NL1037222C2 - METHOD AND DEVICE FOR STERILIZING OR SCREENING OR PROCESSING A POPULATION OF ORGANISMS. - Google Patents

Description

Method and device for sterilizing or screening or breeding a population of organisms

The present invention relates to a method and device for testing whether a fluid and / or object is sterile or can be made sterile, or to screen a population of organisms or to use selection breeding characterized by a container that is filled with at least one organism or germs or spores or seeds of an organism, means for exposing the organism to vibrations including ultrasonic vibrations and / or electric waves and / or magnetic waves and / or electromagnetic waves such as light and radio waves, means to keep the container under controlled conditions such as a climate cabinet and means to investigate whether the exposure of the container and / or the contents of the container is influenced by the vibrations.

preface

There is a growing need for sustainable technology that makes it possible to produce and / or disinfect and / or optimize and / or refine products without the use of chemicals and at low energy costs. Non-limiting examples of products for which this is interesting are: water, foodstuffs, pharmaceutical products including medicines and intermediates from which medicines can be made, enzymes used in the pharmaceutical industry, plants including greenhouse plants, bulbs, trees and algae, fish, shellfish , aquariums, ponds, populations of bacteria, fungi and yeasts.

Examples of new emerging technologies that make it possible to produce or treat products in a sustainable manner are the treatment of a fluid or object with vibrations such as electrical vibrations, magnetic vibrations, electromagnetic vibrations and ultrasonic vibrations.

The present invention relates to a method and device with which it is possible to quickly, cheaply, efficiently and automatically investigate the influence of vibrations on organisms in a fluid or on an object or to screen organisms and to use breeding in this way.

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Description of the technology according to the present invention

According to a first aspect, the present invention consists of a container in which an object and / or an organism and / or a fluid can be placed. In the present invention, organism is also understood to mean spores, seeds, germs of an organism, and virus particles. Non-limiting examples of a container are a test tube, a petri dish. The container is preferably designed such that it can be mass-produced, internally sterile and long-lasting and prepared in such a way that it can be sold separately as standard starting material for use in combination with the present invention.

According to a second aspect, the present invention consists of means for treating at least a part of the holder and / or the contents of the holder with vibrations, electromagnetic waves including light, UV light, infrared light, pulsed light, radio waves including frequency, amplitude , phase modulated radio waves, alpha, beta and gamma radiation, electric waves, magnetic waves, ultrasonic vibrations. The device preferably comprises a large number of placement options for containers, so that a large number of containers can be examined and / or treated automatically or simultaneously or successively. Preferably, the treatment is fully automated and software-driven.

According to a third aspect, the present invention consists of means for growing at least one container with contents in a controlled manner in an incubator or climate cabinet. The device, i.e. the incubator or climate cabinet, preferably comprises a large number of placement options for containers, so that a large number of containers can be treated automatically or simultaneously at the same time. Preferably, the treatment is fully automated and software-driven.

According to a fourth aspect, the present invention consists of means such as a microscope, an oxygen sensor, a carbon dioxide sensor, a pH sensor but not limited to examining at least one container with content so that it can be determined what the influence of the vibrations on the organism has been . The device for determining the influence of the vibrations on the organism preferably comprises a large number of placement options for containers, so that a large number of containers can be automatically or simultaneously examined in succession. The testing of the samples is preferably carried out fully automatically and with software control.

Now that the core of the present invention has been briefly described, a first non-limiting embodiment of the present invention is described for further clarification with which it is possible to test whether a fluid is sterile. A number of other preferred embodiments are then described.

Description of the first embodiment: In microbiology, it is often desirable to quickly test whether a fluid in general or a fluid in particular is sterile. In a standard test method to investigate whether a liquid is sterile, use is made of sterile petri dishes containing a culture medium for microorganisms. These Petri dishes are available in diameters of a few centimeters up to a diameter of approximately 15 35 centimeters and have a characteristic height of 1 to 2 cm. The Petri dishes are provided with a lid to prevent the ingress of contaminating spores. To examine a liquid for the presence of microorganisms, the lid of a sterile Petri dish with culture medium is removed and a small amount of the liquid to be tested is applied to the culture medium in the Petri dish. The Petri dish is then covered again with the lid and placed in the incubator for a few days to promote the multiplication of any micro-organisms present in the liquid. If colonies of microorganisms are visible after a few days in the form of colored or non-colored spots on the culture medium, the investigator knows that the liquid applied to the culture medium was not sterile.

It is known to the person skilled in the art that petri dishes often become infected with (traces of) microorganisms when the lid is lifted from the sterile petri dish.

Especially if the petri dishes are opened in an environment that contains a lot of dust and / or traces / and / or very finely dispersed droplets, the test described above becomes very quickly unreliable. Thus, the inventor of the present invention has found that opening a petri dish with a sterile culture medium as contents, for 15 seconds in a well-ventilated space of a workplace, led to infection of the culture medium in about 10% of the cases. One could perform the preparation of the petri dishes i.e. applying the water samples to be examined on the petri dishes in a suitable laboratory room where measures have been taken to minimize the number of microorganisms per cubic meter of air but this often entails additional costs. Furthermore, it is difficult to test an amount of air instead of liquid for 20 (traces of) microorganisms. In addition, petri dishes are unnecessarily large and unnecessarily expensive for a large number of applications. Petri dishes with culture medium are also not suitable devices for investigating the influence of ultrasonic vibrations and / or electric waves and / or magnetic waves and / or electromagnetic waves on the chances of survival of these microorganisms due to respectively vibrating lids and suboptimal geometry around electric waves. and / or transfer magnetic waves and / or electromagnetic waves to the medium in the petri dishes.

The present invention relates to a method and device for examining both air and water samples in an inexpensive and rapid manner for the presence of (traces of) microorganisms in which the risk of contamination of the culture medium by (traces of) microorganisms from the organisms environment is considerably smaller than when petri dishes are used in the same environment. The present invention also relates to a method and device for studying the chance of survival of microorganisms under the influence of ultrasonic vibrations and / or electric waves and / or magnetic waves and / or electromagnetic waves. An important distinguishing aspect of the present invention compared to the prior art is that both the treatment of the samples with vibrations, the treatment in the climate cabinet and the analysis of the samples take place in an entirely automatic manner. This makes it possible to use a so-called "high throughput" strategy of experimentation 4 in which a very large number of samples are treated differently and analyzed in a fully automatic manner. Automation is of particular importance for screening purposes, breeding cultures or investigating which vibrations (both in terms of amplitude, frequency and modulation form) are the most effective. It is noted here that the automation can be realized cheaply and effectively by providing the device with one or more microcontrollers which preferably communicate with each other. Non-limiting examples of such microcontrollers are PIC16F84A, PIC12F629. Of course you can also opt for a PC in combination with interface and software. It is known to those skilled in the art that stepper motors form an important part of a fully automated unit according to the technology of the present invention. As a basic and non-limiting example, small glass test tubes are used in the first embodiment, preferably with a diameter of less than 20 mm and a length of less than 200 mm. A standard gelatin and / or agar nutrient medium for counting plates is poured into these test tubes. Preferably, an amount of nutrient medium is poured into the tubes such that they are filled for approximately 70%. The test tubes including contents are then sterilized, preferably by placing the test tubes in an incubator at a temperature of 100 degrees Celsius for 30 minutes. After cooling, the opening at the top of the sterilized test tubes with the solidified gelatin and / or agar culture medium is glued up, preferably with a thermoplastic glue but not limited thereto. A particularly suitable glue for use in the present invention is ethylene-vinyl acetate copolymer-based glue as commercially available in the form of "Pattex Hot Glue Cartridges" to be used in combination with a "Pattex Hot Glue Gun". Since this glue, at the moment it is applied to the top of the test tube by means of a glue gun, has a temperature that is higher than 100 degrees Celsius, the glue is sterile and eminently suitable for sealing off the test tube without sealing it. to infect the nutrient medium. Furthermore, this glue appears to contain virtually no solvents, so that the glue has no influence on any chance of survival of (traces of) microorganisms in the liquid to be investigated. After the glue has cooled, the disinfected culture medium in the test tube 30 is hermetically sealed from the environment. Experiments with test tubes prepared in this way show that the culture medium in these test tubes, after being in a living room for a few months or in an incubator at 30 degrees Celsius, is still sterile. If it is necessary to examine whether a liquid is sterile, an amount of the liquid to be tested is drawn up with a sterile syringe. Subsequently, a test tube with sterile culture medium sealed with thermoplastic glue is taken as contents, the needle of the syringe is punctured by the thermoplastic glue and a few drops of liquid are introduced into the test tube. After this, the injection needle is withdrawn and the hole arranged in the thermoplastic glue is sealed with the glue gun. The test tube is then placed in an incubator at 30 degrees Celsius and after a few days the contents of the test tube are subjected to a visual inspection. If the culture medium visibly changed color relative to a blank measurement, the liquid sample to be examined was not sterile.

It is clear to the skilled person that when this procedure is applied the chance of infection of the culture medium with (traces of) microorganisms from the environment is minimal and that this procedure, in contrast to the procedure with the petri dishes, is suitable for use outside a laboratory environment to fit. Furthermore, it is clear to the person skilled in the art that the procedure and the device with the test tube are also suitable for testing gases for sterility, while this is virtually impossible in the case of petri dishes. It is also clear to those skilled in the art that the preparation of test tubes according to the above procedure so that these test tubes can be used to test whether or not a fluid is sterile can be easily automated. In this way it becomes possible to produce test tubes as a mass product in a highly reproducible and low-cost manner which, in combination with a simple glue gun for thermal glue, can also be used outside the laboratory to collect samples and set them to culture. The above method for making test tubes in a manual manner and / or in an automated manner and the above-described arrangement of test tubes form an explicit part of the present invention. The test tubes can also be used to study the influence of electric waves and / or magnetic waves and / or radio waves on the chances of survival of (traces of) microorganisms. To this end, a number of test tubes are deliberately infected with a microorganism of choice according to the above procedure with the syringe. After the hole of the syringe with the glue gun has been sealed and the test tube has been hermetically sealed off from the environment, a coil is slid around the tube. This coil preferably occupies the entire length of the tube and the number of turns of the coil depends inter alia on the applied frequency of the electric wave and / or magnetic wave and / or radio wave. The coil is then connected to the output stage of a transmitter and / or to a current source and in this way the microorganisms in the tube are exposed to the waves. Optionally, the coil is not connected directly to the output stage of a transmitter or coupled to a power source, but energy transfer takes place to the coil around the test tube via an antenna device or by induction. After the treatment with the electric waves and / or magnetic waves and / or radio waves, the tubes are optionally combined with tubes that have not undergone treatment with waves and act as a blank, preferably a number of days placed in the incubator at a temperature of at preferably 30 degrees Celsius but not limited to that.

After a few days the tubes are visually inspected and a conclusion can be drawn about the influence of the electric waves and / or the magnetic waves and / or the radio waves on the chances of survival of the microorganisms. It is clear to a person skilled in the art that the geometry of the test tubes with culture medium according to the present invention is eminently suitable for transfer of electric waves and / or magnetic waves and / or radio waves to the microorganisms in the test tube, whereby the method and device according to The present invention has major advantages over systems based on Petri dishes.

The test tubes can also be used to study the influence of ultrasonic vibrations on the chances of survival of microorganisms. To this end, the test tubes are vibrated using a commercially available ultrasonic water bath and / or an ultrasonic water bath with adjustable power and / or an ultrasonic water bath with adjustable frequency and / or an ultrasonic transducer with adjustable frequency and / or an ultrasonic transducer with adjustable power and / or an ultrasonic transducer containing one or more piezo elements.

In a third preferred embodiment, the test tubes are used to determine the combined influence of ultrasonic vibrations and electric waves and / or magnetic waves and / or electromagnetic waves on the chances of survival of microorganisms. In the first embodiment, the device for treating the tubes with vibrations is preferably designed such that at the same time a large number of tubes, preferably more than 5, more preferably more than 10, more preferably more than 100 and most preferably preferably more than 500 can be placed. It is clear to a person skilled in the art that a very large number of samples can be treated quickly and efficiently in this way. In particular if the tubes are treated with light or with waves via coils around the tubes, this method is very suitable because it is simple and inexpensive to place a light source (preferably an LED) or a coil at every placement option. Even more preferably, the device for handling the tubes is designed such that tubes are fed fully automatically, for example via a sample carousel or a conveyor belt construction, into the device for treating the sample with vibrations, after which the treatment takes place in a controlled manner, tubes are automatically placed out of the device and a subsequent sample 30 can be processed. It is clear to the skilled person that this embodiment has the advantage that the device for automatic treatment of the samples is small and therefore inexpensive.

In a second embodiment, a container is used in combination with the present invention which is at least partly made of a metal and / or a composite and / or a metal-containing coating such as a so-called high-density coating, and / or a plastic and / or carbon and / or ceramic. By making a container which, for example, partly consists of metal or a metal-containing coating, it is possible to selectively treat samples with vibrations 7 without other samples in the vicinity being also treated by stray fields. By making a container that is only permeable to light in certain places (for example, only the bottom of a test tube), it can be ensured that samples can be treated very selectively while they are close together. If LEDs are used, this selectivity is further enhanced by the small scattering angle that is characteristic of LEDs.

In a third embodiment, the container used in the present invention comprises at least in part a material which is permeable to an injection needle, such as silicone rubber, but not limited to, thermal glue. This makes it possible to inject a fluid into the container automatically and in a reproducible manner, for example in the same way as happens in liquid or gas chromatography analysis equipment. By automatically disinfecting the injection needle before and after each injection, the chance of unwanted infection of the contents of the container is minimized. A device according to the technology of the present invention in which a fluid is automatically injected into a suitable container by means of an injection needle forms an explicit part of the present invention.

In a fourth embodiment, the technology of the present invention is used for so-called "high throughput experiments". In short, this amounts to the automatic treatment of very large quantities of samples with vibrations, whereby the nature of the samples and / or the nature of the vibrations is varied according to a predetermined program. This is preferably done via a test set-up planned using statistics. This makes it possible to perform a very large number of measurements in the short term at low costs and to map the effects of exposure of organisms to vibrations or combinations of vibrations.

In a fifth embodiment, the technology of the present invention is used to screen populations of algae placed in containers. In the present application, screening means: investigating the composition of a population and / or selectively killing a part of the population so that it has fewer undesirable and more desirable properties and / or the generation of mutations in a population as a result 30 exposure to vibrations and / or suppressing the metabolism of at least a part of the population and / or promoting the metabolism of at least a part of the population. Examples are the production of carotenoids that are produced in particular in stress situations of the algae, the fat content, the compositional distribution of the fatty acids in the fats, the amount of phospholipids and the composition of these phospholipids.

In a sixth embodiment, the technology according to the present invention is used to investigate with which (combination of) vibrations parasites of plants can be killed.

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In a seventh embodiment, the technology of the present invention is used to promote the metabolism of microorganisms or to screen or kill microorganisms used in water treatment plants or bioreactors or fermentors or biogas plants or ponds or fish ponds for fish farming for consumption of those fish or milk products or cheese products.

In an eighth embodiment, the technology according to the present invention investigates whether the production of a substance present in plants or another organism can be increased by applying vibrations.

In a ninth embodiment, the device according to the present invention comprises at least one microprocessor for automating at least one step of the sample handling process, at least injection equipment for automatically injecting a fluid into a container and software for controlling the device.

In a tenth embodiment, the technology according to one of the previous embodiments is used from 9 to 9 to selectively cultivate or kill (seed cultures of) algae, bacteria, plants, fish, bacteriophages, fungi, yeasts, to screen and / or treat plant seeds, spores and / or activate them. In a tenth embodiment, one or more combinations of the technologies described in embodiment 1 to 9 are used.

1037222

Claims (28)

Device for testing whether a fluid and / or object is sterile or can be made sterile or for screening a population of organisms or for applying breeding by selection characterized by 5. a container containing means for at least a part of it of the container to be treated with vibrations including ultrasonic vibrations and / or electric waves and / or magnetic waves and / or electromagnetic waves 2. Device as claimed in claim 1, plus means in which at least placement of the holder in the device for treating the holder with vibrations takes place automatically by using at least one microprocessor and / or sensor. Device as claimed in any of the foregoing claims 1 and 2, wherein at least removal of the holder from the device to treat the holder with vibrations takes place automatically. Device as claimed in any of the foregoing claims 1-3, wherein the means for treating the container with vibrations can contain more than one container at a time and wherein at least two containers of a different adjustable duration and / or intensity and / or nature of the vibration (s) can be exposed. 5. Device as claimed in any of the foregoing claims 1-4, wherein this device comprises at least one stepper motor. Device as claimed in any of the foregoing claims 1-5, wherein the holder consists at least partly of metal or contains at least metal particles. Device as claimed in any of the foregoing claims 1-6, wherein the vibration is an ultrasonic vibration. Device as claimed in any of the foregoing claims 1 to 7, wherein the vibration is light from the UV region and / or the infrared region. Device according to one of the preceding claims, wherein the vibration is an electromagnetic vibration of radio waves, modulated radio waves, alpha radiation, beta radiation, gamma radiation. Device as claimed in any of the foregoing claims 1-9, wherein at least a part of the holder is exposed to more than one vibration at the same time. Device as claimed in any of the foregoing claims 1-10, wherein at least a part of the holder is successively exposed to different vibrations. The device of claims 1 to 11 wherein the organism is a plant. The device of claims 1 to 11, wherein the organism is an alga. The device of claims 1 to 11 wherein the organism is a bacterium. The device of claims 1 to 11 wherein the organism is baceriophage. 1037222 Device according to claims 1 to 11, wherein the organism consists of spores or seeds of an organism. Device as claimed in any of the foregoing claims 1 to 16, wherein the holder contains a culture medium with at least gelatin and / or agar. Device as claimed in any of the foregoing claims 1 to 17, wherein the means for treating at least a part of the holder with electric and / or magnetic and / or electromagnetic waves consist of at least one coil wound around the holder or around the holder is placed. 19. Device as claimed in any of the foregoing claims 1-18, wherein the means for treating at least a part of the holder with electric and / or magnetic and / or electromagnetic waves consist of at least one spiral wound coil on which the holder can be placed . 20. Device as claimed in any of the foregoing claims 1-19, wherein the means for treating at least a part of the holder with electric and / or magnetic and / or electromagnetic waves consist of at least one ultrasonic (water) bath in which at least one part of the holder. Device as claimed in any of the foregoing claims 1-20, wherein the means for treating at least a part of the holder with electric and / or magnetic and / or electromagnetic waves consist of at least one ultrasonic transducer 20 which is operatively connected to the holder. Device as claimed in any of the foregoing claims 1 to 21, wherein the container is closed by an object that is permeable with an injection needle so that a fluid to be examined can be injected into the container by means of a syringe. 23. Device as claimed in any of the foregoing claims 1-22, wherein the holder is closed by a cap or stopper or other object which consists at least in part of a material which is permeable to a syringe. 24. Device as claimed in any of the foregoing claims 1-23, wherein the means for supplying energy to the coil consist of at least one transmitter or a power source or a device for transferring energy via induction to the coil around the test tube or an antenna device for transferring energy to the coil around the test tube. A method for testing whether a fluid is sterile or can be made sterile and / or the metabolism of organisms in the fluid can be changed with a device according to any one of the preceding claims 1 to 24, characterized by 35. Placing a gelatin and / or agar-based culture medium in a container and • sealing off the contents of the container from the environment by means of an object permeable with an injection needle • sterilizing the contents of the container and • applying a sample of a test fluid to the container with a sterile syringe provided with an injection needle and 5. exposing the container containing the test sample to ultrasonic and / or electromagnetic waves and / or electric waves and / or magnetic waves and • putting the thus prepared samples on culture by storing the container under controlled conditions for more than 1 minute and less than 1 10 years and • subjecting the sample thus grown to an inspection to assess whether microorganisms have multiplied in the tube. • The automatic execution of at least one of these steps using a microprocessor or a PC with an interface and software. The method of claim 25 wherein a coil is disposed around the test tube containing the sample to be examined to study the influence of electrical and / or magnetic and / or electromagnetic waves on the chances of survival of microorganisms wherein the coil is supplied with energy by connecting this coil 20 directly to the output of a transmitter or a power source or indirectly via induction or an antenna device. A method according to claim 25, wherein the test tube with the sample to be tested is subjected to ultrasonic vibrations to investigate the influence of these ultrasonic vibrations on the chances of survival of microorganisms. A method according to claim 25, wherein the test tube with the sample to be tested is exposed to both ultrasonic vibrations and to electric and / or magnetic and / or electromagnetic waves to study the combined influence of these types of vibrations on the chances of survival of microorganisms . 1037222