Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/02—Devices for withdrawing samples
  • G01N1/22—Devices for withdrawing samples in the gaseous state
  • G01N1/2202—Devices for withdrawing samples in the gaseous state involving separation of sample components during sampling
  • G01N1/2208—Devices for withdrawing samples in the gaseous state involving separation of sample components during sampling with impactors
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D45/00—Separating dispersed particles from gases or vapours by gravity, inertia, or centrifugal forces
  • B01D45/04—Separating dispersed particles from gases or vapours by gravity, inertia, or centrifugal forces by utilising inertia
  • B01D45/08—Separating dispersed particles from gases or vapours by gravity, inertia, or centrifugal forces by utilising inertia by impingement against baffle separators
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/02—Devices for withdrawing samples
  • G01N1/22—Devices for withdrawing samples in the gaseous state
  • G01N1/2202—Devices for withdrawing samples in the gaseous state involving separation of sample components during sampling
  • G01N2001/222—Other features
  • G01N2001/2223—Other features aerosol sampling devices

Definitions

• the invention belongs to the field of production, applications and analysis of gases.
• the filtration method consists in passing a gas through a filter holder fitted with a filter with a porosity of between approximately 0.22 ⁇ m and 0.45 ⁇ m. The microorganisms carried by the gas flow, and whose size is larger than that of the pores, are retained by the filter.
• the filter is then collected under aseptic conditions, and it is either washed to recover the microorganisms, or placed directly in culture on a petri dish. After a determined incubation period in the oven, the number of colony forming units (CFU) is counted, the germs present are identified and a number of colony forming units per volume of gas withdrawn expressed in CFU is deduced therefrom. / m 3 .
• CFU colony forming units
• a water-soluble gelatin-based filter is used, which after adding water makes it possible to count and identify the microorganisms contained in the liquid using molecular biology tools.
• the impaction method consists in strongly accelerating the flow of a gas and directing it towards a target coated with agar. Particles and microorganisms with sufficient momentum leave the air flow and are projected onto the surface of this target. There are two variations of this method:
• bio-impactors operating according to this same method, to control the quality of compressed gases and designed to work at the pressure of the network to which it is connected, with a constant gas flow rate of the order of 28 liters / minute.
• bio-impactors operating according to this same method, to control the quality of compressed gases and designed to work at the pressure of the network to which it is connected, with a constant gas flow rate of the order of 28 liters / minute.
• the results obtained are not very reproducible because they depend on a large number of factors, such as the size of the viable particles to be analyzed, the type and sensitivity of the microorganisms, the level of contamination, the surrounding conditions, the precision and the efficiency of the operator's sample, the type and height of agar covering the target, the impaction speed of the particles and the volume of gas sampled by its ability to more or less dry the agar.
• the Applicant has therefore sought to develop a method for separating particles from a gas or from a mixture of gases. This is why the subject of the invention is a process for the separation by impaction, of viable and reviviscent particles of a gas or a mixture of gases containing it, flowing at a non-zero speed v 0 , characterized in that that he understands
• step (a) of acceleration in one stage and along an axis (x), of the gas flow or of the gas mixture in a confined volume (V), to reach an axial speed v x greater than v 0 , subsonic, sonic or supersonic,
• step (b) of abrupt deceleration of said flow from step (a) in said volume, along substantially the same axis (x), at atmospheric pressure,
• step (c) of changing the direction of the gas flow or of the gas mixture at the outlet of the volume (V), which becomes radial on average,
• step (d) of trapping the particles, separated from the flow during step (c), on a target
• step (e) exhausting the gas flow or the gas mixture from volume (V) so that there is no contact between the surrounding atmosphere and the target used in step (d).
• a gas or a mixture of gases is designated at a total pressure greater than 1 atmosphere (approximately 10 5 Pa ), generally at a total pressure less than or equal to 200 atmospheres, and more particularly at a total pressure less than or equal to 10 atmospheres.
• a deceleration which makes it possible to reduce the speed of a gas flow, to a zero axial speed on the target.
• confined volume (V) in which the stages (a) and (b) take place and at the end of which the stage (c) takes place, is meant in the present description, a finished volume delimited by a lateral surface finite, which is preferably a surface of revolution around the axis (x).
• the gas flow rate for which it is desired to determine the microbiological quality varies according to the source and / or the use which is made of said gas. It is often between 50 liters per minute and 1000 liters per minute and more particularly between 100 liters per minute (subsonic jet mode) and 300 liters per minute (sonic jet mode for most gases).
• the process which is the subject of the present invention can be carried out with any gas or mixture of gases.
• a gas or gas mixture there are, for example, air, oxygen (0 2 ), nitrogen (N 2 ), argon (Ar), carbon dioxide (CO 2 ), l helium (He), nitrous oxide (N 2 0), nitric oxide (NO), mixtures of nitrous oxide and oxygen, carbon dioxide and oxygen, nitrogen and nitrogen or helium monoxide and oxygen, and more particularly mixtures (50% by volume (v / v) N 2 0 + 50% v / v O U ), (5% v / v C0 2 + 95% v / v 0 2 ), (200 ppm to 800 ppm NO in N 2 ), (78% v / v He + 22% v / v 0 2 ), (65% He +35% 0 2 ) and (80% v / v He + 20% v / v 0 2 ), used in medicine or even mixtures of nitrogen and carbon dioxide (N 2 + C0 2 ) used in the food industry.
• the confined volume (V) consists of a first volume fraction (V a ) in which step (a) is implemented, delimited by two bases (Bi) and (B 2 ), of surfaces (Si) and (S 2 ), with (Si) greater than or equal to (S 2 ) and a second volume fraction (Vb) contiguous to (V a ), in which step (b) is implemented bounded by (B 2 ) and a base (B 3 ) with a surface (S 3 ) greater than or equal to (S 2 ).
• (Si) and (S 2 ) are equal, (Si) is less than the section of the gas flow circulating upstream of the volume (V).
• step (d) of trapping the particles is implemented on a target surface capable of fixing them.
• viable and reviviscent particles microorganisms of all kinds, capable of multiplying under conditions of appropriate pressure, humidity and temperature.
• the target surface is that of a nutritive medium allowing the development and multiplication of the trapped microorganisms.
• a nutritive medium such as a medium comprising agar.
• said medium will either be specific that the growth of one or more species of microorganisms is non-specific.
• solid nutritive medium used to trap the particles there are:
• TCS TM medium (AES laboratory reference: AEB 522 859), which is a selective medium for bacteria comprising 15 g / l (gram per liter) of pastone, 5 g / l of papain peptone from soy , 5 g / l of sodium chloride and 15 g / l of Agar. This medium requires a 24 to 48 hour incubation at 37 ° C.
• the R2A TM medium (AES laboratory, reference AEB 523 480), which is a non-selective medium generally used for the enumeration of so-called "stressed" microorganisms, present in treated water poor in nutrients; it comprises 1.0 g / l of a mixture of peptones (proteose peptone), 0.5 g / l of casein acid hydrolyzate, 0.5 g / l of dextrose, 0.5 g / l of starch soluble, 0.3 g / l of dipotassium phosphate (K 2 HP0), 0.024 g / l of magnesium sulphate, 0.3 g / l of sodium pyruvate and 15 g / l of Agar.
• peptones proteose peptone
• K 2 HP0 dipotassium phosphate
• K 2 HP0 dipotassium phosphate
• - MOSSEL TM medium (AES laboratory, reference AEB 521 740), which is a medium specific for the growth of Bacillus cereus and Bacillus spp. ; in general it includes 10 g / l of meat peptone, 1 g / l of meat extract, 10 g / l of sodium chloride, 10 g / l of mannitol, 0.025 g / l of phenol red and 15 g / l of Agar.
• the Schaedler TM medium with sheep blood (%) (Biomérieux, reference 43273), which is particularly suitable for the culture of anaerobic bacteria and allows the growth of demanding species; it includes 5.7 g / l of casein peptone, 1 g / l of soy peptone, 5 g / l of casein and meat peptone, 5 g / l of yeast extract, 5.83 g / l glucose, 1.7 g / l sodium chloride, 3 g / l bipotassium phosphate, 0.01 g / l Tris (hydroxymethyl) amino methane, 0.01 g / l hemin, 0.4 g / l cysteine, 0.0005 g / l L-cystine, 13.5 g / l Agar and 50 ml / l sheep's blood.
• the hardness at impaction of these media can be modified by changing the concentration of Agar or Agar-agar.
• the invention also relates to a variant of the method as defined above, further comprising a step (f) of counting the particles trapped during step (d).
• Step (f) preferably consists of a step (fi) of cultivating viable and reviving particles trapped at the end of step (d), followed by a step (fg) of counting the colony forming units.
• the culture step consists, for example, in maintaining the nutrient medium containing the trapped particles, at a temperature allowing the multiplication of microorganisms for a time determined by the culture medium used (24 to 48 hours at 37 ° C. for the medium TCS TM, 5 to 15 days at 25 ° C for R2A TM or R3A TM media, 24 hours at 37 ° C for Mossel TM media or 5 days at 37 ° C for Schaedler TM sheep blood medium).
• the method as defined above makes it possible to effectively separate microorganisms having dimensions between 0.05 ⁇ m and 25 ⁇ m and more particularly between 0.2 ⁇ m and 12 ⁇ m.
• microorganisms which have been shown to be present in a compressed or liquefied gas after isolation from a nutrient medium, by the variant of the process as described above, there are molds and yeasts such as, Mucor indicus, Graphium spp, Penicillium spp .
• Gram positive bacteria such as Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus aureus, Bacillus brevis; Gram negative bacteria such as Pantoea spp.
• the subject of the invention is also a process for determining the microbiological quality of a gas or a mixture of gases under pressure, by implementing the variant of the separation process as described above.
• the method and its variant allow the collection, on solid nutritive medium, of the germs present in a gas, compressed or liquefied, under pressure.
• the gas analyzed can come from cylinders, frames, storage tanks, primary or secondary, from a gas supply network, primary or secondary, and more in particular, either a medical gas supply network as found in healthcare establishments, such as hospitals, dispensaries or clinics, or a gas distribution network as there is a in drug production factories or in factories in the food industry.
• the gas does not need to be expanded to atmospheric pressure to be analyzed by the method described above.
• a device (D) suitable for the implementation of the method and its variant as defined above.
• the device (D) comprises:
• the element (Di) of the device (D) is a hollow solid, comprising an inlet orifice, an outlet orifice, a lower lateral surface SL, defining a confined volume (V) having orthogonal, polygonal, elliptical or cylindrical, in which the steps (a) and (b) of the process as defined above take place, at the outlet orifice of which the step (c) takes place.
• the surface SL is more particularly, either a surface of revolution around an axis (x) or a lateral surface of a regular polyhedron of axis of symmetry (x), or a set of one or more surfaces of revolution and / or one or more surfaces of regular polyhedra, with the same axis of symmetry (x), defining the volume (V).
• the two orifices are preferably co-axial with respect to the axis (x).
• the element (Di) can be of any material, be it a plastic, a metal or a metal alloy. It is preferably made of a material which can be sterilized by dry heat (3 hours at 180 ° C) or by wet heat (20 minutes at 130 ° C).
• the element (Di) of the device (D) more particularly comprises an internal lateral surface SL defining a first volume fraction (V a ) of frustoconical or cylindrical shape, in which step (a) is implemented, and a second volume fraction (V b ) contiguous to (V a ), of frustoconical shape, in which step (b) is implemented.
• the element (D) of the device (D) has very particularly a shape defining a first fraction of cylindrical shape of height h and diameter w, in which step (a) is implemented, and a second fraction of shape frustoconical with height H of small base of diameter w and of large base of diameter W, in which step (b) is implemented.
• the inlet orifice is in this case of circular section of diameter w and the outlet orifice is of circular section and of diameter W.
• the h / w ratio is generally between 10/1 and 1/10. It is more particularly between 10/2 and 2/10.
• the W / w ratio is generally between 50/1 and 5/1. It is more particularly between 25/1 and 10/1.
• the H / w ratio is generally between 5/1 and 50/1. It is more particularly between 10/1 and 40/1.
• H is generally between 10 mm and 200 mm, more particularly between 30 mm and 120 mm and very particularly between 40 mm and 100 mm.
• the value of w is preferably between 2 and 5 mm approximately and it is more particularly equal to approximately 4 mm
• the element (D 2 ) of the device (D) is preferably of cylindrical shape and of diameter Wi greater than or equal to W. In general, Wi is less than or equal 90 mm. W is generally comprised between 50 mm and 90 mm. It is preferably a Petri dish. Element (D 2 ) contains a nutritive medium for microorganisms and more particularly a gelicic medium.
• the element (D 3 ) of the device (D) is a hollow solid comprising an inlet orifice, an outlet orifice, a lower lateral surface S 3.
• the surface S 3 is more particularly, that is a surface of revolution around of the axis (x), i.e. a lateral surface of a regular polyhedron with axis of symmetry (x).
• the element (D 3 ) more particularly has the same axis of symmetry as the element (Di). This element D3 can of course be made up of one or more parts.
• the element (D 3 ) defines with the element (Di), a volume through which the gas released from the particles trapped on the element (D 2 ) flows towards the outlet orifices of the device (D) to a sufficient speed so that the microorganisms present in the ambient atmosphere do not reach the internal surface of said element D 2 .
• the element (D 3 ) can be of any material, be it a plastic, a metal or a metal alloy. It is preferably made of a material which can be sterilized by dry heat (3 hours at 180 ° C) or by wet heat (20 minutes at 121 ° C).
• the element (D 3 ) of the device (D) also makes it possible to maintain the element D 2 at a distance d from the large base of (D 1 ), sufficient to allow the entire flow of the gas or the mixture of gas at the outlet of the element (D 1 ), radially on average at the axis (x).
• the H / d ratio is generally between 2/1 and 20/1. It is more particularly between 3/1 and 15/1.
• the opening system of the element (D 3 ) preferably allows the release of the elements (D 2 ) and (D ⁇ , in order to more easily carry out the culture of the viable and reviviscent particles.
• the device (D) as defined above may further comprise a connection element (D 0 ), such as for example a notched plug capable of adapting to a medical gas outlet, a small diameter tube capable of adapt to the outlet of a pressure regulator and more generally any connection capable of adapting to any device for delivering compressed or liquefied gas.
• a connection element such as for example a notched plug capable of adapting to a medical gas outlet, a small diameter tube capable of adapt to the outlet of a pressure regulator and more generally any connection capable of adapting to any device for delivering compressed or liquefied gas.
• the device (D) as defined above may further comprise a pressure regulator placed between the connection element (Do) and the element (D 1 ).
• the device (D) as defined above can be connected to the supply of gas to be analyzed, sojt by being connected to a wall outlet, sojt by being connected to the supply line for the gas to be analyzed, preferably downstream of '' a 1/4 turn valve.
• the method and the device, objects of the present invention are used to determine the microbiological quality of gases or gas mixtures contained in bottles, whether medical bottles or industrial bottles, such as those intended to the manufacturing industry of electronic components, to the food industry or to the pharmaceutical industry.
• the method and the device, objects of the present invention are also implemented for determining the microbiological quality of gas or mixtures of gases supplied by supply networks, such as hospital supply networks allowing in particular the delivery of gases in operating rooms or operating theaters, supply networks for frozen food manufacturing chains, very high purity gas supply networks for the manufacture of electronic components or gas supply networks necessary for the manufacture and / or packaging of pharmaceutical products and formulations.
• supply networks such as hospital supply networks allowing in particular the delivery of gases in operating rooms or operating theaters, supply networks for frozen food manufacturing chains, very high purity gas supply networks for the manufacture of electronic components or gas supply networks necessary for the manufacture and / or packaging of pharmaceutical products and formulations.
• the method and the device, objects of the present invention are also implemented for determining the microbiological quality of gases or gas mixtures at the outlet of a unit for producing the gas or the gas mixture, such as for example, a compressor, a cryogenic distillation column, a gas separation column by adsorption, whether it is a PSA column (pressure swing adsorption), a column VSA (modulated volume adsorption; in English: Volume Swing Adsorption) or a TSA column (modulated pressure adsorption; in English: Temperature Swing Adsorption) or a gas separation unit by permeation through membranes polymers such as for example a FLOXAL TM unit.
• a unit for producing the gas or the gas mixture such as for example, a compressor, a cryogenic distillation column, a gas separation column by adsorption, whether it is a PSA column (pressure swing adsorption), a column VSA (modulated volume adsorption; in English: Volume Swing Adsorption) or a TSA column (modulated
• the method and the device, objects of the present invention are also implemented for determining the microbiological quality of the ambient air in premises, and more particularly in the clean rooms of drug manufacturing sites or manufacturing sites of electronic components or in document storage sites.
• its subject is a method for determining the ability of a device capable of trapping viable and reviviscent particles, to determine the microbiological quality of a gas or of a mixture of gas under pressure with respect to a reference device as defined above, comprising the injection of a determined quantity of one or more known microorganisms into the flow upstream of the device to be qualified and the quantification of the microorganisms trapped by the latter device.
• a calibration bench comprising:
• a nebulizer operating on the venturi principle comprising:
• a spray head connected to said chamber, of frustoconical shape with an axis of symmetry (y), comprising at its small base, a spray orifice and (iii) a cylindrical capillary tube (y), of diameter less than diameter of said spray orifice, one end of which is inserted into said orifice coaxially with the spray head,
• a casing completely enveloping said nebulizer to which are connected a line for supplying carrier gas and a line for distributing the gas flow from the nebulizer-casing assembly, containing the microorganisms to the device for which it is desired to determine its ability to trap them, in which the nebulizer adapts coaxially to said distribution line.
• the nebulizer is non-removable. According to another particular and preferred aspect of the nebulizer as described above, the nebulizer is removable in that at least the spray head, the cylindrical chamber and the capillary tube can be separated from each other.
• the invention relates to a nebulizer as well as a set, nebulizer-casing, capable of being implemented in the method described above.
• Figure 1A shows a diagram of a device according to the invention. It consists of a neck (cylindrical fraction of the element (Di)), a diffuser (frustoconical fraction of the element Di) and a Petri dish (element (D 2 )).
• w represents the inside diameter of the neck and is equal to 4 mm
• h represents the length of the neck and is equal to 10 mm
• H represents the height of the diffuser and is equal to 80 mm
• W represents the diameter of the large base of the diffuser and is equal to 75 mm
• Wi represents the diameter of the Petri dish and is equal to 90 mm
• d represents the distance between the large base of the diffuser and the external surface of the agar of the Petri dish and is equal to 13 mm;
• the device operates so that the speed of the jet continues to increase, as long as its pressure has not reached atmospheric pressure.
• the total supply pressure is between approximately 2.10 5 Pa and 10 6 Pa.
• the fluid accelerates until it leaves the cervix. Then there are areas of low pressure / expansion where the flow is alternately accelerated and slowed down.
• the calibration bench for compressed or liquefied gas presented in the photo in FIG. 2 is used, fitted with the device according to the invention, in order to qualify its biological efficiency and to compare it with that of existing biocollectors.
• the carrier gas in this case medical air from a cylinder sold by the plaintiffs and connected to a pressure regulator, single expansion Air Liquide referenced: AHL 16-25, circulates in line d 'supply of the crankcase-nebulizer assembly, at a pressure P' of between approximately 10 5 and 5.10 5 Pa relative and the nebulizer gas, in this case medical air from a bottle sold by the applicants and connected to an Air Liquide pressure reducer, simple expansion referenced: HBS 300/8, circulates in the supply line of the crankcase - nebulizer assembly, at a pressure P greater than> P 'between 4.10 5 Pa and 6.10 5 Pa relative.
• the complexity of such a device resides in the nebulization under pressure of microorganisms, the nebulizers of the state of the art operating only rarely under pressure.
• TR50-1C1 TM nebulizer marketed by the company ANALAB, capable of nebulizing microorganisms under pressure (between 10 5 Pa and 4.10 5 Pa) without destroying them, was carried out with the calibration bench and was found to be unsatisfactory due, on the one hand, to the fragility of the nebulizer and, on the other hand, to significant losses during nebulization.
• the nebulizer comprises a tube allowing it to be supplied with microbiological inoculum and a tube for supplying nebulizer gas at the pressure P 'greater than the pressure P, to obtain gas flows at the neck of 100 to 300 liters per minute (l / min).
• nebulizer gas at the pressure P 'greater than the pressure P, to obtain gas flows at the neck of 100 to 300 liters per minute (l / min).
• two plastic three-way valves connected together by a flexible hose and located upstream, on the two nebulizer supply tubes, allow the circulation of nebulizer gas in the Pinoculum injection tube. . This avoids stagnation of droplets of inoculum in the nebulizer.
• the casing, the straight pipe and the device (D) composed of the elements (Di) and (D 3 ) are sterilized by humid heat in an autoclave for 20 minutes at 120 ° C.
• the nebulizer is disinfected with 70 ° alcohol before mounting on the housing.
• the system for injecting microorganisms into the nebulizer (the 2 plastic 3-way valves, the pipes connecting them and the syringe) is a sterile device for single use.
• Method of injecting a microbiological inoculum of known concentration It includes the following steps:
• a known volume of a suspension of microorganisms in determined quantity is nebulized while a defined volume of gas is injected into the calibration bench. At the outlet of the bench, there is therefore a known volume of gas containing a perfectly determined number of microorganisms in suspension in this volume.
• Device D can be used independently at a gas flow rate below 300 l / min (subsonic gas speed) or at a gas flow rate above 300 l / min (supersonic speed), while providing a constant recovery rate and reproducible by around 40% for Bacillus subtilis spores. The only limit is the eventual destruction of the agar at supersonic speed.
• the device (D) as defined above can be connected to the supply of gas to be analyzed, either by being connected to a wall outlet, or by being connected to the supply line for the gas to be analyzed, preferably downstream a 1/4 turn valve.
• Figure 6 schematically illustrates a bench and nebulizer structure according to the invention and therefore allows a better understanding of their operation.

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• 2002
  • 2002-05-24 FR FR0206324A patent/FR2839897B1/fr not_active Expired - Fee Related
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