US 3617178 A
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United States Patent John Gannon Clouston SENSITIZATION 0F BACTERIAL SPORES TO THE LETIIAL EFFECTS OF CERTAIN TREATMENTS 12 Claims, No Drawings u.s. Cl 21/2, 21/60, 99/211, 99/219,195/96 Int. Cl A611 l/00 Field of Search. 21/1, 2, DIG. 3, 58, 54, 60,101,102;99/211, 219; 195/96 References Cited UNITED STATES PATENTS 4/1922 Crowther 21/60 1,711,097 4/1929 Kratzer 99/219 1,746,731 2/1930 Koehler... 99/219 2,069,820 2/1937 Dodge 21/60 2,356,505 8/1944 Christensen 21/2 2,374,805 5/1945 Camelford 21/60 2,899,266 8/1959 Gewalt et a1. 21/58 3,276,840 10/1966 Sierra 21/2 3,457,031 7/1969 Linder et a1. 21/58 3,494,722 2/1970 Gray 21/54 FOREIGN PATENTS 418,127 10/1934 Great Britain 99/219 Primary Examiner-Morris 0. Wolk Assistant Examiner-Joseph T. Zatarga Attorney-Waters, Roditi, Schwartz & Nissen ABSTRACT: A method for sterilizing, disinfecting or preserving substances, whether fluid or solid, by subjecting them to hydrostatic pressure for a duration sufficient to germinate bacterial spores present in the substances either before or dur ing the usual treatments such as radiation, heating or chemical treatment.
SENSITIZATION OFBACTERIAL SPORES TO THE LETHAL EFFECTS OF CERTAIN TREATMENTS This application is a continuation-in-part application of my copending application Ser. No. 75 I055 filed 8th Aug., 1968, now abandoned.
BACKGROUND OF THE INVENTION This invention relates to methods for sterilizing, disinfecting and/or preserving fluid or solid substances. It is well known. as for'example, in US. Pat. No. l4l,3006 and US. Pat. No. 206,9820, that germs or bacteria may be killed by the application of pressure followed by the sudden release of the pressure. In these patents it is not the pressure but the release of pressure which disrupts the micro-organisms but this method is not efficient for the killing of bacterial spores. ln. accordance with this invention it has been found that the application of pressure to fluid or solid substances contaminated with bacterial spores causes germination of the spores, thereby reducing their resistance to sterilizing, disinfecting and/or preserving treatments. In accordance with the invention known sterilizing, disinfecting, and/or preserving treatments such as ionizing radiation, ultraviolet radiation, chemicalor gaseous treatment or heating may be employed for killing spores which have beengerminated by the application of pressure.
As a result of this invention, the quantity of heat, ionizing radiation or ultraviolet radiation, chemicals or any other treatment that proves lethal required to sterilize, disinfect and/or preserve said fluid or solid substances is reduced.
SUMMARY OF THE INVENTION The invention providesan improved method of sterilizing, disinfecting and/or preserving fluid or solid substances, wherein bacterial spores are destroyed by germinating them by the application of hydrostatic compression, in the pressure range 100 p.s.i. to 20,000 p.s.i. for a period of time depending on the germinating pressure and temperature employed to the substances prior to or during per se known sterilizing and/or disinfecting treatments. In .the. case of heating the maximum effect is obtained by applying pressure before heating.
As a result of the pressure application a physiological change is induced in the bacterial spores, which remain viable, but consequently are more susceptible to the usual sterilizing and disinfecting treatments mentioned above. The physiological change can be termed initiation-of-germination, initiation or germination as defined by W. G. Murrel (I961). (Symp.
Soc. Gen. Microbiol, 1l,l l0) and is identified according to the properties listed by L. J. Rode and J. W. Foster (Proc. Natl. AcadSci. (Wash.) l 18).
The fraction ofspores which initiate germination depends on the period of compression as well as the applied pressure and temperature. The fraction is defined as the ratio-of the number of surviving spores resistant to the lethal effects of heat, radiation, ultraviolet light and chemicals to the initial number of resistant spores in unit of spores per millilitre or spores per gram or total spore count. The same residual population or resistant spores will resultby applying a high-pressure for a short time or a lower pressure for a longer time.
The invention can be used for the germination of any species of bacterial spores of the genus Bacillus and Clostridium. The duration of the treatment depends on the pressure and temperature employed for germination. At ambient temperatures the degree of germination caused by a pressure of 20,000 p.s.i. applied to Bacillus Pumilus spores for 15 minutes at 25 C. is 99.9 percent whereas at a pressure of 9,600 p.s.i. 85 percent germinate in IS minutes. At a temperature of 48 C. and a pressure of 9,600 p.s.i. 99.5 percent ofthe spores germinate in 15 minutes.
The method applies to the pH range 1 to 10 and is effective at ambient temperature and at temperatures less than or greater than ambient, and also applies to both aerobic and anaerobic conditions.
reached above which the percent germination decreases with increasing temperature. As the pressure is increased the optimum temperature for germination increases but for the pressure range specified the optimum temperature is less than C. At a pressure of 9,600 p.s.i. the optimum temperature for Bacillus pumilus is 48 C. At a pressure of 3,750 p.s.i. the bptimum temperature for B. cereus is 38 C. whereas at 7,500 p.s.i. the optimum temperature is 42 C. The optimum temperature for Bacillus subtilis is 50 C. at 7,500 p.s.i. and 70 C. at 15,000 p.s.i. Also, the hydraulic fluid used in the compression can be a liquid ora moist gas, or,' the material being treated can itself be the hydraulic fluid.
The present invention can be further improved by the presence of exogenous compounds inthe form of ionic inorganic, or organic solutes, which enhance the physiological change in the bacterial spore.
Fluid and solids each require a slight modification in the application of compression through the difference in handling problems thereof. Each treatment is considered below butit will be appreciated that the description is exemplary only and the invention is not intended to be limited to the exemplary methods described.
The pressure process applied to liquids contaminated with bacterial sporesinvolves placement of theliquid in a pressure vessel which is a simple piston and cylinder device. Pressure is generated for the required time by applying sufficient force to the piston to obtain the required pressure for the required time.
Alternatively, the pressure can be generated using a hydraulic intensifying pump. Several types of pumps suitable for pressurizing fluids to pressures up to 20,000 p.s.i. using inlet-air pressures up to p.s.i. are available commercially. The fluid which is to betreated can be the hydraulic fluid or it can be separated from the hydraulic fluid by means of an elastic membrane such as rubber or plastic material.
The treatment of solid substances involves placing the contaminated object in a pressure vessel which is connected to a compressed gas'cylinder and is subjected to the required gas pressure for the required period of time.
The object is placed in a pressure vessel which is connected to a gas compressing device via a heat exchanger to control the temperatureof the gas in the pressure vessel which will rise dueto the heat of compression. The required pressure is maintained for the requiredtime. The method is more effective when the solid is pressurized by a moist gas. At high-pressures a trace of water vapor in the gas or vessel will condense onto the solid and assist the physiological change initiated by compression. 1
DESCRIPTION OF THE PREFERRED EMBODIMENTS The invention is further illustrated by the following nonlimiting examples:
EXAMPLE I A suspension of Bacillus pumilus spores in distilled water to a concentration of between five and ten x 10 organisms per ml. was compressed to 18,000 p.s.i. for 270 minutes (sample 1). An equivalent (sample 2) was compressed simultaneously while being irradiated by cobalt 60 gamma rays. A further equivalent sample was maintained at atmospheric pressure and 25 C. and irradiated (sample 3). A control sample was maintained at 25 C. and one atmosphere but not pressurized or irradiated.
Immediately following decompression, the survival was measured by the drop count method using oxoid nutrient broth containing 1 percent oxoid agar No. 3 gas as the nutritive medium. After 24 to 48 hours incubation at 37 C. colonies were counted using a Brunswick colony counter. Further incubation did not increase the number of colonies. The total viable count wasestimated from at least two different dilutions by the method of Farmiloe, F. J. Cornford, S. .1. Coppock, J. B. M. and Ingram, M. .1. Sci. Food Agric. 5, 292,304 (1954).
The number of surviving resistant spores and the number of surviving forms sensitive to the treatment was established by heating the spore dilutions used in estimating the total viable count at 80 C. for 15 minutes. After cooling, drop counts were made as described above and the number of forms which had initiated germination calculated by difference.
The results expressed as the number of surviving heat stable spores initial number of heat stable spores were as follows:
Sample Medium T t Radiation Dose Result C. p.s.i. min. rads 1 Water 25 18,000 270 nil.
2 Water 25 18.000 270 247.000
3 Water 25 14.7 270 247.000
Control 4 25 14.7 270 nil Heating at 80 C. for 15 min. and 14.7 p.s.i. reduced the spore count at most by percent. Compression to 18,000 p.s.i. for 270 min., followed by heating to 80 C. for min. reduced the spore count by 94.8 percent. Irradiation for 270 min. at 14.7 p.s.i. reduced the spore count by at most 87 percent. Compression during irradiation reduced the spore count by 99.3 percent.
EXAMPLE 2 A suspension of Bacillus pumilus spores in 0.067M potassium mono and dihydrogen phosphate pI-16.8 was compressed to 16,000 p.s.i. for 240 min. (sample 1). Sample (2) was compressed simultaneously while being irradiated by cobalt 60 gamma rays. Sample (3) was irradiated at atmospheric pressure. Sample (4) the control was maintained at C. and measurements made as before.
Results were as follows expressed as above.
Sample Medium T p t Radiation Result C. p.s.i. min. Dose rads I 0.067M potas- 25 16.000 240 nil 0.000155 2 sium monoand 25 16.000 40 110.000 0.000037 3 dihydrogen 25 14.7 240 220.000 0.143
4 phosphate 25 14 7 40 nil Heating at 80 C. for 15 min. did not alter the spore count. Compression to 16,000 p.s.i. for 240 min. followed by heating to 80 C. for 15 min. reduced the spore count by 99.985 percent. Irradiation for 240 min. reduced the spore count by at most 86 percent. Irradiation during compression reduced the spore count by 99.996 percent.
EXAMPLE 3 LII The result shows that presence of exogenous solutes increases the rate of decrease of the resistant bacterial spore count and by suitable choice of additive a similar but greater effect can be obtained at a pressure of 6,600 p.s.i. for 30 min. as is obtained by a pressure of 16,000 p.s.i. for 30 min.
Compounds such as tyrosine, adenosine. glucose, mannose, I-arginine, l-phenylalanine, l-alanine, 1-cysteine,inosine and yeast extract, when added in suitable proportions will initiate germination at ambient temperature and pressures exceeding 100 p.s.i.
EXAMPLE 4 Effect ofTemperature at Constant Pressure Organism T P l Medium Result C. p.s.i. mins. '1 germination B.subtilis 38.7 7.000 30 Potassium phosphate buffer 405 7.000 30 60 41.2 7.000 30 43.0 7,000 30 B.subtilis 35.0 8.000 30 Distilled \LLIICI EXAMPLES Effect ofTime of Compression at Different Temperatures Organism T P 7tgcrm Medium Result C. inution Time for 50;
germination B.subtilis 32,6 9 280 50 Potassium 60 mins.
bulTer 38.7 9.280 50 30 mins.
40.8 9.280 50 25 mins.
42.) 9.280 50 18 mins.
EXAMPLE 6 Effect ofincreasing pressure at constant temperature and duration of compression Organism T P t Medium Result C. p.s.i. mins. 1 germination B.cereusT 25 7.000 10 Potassium phosphate buffer EXAMPLE 7 Effect of compression on different organisms Result,
percent T, P, t, germi- Organism C. p.s.i. min. Medium nation B. subtilis 25 15,000 30 Phosphate bufler 90 B.cagulan. 25 15,000 80 B. pumilua 25 15,000 00 Cl. sporoaenea... 25 20,000 70 EXAMPLE 8 Effect of exogenous compounds on initlatiou or germination by hydro static pressure at constant temperature iie pafiic ularly useful pui pose application of the method of the present invention is in the sterilizing of surgical sutures. The sutures in their final sealed containers may be subjected to hydrostatic pressure and temperatures in the range 25 C. to 70C., before or during sterilizing treatment. The method involves placing the sutures after manufacture in a container or containers which may be metal, plastic or glass containing an aqueous solution. The container may be flexible or rigid, but if rigid must contain means such as a diaphragm for transmitting the pressure to the aqueous solution surrounding the sutures.
The container is placed in a pressure vessel, the pressure vessel sealed and using water as the hydraulic fluid and conventional methods of generating hydrostatic pressure, the container is compressed by pressures in the range 100 to 20,000 p.s.i. for a period of time depending on the temperature and the nature of exogenous compound (if any) in the solution. The container may simultaneously be subjected to the sterilizing effect of ionizing radiation or subsequently treated by radiation, heat, ultraviolet light or gaseous treatment. One suitable container would be a plastic container filled with the aqueous solution and sealed with a stopper in such a way that air is excluded and the stopper and solution are in intimate contact. The stopper, especially in the case of a rigid container, should be of rubber or plastic to ensure that the hydrostatic pressure is transmitted to the solution in the container.
What i claim is:
1. in a method of sterilizing, disinfecting and/or preserving fluid or solid substances contaminated with bacterial spores of the genus Bacillus and Clostridium. the improvement comprising germinating said spores by hydrostatic compression applied to said substance in the pressure range of 100 psi. to 20,000 psi. and at a temperature less than C, said pressure and temperature being maintained for a period of time sufficient to germinate said spores, and either simultaneously with or subsequently to said germinating step exposing said substance to a sterilizing, disinfecting and/or preserving treatment to destroy said spores.
2. A method according to claim 1 wherein the sterilizing. disinfecting and/or preserving is effected by ionizing radiation.
3. A method according to claim 1 wherein the sterilizing, disinfecting and/or preserving is effected by ultraviolet radiation.
4. A method according to claim 1 wherein the sterilizing, disinfecting and/or preserving is effected by chemical treatment.
5. A method according to claim 1 wherein the sterilizing, disinfecting and/or preserving is effected by gaseous treat ment.
6. A method according to claim I wherein the sterilizing, disinfecting and/or preserving is effected by heating.
7. A method as claimed in claim 6 wherein the hydrostatic compression is applied before heating.
8. A method as claimed in claim 1 comprising adding exogenous compounds in the form of ionic inorganic or organic solutes to the substances before they are subjected to the hydrostatic compression.
9. A method as claimed in claim 1 wherein the substance is a liquid substance and same is placed in a pressure vessel comprising a piston and cylinder and the compression is applied by applying sufficient force to the piston to obtain the desired pressure for the desired time.
10. A method as claimed in claim 1 wherein the substance is a liquid substance and the compression is generated by using a hydraulic intensifying pump.
11. A method as claimed in claim 1 wherein the substance is a solid substance and the compression is applied by a gas pressure on the substance placed in a pressure vessel.
12. A method as claimed in claim 11 wherein the solid is pressurized by a moist gas.
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