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Sterilizing effect of high intensity airborne sound and ultrasound
STERILIZING EFFECT OF HIGH INTENSITY AIRBORNE SOUND AND ULTRASOUND by R. M. Boucher* and A. Pisano* A study of the lethal effects of high intensity airborne sound (9. 9kc/s) and ultrasound (30. 4kc/s) on spores of B.Subtilis var niger ATCC 9372 deposited on paper strips was conducted in an experimental chamber. From the first series of collected data it appears that the acoustic intensity level and the irradiation time are the main governing factors in airborne acoustic sterilization. Preliminary observations in the 155-156 dB level showed that large amplitude sonic waves were more lethal than ultrasonic waves. Thermal. effects and acoustic turbulence at the micro-organism interface appear to be the main physical mechanisms responsible for spore destruction. As expected theorectically, the death rate is greater at pressure antinodes under standing wave conditions.
Among the various problems which are at present faced in the N.A.S.A. Ranger programme, the reduction of time and temperature during sterilization procedures is one of the most important. 1 At the moment it is the intention to combine heat treatment at 135°C with ethylene oxide decontamination within the 4050°C range. Both treatments require long exposure times and any reduction of the heat or ethylene oxide cycle would greatly increase the reliability of the delicate electronic components of the space probe.
MATERIALS AND METHODS Before starting our investigation a very thoroug!J. survey had been made of all types of high intensity airborne sound source which would deliver a high acoustic energy density beam (at least 150 dB at 12 in) at various frequencies without introducing any gas in the irradiating chamber. It was soon discovered that no existing sound source
would fulfil our requirements, specially in the ultrasonic region of the frequency spectrum. At very low With a view to decreasing the contact time during frequencies (Le. up to 1100 cis) foghorns of the vibrachemical gas sterilization we suggested in 1962 the ting membrane type would give a partial answer but combination of the sterilizing effects of large amplieach irradiating frequency would require a different tude sonic and ultrasonic waves with the bacteriaexponential horn. At high frequency the excited quartz killing power of ethylene oxide. Previous experiments described by Kritz2 would only give a partial answer conducted by one of us in France in 1953 with the col- since this sould source can only be driven at specific laboration of the Institute Pasteur had shown that the quartz resonant frequencies. The only promising decombined action of an hexyl-resorcinol (1-n hexylvice was the Friedman horn described in August 1962 2:4-resorcinol) submicronic aerosol with high intenat the Fourth International Congress on Acoustics in sity airborne sound ·waves in the 11 kc/s range greatly Copenhagen. 3 This author described a hollow exponenboosted the killing rate of several types of bacteria. tially sliaped velocity transformer which was driven In other words, the goal of the present work is the by a powerful magneto-strictive transducer. development of a sonochemical technique (low temperature sterilization) which will take advantage of It was said to produce a strong emission at 3.4 kC/s (158 dB at 14 in) and 23.8 kc/s (152 dB at 14 in). the synergetic effect observed when combining large Through design modification of the velocity transamplitude acoustic waves in a gas phase with chemiformer shape and replacing the magneto-strictive cal sterilization mechanisms. transducer by two lead zirconate titanate crystals Owing to the lack of literature data on the effect of mounted as in a Langevin sandwich we developed a large amplitude airborne sound waves on micronew powerful airborne sound source which can be organisms our first report will deal exclusively with activated from a few kilocycles up to 150 kC/S. With the problem of high intensity airborne irradiation of a view to making possible irradiation at high intensity spores. It will be followed by a second report on the over extended periods of time, the crystals were coolcombined action of airborne sound and ultrasound ed at 20°C through continuous recycling of a constantwith ethylene oxide and other chemical sterilizing ly refrigerated dielectric oil (5 gal/min). gases. During the July 1962 conference on spacecraft The powerful sonic and ultrasonic airborne waves sterilization Bruch rightly stressed how the microproduced by this device are mainly due to the large organisms' carriers could influence the results in amplitude excursions (20-200 /lm) of the walls of the sterilization procedures. For instance, he pOinted out velocity transformer (specially at the level of the the fact that the resistance of the same species of lips) which alternately compress and dilate the spore deposited on a piece of paper could be greater pocket of gas inside the hollow resonant cavity. than on a sand sample. For this reason our irradiation work was conducted with spores of Bacillus SubA second type of horn of a different design (see Fig. 1) tHis var. niger ATCC 9372 deposited on Whatman was also used during our low frequency (9.9 kc/s (No.1) paper strips. study. This horn was made of a 9 1/2 inch long aluminium conical velocity transformer which ended into a cup (radius of curvature 11-2 inch) with a 21/4 in ex* Macrosonics Corporation, 1001 Roosevelt Avenue, ternal diameter. Through the thinning of the cup walls Cartaret, N. J., U.S.A. t St. John's University, Brooklyn, New York, U.S.A. and the increase in radiating area we were able to ULTRASONICS October 1966
199
better the poor impedance mismatch from sond to gas, thus achieving high airborne intensities. Below are given the characteristics and power outputs of the two horns when driven by a broadband 500 W generator under conditions indentical to those used during our irradiation tests. Hollow horn
Fig.1. Low frequency (9.9 kc/s) horn with its transducer and Plexiglass cooling chamber. On the extreme right are seen the two electrical leads which are connected to the r.f. broadband generator
When using a 100 n impedance the total power output computed from series of ten point measurements within the 4 in diameter cross-section of the irradiating chamber was 1. 25 W at 12 in (no resonant conditions). The frequency of emission, controlled with the probescope spectrum analyzer (type SS 300) was 30.4 kc/s (±0.1 kC/S). The average intensity at 12 in on the axis within a 1/2 in radius circle was 155 dB (±1/2 dB). Focusing horn
Fig. 2. Experimental set-up for airborne irradiation with a hollow horn Cooling chamber
Oil out
-~Eii~w===e @
Oil in
.~~
When using a 400 n impedance the total power output computed from series of ten point measurements within the 4 in diameter cross-section of the irradiating chamber was 2.37 W at 12 in (no resonant conditions). The frequency of emission, controlled with the probescope spectrum analyzer was 9.9 kc/s (±0.1 kC/S). The average intensity at 12 in on the axis within a 102 in radius circle was 151 dB (±102 dB). The horns were fastened at the top of the cylindrical glass irradiation chamber (18 in long x 4 in diameter). The insonated sample was inside a petri dish supported by a metal plate (see Fig.2) which was mechanicalally adjusted at the desired level in the irradiating chamber. The entire test set-up was located within a polyethylene enclosure measuring 41 x 18 x 18 in. Intensity measurements were made with the Massa M-213 (adp crystal) high intensity microphone which fed its signal, after amplification, to a Ballantine voltmeter (type 310-A).
To 500w r.f.Generator Gas out
Hollow horn
Preparation of spore suspension Glass - - _.....01•.1 irradiating chamber
The organism, Bacillus Subtilis var. niger ATCC 9372, was grown on slants of nutrient agar which was supplemented with MnS0 4 . H2 0 in a concentration of 10 parts per million. Maximal sporulation occurred after 5 days' incubation at 37°C at which time the spores from each slant were suspended in 5 ml of distilled water. The spore suspensions were then combined, washed three times in distilled water, and reconstituted to the initial volume. The spores were heat-shocked at 80°C for 20 min. Viable counts were made by appropriate plating techniques using trypticase soy agar.
igh intensity sonic field tri dish
Sample
rfff9~~F9~ij;~~~~:"Gas
Preparation of spore-treated paper strips in
Strips of filter paper (Whatman No. 1) measuring 1 x 1/4 in were sterilized at 121°C fOF 15 min and then inoculated with 0.01 ml of a spore suspension containing 1. 6 x 10 8 spores/ml. The strips were dried in sterile glass petri dishes for 24 h at 37°C and then stored in sterile glass jars at 25°C. Insonation of paper strips Three spore-treated paper strips were secured, with sterile stainless steel pins, to the centre of the bottom half of a sterile, plastic petri dish. The dish was placed on the metaY plate, adjusted to the height desired and located in the test chamber. The latter was then sealed tight by means of bolts attached to its lower extremity. Air, dried by passage through a sili-
200
ULTRASONICS October 1966
ca gel column (28-200 mesh) was recirculated through the chamber by means of a small pump while the temperature was simultaneously raised to 40°C. Mter stabilizing for 15 min, the chamber was evacuated to approximately 3 in of mercury, and an appropriate volume of sterile, distilled water, designed to achieve a relative humidity of 40% was introduced. The paper strips were exposed to sound waves for periods of 1, 2,4, or 8 h. Following irradiation, the three paper strips were removed from the chamber, extracted with 30 ml of sterile and distilled water for one minute at 8000 rev/min in a sterile blending apparatus. The number of viable cells present in the extract was determined by plating on trypticase soy agar.
Controls The number of spores present on paper strips not subjected to sound waves was determined by plate counts at the initiation, mid -pOint and termination of the experiments. Ten plate counts, using three strips per count, were performed at each time period.
Statistical methods The means of the plate counts of irradiated paper strips were compared to the means of the controls to determine if significant population differences, attributable to sound, were discernible. The student's t-test, as applied to non-paired experiments was employed as an index of significance between soundirradiated populations and non-irradiated controls. A level of significance of 0.11'0 was selected. RESULTS As already indicated, control counts were made throughout the -period of experimentation, approximately six months. The paper strips gave no evidence of detrimental effects, attributable to storage, with regard to cell counts. Controls plated at the initiation of the experiments and those plated midway and at the end of the testing period showed similar spreads between individual counts. The mean value of all control paper strips was 1. 73 x 10 5 /ml. This figure served
as a basis of comparison with counts obtained from paper strips exposed to sound. Irradiation of spores of B. Subtilis var. niger with sonic waves (9.9 kC/S) resulted in Significant decreases in viable counts under certain conditions (Table 1). Meaningful reductions in cell population, at the 0.11'0 level of significance, were always obtained when spore-treated paper strips were irradiated for 8 h regardless of the distance of the specimen from the sound source. Reducing the period of irradiation to less than 8 h, however, yielded values which were not significant except when the strips were irradiated at a distance of 1 in from the transducer during four hours. Irradiation periods of 2 h or less did not result in significant decreases in the viable count no matter what the intensity level. It is interesting to mention that the sharpest decrease in viable spores count was observed after 8 h at the 91-2 inch distance which corresponded to resonant conditions (a whole number of half wavelengths). The simultaneous decrease in viable organisms and increase in the acoustic energy level near respnant conditions clearly demonstrates that the b0und wave intensity is a key factor in sonic sterilization. The fact that a corresponding decrease was not always observed at 911.! in for shorter irradiation time could be explained by the fact that it is extremely delicate to set up perfect standing waves through accurate sample support pOSitioning. Treatment of the test organisms with ultrasonic waves (30.4 kC/S) also resulted in a significant reduction in viable counts (Table 2). As was the case with sonic waves, meaningful reductions in plate counts occurred after 8 h irradiation with test strips placed at each of the four distances employed. In addition, Significant results were also obtained after 4 h irradiation when the specimens were placed at distances of 5 in, 3. 5 in, and 1 in from the transducer. Beyond this the most striking reduction in the viable count was recorded when the test strips were located 1 in from the sound source and exposed to ultrasonic waves for only 2 h. Here again we found a sharper decrease in viable spores count at the three positions (1 in, 3. 5 in and 5
Table 1. Means of plate counts (X10 5 ) of spores of bacillus subtilis var. niger irradiated with airborne sound (9.9 kc/s) and corresponding t values (in parentheses) Irradiation time [h]
Sample position [inches and number of wavelengths] 9.5 7"- 1
5.0 3.6"-
3.5 2.54"-
1.0 0.76"-
spores/ml
spores/ml
spores/ml
spores/ml
1
1.77(0.12)
1. 56(1.17)
1. 69(0. 50)
1. 45(2.37)
2
1. 77(0. 44)
1.52(0.81)
1. 51(0. 83)
1. 41(2.66)
4
1. 46(1. 83)
1. 56(1. 00)
1.37(2.48)
1. 22(3.48*)
8
0.89(3.59*)
1. 09( 6.40*)
1.18(4.90*)
1. 14( 6 . 21 * )
Intensity level at sample [db]
155
153
153 1i2
154
* Indicates level of significance of O. 1'70 t Pressure antinodal position ULTRASONICS October 1966
201
Table 2. Means of plate counts (X 10 5 ) of spores of bacillus subtilis var. niger irradiated with airborne ultrasounds (30.4 kC/s) and corresponding t values (in parentheses) . Irradiation time [h]
Sample position [inches and number of wavelengths] 9.5 13.2'\
5.0 7,\ t
3.5 5,\ t
1.0
spores/ml
spores/ml
spores/ml
spores/ml
1
1. 85(1. 38)
1. 66(0. 70)
1. 83(0.43)
1. 82(0. 76)
2
1.53(2.38)
1.56(2.21)
1. 53(2.06)
1. 05(3.54*)
4
1. 27(1. 31)
1. 36(3.08*)
1. 29(3.70*)
0.76(4.85*)
8
1. 11(8.88*)
1. 06(3.86*)
0.91(5.23*)
0.95(6.39*)
Intensity level at sample [dB]
156
159
161
161
* Indicates level of significance of O. 1 t Pressure antinodal position
0 /
0
in) which were the closest to standing wave conditions. As was theoretically expected, comparison of the data in both tables reveals that almost twice as many significant values were obtained in the 156-161 dB level than in the 153-155 dB level. A comparison at nearly equal intensity (155 and 156 dB) after identical irradiation time (8 h) seems to indicate that sonic waves (9.9 kC/S) are more efficient for spore destruction than ultrasonic waves (30.4 kC/S). However,more tests have to be conducted at the same energy level before drawing any definite conclusions regarding the specifiC influence of frequency. Consideration was also given to the possibility that the insonation chamber itself might adversely affect the viability of spores of B. Subtilis var. niger. Accordingly, spore-impregnated paper strips were allowed to remain in the chamber for periods up to 8 h under conditions identical with those employed for the experimental paper strips, except that sound energy was not applied. Plate counts of these strips revealed no significant reductions attributable to the test chamber alon"e. DISCUSSION Exposure of spores of B. Subtilis var. niger to high intensity airborne acoustic waves resulted in statistically significant reductions in viable counts compared to spores not exposed to sound. From the data collected it appears that the killing power of airborne acoustic waves is a function of both the acoustic energy density and the irradiation time. More tests are needed to clarify the respective contribution of these two factors. Preliminary observations in the 155-156 dB level showed that airborne sonic waves (9.9 kC/S) have more destructive power than airborne ultrasonic waves (30.4 kC/S). It is interesting to recall that the same trend has recently been observed in acoustic drying expe riments4, 5 with airborne acoustic waves. If confirmed, this would mean that the amplitude of motion of the gas molecules which impinge against the microorganisms' interface is one of the key factors in acoustic sterilization. Also of para202
ULTRASONICS October 1966
11/2 t
mount importance is the fact that maximum kill was observed at a sample position which corresponded to the standing waves condition, i.e. when the spores are located at pressure antinodes. Let us now review the physical mechanisms which could provide an explanation to the lethal properties of high intenSity acoustic waves. As in the case of liquid irradiation, the degradation of acoustic energy into heat is certainly one of the main factors to be taken into consideration. Our results show beyond doubt that for equal irradiation time the lethal effects are more pronounced at high intensity, i.e. when more thermal energy is available through molecular friction. Let us also recall that the temperature gradient associated with the translational part of the sound energy is given by:
where a is acceleration and R is the gas constant. Greguss and Erdeleyi6 calculated, for instance, that for a sound field excited at 200 kC/s (intensity 1 W/ cm 2 ) the molecular acceleration in air is approximat ely 8.5 10 8 / cm sec-2 • This gives for the temperature gradient of the translational part of the energy a value of 19, 750°C/m. Referred to the complete heat content, this gives a value of 11. 86°C/mm and means that there will be a temperature gradient of ±10°C for each half wavelength of path. Furthermore, taking account of nonlinear distortions at high intensities, one can find even higher values of the acceleration. Another factor to be taken into consideration is the magnitude of the acoustic turbulence with its corresponding effect on diffusion and adsorption rates at the microorganisms' interface. The molecules physically adsorbed on the surface of bacteria are greatly affected during high intenSity airborne insonation. This is easily understood if one remembers that while irradiating we set up a very fast regular succession of
compt"essions and rarefactions. The effect of expansion always predominates over that of compression and as a result we break the superficial layer of molecules which coat the bacteria 7,8. In short, we can remove the superficial layers of adsorbed molecules and thus alter the physicochemical balance of the microorganisms' cortex layer. It is known that the impedance mismatch between air (pc""43) and microorganisms (pc""l. 15 x 10 5 ) is very
poor. In other words a very small amount of the radiated acoustic energy is transmitted into the spore. However, the smaller the thickness of the microorganism (compared to the acoustic wavelength) the greater will be the acoustic absorption. This could partially explain the comparatively more favourable results observed at sonic frequencies. A direct comparison of our data with other reports was not possible because of the lack of similar studies in the scientific literature. It is only recently that high power airborne sound sources with frequency flexibility have become available. Pioneering work in this area was conducted after the war by Frings, Allen and Rudnick 9,10 which described the lethal effects of airborne ultrasonic waves (19 kc/s, J = 1 W/cm 2 ) produced by a rotary siren. These authors stressed the importance of thermal phenomena and regarded them as the principal cause of the death of mice, roaches and various insects, after a few minutes of exposure to the high intensity beam. In France, L. Pimonowl l confirmed the lethal effects of a 26 kC/s (160-165 dB) siren emission on bees,flies and mice after 5 min of irradiation. The relatively long periods of irradiation required to demonstrate the antibacterial effects of airborne sound in the present report may also be a reflection of the nature of the chemical composition of spores. Aside from the possible physical barrier presented by the spores' outer layers, the lower water content of spores might tend to minimize any lethal effects of airborne sound. It is known that culture conditions may affect the lipid content of spores as well as their resistance to toxic agents. In view of the observation that lipoidal substances readily adsorb ultrasonic frequencies and behaviour of spores to airborne sound may, in large measure, be a reflection of their chemical composition at the time of irradiation. As generally observed in most applications which deal with high intensity insonation a critical energy threshold may exist above which the lethal effects will be greatly acceleTated. We have now the possibility of
irradiating spores and micro-organisms at intensity levels one order of magnitude higher than those used in the present work. This new ability in high intensity sound and ultrasound generation will make possible further work in an area where much experimental data is needed before drawing any conclusion. ACKNOWLEDGEMENT The authors wish to acknowledge the assistance of Bohdan S. Polanskyj, Project Engineer at Macrosonics, who designed the transducers and irradiating chamber used in this investigation. His advice during the course of the research were greatly appreciated. The authors also wish to thank the National Aeronautics and Space Administration which supported this investigation through a grant.
REFERENCES 1.
Barney, W., Electronics, 13 December 1965, p.134
2.
Kritz, J., Institute of Radio Engineers Transactions,8 No.1 (1961)
3.
Friedman, W. H., and Nowitzky, B. G., Fourth International Congress on Acoustics, Copenhagen (1962).
4.
Boucher, R. M. G., "Buoyant acoustic drying of yeast", Macrosonics Report M-AB-Ol (August 1964). Unpublished.
5.
Borisov, Y. Y., "Application of ultrasonics to chemical engineering processes", Ts INTI, EP and P Press, USSR (1960).
6.
Greguss, P., and Erdeleyi, I., Acustica, 2, p. 365 (1961).
7.
Boucher, R. M. G., Chemical Engineering, 21 September 1959, p. 151
8.
Boucher, R. M. G., Ultrasonic News, 3, No.2, pp. 8 and· 14 (1959).
9.
Allen, C .H., Frings, H., and Rudnick, I., Journal of the Acoustical SOCiety of America, 20,62 (1948).
10. Frings, H., Allen, C. H. and Rudnick, I., Journal of Cell and Comparative Physiology, 31, p. 339 (1948). 11. Pimonow, L., Ann Telecomm., 6, No. 11, 1 (1951)
ULTRASONICS October 1966
203
Among the various problems which are at present faced in the N.A.S.A. Ranger programme, the reduction of time and temperature during sterilization procedures is one of the most important. 1 At the moment it is the intention to combine heat treatment at 135°C with ethylene oxide decontamination within the 4050°C range. Both treatments require long exposure times and any reduction of the heat or ethylene oxide cycle would greatly increase the reliability of the delicate electronic components of the space probe.
MATERIALS AND METHODS Before starting our investigation a very thoroug!J. survey had been made of all types of high intensity airborne sound source which would deliver a high acoustic energy density beam (at least 150 dB at 12 in) at various frequencies without introducing any gas in the irradiating chamber. It was soon discovered that no existing sound source
would fulfil our requirements, specially in the ultrasonic region of the frequency spectrum. At very low With a view to decreasing the contact time during frequencies (Le. up to 1100 cis) foghorns of the vibrachemical gas sterilization we suggested in 1962 the ting membrane type would give a partial answer but combination of the sterilizing effects of large amplieach irradiating frequency would require a different tude sonic and ultrasonic waves with the bacteriaexponential horn. At high frequency the excited quartz killing power of ethylene oxide. Previous experiments described by Kritz2 would only give a partial answer conducted by one of us in France in 1953 with the col- since this sould source can only be driven at specific laboration of the Institute Pasteur had shown that the quartz resonant frequencies. The only promising decombined action of an hexyl-resorcinol (1-n hexylvice was the Friedman horn described in August 1962 2:4-resorcinol) submicronic aerosol with high intenat the Fourth International Congress on Acoustics in sity airborne sound ·waves in the 11 kc/s range greatly Copenhagen. 3 This author described a hollow exponenboosted the killing rate of several types of bacteria. tially sliaped velocity transformer which was driven In other words, the goal of the present work is the by a powerful magneto-strictive transducer. development of a sonochemical technique (low temperature sterilization) which will take advantage of It was said to produce a strong emission at 3.4 kC/s (158 dB at 14 in) and 23.8 kc/s (152 dB at 14 in). the synergetic effect observed when combining large Through design modification of the velocity transamplitude acoustic waves in a gas phase with chemiformer shape and replacing the magneto-strictive cal sterilization mechanisms. transducer by two lead zirconate titanate crystals Owing to the lack of literature data on the effect of mounted as in a Langevin sandwich we developed a large amplitude airborne sound waves on micronew powerful airborne sound source which can be organisms our first report will deal exclusively with activated from a few kilocycles up to 150 kC/S. With the problem of high intensity airborne irradiation of a view to making possible irradiation at high intensity spores. It will be followed by a second report on the over extended periods of time, the crystals were coolcombined action of airborne sound and ultrasound ed at 20°C through continuous recycling of a constantwith ethylene oxide and other chemical sterilizing ly refrigerated dielectric oil (5 gal/min). gases. During the July 1962 conference on spacecraft The powerful sonic and ultrasonic airborne waves sterilization Bruch rightly stressed how the microproduced by this device are mainly due to the large organisms' carriers could influence the results in amplitude excursions (20-200 /lm) of the walls of the sterilization procedures. For instance, he pOinted out velocity transformer (specially at the level of the the fact that the resistance of the same species of lips) which alternately compress and dilate the spore deposited on a piece of paper could be greater pocket of gas inside the hollow resonant cavity. than on a sand sample. For this reason our irradiation work was conducted with spores of Bacillus SubA second type of horn of a different design (see Fig. 1) tHis var. niger ATCC 9372 deposited on Whatman was also used during our low frequency (9.9 kc/s (No.1) paper strips. study. This horn was made of a 9 1/2 inch long aluminium conical velocity transformer which ended into a cup (radius of curvature 11-2 inch) with a 21/4 in ex* Macrosonics Corporation, 1001 Roosevelt Avenue, ternal diameter. Through the thinning of the cup walls Cartaret, N. J., U.S.A. t St. John's University, Brooklyn, New York, U.S.A. and the increase in radiating area we were able to ULTRASONICS October 1966
199
better the poor impedance mismatch from sond to gas, thus achieving high airborne intensities. Below are given the characteristics and power outputs of the two horns when driven by a broadband 500 W generator under conditions indentical to those used during our irradiation tests. Hollow horn
Fig.1. Low frequency (9.9 kc/s) horn with its transducer and Plexiglass cooling chamber. On the extreme right are seen the two electrical leads which are connected to the r.f. broadband generator
When using a 100 n impedance the total power output computed from series of ten point measurements within the 4 in diameter cross-section of the irradiating chamber was 1. 25 W at 12 in (no resonant conditions). The frequency of emission, controlled with the probescope spectrum analyzer (type SS 300) was 30.4 kc/s (±0.1 kC/S). The average intensity at 12 in on the axis within a 1/2 in radius circle was 155 dB (±1/2 dB). Focusing horn
Fig. 2. Experimental set-up for airborne irradiation with a hollow horn Cooling chamber
Oil out
-~Eii~w===e @
Oil in
.~~
When using a 400 n impedance the total power output computed from series of ten point measurements within the 4 in diameter cross-section of the irradiating chamber was 2.37 W at 12 in (no resonant conditions). The frequency of emission, controlled with the probescope spectrum analyzer was 9.9 kc/s (±0.1 kC/S). The average intensity at 12 in on the axis within a 102 in radius circle was 151 dB (±102 dB). The horns were fastened at the top of the cylindrical glass irradiation chamber (18 in long x 4 in diameter). The insonated sample was inside a petri dish supported by a metal plate (see Fig.2) which was mechanicalally adjusted at the desired level in the irradiating chamber. The entire test set-up was located within a polyethylene enclosure measuring 41 x 18 x 18 in. Intensity measurements were made with the Massa M-213 (adp crystal) high intensity microphone which fed its signal, after amplification, to a Ballantine voltmeter (type 310-A).
To 500w r.f.Generator Gas out
Hollow horn
Preparation of spore suspension Glass - - _.....01•.1 irradiating chamber
The organism, Bacillus Subtilis var. niger ATCC 9372, was grown on slants of nutrient agar which was supplemented with MnS0 4 . H2 0 in a concentration of 10 parts per million. Maximal sporulation occurred after 5 days' incubation at 37°C at which time the spores from each slant were suspended in 5 ml of distilled water. The spore suspensions were then combined, washed three times in distilled water, and reconstituted to the initial volume. The spores were heat-shocked at 80°C for 20 min. Viable counts were made by appropriate plating techniques using trypticase soy agar.
igh intensity sonic field tri dish
Sample
rfff9~~F9~ij;~~~~:"Gas
Preparation of spore-treated paper strips in
Strips of filter paper (Whatman No. 1) measuring 1 x 1/4 in were sterilized at 121°C fOF 15 min and then inoculated with 0.01 ml of a spore suspension containing 1. 6 x 10 8 spores/ml. The strips were dried in sterile glass petri dishes for 24 h at 37°C and then stored in sterile glass jars at 25°C. Insonation of paper strips Three spore-treated paper strips were secured, with sterile stainless steel pins, to the centre of the bottom half of a sterile, plastic petri dish. The dish was placed on the metaY plate, adjusted to the height desired and located in the test chamber. The latter was then sealed tight by means of bolts attached to its lower extremity. Air, dried by passage through a sili-
200
ULTRASONICS October 1966
ca gel column (28-200 mesh) was recirculated through the chamber by means of a small pump while the temperature was simultaneously raised to 40°C. Mter stabilizing for 15 min, the chamber was evacuated to approximately 3 in of mercury, and an appropriate volume of sterile, distilled water, designed to achieve a relative humidity of 40% was introduced. The paper strips were exposed to sound waves for periods of 1, 2,4, or 8 h. Following irradiation, the three paper strips were removed from the chamber, extracted with 30 ml of sterile and distilled water for one minute at 8000 rev/min in a sterile blending apparatus. The number of viable cells present in the extract was determined by plating on trypticase soy agar.
Controls The number of spores present on paper strips not subjected to sound waves was determined by plate counts at the initiation, mid -pOint and termination of the experiments. Ten plate counts, using three strips per count, were performed at each time period.
Statistical methods The means of the plate counts of irradiated paper strips were compared to the means of the controls to determine if significant population differences, attributable to sound, were discernible. The student's t-test, as applied to non-paired experiments was employed as an index of significance between soundirradiated populations and non-irradiated controls. A level of significance of 0.11'0 was selected. RESULTS As already indicated, control counts were made throughout the -period of experimentation, approximately six months. The paper strips gave no evidence of detrimental effects, attributable to storage, with regard to cell counts. Controls plated at the initiation of the experiments and those plated midway and at the end of the testing period showed similar spreads between individual counts. The mean value of all control paper strips was 1. 73 x 10 5 /ml. This figure served
as a basis of comparison with counts obtained from paper strips exposed to sound. Irradiation of spores of B. Subtilis var. niger with sonic waves (9.9 kC/S) resulted in Significant decreases in viable counts under certain conditions (Table 1). Meaningful reductions in cell population, at the 0.11'0 level of significance, were always obtained when spore-treated paper strips were irradiated for 8 h regardless of the distance of the specimen from the sound source. Reducing the period of irradiation to less than 8 h, however, yielded values which were not significant except when the strips were irradiated at a distance of 1 in from the transducer during four hours. Irradiation periods of 2 h or less did not result in significant decreases in the viable count no matter what the intensity level. It is interesting to mention that the sharpest decrease in viable spores count was observed after 8 h at the 91-2 inch distance which corresponded to resonant conditions (a whole number of half wavelengths). The simultaneous decrease in viable organisms and increase in the acoustic energy level near respnant conditions clearly demonstrates that the b0und wave intensity is a key factor in sonic sterilization. The fact that a corresponding decrease was not always observed at 911.! in for shorter irradiation time could be explained by the fact that it is extremely delicate to set up perfect standing waves through accurate sample support pOSitioning. Treatment of the test organisms with ultrasonic waves (30.4 kC/S) also resulted in a significant reduction in viable counts (Table 2). As was the case with sonic waves, meaningful reductions in plate counts occurred after 8 h irradiation with test strips placed at each of the four distances employed. In addition, Significant results were also obtained after 4 h irradiation when the specimens were placed at distances of 5 in, 3. 5 in, and 1 in from the transducer. Beyond this the most striking reduction in the viable count was recorded when the test strips were located 1 in from the sound source and exposed to ultrasonic waves for only 2 h. Here again we found a sharper decrease in viable spores count at the three positions (1 in, 3. 5 in and 5
Table 1. Means of plate counts (X10 5 ) of spores of bacillus subtilis var. niger irradiated with airborne sound (9.9 kc/s) and corresponding t values (in parentheses) Irradiation time [h]
Sample position [inches and number of wavelengths] 9.5 7"- 1
5.0 3.6"-
3.5 2.54"-
1.0 0.76"-
spores/ml
spores/ml
spores/ml
spores/ml
1
1.77(0.12)
1. 56(1.17)
1. 69(0. 50)
1. 45(2.37)
2
1. 77(0. 44)
1.52(0.81)
1. 51(0. 83)
1. 41(2.66)
4
1. 46(1. 83)
1. 56(1. 00)
1.37(2.48)
1. 22(3.48*)
8
0.89(3.59*)
1. 09( 6.40*)
1.18(4.90*)
1. 14( 6 . 21 * )
Intensity level at sample [db]
155
153
153 1i2
154
* Indicates level of significance of O. 1'70 t Pressure antinodal position ULTRASONICS October 1966
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Table 2. Means of plate counts (X 10 5 ) of spores of bacillus subtilis var. niger irradiated with airborne ultrasounds (30.4 kC/s) and corresponding t values (in parentheses) . Irradiation time [h]
Sample position [inches and number of wavelengths] 9.5 13.2'\
5.0 7,\ t
3.5 5,\ t
1.0
spores/ml
spores/ml
spores/ml
spores/ml
1
1. 85(1. 38)
1. 66(0. 70)
1. 83(0.43)
1. 82(0. 76)
2
1.53(2.38)
1.56(2.21)
1. 53(2.06)
1. 05(3.54*)
4
1. 27(1. 31)
1. 36(3.08*)
1. 29(3.70*)
0.76(4.85*)
8
1. 11(8.88*)
1. 06(3.86*)
0.91(5.23*)
0.95(6.39*)
Intensity level at sample [dB]
156
159
161
161
* Indicates level of significance of O. 1 t Pressure antinodal position
0 /
0
in) which were the closest to standing wave conditions. As was theoretically expected, comparison of the data in both tables reveals that almost twice as many significant values were obtained in the 156-161 dB level than in the 153-155 dB level. A comparison at nearly equal intensity (155 and 156 dB) after identical irradiation time (8 h) seems to indicate that sonic waves (9.9 kC/S) are more efficient for spore destruction than ultrasonic waves (30.4 kC/S). However,more tests have to be conducted at the same energy level before drawing any definite conclusions regarding the specifiC influence of frequency. Consideration was also given to the possibility that the insonation chamber itself might adversely affect the viability of spores of B. Subtilis var. niger. Accordingly, spore-impregnated paper strips were allowed to remain in the chamber for periods up to 8 h under conditions identical with those employed for the experimental paper strips, except that sound energy was not applied. Plate counts of these strips revealed no significant reductions attributable to the test chamber alon"e. DISCUSSION Exposure of spores of B. Subtilis var. niger to high intensity airborne acoustic waves resulted in statistically significant reductions in viable counts compared to spores not exposed to sound. From the data collected it appears that the killing power of airborne acoustic waves is a function of both the acoustic energy density and the irradiation time. More tests are needed to clarify the respective contribution of these two factors. Preliminary observations in the 155-156 dB level showed that airborne sonic waves (9.9 kC/S) have more destructive power than airborne ultrasonic waves (30.4 kC/S). It is interesting to recall that the same trend has recently been observed in acoustic drying expe riments4, 5 with airborne acoustic waves. If confirmed, this would mean that the amplitude of motion of the gas molecules which impinge against the microorganisms' interface is one of the key factors in acoustic sterilization. Also of para202
ULTRASONICS October 1966
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mount importance is the fact that maximum kill was observed at a sample position which corresponded to the standing waves condition, i.e. when the spores are located at pressure antinodes. Let us now review the physical mechanisms which could provide an explanation to the lethal properties of high intenSity acoustic waves. As in the case of liquid irradiation, the degradation of acoustic energy into heat is certainly one of the main factors to be taken into consideration. Our results show beyond doubt that for equal irradiation time the lethal effects are more pronounced at high intensity, i.e. when more thermal energy is available through molecular friction. Let us also recall that the temperature gradient associated with the translational part of the sound energy is given by:
where a is acceleration and R is the gas constant. Greguss and Erdeleyi6 calculated, for instance, that for a sound field excited at 200 kC/s (intensity 1 W/ cm 2 ) the molecular acceleration in air is approximat ely 8.5 10 8 / cm sec-2 • This gives for the temperature gradient of the translational part of the energy a value of 19, 750°C/m. Referred to the complete heat content, this gives a value of 11. 86°C/mm and means that there will be a temperature gradient of ±10°C for each half wavelength of path. Furthermore, taking account of nonlinear distortions at high intensities, one can find even higher values of the acceleration. Another factor to be taken into consideration is the magnitude of the acoustic turbulence with its corresponding effect on diffusion and adsorption rates at the microorganisms' interface. The molecules physically adsorbed on the surface of bacteria are greatly affected during high intenSity airborne insonation. This is easily understood if one remembers that while irradiating we set up a very fast regular succession of
compt"essions and rarefactions. The effect of expansion always predominates over that of compression and as a result we break the superficial layer of molecules which coat the bacteria 7,8. In short, we can remove the superficial layers of adsorbed molecules and thus alter the physicochemical balance of the microorganisms' cortex layer. It is known that the impedance mismatch between air (pc""43) and microorganisms (pc""l. 15 x 10 5 ) is very
poor. In other words a very small amount of the radiated acoustic energy is transmitted into the spore. However, the smaller the thickness of the microorganism (compared to the acoustic wavelength) the greater will be the acoustic absorption. This could partially explain the comparatively more favourable results observed at sonic frequencies. A direct comparison of our data with other reports was not possible because of the lack of similar studies in the scientific literature. It is only recently that high power airborne sound sources with frequency flexibility have become available. Pioneering work in this area was conducted after the war by Frings, Allen and Rudnick 9,10 which described the lethal effects of airborne ultrasonic waves (19 kc/s, J = 1 W/cm 2 ) produced by a rotary siren. These authors stressed the importance of thermal phenomena and regarded them as the principal cause of the death of mice, roaches and various insects, after a few minutes of exposure to the high intensity beam. In France, L. Pimonowl l confirmed the lethal effects of a 26 kC/s (160-165 dB) siren emission on bees,flies and mice after 5 min of irradiation. The relatively long periods of irradiation required to demonstrate the antibacterial effects of airborne sound in the present report may also be a reflection of the nature of the chemical composition of spores. Aside from the possible physical barrier presented by the spores' outer layers, the lower water content of spores might tend to minimize any lethal effects of airborne sound. It is known that culture conditions may affect the lipid content of spores as well as their resistance to toxic agents. In view of the observation that lipoidal substances readily adsorb ultrasonic frequencies and behaviour of spores to airborne sound may, in large measure, be a reflection of their chemical composition at the time of irradiation. As generally observed in most applications which deal with high intensity insonation a critical energy threshold may exist above which the lethal effects will be greatly acceleTated. We have now the possibility of
irradiating spores and micro-organisms at intensity levels one order of magnitude higher than those used in the present work. This new ability in high intensity sound and ultrasound generation will make possible further work in an area where much experimental data is needed before drawing any conclusion. ACKNOWLEDGEMENT The authors wish to acknowledge the assistance of Bohdan S. Polanskyj, Project Engineer at Macrosonics, who designed the transducers and irradiating chamber used in this investigation. His advice during the course of the research were greatly appreciated. The authors also wish to thank the National Aeronautics and Space Administration which supported this investigation through a grant.
REFERENCES 1.
Barney, W., Electronics, 13 December 1965, p.134
2.
Kritz, J., Institute of Radio Engineers Transactions,8 No.1 (1961)
3.
Friedman, W. H., and Nowitzky, B. G., Fourth International Congress on Acoustics, Copenhagen (1962).
4.
Boucher, R. M. G., "Buoyant acoustic drying of yeast", Macrosonics Report M-AB-Ol (August 1964). Unpublished.
5.
Borisov, Y. Y., "Application of ultrasonics to chemical engineering processes", Ts INTI, EP and P Press, USSR (1960).
6.
Greguss, P., and Erdeleyi, I., Acustica, 2, p. 365 (1961).
7.
Boucher, R. M. G., Chemical Engineering, 21 September 1959, p. 151
8.
Boucher, R. M. G., Ultrasonic News, 3, No.2, pp. 8 and· 14 (1959).
9.
Allen, C .H., Frings, H., and Rudnick, I., Journal of the Acoustical SOCiety of America, 20,62 (1948).
10. Frings, H., Allen, C. H. and Rudnick, I., Journal of Cell and Comparative Physiology, 31, p. 339 (1948). 11. Pimonow, L., Ann Telecomm., 6, No. 11, 1 (1951)
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