Method of killing microorganisms present on surface of a solid body

The subject of the invention is a method of destroying microorganisms on a solid surface using UV irradiation of this surface.

It is known to sterilize packaging material, for example, the inner surfaces of cardboard boxes, by heating with hot air or steam at a temperature of about 100 ° C for a few seconds. Another known method uses ultraviolet radiation with bactericidal wavelength, e.g., 254 nm. An average energy density on the illuminated surface of about 8 mW / cm is ensured²for a few seconds. For surface sterilization, a relatively high concentration H2O2 solution is also used for a few seconds. Preferably, known methods are used together, which makes it possible to reduce certain processing parameters, e.g. temperature, treatment time and / or concentration of H2O2.

For example, WO-A-80/01457 describes a method of sterilizing liquids, e.g. waste water and cooling water, at a canning factory, in particular for

170 455 sterilizing surfaces, such as hospital walls and food container surfaces. The surfaces of the food containers are treated with H 2 O 2 at a concentration of not more than 10% by weight, for example by moving the container or material from which the container will be made, through a container containing the H2O2 solution. You can also spray the surface of the container or material with this solution. Irradiation of the surface with ultraviolet with a wavelength less than 325 nm, is carried out with the help of ultraviolet lamps so arranged that the container or materials that emerge from this tank or the sprayed liquid are subjected to ultraviolet, completely or for the most part, rendering microorganisms incapable of life, thanks to the interaction between ultraviolet and H 2 O2.

The use of ultraviolet radiation as a physical agent for microbial reduction is known. Cellular DNA absorbs radiation energy with a wavelength between 250 and 260 nm, which leads to the formation of chemical bonds between neighboring nucleotide bases - thymine residues. This change distorts DNA chains, interfering with replication and transcription, and thus gene expression. Cell death is unavoidable if essential genes are blocked or dNa replication inhibited. The number of vegetative cells killed by ultraviolet radiation depends on exposure and dose, respectively in mW / cm 1 mJ / cm², and ultraviolet is often ineffective in killing bacterial spores, especially because of the poor ultraviolet penetration power. In the combined use of ultraviolet radiation, hydrogen peroxide (the peroxide radical reacts with most chemical bonds) and heat, sterilization is achieved, but at a high cost.

WO-A-88/03369 describes a method of sterilizing air, water, food and food packaging by means of an intermittent, very intense light pulse of very short duration, in the range of visible and near visible light frequencies. Such light is produced, for example, by flash lights. The components of such an impulse cover a wide spectral range and can include far and near ultraviolet light to deactivate microorganisms by photochemical effects. Such ultraviolet-rich pulses have at least 15%, and preferably at least about 30% of their energy in the wavelength range shorter than 300 nm. Such ultraviolet-rich pulses can usually have a relatively low overall energy density, in the range of from about 0.01 to 15 J / cm2, and usually from about 0.1 to 3 J / cm2. However, for sterilizing the surface of a food product it may be desirable to filter a portion of this spectrum such that at least 90% of its energy is distributed in the wavelength range between 300 and 2500 nm. In such methods, in which the component of the ultraviolet flashes of pulsed light is suppressed, or completely eliminated, the intensity of the pulsed light should be sufficient to heat the surface layer of the foodstuff or packaging material having a thickness less than 10 microns, from a temperature of at least 50 ° C to a temperature of at least 75 ° C and preferably at least about 100 ° C.

Patent No. EP-A-0201650 describes a method of laser disinfection of liquids in which a laser beam radiating light in the ultraviolet range is directed at a stream of disinfected liquid. This method is particularly advantageous for water disinfection and uses a gas pulse laser with adjustable medium intensity of ultraviolet light. The UV beam covers the cross section of the water jet. The frequency changes from slow to so fast that it actually becomes a continuous beam, with the possibility of adjustment depending on changes in the speed of water flow through the laser beam and changes in water turbidity. You can also change the speed of water flow by maintaining a constant intensity of the ultraviolet beam. At least one of the walls of the water conducting conduit may have a relatively high reflection coefficient for ultraviolet to limit the dispersion of this beam due to particles suspended in water and reduce the cross-sectional area of ​​the beam, to compensate for the change of this beam to maintain its uniform intensity in the area of ​​impact on the stream. The angle of penetration of the beam into the stream is adjusted under the influence of changes in water turbidity. In addition, you can change the geometry of this beam, especially its length, by changing water parameters such as organic content.

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An excimer fluorine krypton laser generating 249 nm wavelength is preferred.

US-A-3817703 describes a method of destroying a live substance suspended in a light-permeable material, with the possibility of simultaneously sterilizing the containers and the materials stored therein, using a laser beam directed at the container so that it touches all its internal surfaces during sterilization. At the same time, the entire contents of the container are exposed to light rays. In this way, a living substance in the material or adhering to the inner walls of the container is destroyed. One or more oscillating mirrors are used to cause one or more laser beams to wrap around the container.

US-A-3941670 discloses the use of an infrared laser whose target is searched by a laser beam using an oscillating mirror. Light changes the biological activity of an irradiated macromolecular species by means of excitation of vibrational and torsional states. Various purposes can be sterilized, for example air or other liquid media, plastics and metals, such as aluminum foil tape or ribbon.

In addition, DE-A-2914075 describes a method for sterilizing the internal surfaces of pre-shaped cardboard boxes. Ultraviolet sources, for example vertical sources with a mercury lamp, are inserted from below into each cardboard box, at different positions relative to the side walls of this box. Ultraviolet radiation of sufficient intensity reaches differently obscured angles formed by the bottom and side seams of this cardboard box. In a preferred embodiment, ultraviolet sterilization and heat sterilization are combined so that heat-sensitive yeast and mold are eliminated. This thermal sterilization is carried out using two radiation beams. Infrared radiation acts on cardboard boxes before or after ultraviolet radiation.

US-A-5 068 514 describes a method of polishing the surface of polymer contact lenses with ultraviolet laser radiation at a wavelength of 193 nm to achieve a controlled level of the lens surface, but without excessive photodecomposition, where the lens surface is smoothed.

JP-A-60 28235 describes a method of cleaning the surface of a semiconductor wafer plate to remove chemical particles and foreign atoms from the wafer surface. In a preferred embodiment in which a shot key diode is formed on the upper surface of the N-type silicon wafer, thermoxidation is previously used to form a very thin silicon oxide film on the upper surface of the silicon wafer. A hollow is then made in the silicon oxide film to expose the silicon base plate. The plate is then chemically cleaned using a mixture of liquid ammonia, hydrogen peroxide and water. The plate is then washed in clean water for ten minutes and dried. The plate is then introduced into the vacuum chamber and irradiated with ultraviolet radiation lamps with radiation energy of about 10 mW / cm². After irradiation for a certain time in a vacuum chamber, the plate is transferred to a spray chamber connected to the vacuum chamber and the plate is sprayed with platinum.

In JP-A-60-28235 it is explained that the OH binding energy of water molecules is 4.8 eV and the binding energy CH of the organic substance is 4.5 eV. To clean the surface of a semiconductor wafer, a portion of light with a higher energy than the above energy combination is needed, and ultraviolet radiation with a wavelength below 300 nm is more effective.

An alternative chamber device uses a mercury lamp with ultraviolet radiation and a xenon flash lamp with infrared radiation. The mercury lamp is used to irradiate the surface of the semiconductor wafer with ultraviolet radiation, while the flash lamp is used to heat the surface. Ultraviolet radiation interrupts the binding of water particles and CH bonds at the same time as the surface is heated by infrared radiation of the flash. Specific kinetic energy is transferred to small particles causing them

170 455 quick lowering of the tile surface. It was found that the condition for purification is ultraviolet radiation 10mW / cm², flash exposure at 4 J / cm²at a pulse width of 50 microseconds and an interval of 5 minutes between pulses.

The method of the invention is intended to destroy microbes on a solid surface in which the surface is irradiated with ultraviolet radiation at bactericidal wavelength. This method is characterized by the fact that in the process of destroying microorganisms, the surface is irradiated with ultraviolet laser radiation of bactericidal wavelength.

Preferably, the power density of bactericidal ultraviolet laser radiation is used that does not damage the solid surface. The irradiated surface is preferably the surface of the packaging material in contact with the contents of the packaging. The surface in contact with the contents of the package is the surface of the thermoplastic material. Preferably, the surface in contact with the contents of the package is a thin polymer layer.

The surface is additionally irradiated with infrared radiation. Preferably, the surface is irradiated with infrared laser radiation. The surface is irradiated with laser ultraviolet radiation and infrared radiation simultaneously. In addition, the surface is irradiated with laser ultraviolet and infrared radiation to obtain a synergistic effect on the irradiated surface.

In a preferred embodiment of the invention, the surface of the three-dimensional solid is irradiated, the direction and intensity of the laser radiation are regulated, and the radiation intensity is uniformly maintained on the irradiated surface. Laser radiation is applied using a hologram and the radiation intensity is kept uniform on the irradiated surface.

It is advantageous if the UV radiation irradiates the surface on which the hydrogen peroxide is. In addition, the surface is sterilized with ultraviolet laser radiation and hydrogen peroxide to obtain a synergistic effect on the irradiated surface.

Based on the conducted research, it was found that pulsed ultraviolet radiation is particularly advantageous in the solution according to the invention, as more effective for the destruction of microorganisms on the surface of a solid, compared to continuous ultraviolet radiation.

The recommended ultraviolet wavelength used is a length in the range from 150 to 320 nm, preferably 240 to 280 nm, especially about 250 nm. The recommended infrared wavelength is between 1,000 and 10,000 nm.

It has been found that the use of a laser to deliver ultraviolet, in contrast to a bactericidal ultraviolet emitting lamp, provides certain benefits, such as the relatively high density of absolute energy and the small divergence of the highly directed output beam, which ensures its special suitability for sterilization of specific areas inside a cardboard box, where there may be strong contamination, for example corners and folds in the cardboard box.

The method according to the invention is explained in more detail in the examples with reference to the drawing, in which Fig. 1 is a diagram of the device used for irradiation of the UV of the tested strips, and Figs. 2 and 3 schematically show two different systems for obtaining uniformly distributed laser ultraviolet energy on the internal the surface of the cardboard box with an open top.

Example I. As a test organism in the process of laser sterilization, a bacterial spore forming the bacterium: Bacillus subtilis var. globigii. This bacterium is classified as Gram-positive, aerobic, rod-like, mobile and non-pathogenic. It is ubiquitous in the living environment and can be found in soil, feces, hay dust, milk and water. The formation of spores allows the bacteria to survive in a latent (dormant and viable) state for a long time. In this connection, it is particularly important that the sterilization method is capable of killing these bacterial spores. Because spores are resistant to temperatures over 100 ° C and other destructive

170 455 agents (chemicals) are useful indicators of sterilization efficiency and are systematically used for chemical sterilization control and heat treatment. Standard microbiological tests and microscopic tests are used to evaluate the new sterilization procedure. Identification of spores under the microscope is facilitated by their high refractive index and reduced susceptibility to staining, and due to their specific reaction to specific dyes.

The research used a pure semi-finished product for cardboard boxes (a cardboard layer laminated with polyethylene layers, with or without an aluminum barrier layer) and a spore suspension of Bacillus subtilis var. globiga, obtained from Elopak A / S in Norway. A second bacterial culture (NCIMP 8649) was used for control, serving as independent confirmation for this species. The methodological procedure was divided into three main stages, namely coverage of the test strips with bacteria and all associated pre-coating procedures, exposure of these strips to laser ultraviolet and subsequent analysis of these strips.

Before laser irradiation, a system for applying the culture to the cardboard material in the form of test strips was prepared, with particular regard to maintaining the sterility of these strips before covering and placing the bacteria on the sterile surface of the strips while maintaining their sterility.

The cardboard material was cut with scalpel blades to obtain 6 cm and 2 cm test pieces. These test strips were subjected to several different sterilization processes: 35% hydrogen peroxide, 1 ml / test strip, for 2 min; 2% hydrogen peroxide, 1 ml / tasting strip, for 2 s and 2 min; autoclave steam sterilization at 121 ° C for 20 min.

All strips were well dried before application of the bacterial culture, and a number of sterile strips were set aside to serve as controls.

The sterilized strips were kept in autoclave bags at room temperature for 4 days before use. Coated test strips were stored in Petri dishes at 4 ° C for 4 days.

Spore suspension of Bacillus subtilis var. globigii were grown as a subculture in broth medium (beef extract, peptone, water, pH 6.8) at 37 ° C and tested after 24 hours. A standard pour-plate isolation and viable counting method was used. Sterile distilled water broth medium was used, depending on the plate method used. Agar medium (beef extract, peptone, pH 6.8 agar) served as a solid medium for growing this organism. Visual observations and counting of colonies were carried out after 36 hours, after incubation at 37 ° C. Admixed cultures were used to prepare the smear and then to the differential staining procedures. The dyes used were Gram dye and Ziehl-Nielsen bacterial spore dye, and the dyed preparations were analyzed by light microscopy (10 x 100).

100 gl sample from serial dilution of the stock culture in distilled water, 10³ and 10⁶ colonizing units / ml, are aseptically placed on a sterile cardboard test strip, spread over min and allowed to dry for several hours. The test strips during drying were stored covered in sterile Petri dishes at room temperature and then stored at 4 ° C. Particular care has been taken to keep the Petri dish up at all times to avoid accidental contamination of the unlaminated surface.

One test strip for serial dilution and two sterile samples were separately placed in 10 ml broth and the growth tested after 36 hours at 37 ° C. Turbidity was visually assessed and 100 g culture medium was placed on agar plates for further incubation for 36 hours at 37 ° C and the slants were observed as described above.

The test strips were then irradiated with ultraviolet laser radiation. The Q-hole laser used as shown in Figure 1 was the Questek 2000 Krypton-Fluorine (KrF) laser excimer laser. The laser generated ultraviolet radiation with a wavelength of 248 nm. This laser is a pulsed laser, giving 10-20 nS pulses at repetition frequencies up to several hundred Hertz.

The following operating parameters were used: pulse energy 200 mJ repetition frequency 100 Hz

The laser sent a beam that has approximately 3x2 cm dimensions (horizontal x vertical).

The optical system used is shown in Figure 1. The energy density directed at the sample was changed by changing the bonded silicate lens L, and / or the distance d from the lens to the aperture M, i.e. from the lens to the sample.

Initially, a 25 cm cylindrical lens was used to stretch the beam in the horizontal direction, giving an irradiation field of approximately 3x3 cm on the M iris. The resulting energy density of the shot per sample was 40 mJ / cm², giving an average power density of 4 W / cm2 at 100 Hz.

To produce a higher power density per sample, the distance to the sample was reduced and the sample size was appropriately reduced from 3x3cm to 1 x 1cm. Shot energy and average power densities in this system were 130 mJ / cm2 and 13 W / cm2, respectively.

To enable the radiation density to be reduced, the cylindrical lens was replaced with a 20 cm spherical lens. The resulting beam field on the sample, significantly exceeding the aperture opening, results in energy and average power densities on the sample of 10 mJ / cm and 1 W / cm2, respectively.

In all cases, the energy density was measured in the plane of the shutter's aperture. The block of samples was a few centimeters off this plane. However, the difference in exposure introduced by this was small because the shift was small compared to the distance d, which is usually in the range of 40 - 70 cm.

Different irradiation times were used, depending on the test energy density and power: 10 mJ / shot / cm ^: 1 s and 6 s 40 mJ / shot / cm: s and 6 s

130 mJ / shot / cm2: 15 p

The crafted test strips were mounted, using sterilized pins, on a balsa block covered with aluminum foil. Both the block and the foil were sterilized by steam for 20 min at 121 ° C before use. Depending on the size of the test strips, twelve to sixteen samples were mounted on one block, the block was mounted on a J laboratory jack to provide scanning. The samples were mounted on the block under hygienic conditions, using sterile pliers to attach the strips.

The experiments with H2O 2 were carried out by two methods.

In the first one, 100 (tl 2% H2O2) was added to the test strip (one for each test solution), spread over the surface of the cardboard box, dried in heat, then irradiated with a laser beam. The second experiment was carried out as the first, but without drying in heat before irradiation with a laser beam In contrast, test strips were layered between sterile quartz glass plates, irradiated with a laser beam, and then heat dried.

The temperature used was approximately 100 ° C and the heat was applied through the contact of the bottom silicate plate with the laboratory radiator plate. The top silicate plate was removed during the drying process.

All samples irradiated with laser beams, including control samples, were immediately placed in separate universal containers with 10 ml sterile broth medium and incubated for 36 hours at 37 ° C. Sterility was tested by estimating the turbidity of the solution after the incubation period after which the subculture was carried out on agar medium plates and a second incubation for 36 hours at 37 ° C. The growth was examined under a microscope for the presence of an indicator strain.

The results of conducted experiments before laser irradiation were considered. Test strip sterilization processes show that the autoclave method is the most reliable and successful procedure.

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The method of drying the bacterial culture on test strips was successful with samples of propagated viable colonies, up to 4 days (test strips older than 4 days were not tested). Bacterial culture containing 2x10 was obtained by serial dilution and then the poured plate method⁷ colonization units / ml (cfu / ml). To determine the average number of colonies, 5 dilution agar plates were used. It was found that sterile strips without bacterial addition, used for inspection during testing and indicating successful application of aseptic techniques, remained sterile in most experiments. In two cases where these cards were in contact with the non-sterile surface of the test strips, culture and Gram-negative organism were identified.

In all experiments the use of Gram dye and staining of Bacillus subSihs var. globigii clearly indicated that the organism is of the species Bacillus subtilis.

The classification of the results into sterile and non-sterile, as well as the distinction between the test organism and other potential contaminants, were unambiguous.

A summary of the results of the experiments in which the laser irradiation was used is presented in Table 1.

In Example I, encouraging results were obtained demonstrating that effective sterilization using laser ultraviolet could be achieved. At an average power density of 4 W / cm², for an exposure time of 6 seconds, the microbial killing efficiency (defined as the number of sterile test strips to the total number of test strips increased x 100) was about 30%. The inclusion of hydrogen peroxide on the test strip improves the result to almost 100%. It should be noted that an excellent result with H2 O2 was achieved with a lighting time of only 1 second.

Table 1

Comparison of results after laser exposure

<td>Density power (W / cm²)</td><td>Laser processing conditions, exposure time (S)</td><td>Use H2O2?</td><td>Test strip size (cm x cm)</td><td>number replica</td><td>Degree of sterility after 36 hours and initial cell density</td>

<td> 13</td><td> 15</td><td>No</td><td>1 x 1</td><td>2 each</td><td>1 at 10⁶</td>

<td> 4</td><td> 1</td><td>Yes</td><td>1 x 1</td><td>5 each</td><td>1 at 10⁶</td>

<td> 4</td><td> 6</td><td>Yes</td><td>1 x 1</td><td>5 each</td><td>1 at 10³3 at 10⁴</td>

<td> 4</td><td> 1</td><td>No</td><td>1 x 1</td><td>2 each</td><td>1 at 10⁴</td>

<td></td><td> 6</td><td></td><td></td><td></td><td>2 at 10⁴, 6 p</td>

<td> 4</td><td>1 f</td><td>No</td><td>2.5 x 2.5</td><td>2 each</td><td>no</td>

<td> 4</td><td> 1</td><td>Yes</td><td>2.5 x 2.5</td><td>2 each</td><td>2 at 10³, 6 p</td>

<td></td><td> 6</td><td></td><td></td><td></td><td>1 at 10⁶, 6 p 1 at 10³, 1 p</td>

<td> 4</td><td> 1</td><td>Yes</td><td>2.5 x 2.5</td><td>2 each</td><td>2 at 10³, 1 p</td>

<td></td><td> 6</td><td>(quartz glass)</td><td></td><td></td><td>2 at 10⁶. 6 seconds 1 * at 10⁶, 1 p 2 at 10³, 6 p</td>

<td> 13</td><td> 5</td><td>Yes</td><td>1 x 1</td><td>4 each</td><td>4 at 10⁶</td>

<td></td><td></td><td>(quartz glass)</td><td></td><td></td><td>3 at 1 (0 '</td>

<td> 1</td><td> 1 6</td><td>No</td><td>3x3</td><td>5 each</td><td>no</td>

hand contamination on one sample

Example II The results of Example 1 indicated successful laser ultraviolet treatment to neutralize microorganisms such as Bacillus subtilis. Exposure periods

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2 3 lasers from 1 second to 6 seconds, at 40 mJ / shot / cm (4 Wcm), showed killing 10 to 10⁶ bacterial cells in several samples.

Quantitative estimation of how many organisms were killed by specific laser treatment was further examined in Example II. A large number of samples were tested to control killing efficiency using various laser irradiation procedures, with particular emphasis on changes in power density and repetition frequency. The effectiveness of the laser light sterilization process was measured by observing the survival rates of bacteria after various treatments.

Several other experimental problems were considered during the study. It was considered in turn to what extent the base film on which the strips were placed affects the heat generated and how this additional heat, if present, affects the laser killing potential; whether the wood used for assembling the strips releases the substance onto those strips that can assist in the destruction of bacteria, whether this substance, if present, is activated by the autoclave and is taken over by the strip; whether the strip itself releases substances that can affect the degree of bacterial killing, or whether the heat generated by the laser is necessary to actually kill the bacteria; as reducing the power density (W / cm²) and / or reducing the repetition rate (Hz) affects the killing efficiency of UV laser lighting.

The study used a semi-finished product for cardboard boxes (laminate with aluminum foil) and StUDLAND cardboard assembly card (non-laminated). Bacillus subtilis var. Spore suspension was used to prepare serial dilution in distilled water. globigii. This dilution was distributed in equal amounts into test strips, which were then subjected to drying and UV irradiation.

The procedure was carried out in three main stages, namely, a bacterial layer was applied to the sterilized test strips, the strips were irradiated with UV radiation and then these strips were analyzed.

The cardboard material was cut with a scalpel blade to obtain 1.3 cm x 1.3 cm test strips. 18 test strips were used on a balsa block (the wood was covered with Al foil or left uncoated). The arrangement of stripes on wood was arranged in three rows into a block, with six stripes in a row.

All strips were held in place with autoclave tape so that 1 cm2 of carton area on the strip remained exposed. Each block was placed in a separate autoclave bag and autoclaved at 121 ° C for 20 minutes.

jxl of each prepared dilution (10 '¹, 10'2, 10'³, In⁴) each stock culture of Bacillus subtilis var. globigii was distributed in equal quantities into strips and allowed to dry under laminar airflow for several hours. Two strips per block were used for control, i.e. without UV exposure and one strip per block was used as empty, i.e. without adding bacterial dilution. Then, each block of balsa with stripes returned to a sterile autoclave bag and was sealed until it was irradiated with UV light.

To provide a reference point from which bacterial neutralization is estimated, the number of colonizing units arranged on the strips was estimated at different dilutions using the detergent recovery method as follows:

Prepared strips with appropriate bacterial dilution immersed in 0.1% Tween 20 (sterile, diluted in distilled water). The effectiveness of Tween 20 for the recovery of bacterial culture from the strips was compared with the recovery of sterile water so that confirmation of the principle of this method could be established.

Other methods were changed to see if there was an effect on bacterial counts, including a comparison between balsa-mounted strips or left unmounted on sterile Petri dishes prior to recovery.

The test strips were then irradiated with UV light. A laser was used as in Example 1, but with an increased pulse energy of up to 250 mJ. The only changes introduced were the repetition rate (Hz), exposure times and the energy density used. The tested varieties are listed in the Laser Parameters column in Table 2.

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25 cm cylindrical lenses were used to reduce the vertical beam dimension to achieve an energy density of 40 mJ / shot / cm2. 35 cm focal length lenses were used to stretch this beam so that a reduced energy density of up to 6 mJ / shot / cm2 could be obtained.

Up to 18 strips were exposed to a single block, the block was mounted on a laboratory J lift to ensure scanning. The scanning procedure was carried out under hygienic conditions, and after treatment, the blocks were placed in sterile autoclave bags. These bags were sealed and were stored at room temperature until the next analytical procedures. Most blocks had three stripes that were not exposed to UV light. Two of these samples were used as controls, and one - without bacteria - was used for controls.

After laser irradiation, all strips were aseptically removed from the blocks, put into separate bottles containing 5 ml of diluent and centrifuged for a minimum of 30 seconds. 100 g of each sample was pipetted into separate agar plates, spread out into bands and incubated at 37 ° C for a minimum of 24 hours.

The growth of the colonies on plates was visually examined and the number of colonies was counted.

The results obtained without laser irradiation indicate that the procedure for plating the bacteria on a serial dilution agar medium resulted in a reproducible number of colonies per bacterial concentration and allowed the calculation of the recovery factor.

It was found that the best way to recover bacteria from the strips was to use a 1 minute centrifugation in the diluent. The recovery varied between 50% and 90% of the bacteria on the strip. A serial dilution experiment indicated that the stock culture contained 1.5 x 10⁷colonizing units in ml.

The two types of tested materials did not show any differences in the degree of recovery obtained. The experiment with balsa test strips also showed no difference in results compared to the strips that were left on the Petri dishes, thus excluding some potential chemical influence on their substrate.

The results of the experiments prior to laser irradiation, such as studies of potential bactericidal substances in the material or in wood, were further considered with test strips subjected to laser UV irradiation, but no effect of this radiation was found. The effect of potential heating on the degree of bacterial killing was decisively negated when the intensity of laser ultraviolet heating was reduced to exclude thermal effects. The exposure time was set so as to obtain the same total number of photons, for example 10 pulses per second for 10 seconds, instead of 100 pulses in one second.

Table 2

Summary of results for example II

<td>Laser parameters</td><td>, 2 Total energy (J / cm)</td><td>Number of samples; *</td><td>** Reduction logio</td>

<td>2 40 mJ / shot / cm 100 Hz, 1 second</td><td> 4'</td><td> 36</td><td> &gt;5,2</td>

<td>40 mJ / shot / cm2 30 Hz, 1 second</td><td> 1,2</td><td> 1</td><td> &gt;5,2</td>

<td>6 mJ / shot / cm 100 Hz, 1 second</td><td> 0,6</td><td> 3</td><td> 2,7</td>

<td>40 mJ / shot / cm 2 Hz, 5 s</td><td> 0,4</td><td> 2</td><td> &lt;1,0</td>

<td>40 mJ / shot / cm2 3 Hz, 1 s</td><td> 0,12</td><td> 2</td><td> 3,3</td>

<td>6 mJ / shot / cm 2 Hz, 5 s</td><td> 0,06</td><td> 3</td><td> 1,8</td>

* Each sample had 1.5 x 10 spores on a 1 cm target surface. ** Specified as logio (1.5 x 10r / number of survivors).

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The use of an energy density of 55 mJ / shot / cm for more than 2 seconds caused physical damage to the stripes (bubble and browning).

Experimental results demonstrate that the effectiveness of killing laser beams of UV radiation depends on the laser parameters used and the number of existing bacterial microorganisms, but is independent of the substrate material. Most of the tested laser parameters indicate the effectiveness of killing the number of bacteria because 10⁶ colonizing units. Illumination period of one second at 40 mJ / shot / cm² and 100 Hz gave 100% neutralization of bacterial concentration 1.5 x 106 colonizing units. High 1.5 x 10 killing efficiency⁵colonizing units at lighting times of one second and relatively low energy parameters or repetition rates, such as 6 mJ / shot / cm2 at 100 Hz and 40 mJ / shot / cm at 3 Hz, is particularly important for sterilizing cardboard boxes.

The two systems illustrated in Figs. 2 and 3 of the drawing are possible variations of solutions that ensure the planned distribution of energy to obtain evenly distributed energy across the inner surface of the carton box.

In the example shown in Fig. 2, a computer generated H hologram is used which causes the diffraction of the ultraviolet light beam B with a particular pattern. It consists of a complex surface structure or mesh that is formed on the surface of a piece of glass. Its design requires computer devices that can predict how light will interact with the mesh structure. This mesh is created by covering the glass with a photolithographic mask and recording the pattern on this photolithographic mask with an electron beam. Invocation of photolithographic mask removes exposed fields. The glass is then etched to reproduce the pattern existing in the photolithographic mask on the glass.

The second system, illustrated in Fig. 3, uses two galvanometers (not shown) to drive N mirrors, which are rotatably installed around their axes, thereby deflecting the feeding laser beam B. The combination of two scanners allows the beam to be directed in two axes. The addition of a third galvanometer (not shown) driving the linear shift of the station to the focal length movement F is preferably used to change the separation of two lenses, which changes the beam divergence.

It should be emphasized that these two systems are fundamentally different. The H hologram directs radiation to each point of container C simultaneously, while the galvanometer selects the field directions in turn. In addition, the hologram system has no moving parts.

Example III. This example describes the scanning of the entire cardboard box, especially the microbiological methods and tests used in testing the efficiency and efficiency of ultraviolet laser irradiation in the field of laser energy and bacterial concentration.

The task of the basic test includes spraying of empty, top-open, double-covered ELOPAK-type cardboard boxes with a specific indicator organism, irradiation of these cardboard boxes with laser energy and testing the number of bacterial cells that survived after irradiation.

The indicator organism used in these studies was Bacillus subtilis var. globigii. To ensure a sufficient supply of the stock spore solution, a 0.5 ml sample was grown as a derivative in broth medium and was able to multiply at 37 ° C for 48 hours before centrifugation and harvesting of the bacterial culture in sterile Ringers solution. This solution was left at 4 ° C for a minimum of seven days before being used in the experiments.

The five inner surfaces of the cardboard box were sprayed with the indicator solution. The spraying device was a spray cannon held in hand, which spread the cells evenly over the surface of the cardboard box. The bacterial concentration range was in the order of 10⁷ up to 10⁸ cells on a cardboard box. The sprayed cardboard boxes were allowed to dry, at room temperature, in a laminar air hood for a maximum of 18 hours before use.

A wash test was used to detect surviving bacteria.

For testing, the spore suspension was diluted in Ringers solution to ensure serial dilution of this material from 10 '¹ up to 108 100 gl each

170 455 dilutions were applied and pipetted to the agar flakes, the plate was inverted and incubated at 37 ° C for 24 hours. Then a visual bacterial multiplication check was carried out. 2 ml of each bacterial dilution was sprayed onto the test carton boxes, which were allowed to dry. All reagent containers and Petri dishes were sterile before use and were aseptically operated under LPP laminam air flow conditions. Ringers' solution was autoclaved before adding Tween 20.

Patterns for spraying and loading the number of cells onto a cardboard box were checked by means of water test runs and opening the sides of the cardboard box. They were also confirmed by bacterial spraying and application to agar as well as in recovery tests. The spray cannon was cleaned with 70% ethanol before use and was able to dry under laminam airflow conditions. The cardboard box covers were prepared by soaking with 70% ethanol followed by protection with 00% ethanol and subsequent drying. Several lid samples were tested for sterility in ethanol treated broth.

Each rinse test experiment included checking for a non-machining condition, i.e. cardboard boxes without laser irradiation and cardboard boxes not sprayed with bacterial spores. The latter served as a control for background contamination. Most experiments included testing at least two bacterial dilutions. A typical course of the experiment was as follows: 3 cardboard boxes were sprayed with a 10 'dilution¹ spore suspensions, 3 further cardboard boxes were sprayed with a 10 'dilution² spore suspensions. For 10'1 dilution, cardboard boxes 1, 2 and 3 were used for the rinsing test, with boxes 1 and 2 being tested and box 3 control, i.e. the box was not irradiated with laser. The same was done with cartons sprayed with dilution 10 '2 spore suspensions.

Carton boxes were placed (each at a specified time) in a special laser assembly holder. After the irradiation time, the box was removed from this holder, covered with a sterilized cover, and the box was transported in laminar air flow. Handling of a cardboard box in a non-sterile environment is kept to an absolute minimum. There was only a potential risk of contamination when the cardboard box was removed from the handle and then moved.

ml of the test rinse solution was pipetted onto each test rinse box. The sterilized cover was placed on each cardboard box and these boxes were shaken vigorously at least 30 times on each side. Then they were able to stand for a short time, after which their contents were poured into separate, sterile containers. Serial dilutions were prepared from the recovered content using sterile Ringers solution. Volume 250 g or 500 g of each dilution and recovered undiluted content were spread on agar plates and incubated at 37 ° C for 24 hours.

Then laser tests were carried out. The range of tested energies varied from 1.0 Joule to 4.0 Jouli per cm2, with irradiation times of approximately 10 minutes. This period of time, which is sixty times the maximum time of ten seconds per cardboard box, justified by commercial considerations, was chosen for real experimentation in order to obtain a total dose equal to the commercially desired dose, used for ten seconds. Each cardboard box was placed in the handle in exactly the same position, with the code print of the box on the back of the handle. Attempts were made to keep the top of the cardboard box completely open, but in some cases it was slightly bent. The irradiation procedure was automated and computer controlled.

Initial attempts to test the test method showed that the spray system caused even distribution of bacteria, and that recovery tests were in most experiments at the same concentration level as those initially introduced. The description is given below

170 455 typical examples of individual testing methods followed by a summary of the most relevant experimental results.

Serial dilutions of the spore stock solution were prepared for each experimental run to correctly assess the bacterial concentration of each sample tested.

Then a rinsing test was carried out. The results for serial dilutions were used to qualify the concentration of bacteria applied to cardboard boxes and to check the accuracy of the recovery test. A control recovery test was included in each experimental run and the percentage of recovery was calculated for each bacterial concentration. Since the recovery method was found to be correct, the effectiveness of laser irradiation can be measured on the basis of recorded recovery results. The actual number of recovered colonies in the test sample was compared with the expected number of specific bacterial concentrations. Typical recovery results were slightly below the expected results.

Example of rinsing test:

<td>tests</td><td>Sample 10'1 rinsing test</td><td>Rinse control sample 101</td><td>Serial dilution</td>

<td>undiluted</td><td> &gt;200</td><td>bacteria coat</td><td>bacteria coat</td>

<td> 10‘1</td><td> 50</td><td>goatskin bacteria</td><td>bacteria coat</td>

<td> 10-2</td><td> 4</td><td>uncountable</td><td>bacteria coat</td>

<td> 10-3</td><td> 1</td><td> 278</td><td> 1315</td>

<td> 10⁴</td><td> 0</td><td> 27</td><td> 220</td>

Calculation example:

The concentration of the base material was calculated from the serial dilution result:

220 colonies at a dilution of 10 ^ for a 100 g sample on the plate, so for the 2.2 x 10 base material⁸.

10 'dilution of base material¹ so it should be 2.2 x 10⁷, but because 2 ml was used, the carton concentration was 4.4 x 107. Recovery in 20 ml of Ringers / T ween solution 20 and 100 g of measured aliquot per plate means that each result for dilution of the rinsing test must be multiplied by coefficients. The control sample shows 278 colonies in 10 'diluent³ recovered volume, so the calculation of the recovery concentration is 278 x 10³ x 10 x 20 = 5.6 x 107. This differs from the charge concentration by 1.2 x 107, which is a slight difference, and does not indicate any significant recovery problem. The actual sample of the laser irradiation test left approximately 200 colonies from a measured quantity of 100 g with 20 ml of volume of undiluted recovery, thus indicating that it survived 4.0 x 10⁴ colonies in this particular sample, giving a reduction log &lt;3.04, i.e. log (4.4 x 10⁷: 4.0 x 10⁴).

For a 250 g sample taken from a 10 ml wash test volume, the calculation factor is x40 (i.e. x4 x 10), for a 500 g sample taken from a 10 ml wash test volume, the calculation factor is x20 (i.e. x2 x 10).

The definition of Log reduction is as follows:

logio (cells per cardboard box: number of cells per box that survived).

Cardboard boxes were sprayed with 1-7-108 cells, irradiated with UV at 1, 2 or 4 Jouli / cm² and surviving spores were determined by washing rinsing using 10 ml rinsing solution and taking 500 g of bacterial culture. The kill rates found are given in Table 3.

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Table 3

Statement of results 10 ml volume of rinse test / 500 g or 250 g sample

<td>Cargo cells / box</td><td>Cells recovered rinsing test</td><td>Cells calculated for 10 ml</td><td>Energy J / cm²</td><td>Reduction logio</td>

<td>2.4 x 10⁷</td><td> 80**</td><td>1.6 x 10³</td><td> 1</td><td> 4,18</td>

<td>2.4 x 10⁷</td><td>2.4 x 10³ *'</td><td>4.8 x 104</td><td> 1</td><td> 2,7</td>

<td>2.4 x 10⁸</td><td>2.2 x 104 * 1</td><td>4.4 x 105</td><td> 1</td><td> 2,7</td>

<td>1.3 x 108</td><td>8.6 x 10³ *²</td><td>3.4 x 105</td><td> 1</td><td> 2,58</td>

<td>3.3 x 108</td><td>8.8 x 10³ *2</td><td>3.5 x 105</td><td> 1</td><td> 2,57</td>

<td>1.3 x 10⁷</td><td>1.12 x 102 * 2</td><td>4.48 x 10³</td><td> 1</td><td> 3,46</td>

<td>2.6 x 10⁸</td><td> 65 *2</td><td>2.6 x 10³</td><td> 2</td><td> 5,0</td>

<td>2.6 x 10⁸</td><td>1.44 x 102 * 2</td><td>5.76 x 10³</td><td> 2</td><td> 4,7</td>

<td>2.6 x 10⁸</td><td> 85 *2</td><td>3.4 x 10³</td><td> 2</td><td> 4,9</td>

<td>2.6 x 10⁸</td><td> 27 *2</td><td>1.08 x 10³</td><td> 4</td><td> 5,38</td>

<td>2.6 x 10⁸</td><td> 27,5 *2</td><td>1.10 x 10³</td><td> 4</td><td> 5,37</td>

* 1 (500 ul) 2 (250 gl)

Irradiation of 2 J / cm gives a reduction log greater than 5. The following conclusions have been drawn from the compilation for experimental results: Ultraviolet laser scanning of entire cardboard boxes sterilizes these boxes. Irradiation 2 J / cm2 gives a reduction log of Bacillus subtilis var. globigii equal to 5. The internal field of a 750 cm2 carton box requires 1500 Jouli for energy sterilization. Achieving a log reduction of 5 takes 10 seconds with a 150 W ultraviolet laser beam. It is possible to use very low concentration H2O 2 in combination with laser irradiation, both to reduce the required energy level for the same time as the cardboard box and to reduce the time for a cardboard box for the same energy level.

Example IV In this example, a qualitative estimation of the killing factor was investigated when the combined effects of laser UV and laser infrared were applied simultaneously. The experimental set was designed to have additional information about the heat generated during exposure and also about the impact of each of these samples separately.

A semi-finished product was used for a cardboard box (laminate of Al foil). The indicator organism was Bacillus subtilis var. globigii. A spore stock solution of this strain was used for the cardboard box material.

The trial contained three main phases, namely bacterial coverage on pre-sterilized cardboard strips, exposure of these strips to radiation, and analysis of spores that survived on these strips.

Prior to laser exposure, the cardboard material was cut with a scalpel to obtain 2 cm x 2 cm test strips. 10-fold and 100-fold dilution of a 4.5 x 10 stock spore suspension⁷ colonization units / ml, made in sterile saline (0.85% NaCl containing 0.02% Tween 20 to facilitate spattering), These dilutions contained 4.5 x 10 respectively⁶ and 4.5 x 10⁵ colonization units / ml.

Measured amounts of 100 g of each diluted suspension were spread on stripes to give 4.5 x 10 charges respectively⁵ and 4.5 x 10⁴.

The strips were allowed to dry at room temperature overnight. Some strips were left uncoated for sterility testing. After drying, each strip was placed in a sterile plastic bottle.

During irradiation, the strips were held vertically on a previously sterilized crocodile clip.

The 15W CO 2 laser used for these tests was an infrared CO 2 laser. It was controlled from a unit that provided radiation pulses lasting up to 6.6 seconds and variable output power in the range of 0-80% of the total output power.

170 455

This system was calibrated by measuring the temperature rise of test pieces of cardboard material of the same size as used for bioassays. The temperature was measured using a thermocouple that was attached to the front surface of the card.

Infrared radiation had two pulses of 6.6 seconds at 80% (approximately 40 J / cm2).

The highest surface temperature that could be achieved with front lighting was around 40 ° C. This was due to the presence of thin aluminum foil under the polyethylene cover on this side of the card that reflected a significant portion of the infrared radiation, preventing it from being absorbed into the mass of the card.

The excimer laser used a gas mixture of krypton and fluorine and emitted a light beam at a wavelength of 248 nm. The laser pulse energy was about 300 mJ. The laser radiation incident on the sample was calibrated using a detector placed outside the 2 cm x 2 cm opening. The pulse energy in this hole was 125 mJ. In this way, it was determined that the energy density of 1 J / cm2 required 32 pulses. The following tests were carried out using laser radiation:

1. Only 1.0 Jcm2 ultraviolet

2. Infrared on the front of the carton

3. 1.0 Jcm2 ultraviolet and infrared (simultaneously)

4. Ultraviolet 1.0 Jcm2, then a few seconds (2 to 3) later infrared (front)

5. Infrared (front) followed by 1.0 Jcm2 (30) seconds later.

Cards covered with bacteria were left without irradiation to determine maximum recovery. Cards not covered with bacteria were exposed to the atmosphere for about 30 seconds to check for accidental contamination. Then the number of surviving bacteria was determined. After irradiation, the cards returned immediately to their bottles and were stored overnight at 4 ° C.

10 ml sterile saline containing 0.1% Tween-20 was added to each bottle. The bottles were shaken vigorously for 30 seconds, after which the cards were removed and discarded. Secondary wash samples (100 g or 200 tl) were aseptically removed and spread on the surface of the agar plate. Some samples have been serially diluted to facilitate counting. Plates were incubated at 37 ° C for 24 hours. and colonies were counted. The total number of colonizing units in 10 ml washes was calculated from this, which gives the number of colonizing units recovered from this card.

The results are shown in Table 4.

Table 4

<td>Treatment</td><td>Loaded jk / ml</td><td>Recovered JK</td><td>Log. reduction</td>

<td>1 (i) Ultraviolet only 00</td><td>4.7 x 10⁵II</td><td>4.4 x 10³5.6 x 10³</td><td> 2,03 1,92 = 1,98</td>

<td>2 (i) Only infrared (ii)</td><td>11 II</td><td>4.5 x 10⁵4.5 x 10⁵</td><td> 0,02 0,02 = 0,02</td>

<td>3 (i) Ultraviolet and infrared simultaneously (Ii)</td><td> ,,</td><td>2.4 x 10³1.3 x 10³</td><td> 2,29 2,56 = 2,43</td>

<td>4 (i) Ultraviolet-infrared successively (ii)</td><td> ,,</td><td>1.40 x 10³2.2 x 10³</td><td> 2,53 2, 33 = 2,43</td>

<td>5 (i) Infrared-ultraviolet, followed by (ii)</td><td> ,,</td><td>7.2 x 10³contaminated</td><td> 1,81 2,56 = 1,81</td>

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According to the conducted experiments, infrared radiation alone did not cause bacterial killing. It is also clear that all treatments requiring essentially simultaneous infrared and ultraviolet at 1.0 Jem2 (No. 3 and 4), give a greater reduction logio than ultraviolet alone, infrared alone, or the sum of ultraviolet alone and infrared alone. It follows from these experiments that there is a cooperative effect when infrared and ultraviolet are used together.

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<img file="PL170455B1_D0001.tif" />

Figure 3

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