WO2004022245A1

WO2004022245A1 - Inoculation method and related apparatus

Info

Publication number: WO2004022245A1
Application number: PCT/GB2003/003749
Authority: WO
Inventors: Richard John Beedham; Michael Green; Thomas James Parry Hawkyard; Stephen James Moore; Anthony Jhon Stagg
Original assignee: The Secretary Of State For Defence
Priority date: 2002-09-06
Filing date: 2003-08-29
Publication date: 2004-03-18
Also published as: GB0220735D0; AU2003260753A1

Abstract

An apparatus for inoculation of a substrate with an aerosolised material having a sealed chamber, said chamber comprising: an inlet port through which an aerosolised material is able to pass into the chamber; and an outlet port, connected to a means for generating a vacuum, through which an aerosolised material is able to pass out of the chamber; wherein said inlet port and said outlet port are arranged such that aerosolised material flowing through the chamber passes over the surface of the substrate and material within the aerosol is deposited thereon. The invention also relates to a method and use of the same. This invention has several advantages including allowing for the fast and efficient inoculation of a substrate, particularly a solid substrate, with a wide variety of different chemical and biological materials from an aerosol, the apparatus can be easily used with protective clothing or in a containment chamber and that the apparatus is reusable.

Description

Inoculation Method and Related Apparatus

This invention relates to a method, and related apparatus, for the inoculation of a chemical or biological material onto a given substrate. More particularly this invention relates to a method, and related apparatus, whereby an aerosolised biological material can be successfully deposited onto a solid substrate.

In environmental assessment it is important to understand how a wide variety of different materials, including biological and chemical materials, are deposited, continue and degrade on different substrates. This can help in developing new and effective deposition techniques, in understanding the success of the deposition and also in assessing long-term degradation and therefore the extent and duration of pollution. To study such factors it is useful to establish laboratory models that simulate deposition and degradation. Such models can be used particularly to conduct trials to assess pollution in a controlled manner.

For many years it has been general practice to use spraying, including from a aeroplane or a helicopter, to quickly and effectively deposit a given chemical or biological material over a large area. The development of sophisticated aerosol technology has been used to improve the efficiency of spraying techniques. Although such techniques have many civilian applications, including spraying of farmland and crops, recently a concern has developed that they may be used as an act of war or terror to contaminate an area of land with warfare agents. As such it would be useful to develop a model which could be used to effectively mimic the deposition of one or more materials on a wide variety of substrates, including mimics for different types of terrain, to aid in understanding the likely outcome of a potential attack and the extent and duration of any resultant contamination. In addition such a model would have a variety of civilian uses in understanding the degradation patterns of materials that have been sprayed on land and the like. Ideally such a model system should be flexible so that it can easily be used for different types of studies; quick and efficient to use; and designed so that it can be operated in a containment chamber if required to minimise human exposure when dealing with toxic materials.

A wide variety of different nebulisers are already in use in laboratories to generate aerosols comprising either a biological or a chemical material and various apparatus, including the Henderson apparatus, are known for conditioning such aerosols. To date, settling chambers are also used in laboratories to monitor the properties of an aerosol over time. However, a problem remains of how to effectively deposit material from an aerosol onto a substrate such that further investigations into the duration and extent of substrate contamination can be conducted.

Unfortunately it is not practical to simply use a known settling chamber to model aerosol deposition onto different substrates. Problems include the likely unacceptably long time required for a material to settle; unreliable and inconsistent settling patterns caused by settling time variation; the maximum concentration of material deposited limited based on the concentration of material achievable within the aerosol; and insufficient safety for use with highly toxic materials. Potential improvements to using a crude chamber include using an airbrush spray device or using an Anderson sampler that is known for impacting aerosol samples onto agar plates. However the former is insufficiently safe for use with the most toxic chemical and biological materials and the latter is unsuitable for use with substrates that comprise small particulate.

It is therefore desirable to develop an apparatus that allows the fast deposition of aerosolised materials onto a wide variety of substrates, especially solid and particulate substrates. Such an apparatus should be safe to use with an aerosol comprising even the most toxic chemical and biological materials and as such, including that it should be effectively sealed so as not to leak. Ideally such an apparatus would be designed so that the deposition could be controlled allowing deposition levels to be varied and also allowing deposition to build up over time such that the concentration of deposition achieved is not limited by the maximum achievable concentration of material within the aerosol. In addition, the apparatus should be easy to handle so that it can be used inside a variety of containment facilities, including Class III microbiological safety cabinets. Furthermore the apparatus should be able to be readily cleansed and sterilised such that it can be repeatedly used in a wide variety of experiments without any concern of cross contamination. It would be even more desirable if this apparatus were designed such that the inoculated substrate could be immediately subjected to a variety of environmental stresses including degradation profiling by exposure to radiation, for example simulated solar radiation, without the need for the substrate to be first removed from the inoculation apparatus, This would simplify the procedures related to further analysis thus minimising exposure risk when hazardous or toxic materials are being used. A simple apparatus has now been developed which is capable of allowing a wide variety of substrates to be quickly and efficiently inoculated with a material from an aerosol. The apparatus comprises a sealed chamber with an inlet port and an outlet port. The chosen substrate is placed into the chamber, and the chamber is then placed under a small negative pressure using a vacuum pump attached to the outlet port. The aerosol, comprising the chemical or biological material for inoculation, enters the chamber through the inlet port and, without wishing to be bound by theory, it is believed that it then expands and decelerates. This deceleration minimises disruption of the substrate material, a factor which is particularly important when the substrate comprises many small particulates for example sand. As a result of the negative pressure applied via the outlet port, the aerosolised material is drawn towards the outlet port in a trajectory in which it flows over the substrate surface. The material in the aerosol is then successfully deposited on the substrate. By controlling one or more of the following factors - the concentration of material in the aerosol, the length of time the substrate is exposed to the aerosolised material and the flow rate of the aerosolised material over the substrate - it is possible to control and optimise the deposition conditions for accurate deposition of a pre-determined level of chemical or biological material onto a variety of different substrates. The apparatus can be successfully used for the fast and effective inoculation of a wide variety of substrates including solid substrates and more particularly including solid substrates which comprise many small particulates eg sand, with one or more chemical or biological materials from an aerosol. After use, the apparatus can be dismantled, the substrate removed for testing and the apparatus sterilised for future use. In addition the apparatus has been designed so that it can be easily handled, assembled and dismantled while wearing protective clothing, including within a containment facility, and so that it has no sharp edges which could cause tears. This means that the apparatus can be used safely with even the most hazardous or toxic of chemical or biological materials.

The apparatus can optionally be further improved by providing an area of the apparatus that is transparent to radiation so that once the substrate has been inoculated it can be exposed directly to, for example, solar radiation and the degradation of the contaminant can be assessed over time. The apparatus can also be modified to comprise one or more different probes for measuring the atmospheric conditions within the apparatus. This allows the conditions within the chamber to be monitored so that degradation under different environmental conditions can be assessed if needed.

It is an object of the present invention to develop a method, and associated apparatus, for the fast and efficient inoculation of a substrate, particularly a solid substrate, with a wide variety of different chemical and biological materials from an aerosol. Furthermore it is an object of the present invention to develop an apparatus that can be easily used with protective clothing or in a containment chamber. It is also an object of the present invention to develop a reusable apparatus and as such it should be able to be cleansed by sterilisation at high temperature and pressure to eliminate cross contamination. Finally it is an object of the present invention to develop an apparatus whereby, once the substrate has been inoculated, it can be subjected to further testing without the need for further handling. These, and other objects of this invention, will become apparent in light of the following disclosure. Summary of the Invention

According to a first aspect this invention relates to an apparatus for inoculation of a

substrate with an aerosolised material having a sealed chamber, said chamber comprising: an inlet port through which an aerosolised material is able to pass into the chamber; and an outlet port, connected to a means for generating a vacuum, through which an aerosolised material is able to pass out of the chamber; wherein said inlet port and said outlet port are arranged such that aerosolised material flowing through the chamber passes over the surface of the substrate and material within the aerosol is deposited thereon.

According to a second aspect, this invention relates to a method of inoculating a substrate with an aerosolised material comprising:

(i) placing a substrate material in an apparatus according to the present invention;

(ii) applying a vacuum via the outlet port;

(iii) introducing a aerosolised material into the chamber via the inlet port.

According to a third aspect, this invention relates to use of an apparatus according to this invention for the inoculation of a substrate with a biological material. Detailed Description of the Invention

All publications cited herein are hereby incorporated by reference in their entirety, unless otherwise indicated.

As used herein the term biological material means any material which is related to a biological or physiological process or which is produced by such a process. As used herein the term biological material includes any microbiological material. The term microbiological material should be understood as relating to any material from the group comprising microscopic organisms that shall be considered to include, but is not limited to, bacteria, viruses, fungi and toxins derived therefrom.

As used herein the term chemical material means any compound, either natural or synthetic, which comprises one or more elements of the periodic table. This group shall include, but not be limited to, organic material, inorganic material and elemental material.

As used herein the term inoculation means the practice of introducing one or more biological material or chemical material onto a substrate, preferably such that there is a detectable level of the new material on the surface of the substrate. The introduced material can be detected by a wide range of chemical and biological assays that are well known to one skilled in the art.

As used herein the term aerosol means a colloidal system comprising solid or liquid particles dispersed in a gas phase.

As used herein the term aerosolised material means an aerosol that comprises one or more of a biological or chemical material, preferably at a known concentration.

The elements of the apparatus are described in more detail below. According to the present invention the apparatus comprises a sealed chamber. The chamber itself comprises an inlet port through which an aerosolised material enters the chamber and an outlet port through which the aerosolised material leaves the chamber. The aerosolised material can be generated by any suitable means and such means would be known to one skilled in the art including using a nebuliser. The aerosolised material can be conditioned outside the chamber to have a specific temperature and humidity. One example of an apparatus that can be used to condition an aerosol is a Henderson apparatus. The advantage of being able to condition an aerosolised material is that when the conditioned aerosol then enters the chamber the temperature and humidity of the chamber will equilibrate with that of the aerosolised material. The aerosolised material can be introduced into the chamber using pulsing or as a steady stream. It is preferred that the aerosolised material is introduced into the chamber using a steady stream.

The inlet port is designed such that an aerosolised material can pass through the inlet port and into the chamber itself. The inlet port is defined as a position within the chamber whereby one or more openings exist allowing aerosolised material to be released into the interior of the chamber. There are many different positions in which the inlet port can be placed in the chamber. Preferably it is arranged such that the aerosolised material enters the chamber above the substrate. In addition it is preferred that the inlet port is arranged such that the force of the aerosolised material entering the chamber does not disturb the substrate and it is therefore preferred that the inlet port is not arranged to directly face the substrate. To this end it is preferred that the inlet port is arranged on the side of the chamber. This consideration is particularly important if the substrate comprises a liquid or a solid made up of many small particles for example sand. The inlet port can have a wide variety of different sizes and shapes. It is preferred that the dimensions of the inlet port are such that as the aerosolised material enters the chamber it is not channelled into a stream which may be able to disturb the substrate and may lead to uneven dispersion of the aerosolised material across the substrate surface. It is preferred that that inlet port openings has a diameter of from about 1mm to about 10mm, preferably of from about 3mm to about 8mm and more preferably of from about 5mm to about 7mm. It is possible that a single inlet port may have a more than one opening into the chamber. This can be used to prevent the aerosolised material being channelled into a stream and can be used to scatter or spray the aerosolised material into the chamber in several directions such that it is dispersed evenly within the chamber. Furthermore, the chamber may comprise more than one inlet port with each port located in a different position around the inside of the chamber and with each inlet port itself optionally having more than one opening. If the chamber comprises more than one inlet port it is preferred that the inlet ports are spaced evenly throughout the chamber such that the aerosolised material enters evenly from each direction with the result that it is evenly dispersed across the surface of the substrate. In order to simplify the apparatus it is preferred that the inlet port can be readily attached to the aerosolised material source. Preferably the inlet port can be readily attached to a nebuliser or to an aerosol generating / conditioning apparatus such as an Henderson apparatus. If the chamber comprises more than one inlet port, all inlet ports can optionally be attached to the same aerosol source, or optionally the ports could each be attached to different aerosol sources such that the chamber could be used to either simultaneously or sequentially inoculate a single substrate with more than one different type of aerosolised material. The chamber according to the present invention also comprises an outlet port. The outlet port is defined as a position within the chamber whereby one or more openings exist allowing aerosolised material to leave the interior of the chamber. The outlet port is connected to a means for applying a vacuum, for example a simple pump. This vacuum, when applied via the outlet port, ensures that the chamber is placed at a small negative pressure, preferably from about -O.lmBar to about -lOmBar, more preferably from about -lmBar to about -5mBar and even more preferably about - 2.5mBar. This has two effects. This first is that, as the aerosolised material enters the chamber from the inlet port it expands and slows down which reduces the potential for the flow of the aerosolised material to disturb substrate. The second effect of the vacuum applied via the outlet port is that it draws the aerosolised material through the chamber towards the outlet port. Therefore the speed with which the aerosolised material flows through the chamber is controlled by the strength of the vacuum applied via the outlet port. It is preferred that the vacuum means comprises a valve such that the level of the vacuum, and net the flow of aerosolised material through the chamber, can be regulated. It is further preferred that the flow rate of the aerosolised material through the chamber is from about 51min^"! to about 201mm ¹, preferably from about 81mm ¹ to about 151mm ¹ and more preferably from about lOlmin^"1 to about lSlmin^"1.

The direction of flow of the aerosolised material through the chamber is controlled by the relative position of the outlet port and the inlet port. In order for the apparatus of the present invention to function correctly, it is necessary to ensure that the inlet port and the outlet port are arranged relative to each other such that the aerosolised material flows over the surface of the substrate as it passes from through the apparatus. It is preferred that the inlet port and the outlet port are arranged such that the aerosolised material flows evenly across the whole surface area of the substrate thus leading to even deposition of the material on the substrate surface. In order to achieve the desired flow pattern of the aerosolised material through the chamber it is preferred that the outlet port is positioned within the apparatus at an equal height to the substrate or below the substrate, preferably the outlet port is positioned below the surface of the substrate. It is even more preferred that the outlet port is positioned directly underneath the substrate, even more preferably with the substrate centred above the outlet port. The outlet port may be designed to have a wide variety of different sizes and shapes provided that it enables an unrestricted flow of aerosolised material through the chamber. It is preferred that that outlet port openings has a diameter of from about 1mm to about 10mm, preferably of from about 3mm to about 8mm and more preferably of from about 5mm to about 7mm. As with the inlet port, a single outlet port can be designed to have several openings. It is preferred that the outlet port comprises a round cylinder which has several different openings evenly placed around the cylinder. This ensures that the negative pressure drawing the aerosolised material through the chamber is applied evenly from all directions around the chamber thus ensuring that the material flows evenly over the surface of the substrate. Alternatively it is also possible to have several outlet ports arranged within the chamber. Each of these ports can be connected to one or more means for applying a vacuum and, in order to ensure even flow of aerosolised material through the chamber, it is preferred that the vacuum applied to each port is of the same strength. Both the inlet port and the outlet port may be attached to internal pipe work or channelling within the chamber such that the opening of the port into the chamber need not necessarily be the same point at which the port breaches the walls of the sealed chamber. This is particularly useful in the case of the outlet port where the preferred position of the outlet port is centred underneath the substrate on the base of the chamber but where the use of internal pipe work means that the port can leave the chamber on a side wall thus ensuring that the chamber retains a flat base.

The chamber can be made from a wide variety of materials including metals, plastics and glasses. It is possible that the internal walls of the chamber can be coated with a material that has a low surface energy for example Teflon™. This helps to prevent deposition of the material from the aerosolised material onto the internal walls of the chamber. The chamber material should be chosen by taking into consideration the substrate, the aerosolised material and the strength of the negative pressure that will be applied via the outlet port. If the aerosolised material is charged it is important that the chamber is not made of a material which itself carries an electrostatic charge, for example plastics, since this would result in the aerosolised material depositing on the internal walls of the chamber rather than on the substrate. It is preferred that the chamber is made of a non reactive metal or metal alloy material or a silicone oxide material such as glass or quartz. The most preferred material for making the chamber is stainless steel. Such materials are preferred since they are easily modelled to form the chamber, they can be used with a wide variety of different biological and chemical materials, and they can withstand a negative internal pressure within the chamber. It is also preferred to use a material, which can be easily planed to have a smooth surface finish such that the apparatus will not snag or tear clothing, if it is used either with protective clothing or in a containment facility. It is also preferred that the material chosen for the chamber is suitable for cleaning such that the chamber can be reused. Preferably the chamber is durable enough to be cleaned by sterilisation in an

autoclave at temperatures of up to 200°C and pressures of up to 5Bar, or by formaldehyde fumigation.

The chamber itself can have a wide variety of different sizes and shapes with the exact dimensions being readily determined by one skilled in the art for the specific purpose. The volume of the chamber should be designed such that it is able to readily accept the size of the substrate to be inoculated. In addition the volume should be chosen such that, when the aerosolised material enters the chamber, it is able to expand thus decreasing its flow rate to and extent such that it does not disturb the substrate. However, it is important that the volume of the chamber is not so large that the aerosolised material is diluted substantially when it enters the chamber since this will decrease the efficiency with which the substrate can be inoculated. It has been shown that if the volume of the chamber is too large then the deposition of material in the centre of the substrate can be reduced and an uneven deposition pattern is achieved. An additional advantage of having a smaller internal chamber volume is that the internal temperature and humidity conditions can be more easily and quickly adjusted to mimic different external environmental conditions. In order to inoculate substrate for laboratory based work it is likely that a chamber with a diameter of from about 10mm to about 250mm, preferably of from about 30mm to about 200mm and more preferably of from about 50mm to about 150mm and with a height of from about 20mm to about 200mm, preferably of from about 50mm to about 150mm and more preferably from about 60mm to about 100mm is likely to be sufficient. However for larger scale trials a larger chamber may be required. It is preferred that the internal volume of the chamber is from about 1cm³ to about 2000cm³, preferably

from about 50cm³ to about 1000cm³ and more preferably from about 100cm³ to about 700cm³. This volume makes the chamber ideal for use as a model system.

In theory the chamber may have any possible internal shape. It is however preferred that that chamber is round or oval in shape to facilitate the flow of aerosolised material through the chamber and across the surface of the substrate. It is preferred that the chamber is designed to comprise a first area and a second area. The first area of the chamber comprises the inlet port and preferably has a diameter that is smaller than that diameter of the surface area of the substrate. The second area of the chamber comprises the substrate, the outlet port and preferably has a diameter that is larger than the diameter of the surface area of the substrate. It is preferred that the ' diameter of the first area of the chamber is approximately about 20%, preferably about 25% and more preferably about 30% smaller than the diameter of the second area of the chamber. For example it is prefened that the first area has a diameter of from about 10mm to about 100mm, preferably of from about 30mm to about 90mm and more preferably of from about 50mm to about 80mm. It is preferred that the second area has a diameter of from about 50mm to about 200mm, preferably of from about 70mm to about 150mm and more preferably of from about 80mm to about 120mm. It is prefened that the first area of the chamber is approximately the same height as the second area of the chamber. The internal walls of the first area of the chamber preferably extend into the second area of the chamber such that there is only a small gap between the base of the walls of the first area of the chamber and the surface area of the substrate. In a chamber which has a total height of about 80mm, it is prefened that the gap between the bottom of the inner walls of the first area and the surface of the substrate is from about 10mm to about 1mm, preferably from about 8mm to about 3mm and more preferably from about 6mm to about 4mm. The small level of the gap between the base of the walls of the first area of the chamber and the surface area of the substrate ensures that the flow of aerosolised material as it passes from the first area of the chamber to the second area of the chamber is across the surface area of the substrate thus maximising the deposition of aerosolised material.

The apparatus of the present invention is designed for the inoculation of one or more substrates with one or more of a biological or chemical material. The substrate for use in the present invention can be either solid or liquid or a semi-solid material such a gel. It is prefened that the substrate is solid or semi-solid. The present invention is suitable for use with a wide range of different solid substrates including solid substrates that comprise many small particles. The apparatus is particularly useful for inoculating concrete, soil, sand, concrete, wood, agar gel and the like. The apparatus of the present invention can be designed to comprise one or more substrates simultaneously.

It is important that the substrate does not block the outlet port preventing the flow of aerosolised material through the chamber and as such it is prefened that the substrate is suspended within the chamber on a frame, preferably suspended on a frame and centred above the outlet port. It is also prefened that the apparatus comprise a dish into which the substrate is placed which allows for easier handling, and that in turn the dish is suspended on a frame. If the apparatus is to be used to inoculate one or more substrates simultaneously it is prefened that each substrate is placed into a separate dish or that the dish is sub-divided. The size of the dish should be large enough such that it holds the substrate but should not be so large as to inhibit the flow of the aerosolised material around the dish to the outlet port. It is prefened that the diameter of the dish is from about 5% to about 20% smaller than the diameter of the chamber. If the chamber of the apparatus comprises a first area and a second area the substrate is held within a dish in the second area of the chamber. In such an embodiment it is prefened that the diameter of the dish is larger than the diameter of the first area of the chamber but smaller than the diameter of the second area of the chamber. Furthermore it is prefened that dish has sufficient depth such that the walls of the dish extend beyond the inner walls of the first area of the chamber so that there is an overlap between the walls of the dish and the inner walls of the first area of the chamber. In a chamber with a total height of approximately 80mm, it is prefened that the overlap between the inner walls of the first area of the chamber and the outer walls of the dish is about 5mm. This ensures that the aerosolised material flows underneath the walls of the first area of the chamber, across the surface area of the substrate in the dish and then up over the outer walls of the dish improving deposition. However, it is important that the overlap is not so great that the flow of the aerosolised material is directed away from the surface area of the substrate which may have the result that those parts of the substrate close to the walls of the dish are not subject to aerosolised material deposition.

The chamber should comprise a means for opening the chamber such that the substrate can be placed within the chamber and removed after inoculation for further analysis. In order to ensure that the chamber works effectively it is important that the opening can be securely sealed so that the vacuum can be applied to the chamber. To ensure this seal it is prefened to use an "O"-Ring preferably made of a rubber material, a silicone material or a mixture of a rubber material or a silicone material. The opening should be designed such that is can be readily handled and manipulated by users with reduced dexterity who are wearing protective clothing or operating within a containment facility. As such it is prefened that the lid attaches to the rest of the chamber by a simple screw action. However other options for fitting the lid include clamps and the like.

Optionally the chamber can also comprise other features such that the conditions within the chamber can be modified and the material can be subjected to various different experimental analyses. It is prefened that the chamber comprises an area that is transparent to different types of radiation preferably one or more of solar radiation, UV radiation, IR radiation, microwave and more preferably solar radiation and even more preferably solar radiation from a simulated source. The transparent area may be conveniently orientated in the lid of the chamber such that post inoculation the substrate can be immediately subjected to radiation without the need for any further manipulation. The transparent area can be flat or be designed with a concave or a convex shape to focus the radiation onto a particular area of the substrate. It is prefened that the transparent area is flat. The transparent area can be made of any material that is suitable for the radiation in question. For use with solar radiation and simulated solar radiation it is prefened that the transparent area is made of glass or silicon dioxide quartz, preferably quartz. It is prefened that the transparent area is larger than the surface area of the substrate such that the whole of the substrate can be evenly exposed to degradation. The transparent area should be thick enough to withstand the pressure inside the chamber, it should preferably be designed such that it can be cleansed by sterilisation and or formaldehyde fumigation, and is should be designed to be suitable for use by a user wearing protective clothing or in a

containment facility. Optionally the transparent area can be removed from the chamber and replaced with a different transparent material such that the apparatus can be used to screen the substrate for exposure to several different types of radiation. If the transparent area of the chamber is removable and replaceable then it should be designed to have a good seal with the rest of the chamber such that the chamber can be put under a negative pressure.

Optionally the chamber may comprise one or more probes to monitor the atmosphere within the chamber for example a temperature probe, a humidity probe or a radiation probe. It is prefened that if the chamber comprises one or more of such probes that the probes are positioned within the chamber such as to minimise any disturbance to the flow of the aerosolised material through the chamber. It is prefened that any such probes are located on the side of the chamber such that they do not interfere with the opening of the chamber and also that they are located away from any transparent area of the chamber again to minimise interference. Such probes can be inserted into the chamber using a self-sealing device such that the probe can be removed and changed without breaking the seal of the internal atmosphere of the chamber.

According to a further aspect this invention relates to a method of inoculating a substrate with an aerosolised material comprising:

(ii) applying a vacuum via the outlet port; (iii) introducing a aerosolised material into the chamber via the inlet port.

The aerosolised material can be introduced into the chamber. This apparatus can be preferably used to inoculate a solid substrate with a microbiological material. The concentration of the material to be inoculated on the substrate can vary depending on the desired level of inoculation to be achieved. When inoculating a sold substrate with bacteria it is prefened that the concentration of the material in the aerosol is approximately 10⁹bacteria/ml.

Furthermore, this invention relates to the use of an apparatus according to this invention for the inoculation of a substrate with a biological material, preferably a biological material and more preferably a microbiological material. It is possible to mix the different materials to be used with the present invention but, when dealing with particularly hazardous materials and especially in the case of use of microorganisms any mixing of materials should be in strict compliance with the relevant regulations. The efficiency of the apparatus varies depending on the material in the aerosol and the concentration of the aerosol.

Figures

The following figure illustrates the prefened embodiments within the scope of the present invention. It is given solely for the purpose of illustration and is not to be construed as limitations of the present invention as many variations of the invention are possible without departing from its spirit or scope. This invention will now be described by reference to the following drawings in which; Figure 1 shows a simple embodiment of the apparatus of the present invention; and

Figure 2 shows a preferred embodiment of the apparatus wherein the chamber comprises a first area and a second area.

Figure 1 comprises a chamber 2 into which is placed a dish 4 comprising the substrate 6. The chamber comprises an inlet port 12 and an outlet port 20. The outlet port is directed by internal piping 22 such that it exits the chamber 2 at the side. The chamber also comprises a removable lid 24 in which is a area 26 transparent to solar radiation. The chamber also comprises a frame 28 which suspends a dish 4 within the chamber and above the outlet port 20. The outlet port 20 is attached to a vacuum pump (not shown). The aerosol cloud 8, comprising the material 10 to be inoculated onto the substrate 6, enters the chamber via the inlet port 12. The aerosol 8 is generated by an aerosol generating means (not shown) and conditioned by an aerosol conditioning means (not shown). The vacuum applied via the outlet port 20 draws the aerosol 8 comprising the material 10 towards the outlet port 20. As it is drawn it flows in the direction of the anows as shown across the surface of the substrate 6 and around the dish 4. In doing so the material 10 in the aerosol 8 is able to deposit on the substrate 6.

Figure 2 comprises a chamber 2, which is divided into a first area 30 and a second area 32. The first area 30 comprises an inlet port 12, a transparent area 26 and a temperature probe 34. The second area 32 comprises an outlet port 20, with several openings 36, and which is directed by internal piping 22 to the outside of the chamber 2. The second area also comprises a frame 28 on which is placed a dish 4 containing a substrate 6. The walls of the first area 30 extend to within the second area such that there is only a very small gap between the base of the walls and the surface of the substrate 6. The walls of the dish 4 are tall enough such that they overlap with the walls of the first area 30. The aerosol 8, containing the material to be deposited, 10, and generated by an aerosol generating means (not shown) enters the first area of the chamber 30 through the inlet port 12. A vacuum, generated by a pump (not shown) draws the aerosol in the direction of the arrows as shown across the surface of the substrate 6, under the walls of the first area 30, over the walls of the dish 4 and out of the chamber 2 through the openings 36 in the outlet port 20. As the aerosol 8 passes across the surface of the substrate 6 the material 10 is deposited on the substrate 6.

Examples

The following examples further illustrate the prefened embodiments within the scope of the present invention. These examples are given solely for the purpose of illustration and are not to be construed as limitations of the present invention as many variations of the invention are possible without departing from its spirit or scope.

A petri dish of 90mm in diameter was half filled with sand, the surface of the sand levelled, and the dish placed into an apparatus according to the present invention. Once the substrate was placed into the apparatus a vacuum pump was attached to the outlet port of the apparatus and the apparatus was placed at an internal pressure of - 2.5mBar.

A nebuliser was used to prepare an aerosol comprising bacteria in a concentration of 10⁹ bacteria/ml. An Henderson apparatus was used to condition the aerosol to room temperature and 50-60% relative humidity. The inlet port of the apparatus was attached to the Henderson apparatus such that the conditioned aerosol entered the inlet port. The aerosol was left to flow through the apparatus at a flow rate of approximately 121mm ¹ for a period of 2 minutes. After this time the substrate was removed from the apparatus and testing demonstrated that an average level of 10⁷ bacteria were deposited on the surface of the substrate. Repetition of this method with soil substrate demonstrated similar results.

The above experiment was repeated. On each subsequent test the substrate was exposed to solar radiation for a period of time prior to being removed from the apparatus and tested for bacteria levels. This programme enabled a degradation profile of the bacteria on the substrate to be developed.

Claims

1. An apparatus for inoculation of a substrate with an aerosolised material having a sealed chamber, said chamber comprising: an inlet port through which an aerosolised material is able to pass into the chamber; and an outlet port, connected to a means for generating a vacuum, through which an aerosolised material is able to pass out of the chamber; wherein said inlet port and said outlet port are ananged such that aerosolised material flowing through the chamber passes over the surface of the substrate and material within the aerosol is deposited thereon.

2. An apparatus according to Claim 1 wherein the substrate is a solid substrate.

3. An apparatus according to any of Claims 1 or 2 wherein the inlet port is located above the substrate and on the side of the chamber.

4. An apparatus according to any of Claims 1 to 3 wherein the inlet port can be connected to a Henderson apparatus.

5. An apparatus according to any of Claims 1 to 4 wherein the substrate is centred above the outlet port.

6. An apparatus according to Claim 5 wherein the outlet port comprises greater than 1 opening, preferably greater than 2 openings and more preferably comprises from about 3 to about 5 openings.

7. An apparatus according to any of Claims 1 to 6 wherein the chamber comprises a frame capable of suspending the substrate above the base of the chamber.

8. An apparatus according to Claim 7 wherein substrate material is held in a dish which is in turn suspended above the base of the chamber on a frame.

9. An apparatus according to any of Claims 1 to 8 wherein the chamber comprises a first area and a second area, wherein the first area comprises the inlet port and the second area comprises the substrate and the outlet port and

wherein the internal diameter of the first area is smaller than the internal diameter of the second area.

10. An apparatus according to Claim 9 wherein the internal walls of the first area of the chamber extend into the second area of the chamber such that there is only a small gap between the base of the walls of the first area of the chamber and the surface area of the substrate.

11. An apparatus according to any of Claims 9 or 10 wherein the substrate is held within a dish and wherein the dish has sufficient depth such that the walls of the dish extend beyond the inner walls of the first area of the chamber.

12. An apparatus according to any of Claims 1 to 11 wherein the chamber comprises a window that is transparent to solar radiation, preferably made of silicon dioxide.

13. An apparatus according to any of Claims 1 to 12 wherein the apparatus and each constituent part is made of a material which is durable enough to be cleaned by sterilisation.

14. An apparatus according to any of Claims 1 to 13 wherein the apparatus comprises one or more of a temperature probe, a humidity probe or a radiation probe.

15. A method of inoculating a substrate with an aerosolised material comprising: (i) placing a substrate material in an apparatus according to any of Claims

1 to 14; (ii) applying a vacuum via the outlet port; (iii) introducing a aerosolised material into the chamber via the inlet port.

16. A method according to Claim 15 to wherein the chamber is placed at pressure of from about -O.lmBar to about -lOmBar, preferably from about -lmBar to about -5mBar and more preferably about -2.5mBar.

17. A method according to any of Claims 15 or 16 wherein the aerosol comprises a biological material.

18. A method according to any of Claims 15 to 17 wherein flow rate of the aerosolised material through the chamber is from about 51mm^"1 to about 201mm^"1, preferably from about 81mm^"1 to about 151mm ¹ and more preferably from about lOlmin^"1 to about 131mm^"1.

19. Use of an apparatus according to any of Claims 1 to 14 for the inoculation of a substrate with a biological material.