FR2834719A1 - NOVEL METHOD FOR ANALYSIS, DETERMINATION AND / OR MONITORING OF THE MICROBIOLOGICAL QUALITY OF A GAS, DEVICE FOR IMPLEMENTING THE METHOD AND USE OF SAID DEVICE - Google Patents

Abstract

New process for analyzing, determining and / or controlling the microbiological quality of a gas comprising a step (a), collecting a sample of gas to be analyzed, a step (b), transferring the phase gaseous to the liquid phase, microorganisms contained in the gas sample resulting from step (a), a step (c) of labeling the microorganisms contained in the liquid sample resulting from step (b), a step ( d) detecting the microorganisms present in the labeled liquid sample resulting from step (c); device for implementing the method; use of the device to monitor the microbiological quality of the air in residential premises, hospital premises or industrial premises and / or to detect bio-contamination of the atmosphere in residential premises, hospital premises or industrial premises.

Description

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  The invention relates to the field of microblological analysis of gases.

  Biological contamination by air is an important concern in many sectors of activity, for example, the transmission of nosocomial diseases in hospitals or the epidemics of legionellosis carried by aerosols from air conditioning systems and especially the air coolers. Industrialists in the food, pharmaceutical or cosmetics sectors, who use products that can constitute good growth media for microorganisms, devote a lot of effort to create and maintain in their industrial installations, "clean", even " steri

  "to protect them from airborne contamination.

  Controlling the air quality inside residential, professional or institutional buildings, more particularly by determining the blo contamination of the air, is an operation of great importance with regard to the population there. parking, especially when there are elderly, young children or immunocompromised people who constitute a popula

  tion more fragile than the average, vis-à-vis microbial aggressions.

  By blo-contamination is meant any living organism or product secreted by microorganisms, which may develop harmful health effects or spoilage effects on products, for example bacteria, fungi

  gnons, yeasts, viruses, toxins and by extension allergens.

  There is currently no real-time measuring instrument which is easy to use while making it possible to measure the block contamination of a

  gas phase, nor any reference method for such a measurement.

  Many devices for collecting and analyzing air samples are commercially available, based on the principle of blo-impaction, according to which a controlled flow of the fluid to be analyzed is directed on the surface of a liquid or a viscous or solid culture medium of the "Petri dish" type. Among the many available variants, mention may be made, for reference, of the devices known commercially as follows: RCS Microbial Air Sampler_ from Biotest Diagnos tics Corp .; Air-O-Cell bloaerosol sampling cassettes_ from Zefon Intl .; MicroBio MB1 Plus Air Sampler_ from Spiral Biotech; BioCapture Air Sampler_ from Meso Systems Technology Inc .; or MAS Air Sampler_of Merck; Impactor_ waterfall

by Thermo Andersen.

  In addition to an often questionable collection yield, the method implemented with such devices requires a culture prior to determination,

  which requires a delay of at least one day.

  The methods of detection and / or analysis of microorganisms in solution are generally based on methods of immunofluorescence, bac terial growth, DNA or RNA typing, or ATPmetry, the complexity of which _work depends on the detail of the desired information. For example, for DNA or RNA typing, the preparation time for the analyzable sample is of the order of a few hours if one notably integrates the cell lysis, extraction and purification phases. genetic material and amplification, which makes it impossible to implement them in real time or near real time measurements. A new method of flow cytometry type has been described in the American patent published under the number 6,256,096. This method consists in detecting the fluorescent marked particles by means of a CCD detector (called in English: charge coupled device) coupled with a TDI technique (de

  named in English: time delayed integration).

  This system makes it possible to visualize bacteria on a computer screen and to gain a few multiplicative factors for the signal / noise ratio compared to conventional CCD detectors, thanks to TDI image processing based on photomultiplication, making it possible to detect small amounts of microorganisms. This is why the subject of the invention is a method of analysis, determination and / or control of the microblological quality of a gas comprising the following successive steps: A step (a), of collecting a sample of gas to be analyzed, A step (b), of transfer from the gas phase to the liquid phase, of the microorganisms contained in the gaseous sample resulting from step (a), A step (c) of labeling the microorganisms contained in the liquid sample resulting from step (b), a step (d) for detecting the microorganisms present in the sample

  marked liquid resulting from step (c).

  By gaseous sample, is meant more particularly, either a sample of air from the atmosphere of a room, or a sample from the sky of a reactor or a vessel, or a sample of a cylinder of compressed gas , either a sample from a gas generator using distillation, permeation, adsorption, absorption, complexation, diffusion techniques, or a combination of said methods, or a sample from a reservoir in whichone

  gas is in contact with its liquid.

  The collection liquid is more particularly water, pepto water

  born, water with surfactants or a culture medium for microorganisms.

  According to a first variant of the method as defined above, step (b) consists of a step (b) of transfer from the gas phase to a solid phase, followed by a step (b2) of transfer from the solid phase to a liquid phase; According to a second variant of the method as defined above, it comprises a step (e) of determining the concentration of microorganisms present in the sample, by processing the detected signals, during

  step (d) by means of a computer operating using dedicated software.

  According to a third variant of the method as defined above, that

  - This includes a step of (f) collecting the sample from step (d).

  According to a fourth variant of the method as defined above, this comprises a step (e2) of transformation of the detected signals, lo rs of step (d), binary message "Yes or No" by means of a computer running

using dedicated software.

  A more particular subject of the invention is the method or a variant of the method as described above in which, step (d) is a step

flow cytometry.

  The invention also relates to a device, capable of implementing the pro

  assigned and its variants as defined above.

  The device as defined above comprises: The device as defined above comprises: At least one means for collecting samples of gases to be analyzed; At least one means for transferring the microorganisms contained in the gaseous sample collected by said collection means to a liquid phase, to form a liquid sample;

  At least one means of labeling the microorganisms contained in the-

  said liquid sample; At least one means of detecting microorganisms present in

the labeled liquid sample.

  As a means of collection there are for example cyclone collectors

  in which the gas stream is washed with a liquid.

  For small samples, the collection means there is, for example, a means implementing so-called MEMS technologies (micro electromechanical systems in English) or even micro

1 5 fluidics.

  According to a particular aspect of the device as defined above, that

  this comprises at least one means for processing the detected signals.

  The processing and / or analysis means of the measurement system can include

  carry multiple control systems.

  It is possible to collect the liquid sample after passage through the cell

  measurement to carry out more detailed microbiological analyzes offline.

  Detection of microorganisms or microorganism contamination thresholds can trigger active alarm systems, stop production or trigger quarantine or even activate procedures such as increasing the laminar flow speed or increase in

  extraction ventilation or trigger decontamination.

  The use of a continuous measurement device also makes it possible to optimize the operation of the protection systems which make it possible to keep "gray" "" clean "or" white "zones which do not require sterility. The recurrent archiving of data from the analyzer constitutes an important element in the quality approach as well as in the respect of the procedures linked to the implementation of the procedures called HACCP approach or good practices.

manufacturing issues (BPF).

  According to another aspect of the device as defined above, the detection means is a flow cytometer. According to another particular aspect of the present invention, the device as defined above, detects and analyzes the presence of blocking agents.

  in the sample of gas to be analyzed in real or near real time.

  According to another particular aspect, of the present invention, the device as described above is an integrated, autonomous and self-operating system

1 0 matically.

  The invention also relates to the use of the device as defined above to control the microblological quality of the air in residential premises, hospital premises or industrial premises and / or to detect bio contamination of the atmosphere in residential premises, hospita premises

liers or industrial premises.

  Controlling the air quality inside residential, professional or institutional buildings, more particularly by determining the blo contamination of the air, is an operation of great importance with regard to the population there. parking, especially when there are elderly, young children or immunocompromised people who constitute a popula

  tion more fragile than the average, vis-à-vis microbial aggressions.

  By bio-contamination is meant any living organism or product secreted by microorganisms, which may develop harmful health effects or spoilage effects on products, for example bacteria, fungi

  gnons, yeasts, viruses, toxins and by extension allergens.

  There is currently no real-time measurement instrument which is easy to use while allowing the biocontamination of a

  gas phase, nor any reference method for such a measurement.

  Figure 1 illustrates such a device.

  Means 1 is a collector block whose function is to transfer the microorganisms

  nisms present in the gaseous atmosphere to a liquid or a solid.

  Means 2 is a container for the labeling reagents.

  The means 3 is a container for rinsing and decontamination liquid.

  Means 4 is a cell for measuring the marked species.

  The means 5 is a container used to collect the waste.

  Means 6 is a computer which presents the results obtained.

  The syringes are activated by a system that moves the plunger. The means S1 and S2 are syringes which serve to withdraw liquid from

  from the manifold block and inject it into the reaction pipe (T).

  The means S1 and S2 operate in torque to ensure a continuous flow.

  The means S3 and S4 are syringes which are used to withdraw and inject

  labeling reagent contained in means 2.

  The means T is a reaction pipe. The residence time in T corresponds to

marking time.

  The means V1 to V6 are valves.

  The dotted lines are the pipes that allow the residue to be sent to the

medium 5.

  To bring the liquid to the different means, the syringes in pairs (S1 / S2 and S3 / S4) function like pumps. The coupled syringes form a pump which can run continuously. Besides the sampling and injection valves, there are flushing valves (Vl 1 to V14) and "purge" valves (V5, V6, V9, V10). The use of syringes as pumps allows the system to be compact and therefore unobtrusive in

tight spaces.

  The device illustrated in Figure 1, performs all of the steps of the pro

  assigned and its variants, which are the subject of the present invention.

  Biocollection consists in transferring the microorganisms which are suspended in the gas to a liquid or solid medium. The "cy clone" type biocollectors create a vortex of gas which returns the particles towards the wall of the reader block thanks to their inertia. The wall is then rinsed with liquid in order to recover the microorganisms. The speed of collection generally varies between

1 OOUmin and 1 OOOUmin.

  Block collectors of the "impactor" type return the gas to a certain speed sufficient to ensure the passage of microorganisms from the surface of the collection liquid. The speed of collection of the impactor is of the order of a few tens of liters per minute. The two types of biocollectors described above can be integrated as the first element of the device. The blocco

  chosen reader, has a liquid outlet to syringes S1 and S2.

  Labeling is necessary to make the microorganisms visible in the next step of the device protocol. The marking is chosen according to

type of analysis.

  When you want to enumerate microorganisms without paying attention to their species, a color like LIVE / DEAD Ba cLight for bacteria or LIVE / DEAD Yeast Viability Kit_ by Molecular

Probes are used.

  When more specific detection is desired, anti-bodies or nucleic probes marked with a fluorophore which corresponds to the

  excitation system, which thus allow detection by fluorescence.

  It is also possible to use enzyme labeling, electrochemical detection or mass or optical detection. The labeling reagent is

  injected continuously (when the sample is injected) by syringes S3 and S4.

  Detection is carried out by reception of signals induced by the marking

  microorganisms present in the liquid.

  Depending on the labeling, it is possible to use a flow cytometry system with reading by CCD and acquisition of the intensity by TDI, a conventional flow cytometry system, a system using bioelectronic DNA chips or

  well a classic fluorescence scanner of classic bloats.

  The translation of the received signals is carried out by computer with dedicated software and the result is presented either in binary or (YES / NO) form, or a quantitative result. The liquid is brought through the detection cell (4) by

  continuous pumping of syringes S1 or S2 and S3 or S4.

  A radio communication system could be added for machines placed in places with little traffic or an entire system

autonomous is desired.

Protocol description

  The manifold samples the gas at a flow rate of approximately 5 liters at 1000 li

  very per minute, in approximately 1 ml to 100 ml of liquid.

  The collection liquid is preferably water, peptone water, water with surfactants or a culture medium for microorganisms. When the biocollector has sampled enough gas for the concentration of

  microorganisms in the liquid exceeds the detection threshold of the read module

  ture, approximately from 0.5 ml to 10 ml of liquid is aspirated by one of the two syringes S1 or S2 operating in alternating fashion, to allow a global operation in

  device, depending on the open or closed valve positions. (Van-

  nes V1, V5, V11 closed and V3 open for syringe S1 and valves V4, V6, V12

  closed and V2 open for syringe S2).

  The reagent syringes S3 and S4 also work alternately. One injects the reagent while the other fills with it. The filling speed is higher than the injection speed which results in the relay between the two syringes being made without the flow falling to zero. The flow rate at the detection module is the sum of the sample injection and reagent injection flow rates in volume per unit of time. The volume / flow rate of injected reagent is equal to or less than the volume / flow rate of the sample. The flow rates are determined by the incubation time required for the labeling reaction which generally lasts from 15 minutes to 1 hour. If the volume of the tube before the detection module is fixed, the incubation time will be determined by the sum of the syringe flow rates S1 / S2

and S3 / S4.

  When the liquid is in the detection module it is illuminated by a beam of light of wavelength which corresponds to the length

  excitation of the fluorophore used. If microorganisms are present, a

  photon tector collects the photons corresponding to the emission wavelength of the fluorophore. This photon collector can be a CCD device

  (called: "charged coupled devices" in English) or a photomulti- tube

  plicator. The signals are interpreted by dedicated software which gives the results

  as a concentration of microorganisms in the gaseous atmosphere.

Claims (12)

claims   1. Method for analysis, determination and / or control of the mi croblological quality of a gas comprising the following successive steps: A step (a), for collecting a sample of gas to be analyzed, A step (b ), transfer from the gas phase to the liquid phase, of the microorganisms contained in the gas sample resulting from step (a), A step (c) of labeling the microorganisms contained in the liquid sample resulting from the step (b), step (d) of detecting the microorganisms present in the sample   marked liquid resulting from step (c).   2. Method as defined in claim 1, in which step (b) consists of a step (b,) of transfer from the gas phase to a phase so   lide, followed by a step (b2) of transfer from the solid phase to a liquid phase.   3. Method as defined in one of claims 1 or 2, comprising in   in addition to a step (e) of determining the concentration of microorganisms present in the sample, by processing the signals detected, during step (d)   using a computer running dedicated software.   4. Method as defined in one of claims 1 or 2, comprising in   in addition to a step of (f) collecting the sample from step (d).   5. Method as defined in one of claims 1 or 2, comprising in   in addition to a step (e2) of transformation of the detected signals, during step (d), binary message "Yes or No" by means of a computer operating using a dedicated software.   6. Method as defined in one of claims 1 to 5, in which   step (d) is a measurement step by flow cytometry.   7. Device capable of implementing the work as defined in one of claims 1 to 6. Comprising: At least one means for collecting samples of gases to be analyzed; At least one means for transferring the microorganisms contained in the gaseous sample collected by said collection means to a liquid phase, to form a liquid sample; At least one means for marking the microorganisms contained in said liquid sample; At least one means of detecting microorganisms present in the labeled liquid sample.   8. Device as defined in claim 7, further comprising at   minus a means of processing the detected signals.   9. Device as defined in one of claims 7 or 8, in which the   detection means is a flow cytometer   10. Device as defined in one of claims 7 to 9, characterized in   what it detects and analyzes the presence of biocontaminants in the sample of   gas to be analyzed in real or near real time.   11. Device as defined in one of claims 7 to 10, characterized in   that it is an integrated, autonomous and automatically operating system.   12. Use of the device as defined in one of claims 7 to 10,   to control the microblological quality of the air in residential premises, hospital premises or industrial premises and / or to detect bio-contamination of the atmosphere in residential premises, hospital premises or premises