of different thickness. Because our power was reduced when thinner crystals were used, it is difficult to evaluate the relationship between lethal effects and wave length. If one assumes a power to be a constant, then this should be inversely proportional to the wave length because of the increased pressure gradient in the standing wave system. EXPERIMENTS WITH BACTERIA Certain tests have been made upon bacterial suspensions in tap water to determine whether there is a similar lethal effect when orgasms smaller than yeast are exposed to ultrasonic treatment. These have been pursued under three separate headings. Escherichia coli. For the first group of experiments, a strain of the colon bacillus originally isolated from water was grown for 24 hours in beef-extract broth at 37. This was then diluted

5 SONIC ENERGY AS LETHAL AGENT FOR YEAST from 125,000 to 175,000 times with tap water and treated upon five separate occasions and on different days. The treatment period was of five minutes duration with thickness of the quartz disc varying between 1.5 and 4 mm. Counts were made by dilution and plating of each specimen before and after treatment. The results are shown in table 2. Evidently the vibration rate utilized above is decidedly lethal to the colon bacillus in watery suspension. A further experiment was now conducted to obtain somewhat more exact information regarding the rate of death of these organisms under conditions closely similar. We prepared a suspension of greater concentration of the colon bacillus. The suspension was treated and after 1.5 minutes, the treatment was interrupted for sampling. Treatment was then resumed and continued until an aggregate interval of 5 minutes had elapsed when a final sampling was made. The TABLE 2 Ultrasonic irradiation of water suspensions of B. coli Control counts... 1,325 3,250 3,700 1,550 1,100 5 minutes treatment counts obtained were: Control untreated, 2,000,000 per cubic centimeter; 1.5 minutes, 65,000; 5, minutes, 300. This yields a bactericidal efficiency of slightly less than 100 per cent. Eberthella typhosa. The second series of experiments dealt with ultrasonic treatment of E. typhosa (Hopkins strain), but conditions were not exactly the same as those outlined above. In these experiments, beef-extract broth was placed to a depth of approximately 1.6 cm. in a small flask blown from a Pyrex tube. The diameter was 5 cm. and the bottom was rounded. Thus, a greater portion of the energy was reflected by the contour of the container, and also more was absorbed by the glass than by the cellophane used in the previous experiments. In addition, the suspending medium was now beef-extract broth rather than tap water and as we shall see later, the structure of the suspending medium is of fundamental importance. The preparatory period of incubation at 370 was six hours.

6 366 T. D. BECKWITH AND C. E. WEAVER Two experiments were performed under this heading. In each the period of treatment was 10 minutes, and the temperature was 370 at termination of the treatment. In the first, the initial count before irradiation was 20,500,000 per cubic centimeter; and this had diminished to 1,950,000 per cubic centimeter after 10 minutes' exposure. The efficiency therefore was 90.5 per cent. In the second test, the control count was 5,500,000 per cubic centimeter but after 10 miutes' treatment, this had lessened to 12,500 per cubic centimeter-efficiency 99.8 per cent. From the foregoing, it appears that this type of energy is actively bactericidal both to E. coli and to E. typhosa in suspensions in water or in beef-extract broth. The particle size which may be disrupted may thus be much smaller than a yeast cell. Bacillus mubtili. The third division of this portion of the work deals with the effect of ultrasonic radiation upon the spores of B. subtilis. The organism originally isolated from soil, was grown upon beef-extract agar for 4 days at 37'. Microscopically, many spores were evident. The culture was washed off with tap water and with constant agitation, the resulting suspension was heated to 800C. for 5 minutes to kill vegetative forms. With this material, the three following experiments were performed. It should be stated, however, that conditions obtaining throughout this series of experiments were not strictly comparable. Power input was a variable because of differences in crystal thickness. The suspension to be treated was placed in a tube with cellophane diaphragm to a depth of 1.9 cm. in each instance. The period of exposure was 15 minutes. Counts were made by dilution and duplicate plates of the control suspension before treatment and of each 2f the three aliquot portions following its particular mode of irradiation. These results were obtained: Experiment 1. The thickness of the crystal was 4 mm. and the final thermometer reading was 15. Control count, 3,600,000 per cubic centimeter. Following treatment, 1,100,000 per cubic centimeter. Efficiency, 69.4 per cent. Experiment 2. The thickness of the crystal was 3 mm.; final tempera-

7 SONIC ENERGY AS LETHAL AGENT FOR YEAST 367 ture 15. Control count, 3,600,000. Final count following treatment, 1,650,000. Efficiency, 54.2 per cent. Experiment 3. Same as above except that the thickness of the quartz disc was 1.6 mm. Control count, 3,600,000 and final count 2,050,000. Efficiency 45.8 per cent. Thermophils. The fourth portion of this work upon bacteria deals with certain experiments in connection with thermophils. It had been demonstrated that vegetative forms and spores within mesophilic range are susceptible to supersonic irradiation. It was now desired to extend these observations to other forms thriving at higher temperature. For this purpose, there was obtained from the laboratories of the California and Hawaiian Sugar Company at Crockett, California, a mixed culture of the organisms causing certain difficulties in refining of cane sugar. The predominant form is a Gram-positive aerobe which grows in chains and which is a luxuriant spore former. Multiplication takes place readily within 48 hours at 50TC. in a solution of brown sugar with concentrations varying between 3 and 20 per cent. These forms, however, are not obligate thermophils inasmuch as multiplication at much slower rate will occur at 37. With these a variety of tests were made but the following three were most significant. (1) Five minutes' treatment of a suspension in 5 per cent brown sugar produced a reduction from 840 to 15 per cubic centimeter. (2) Upon another occasion, a 5-minute exposure of a culture in 5 per cent brown sugar solution induced a lessening from 16,100 to 2,050 per cubic centimeter. (3) Another culture grown in a 20 per cent brown sugar solution exposed for 5 minutes decreased the count from 3,000,000 to 125,000 per cubic centimeter. All counts were made upon glucose beef-extract agar after 48 hours incubation at 500. It appears probable then that ultrasonic radiation is an effective germicide for bacteria when suspended in water, broth, or sucrose solution. Usually Gram-positive organisms are more susceptible to deleterious influences such as germicides than are Gram-negative forms. Each class is killed by this type of radiation. Ultrasonic energy is fatal to bacterial spores likewise.

8 368 T. D. BECEWITH AND C. 3E. WEAVER ANTIBODY AND BACTERIOPHAGE Ultrasonic energy must kill because of absorption of it by the cell. If the particle be sufficiently small, absorption of energy may not follow. It was desired then to utilize elements of still smaller dimension for exposure. Certain antibodies and also bacteriophage fulfill this requirement. We have found that rabbit antidysentery (Flexner) agglutinating serum following 15 minutes' exposure reveals no difference in titer from the portion which has been untreated. Also, a strain of bacteriophage active against Salmonella paratyphosum B. was selected for treatment. This had been "stepped up" after isolation until it had become very active. When this strain of bacteriophage was treated for 10 minutes, we were unable to demonstrate any change in its lytic activity. It seems to be proven, therefore, that these agglutinins and this strain of bacteriophage cannot absorb this type of energy. In spite of this assumption, it should be recognized that certain effects which are chemical in character may be exerted by ultrasonic radiation. The work of Olson and Garden (1932) with bromthymol blue indicates this. MILK AND ITS BACTERIAL FLORA The preceding experiments have dealt mainly with particular organisms in a particular environment, and at this point it was determined to widen the field of exploration somewhat so as to include a variety of forms. Cow's milk was selected for this purpose. It contains a flora consisting of a large variety of organisms. Moreover, as a suspending medium it is an emulsion containing lipoid, protein, carbohydrate, together with certain inorganic constituents. The material utilized for this series of over 40 tests was procured from various commercial sources and nearly all of it was certified raw milk. This grade was desired whenever readily available in order to obviate the objectionable feature of the heat treatment of pasteurization, which alters fundamentally the characteristic flora. Following purchase, the milk was allowed to stand at room temperature for lengths of time varying between 2 and 24 hours to permit bacterial multiplication.

9 SONIC ENERGY AS LETHAL AGENT FOR YEAST Table 3 includes the results of six treatments. These are representative of the large group, but the entire series has not been listed in order to save space. All computations were made from dilutions and poured plates according to the Standard Methods of the American Public Health Association. It will be noted that table 3 presents results most uneven in character. The point demonstrated, however, is that after exposure of these bacterial forms to such radiation, there is some degree of lessening of bacterial count. Examination of colonies from treated material in comparison with those from control plates did not reveal macroscopical nor microscopical differences. In other words, no selective killing effect toward any particular group was demonstrated. TABLE 3 Effects of ultrasonic radiation upon bacterial counts in milk 369 NUMBER BEFORE TIME OF AFTER EFCEC TREATMENT TREATMENT TRATMNTICINC minutes 1 1,390, , ,200, ,000, , , , ,500, ,040, , , The effectiveness of ultrasonic treatment is shown by these last experiments to be less than when organisms are suspended in water or in physiological saline. Compare tables 2 and 3. The "dampening" effects of certain components of milk. Bacteria suspended in water are killed by the effects of supersonic energy when this is properly directed and applied. In milk, however, this reduction of numbers of viable bacteria is much less marked. Our next endeavor was to explain this difference. Cow's milk differs from water partly in that it contains lipoid, carbohydrate and protein. We determined to examine the influence of these materials separately. Attention in this aspect of our experiments was devoted first to possible interference offered by lipoid to the effects of supersonic

10 370 T. D. BECKWITH AND C. E. 'WEAVER vibration. The following were prepared. (a) A suspension of olive oil in tap water. This had been emulsified the day previously by ultrasonic treatment and was steamed for 20 minutes. (b) An emulsion of butter fat in tap water, steamed for 20 minutes. (c) Whole milk steamed for 20 minutes. (d) Sterilized tap water. One hundred twenty-five-cubic-centimeter-portions of each were inoculated with 1 cc. of a 1:2000 water dilution of a 24-hour broth culture of the colon bacillus. Enough of each TABLE 4 Effect of supersonic radiation upon B. coli in lipoid suspensions in water Water alone and milk served as controls COUNT PER COUNT PER CUBIC CENTI- CUBIC CENT!- PERCENTAGE DES8CRIPTION OF SUSPENSION METER BEFORE METER AFTER EFFICIENCY TREATMENT TREATMENT Tap water... 1, Olive oil in water Butter fat in water Whole milk... 4,000 1, TABLE 5 Effect of supersonic radiation upon the colon bacillus suspended in S per cent solutions of glucose, lactose and sucrose in tap water COUNT PER CUBIC COUNT PER CUBIC MEDIUM FOR SUSPENSION CENTIMETER CENTIMETER BEFORE TREATMENT AFTER TREATMENT Glucose Lactose Sucrose... 1,350 0 Tap water... 1,300 0 preparation to make a layer 2.5 cm. thick was then irradiated for five minutes; after which agar plate counts were made. The results are listed in table 4. Inspection of table 4 demonstrates that the presence of oil in watery suspension offers no marked degree of resistance to the germicidal effect of supersonic energy. The possible effect of carbohydrate was next considered. For this purpose there were utilized glucose, lactose and sucrose. Each of these was made up as a 3 per cent solution in tap water

11 SONIC ENERGY AS LETHAL AGENT FOR YEAST and steamed before use. Tap water alone was included again for control purposes. The method of inoculation and of treatment was precisely the same as that used in the preceding experiment. The results are presented in table 5, from which it is apparent that watery solutions of carbohydrate do not interfere with supersonic germicidal activity. Attention was next turned to protein as a material which in solution may diminish the germicidal efficiency of supersonic energy. For these experiments, which were two in number, we proceeded in the following manner. There were prepared the following: (a) A 0.75 per cent solution of casein in distilled water. The ph of this solution was adjusted to 7.4 and then it was steamed for 20 minutes. (b) A 3 per cent solution of Difco TABLE 6 Effects of supersonic radiation upon the colon bacillus suspended in solutions of casein and of gelatin Tap water served as a control SUSPENDING SUSPENDING MI{EDIUM COUNT PER COUNT PER PRETG CUmIC CuNT- MEDIUMMETER CBu *rnt1 C or ZEFORE METER AFTER EFFICIENCY TREATMENT TREATMENT 0.75 per cent casein...2, per cent gelatin... 2, Tap water... 1, gelatin in distilled water, steamed for 20 minutes. (c) Tap water, steamed for 20 minutes. These were then inoculated with E. coli as in the experiments previously described and irradiated. Plating afterwards gave the results which are shown in table 6. Results of one experiment only are indicated, but the second was identical in outcome within the expected limits of experimental error. Evidently these protein solutions are much more difficult to sterilize by supersonic treatment than are solutions of carbohydrate or suspensions of lipoid. No attempt to date has been made by us to explain the apparent refractoriness of a protein solution to this treatment. Our supposition, however, is that the physical structure of the fluid constitutes the underlying factor.

12 372 T. D. BECCWITH AND C. E. WEAVER We have shown here that sonic energy is lethal to yeasts and bacteria under certain conditions. The reasons for this destructive activity are not understood definitely, but we advance the theory that physical factors may. underlie this effect. If one assumes a given excitation frequency, f, which is likewise the fundamental frequency of the piezo-crystal; a given velocity of propagation through the material in question, v; then the wave length, a, is given by the equation: f With increase in frequency X becomes smaller, that is, rarefaction and compression approach each other more closely. Therefore the pressure gradient between rarefaction and compression becomes increasingly steeper. We know already that large organisms yield readily to frequencies which are relatively low. To kill bacteria, it has been necessary to increase the frequency for the same power input to the crystal. These facts indicate that compression and its release alternating many times per second produces the killing effect. The problem then becomes one of differential pressure. We desire to express our thanks to Professor A. R. Olson of the Department of Chemistry at the University of California at Berkeley. It was he who made suggestions which led to these experiments. SUMMARY 1. A wine yeast, Saccharomyces ellipsoideus, suspended in grape juice is usually killed by supersonic treatment. 2. Vegetative organisms of still smaller dimension such as the colon bacillus in tap water, Eberthella typhosa in beef broth, or a thermophil in solution of sucrose are killed by this form of energy. 3. Certain particles of still less size such as the agglutinin of Salmonella paratyphosum B. or a strain of bacteriophage are not inactivated by supersonic effect.

13 SONIC ENERGY AS LETHAL AGENT FOR YEAST Suspensions of Bacillus subtilis spores are diminished greatly in viable numbers by supersonic vibration. 5. The Gram reaction of the organism treated has no influence upon the outcome. 6. The presence of protein in solution interferes with the germicidal activity of supersonic treatment. Lipoid in suspension or carbohydrate in solution does not exert similar depressing effects. 7. Supersonic irradiation of milk is less efficient bactericidally than when applied to fluids containing smaller amounts of protein. 8. It is probable that the pressure gradient constitutes the lethal factor of sonic energy if one assumes viscosity and like factors herein mentioned to be a constant. REFERENCES American Public Health Association, Standard Methods of Milk Analysis, CHAMBERS, L. A., AND FLOSDORF, E. W Proc. Soc. Exper. Biol. and Med., 34, HARVEY, E. N., AND LOOMIS, A. L Jour. Bact., 17, OLSON, A. R., AND GARDEN, N. B Jour. Amer. Chem. Soc., 54,3617. WILLIAMS, O. B., AND GAINES, N Jour. Inf. Dis., 47, 485. WOOD, R. W Sci. American, 138, 201. WOOD, R. W., AND LOOMIS, A. L London, Edinburgh and Dublin, Phil. Mag. and Jour. Sci., 7th Ser., 4, No. 22,