Indian Patents. 242083:A PROCESS FOR ISOLATING BIOFILM FORMING MICRO-ORGANISM

Title of Invention	A PROCESS FOR ISOLATING BIOFILM FORMING MICRO-ORGANISM
Abstract	The invention concerns a novel method for isolating micro-organisms based on the capacity of said micro-organisms to adhere to the surface of particles.

Full Text	FORM 2 THE PATENT ACT 1970 (39 of 1970) & The Patents Rules, 2003 COMPLETE SPECIFICATION (See Section 10, and rule 13) 1. TITLE OF INVENTION METHOD AND DEVICE FOR ISOLATING MICRO-ORGANISMS 2. APPLICANT(S) a) Name b) Nationality c) Address BIOFILM CONTROL FRENCH Company BIOPOLE CLERMONT-LIMAGNE, F-63360 SAINT BEAUZIRE, FRANCE 3. PREAMBLE TO THE DESCRIPTION The following specification particularly describes the invention and the manner in which it is to be performed : - The present invention relates to the isolation of micro-organisms. Many micro-organisms develop by synthesizing a biofilm. Besides bacteria, fungi, algae, and protozoa are also organized in biofilms. Biofilms are thus found in numerous fields where they involve health risks and may cause relatively important damages. When a biofilm develops, bacteria first adhere to a support and then colonise the support. When multiplying, the bacteria rapidly form a film constituted of cell bodies layers which secrete a matrix of exopolysaccharides which protect them against the environmental stress (COSTERTON and al., Science, vol. 284 (5418), p: 1318-22, 1999). The kinetics of the formation of a biofilm can be subdivided into 5 steps (Figure 1): surface conditioning: the organic or mineral molecules existing in the liquid phase will adsorb on the surface and will form a "conditioning film". the adherence or reversible adhesion: the existing micro-organisms get closer to the surfaces by gravimetry, Brownian movements or chemiotaxis, if they have flagella. During this first fixing step, wherein only purely physical phenomena and weak physido-chemical interactions are executed, the microorganisms can still be easily disconnected. the adhesion: this step is slower and requires interactions of higher energy as well as the microbial metabolism and the cell appendices of the microorganism (flagella, pili,....). Adhesion is an active and specific phenomenon. The first colonizing micro-organisms will irreversibly cling to the surface, more particularly through the synthesis of exopolysaccharides (EPS). This process is relatively slow and depends on the environmental factors and the existing micro-organisms. 2 the maturation of the biofilm (development and colonisation of the surface): when they have adhered to a surface, the bacteria multiply and gather together to form micro-colonies surrounded by polymers. Such polymer matrix (or glycocalix) will act as a "cement" and reinforce the association of bacteria with one another and with the surface, to finally form a biofilm and reach a state of equilibrium. The biofilm generally develops into a three-dimension structure, which constitutes a confinement place. Such micro-environment will be the head quarters of numerous physiological and molecular modifications with respect to the plankton growth mode. If the conditions are favourable, the thus formed biofilm will fill the whole surface that is available. The maturation of the biofilm is generally correlated to the production of EPS, even though some species of micro-organisms which synthesize few or no polymers can also adhere and form biofilms on surfaces. Disconnection: Biofilms are structures in perpetual dynamic equilibrium which evolve according to the support, the micro-organisms and the environment. Such evolution can result in cells or aggregates disconnection. According to the present invention, the adhesion and fixation property of microorganisms is used to isolate them. The invention principle consists in adding into a more or less liquid raw sample of a medium, the micro-organisms contamination of which is to be studied, magnetic or magnetisable particles and more particularly balls, leave the balls in contact with the medium for a time sufficient for the micro-organisms to adhere to the particles surfaces, isolate said particles using all appropriate means, particularly a magnet and spread said particles on an appropriate solid culture medium so as to obtain a culture of said micro-organisms trapped in the particles. Thus, the object of the present invention is a process for isolating at least a microorganism from the medium which contains them, comprising the following steps: 3 a) introducing a given quantity of magnetic or magnetisable particles into a sample of said medium; b) incubating the particles and the medium for a sufficient time for the microorganisms to adhere to the surface of said particles; c) separating said particles from the medium; d) spreading said particles on a support compatible with the development of the micro-organisms; e) incubating said particles on a support for a sufficient time for the development of colonies corresponding to the isolated micro-organism. According to a particular embodiment of the invention, the process may comprise a prior step of pre-culture of the sample from said medium containing the microorganism to be isolated. Therefor, said sample is heated up to a temperature compatible with the viability of the micro-organisms. It is known that, in addition to the organisms which live at conventional temperatures (20 to 50 degrees Celsius), some micro-organisms live in extreme conditions, as regards temperature, gas partial pressure (Oxygen, nitrogen, carbon dioxide, ...), salinity, pH (acid, basic), redox and or in aerobic or anaerobic conditions. Most currently, culture temperatures may be between 20 and 50 degrees Celsius, preferably between 30 and 40 degrees Celsius. Such step of pre-culture, the object of which is to enrich the culture medium with micro-organisms, can be carried out in an extensively variable time, depending on the micro-organisms, which can extend between 20 minutes and 7 to 10 days, preferably between 1 hour and 48 hours, while possibly being stirred. According to yet another particular embodiment of the invention, the process may include an additional step between steps c) and d) of the invention's process which consists in the optional immersion of the balls obtained in step c) into an 4 advantageously aqueous washing solution, which makes it possible to eliminate the non adherent micro-organisms (which are present in the ball saturation ("imbibition") liquid). Such step makes it possible to select the most adherent microorganisms whose adhesion is the most irreversible. Such step may also be the opportunity of applying rapid or extended treatments with a view to testing the adhesion property of micro-organisms (preventive or curative treatments in the washing solution). One skil in the art knows how to determine without any difficulty the quantity of balls to be introduced into the medium. According to the invention, the incubation of step b) carries on for a time which can last from a few seconds to a few hours, preferably between 15 seconds and 45 minutes, depending on the micro-organisms. After the incubation time, the micro-organisms have had the possibility of adhering to the particles (balls). According to the invention, the separation of the particles and the medium in step c) can be carried out by any method known to one skill in the art. For example, such particles could be sampled by centrifugation and elimination of the culture medium, or further, and preferably according to the invention by using a system generating a magnetic or electric field capable of attracting the particles, particularly a magnet. According to this particularly preferred embodiment, the particles are sampled using a magnet which is advantageously dipped into the sample. According to an advantageous embodiment, the system generating a magnetic or electric field capable of attracting the particles, particularly a magnet, may be protected, by any system, particularly by a removable coating or a cover, made of any material, for example, plastics, which does not interfere with magnetic or electric 5 waves. More advantageously still, said cover is disposable after use. Such magnet could then be used again. According to yet another particular embodiment of the invention, the method may include an additional step of washing the system generating a magnetic or electric field so as to eliminate the non-adherent micro-organisms which are present in the wetting liquid, or the micro-organisms which do not adhere much. During this additional step, said system is dipped into a washing solution which may be a sterile culture medium, for instance. One skill in the art understands that, in principle, such step only lasts for a few seconds, at most a few minutes, which is the time required for eliminating the micro-organisms which did not adhere to the system surface. According to the invention, the spreading of said particles on a support compatible with the development of the micro-organisms can be carried out by a deposition of said particles onto the surface of a micro-organism culture device, for example a Petri dish containing an appropriate culture medium for the development of the micro-organisms. According to a particular embodiment, the deposition can be carried out by taking the magnet out of the plastic cover while placing the plastic cover closer to the micro-organism culture device surface. According to another embodiment of the invention, the balls may be deposited using another magnet placed under the culture device surface. Any system known to one skill in the art, such as a manual spreader, for example, can be used for spreading and dispersing the balls. According to a particular embodiment, a rotating magnet is used and placed under the micro-organism culture device surface. 6 The ball dispersion can also be obtained by means of a liquid vortex generated by the rotation of the micro-organism culture device. When the particles (balls) are dispersed, the culture device is placed into an incubator for a time sufficient for the micro-organisms to develop on the device surface. Then, one skill in the art will adapt the incubation time and the temperature to the micro-organism to be isolated. This time can be between a few hours and several days, preferably between 4 days and 48 days. The incubation temperature can be between 30 and 40 degrees Celsius. Other advantages of the invention will appear in the appended Figures, in which: Figure 1 shows a comparison of conventional methods for sampling microorganisms (A) and the method according to the invention (B); Figure 2 shows the steps of sampling and washing the particles; Figure 3 shows the deposition of particles onto the surface of a Petri box; Figure 4 shows the dispersion of the particles on the surface of the Petri box, using a rotating magnet. 7 WE CLAIM: 1. A process for isolating at least one micro-organism from the medium which contains them, comprising the following steps: a) introducing a given quantity of magnetic or magnetisable particles into a sample of said medium; b) incubating the particles and the medium for a sufficient time for the microorganisms to develop and to adhere to the surface of said particles; c) separating said particles from the medium; d) spreading said particles on a support compatible with the development of the micro-organisms; e) incubating said particles on a support for a sufficient time for the development of colonies corresponding to the isolated micro-organism. 2. A process according to claim 1, characterized in that it further comprises a prior step of pre-culture of the sample from said medium containing the micro-organism to be isolated, possibly while it is stirred. 3. A process according to claim 2, characterized in that said sample is heated up to a temperature compatible with the viability of the micro-organisms, more particularly up to a temperature between 20 and 50 degrees Celsius, and preferably between 30 and 40 degrees Celsius. 4. A process according to claim 2, characterized in that the pre-culture step has a duration between 20 minutes and 10 days, preferably between 1 hour and 48 hours. 8 5. A process according to any one of claims 1 to 4, characterized in that it further includes an additional step between steps c) and d) which consists in the optional immersion of the balls obtained in step c) into an advantageously aqueous washing solution. 6. A process according to any one of claims 1 to 5, characterized in that the incubation of step b) carries on for a time which can last from a few seconds to a few hours, preferably between 15 seconds and 45 minutes, depending on the micro-organisms. 7. A process according to any one of claims 1 to 6, characterized in that the separation of the particles and the medium in step c) is carried out by centrifugation and elimination of the culture medium, or by using a system generating a magnetic or electric field capable of attracting the particles. 8. A process according to any one of claims 1 to 6, characterized in that the separation of the particles and the medium in step c) is carried out using a magnet. 9. A process according to claim 8, characterized in that the magnet is dipped into the sample. 10. A process according to any one of claims 1 to 7, characterized in that the system generating a magnetic or an electric field capable of attracting the particles is protected, particularly by a removable coating or a cover. 9 11. A process according to claim 10, characterized in that said removable coating or cover is disposable. 12. A process according to any one of claims 7 to 10, characterized in that it includes an additional step of washing the system generating a magnetic or electric field. Dated this 19th day of September 2007 10 ABSTRACT The invention concerns a novel method for isolating micro-organisms based on the capacity of said micro-organisms to adhere to the surface of particles. To The Controller of Patent The Patent office Mumbai

Full Text

FORM 2
THE PATENT ACT 1970 (39 of 1970)
&
The Patents Rules, 2003 COMPLETE SPECIFICATION
(See Section 10, and rule 13)
1. TITLE OF INVENTION
METHOD AND DEVICE FOR ISOLATING MICRO-ORGANISMS

2. APPLICANT(S)
a) Name
b) Nationality
c) Address

BIOFILM CONTROL
FRENCH Company
BIOPOLE CLERMONT-LIMAGNE,
F-63360 SAINT BEAUZIRE,
FRANCE

3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed : -

The present invention relates to the isolation of micro-organisms.
Many micro-organisms develop by synthesizing a biofilm. Besides bacteria, fungi, algae, and protozoa are also organized in biofilms.
Biofilms are thus found in numerous fields where they involve health risks and may cause relatively important damages.
When a biofilm develops, bacteria first adhere to a support and then colonise the support. When multiplying, the bacteria rapidly form a film constituted of cell bodies layers which secrete a matrix of exopolysaccharides which protect them against the environmental stress (COSTERTON and al., Science, vol. 284 (5418), p: 1318-22, 1999). The kinetics of the formation of a biofilm can be subdivided into 5 steps (Figure 1):
surface conditioning: the organic or mineral molecules existing in the liquid phase will adsorb on the surface and will form a "conditioning film".
the adherence or reversible adhesion: the existing micro-organisms get closer to the surfaces by gravimetry, Brownian movements or chemiotaxis, if they have flagella. During this first fixing step, wherein only purely physical phenomena and weak physido-chemical interactions are executed, the microorganisms can still be easily disconnected.
the adhesion: this step is slower and requires interactions of higher energy as well as the microbial metabolism and the cell appendices of the microorganism (flagella, pili,....). Adhesion is an active and specific phenomenon. The first colonizing micro-organisms will irreversibly cling to the surface, more particularly through the synthesis of exopolysaccharides (EPS). This process is relatively slow and depends on the environmental factors and the existing micro-organisms.
2

the maturation of the biofilm (development and colonisation of the surface): when they have adhered to a surface, the bacteria multiply and gather together to form micro-colonies surrounded by polymers. Such polymer matrix (or glycocalix) will act as a "cement" and reinforce the association of bacteria with one another and with the surface, to finally form a biofilm and reach a state of equilibrium. The biofilm generally develops into a three-dimension structure, which constitutes a confinement place. Such micro-environment will be the head quarters of numerous physiological and molecular modifications with respect to the plankton growth mode. If the conditions are favourable, the thus formed biofilm will fill the whole surface that is available. The maturation of the biofilm is generally correlated to the production of EPS, even though some species of micro-organisms which synthesize few or no polymers can also adhere and form biofilms on surfaces.
Disconnection: Biofilms are structures in perpetual dynamic equilibrium which evolve according to the support, the micro-organisms and the environment. Such evolution can result in cells or aggregates disconnection.
According to the present invention, the adhesion and fixation property of microorganisms is used to isolate them.
The invention principle consists in adding into a more or less liquid raw sample of a medium, the micro-organisms contamination of which is to be studied, magnetic or magnetisable particles and more particularly balls, leave the balls in contact with the medium for a time sufficient for the micro-organisms to adhere to the particles surfaces, isolate said particles using all appropriate means, particularly a magnet and spread said particles on an appropriate solid culture medium so as to obtain a culture of said micro-organisms trapped in the particles.
Thus, the object of the present invention is a process for isolating at least a microorganism from the medium which contains them, comprising the following steps:
3

a) introducing a given quantity of magnetic or magnetisable particles into a sample of said medium;
b) incubating the particles and the medium for a sufficient time for the microorganisms to adhere to the surface of said particles;
c) separating said particles from the medium;
d) spreading said particles on a support compatible with the development of the micro-organisms;
e) incubating said particles on a support for a sufficient time for the development of colonies corresponding to the isolated micro-organism.
According to a particular embodiment of the invention, the process may comprise a prior step of pre-culture of the sample from said medium containing the microorganism to be isolated. Therefor, said sample is heated up to a temperature compatible with the viability of the micro-organisms. It is known that, in addition to the organisms which live at conventional temperatures (20 to 50 degrees Celsius), some micro-organisms live in extreme conditions, as regards temperature, gas partial pressure (Oxygen, nitrogen, carbon dioxide, ...), salinity, pH (acid, basic), redox and or in aerobic or anaerobic conditions. Most currently, culture temperatures may be between 20 and 50 degrees Celsius, preferably between 30 and 40 degrees Celsius. Such step of pre-culture, the object of which is to enrich the culture medium with micro-organisms, can be carried out in an extensively variable time, depending on the micro-organisms, which can extend between 20 minutes and 7 to 10 days, preferably between 1 hour and 48 hours, while possibly being stirred.
According to yet another particular embodiment of the invention, the process may include an additional step between steps c) and d) of the invention's process which consists in the optional immersion of the balls obtained in step c) into an
4

advantageously aqueous washing solution, which makes it possible to eliminate the non adherent micro-organisms (which are present in the ball saturation ("imbibition") liquid). Such step makes it possible to select the most adherent microorganisms whose adhesion is the most irreversible. Such step may also be the opportunity of applying rapid or extended treatments with a view to testing the adhesion property of micro-organisms (preventive or curative treatments in the washing solution).
One skil in the art knows how to determine without any difficulty the quantity of balls to be introduced into the medium.
According to the invention, the incubation of step b) carries on for a time which can last from a few seconds to a few hours, preferably between 15 seconds and 45 minutes, depending on the micro-organisms.
After the incubation time, the micro-organisms have had the possibility of adhering to the particles (balls).
According to the invention, the separation of the particles and the medium in step c) can be carried out by any method known to one skill in the art. For example, such particles could be sampled by centrifugation and elimination of the culture medium, or further, and preferably according to the invention by using a system generating a magnetic or electric field capable of attracting the particles, particularly a magnet. According to this particularly preferred embodiment, the particles are sampled using a magnet which is advantageously dipped into the sample.
According to an advantageous embodiment, the system generating a magnetic or electric field capable of attracting the particles, particularly a magnet, may be protected, by any system, particularly by a removable coating or a cover, made of any material, for example, plastics, which does not interfere with magnetic or electric
5

waves. More advantageously still, said cover is disposable after use. Such magnet could then be used again.
According to yet another particular embodiment of the invention, the method may include an additional step of washing the system generating a magnetic or electric field so as to eliminate the non-adherent micro-organisms which are present in the wetting liquid, or the micro-organisms which do not adhere much. During this additional step, said system is dipped into a washing solution which may be a sterile culture medium, for instance. One skill in the art understands that, in principle, such step only lasts for a few seconds, at most a few minutes, which is the time required for eliminating the micro-organisms which did not adhere to the system surface.
According to the invention, the spreading of said particles on a support compatible with the development of the micro-organisms can be carried out by a deposition of said particles onto the surface of a micro-organism culture device, for example a Petri dish containing an appropriate culture medium for the development of the micro-organisms.
According to a particular embodiment, the deposition can be carried out by taking the magnet out of the plastic cover while placing the plastic cover closer to the micro-organism culture device surface.
According to another embodiment of the invention, the balls may be deposited using another magnet placed under the culture device surface.
Any system known to one skill in the art, such as a manual spreader, for example, can be used for spreading and dispersing the balls. According to a particular embodiment, a rotating magnet is used and placed under the micro-organism culture device surface.
6

The ball dispersion can also be obtained by means of a liquid vortex generated by the rotation of the micro-organism culture device.
When the particles (balls) are dispersed, the culture device is placed into an incubator for a time sufficient for the micro-organisms to develop on the device surface. Then, one skill in the art will adapt the incubation time and the temperature to the micro-organism to be isolated. This time can be between a few hours and several days, preferably between 4 days and 48 days. The incubation temperature can be between 30 and 40 degrees Celsius.
Other advantages of the invention will appear in the appended Figures, in which:
Figure 1 shows a comparison of conventional methods for sampling microorganisms (A) and the method according to the invention (B);
Figure 2 shows the steps of sampling and washing the particles;
Figure 3 shows the deposition of particles onto the surface of a Petri box;
Figure 4 shows the dispersion of the particles on the surface of the Petri box, using a rotating magnet.
7

WE CLAIM:
1. A process for isolating at least one micro-organism from the medium which
contains them, comprising the following steps:
a) introducing a given quantity of magnetic or magnetisable particles into a sample of said medium;
b) incubating the particles and the medium for a sufficient time for the microorganisms to develop and to adhere to the surface of said particles;
c) separating said particles from the medium;
d) spreading said particles on a support compatible with the development of the micro-organisms;
e) incubating said particles on a support for a sufficient time for the development of colonies corresponding to the isolated micro-organism.

2. A process according to claim 1, characterized in that it further comprises a prior step of pre-culture of the sample from said medium containing the micro-organism to be isolated, possibly while it is stirred.
3. A process according to claim 2, characterized in that said sample is heated up to a temperature compatible with the viability of the micro-organisms, more particularly up to a temperature between 20 and 50 degrees Celsius, and preferably between 30 and 40 degrees Celsius.
4. A process according to claim 2, characterized in that the pre-culture step has a duration between 20 minutes and 10 days, preferably between 1 hour and 48 hours.
8

5. A process according to any one of claims 1 to 4, characterized in that it further includes an additional step between steps c) and d) which consists in the optional immersion of the balls obtained in step c) into an advantageously aqueous washing solution.
6. A process according to any one of claims 1 to 5, characterized in that the incubation of step b) carries on for a time which can last from a few seconds to a few hours, preferably between 15 seconds and 45 minutes, depending on the micro-organisms.
7. A process according to any one of claims 1 to 6, characterized in that the separation of the particles and the medium in step c) is carried out by centrifugation and elimination of the culture medium, or by using a system generating a magnetic or electric field capable of attracting the particles.
8. A process according to any one of claims 1 to 6, characterized in that the separation of the particles and the medium in step c) is carried out using a magnet.
9. A process according to claim 8, characterized in that the magnet is dipped into the sample.
10. A process according to any one of claims 1 to 7, characterized in that the system generating a magnetic or an electric field capable of attracting the particles is protected, particularly by a removable coating or a cover.
9

11. A process according to claim 10, characterized in that said removable coating or cover is disposable.
12. A process according to any one of claims 7 to 10, characterized in that it includes an additional step of washing the system generating a magnetic or electric field.
Dated this 19th day of September 2007

10

ABSTRACT
The invention concerns a novel method for isolating micro-organisms based on the capacity of said micro-organisms to adhere to the surface of particles.
To
The Controller of Patent
The Patent office
Mumbai

A PROCESS FOR ISOLATING BIOFILM FORMING MICRO-ORGANISM

Documents:

Inventors:

PCT Conventions: