US20050170497A1 - Method and apparatus for the non-invasive monitoring of gas exchange by biological material - Google Patents

Method and apparatus for the non-invasive monitoring of gas exchange by biological material Download PDF

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Publication number
US20050170497A1
US20050170497A1 US10/508,081 US50808105A US2005170497A1 US 20050170497 A1 US20050170497 A1 US 20050170497A1 US 50808105 A US50808105 A US 50808105A US 2005170497 A1 US2005170497 A1 US 2005170497A1
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pressure
container
pressure sensing
sensing system
silo
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US10/508,081
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English (en)
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Tony Carr
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Bactest Ltd
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Bactest Ltd
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Priority to US13/102,445 priority Critical patent/US8389274B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • the present invention describes a method and instrument for detecting and monitoring gas exchange in biological preparations.
  • the system is based on the measurement of pressure in a sealed container, partially filled with liquid. Components of the liquid can give rise to gas exchanges between the liquid and container headspace, resulting in primary pressure variations.
  • the sealed container includes a flexible diaphragm at the boundary between a main headspace and a closed secondary chamber that contains compressible gas and a direct connection to an electronic pressure sensor. There is a simple relationship between primary and secondary pressures, where the sensor measures the latter.
  • the complete system is divided into a single use component and a durable instrument.
  • Single use, disposable containers provide containment during the test, followed by sterilisation and disposal after use.
  • the unique design of the container described here is intended to provide optimum conditions for gas exchange.
  • the present invention gives rise to the container design, which is directly linked to the functionalities required in practical applications of the system.
  • the instrument is physically compact, housing one, or more normally two, of the single use containers.
  • the mixing, measurement and temperature control functions are supervised in parallel with data processing to identify significant pressure changes. No part of the instrument comes into contact with biological material.
  • the regions which house the disposables, for test duration, provide precise temperature control but also act as a bund or silo for containment purposes.
  • Methods for monitoring biological activity are particularly valuable in laboratory tests carried out in sealed containers. Unlike open processes (for the conversion of raw materials to valuable end products), the laboratory test generates information about the detection or monitoring of biological activities. Containment is a critical feature both for safety reasons (where the contents are unknown and potentially dangerous) and as a barrier to contamination (where the test outcome could be invalidated by an unknown contaminant to which the result may be attributable).
  • Blood culture instruments described in US5672484 and WO9521241 involve the use of individual pressure sensors connected to individual culture bottles, with a protective filter interposed. This is effective as a measurement system but is unavoidably invasive, due to the use of a hypodermic needle in the connection assembly.
  • the method described in WO9303178 is based on laser height measurement to measure pressure in terms of distension of a large area septum. While non-invasive, the method involves a relatively expensive, high sensitivity sensor. This is only practical when a mechanism is used to move the sensor over the bottles.
  • the current invention recognises that a sealed container, partially filled with liquid can experience gas exchanges, which results in pressure variation in the headspace. Pressure variation could be due to temperature effects, but these can be prevented by good temperature control, which is relatively easy to achieve. Should the container include any significant area of flexible material then barometric pressure changes can exert an influence, however, this is not so in a rigid container. Ideally all pressure variations should be a direct reflection of activity levels in the biological component of the liquid phase.
  • This invention includes a primary sealed container with a closure incorporating a flexible diaphragm closure across a secondary chamber.
  • One side of the diaphragm is in contact with the gas and liquid phases of the main chamber so that the diaphragm forms a barrier isolating the secondary chamber and sensor from the main vessel contents.
  • Pressure variations in the main container headspace are sensed by a pressure transducer fitted to the secondary chamber.
  • This system operates as a respirometer monitoring gas exchange by biological preparations.
  • the flexible diaphragm is a sterility barrier.
  • the liquid phase and any active cells can only exchange gasses with the main headspace (25 mL in this case).
  • the biological preparation has access to the combined headspace volume of 30 mL. approximately.
  • the monitoring system operates at or around atmospheric pressure and changes are normally in the order of 100 millibars positive or negative, about atmospheric pressure.
  • Sensitivity, sterility and impermeability are essential, in addition the material of the diaphragm must be bio-compatible.
  • the characteristics of the diaphragm (for example, diameter, elasticity and thickness) will attenuate the extent of the variation, but the relationship between ‘primary’ and ‘secondary’ pressures is simple.
  • test containers in commercially available systems are based on a bottle or tube, usually of glass.
  • This invention provides a container, which is itself purpose designed as a disposable, single use item, for use with the apparatus of the invention.
  • the crux of any method based on gas exchange is to ensure adequate interaction between the liquid and gas phases.
  • the device includes a major feature intended to promote efficient gas exchange. Since the pressure itself is monitored, the design of the vessel leaves scope to add a series of additional features, each intended to provide management of vessel pressure at various stages of a test procedure.
  • Integral filters ensure that any gas exchange with the environment does not compromise the levels of containment. In blood culture applications these additional facilities are unique, and provide an improved level of safety. This is an attractive feature where a blood sample may include virus such as hepatitis and/or HIV, in addition to any bacterial infection.
  • the invention provides the facility for monitoring gas exchange, and this can be applied in a number of ways.
  • the presence of micro-organisms at low levels can be demonstrated by the addition of a sample to a suitable growth media, followed by a period of incubation. The viable count will rise, and activity will be detected.
  • This detection capability is applicable to blood, serum, spinal fluid and aspirates.
  • the monitoring of activity levels can be applied to specific species or, indeed, complex mixtures (e.g. Activated Sludge). Tests can monitor both enhancement and inhibition of activity. In this way, potential nutrients, preservatives or pollutants are examined for any influence on activity levels, with the test apparatus performing the role of a respirometer.
  • a chemical carbon dioxide scavenger KOH creates an oxygen depletion measurement and, hence, a means to determine the Biological Oxygen Demand (BOD) of, say, a water sample.
  • growth media formulations should be matched to the applications, but with an emphasis on promoting gas exchange.
  • the instrument could take many forms, but must control mixing effectively, through a range of possible regimes, and maintain precise control of temperature. Signals representing pressure are transferred from the sensor, to an Analogue to Digital convertor, and then to a microprocessor where data can be processed, generally using simple algorithms to calculate the rates and significance of changes. Decisions and conclusions can be displayed, or relayed to a PC. Instrument capacity could be small or scaled up as multiples of the small basic unit. In a simple form the unit would process one sample, but it would be more typical to have a basic unit that processes two disposables at a time (typically one aerobic/one anaerobic). This is an unusually low cost package of minimal complexity, very portable, and with virtually no service requirement.
  • the basic unit could process aerobic/anaerobic samples from the same patient, thus providing a self contained, low cost system with no need for large, high capacity machines of major capital cost that would require service back-up.
  • the enhanced level of containment and safety features are of value in the testing of high risk samples, even to the extent of locating units close to the sampling facility. In the event of the unit becoming unsafe, e.g. if leakage has occurred, either the test vessel and its silo can be removed, or the complete unit subjected to sterilisation and disposal.
  • the majority of culture containers used as closed vessels are based on bottles, tubes or flasks generally of a robust grade of glass.
  • the emphasis in this invention is upon the promotion of gas exchange, including a mixing means as a key feature.
  • the vessel is moulded as a trough, of approximately cuboid dimensions, with the lower half having a semicircular section.
  • a cylindrical mixing means Within this, and closely following the wall profile, is a cylindrical mixing means. Since robustness is desirable the preferred material is polycarbonate.
  • the working volume of the container is in the region of 125 ml., where 25 ml is headspace atmosphere and 100 ml. is the liquid phase.
  • the container starts with a media volume of 90 ml.
  • the mixing means is a rotating structure such as a paddle wheel, designed to rotate and cause consistent disturbance to the liquid surface. This is analogous to a constant vortex, essentially improving the interfacial area and promoting gas exchange. Paddle blades leaving and re-entering the liquid phase cause useful disturbance. Paddle blades moving in the liquid phase assist mixing and uniformity of conditions.
  • the rotating structure has a central shaft, the ends of which rotate in simple, plain bearing bushes—in this way the total mixing means follows a defined circular path about a relatively low friction mounting.
  • the rotating element carries two small cylindrical ferrite magnets. This is the basis for a magnetic coupling to two external rare earth high power magnets.
  • the external magnets When the external magnets are rotated by a stepping motor drive, the internal paddle wheel rotates at exactly the same rate. Since the magnetic coupling operates through the wall of the vessel there are no seals, apertures or other potential leakage points.
  • a simple magnetic steering in the form of a rotating bar in a cylindrical bottle is described in WO9402238, but here the mixing action is created by the magnet per se, usually rotating at approximately 200 rpm.
  • the arrangement described in this invention operates at substantially slower speeds and the magnetic coupling is precisely aligned in a ‘captive’ registration of the magnets.
  • the vessel described so far is completed as an assembly by the addition of a “lid” as a closure.
  • the “lid” has a series of features built into the structure which provide additional function.
  • the first of these functions is a link to the main vessel headspace from a secondary chamber of approximately 5 ml capacity.
  • the central feature is a working diameter of 25-30 mm, across which a flexible diaphragm membrane is mounted.
  • the diaphragm provides a containment barrier, whilst allowing pressure variations across the interface between primary and secondary chambers. Diameter, thickness and flexibility can be varied, but the most consistent requirement is that the diaphragm membrane is totally impermeable to gases.
  • the culture vessel with integral diaphragm, is treated as a one-use disposable.
  • a resilient seal eg. made of rubber
  • the container has a formation designed and adapted to receive and locate a “boss” on the sensor assembly which is part of the instrument.
  • a fluid-filed manometer tube with a flexible membrane at the base end.
  • This membrane is physically linked to that of the culture vessel (using a magnetic link or a small area of “hook and loop fastener” (eg. VelcroTM) so that the two membranes move as one. Hence fluid is moved in the manometer for visual observation.
  • a culture container in transit may be shaken or inverted. Consequently the pressure transfer link to the headspace is closed until required, thus avoiding the ingress of fluids into critical regions of the lid assembly.
  • This is easily achieved by a small area foil seal, perforated on demand by pressure on a hollow pin with a sharp, cutting action end. To preserve sterility this action can be performed indirectly via a flexible covering membrane.
  • a similar valve action, or equivalent opening of a rubber seal mechanism can be provided to open a link to a barrier filter venting to the surroundings.
  • the filter must be totally effective in preventing the escape of both bacteria and viruses. Since many cultures, particularly anaerobic bacteria, are capable of establishing appreciable pressures this is a basis for a pressure relief device which operates automatically or on demand. In a preferred version automatic operation is initiated by gas pressure directly, which is fail/safe. On demand operation is controlled by the instrument electronically, and can be triggered for both excess positive pressure or persistent negative pressure.
  • valve operation linked to a filter protected port to atmosphere.
  • Equilibration to atmospheric pressure can be carried out at any time to effectively zero the pressure sensor.
  • this is a useful facility during injection of a sample; opening a port allows progressive displacement of headspace gas to accommodate the incoming volume of sample.
  • opening a port allows progressive displacement of headspace gas to accommodate the incoming volume of sample.
  • this invention matches displacement to sample volume exactly. This has an advantage where the incoming volume is large.
  • Valve operation should again be indirect, via a membrane barrier, to maintain integrity of the culture vessel.
  • the septum should be of minimal diameter and relatively thick material, to avoid any barometric influence.
  • the septum material has excellent characteristics.
  • sample addition or removal is the specific function for which it is used.
  • the culture container as described avoids the incorporation of any components or materials which would be classified as “sharps”.
  • the only operation using a conventional needle is sample addition (or removal for subculture/staining).
  • the region of the lid, directly over the injection septum, is essential for access.
  • the lid includes a chimney-like formation which encircles the needle and provides some shielding of the technicians using the culture units.
  • the possibility of needle stick injuries should be reduced by correct use of this feature.
  • the outer end of the access tube will initially be closed by a cap with integral tamper-evident seal. The cap can be reapplied but provides a clear indication that a culture unit has been partially opened or inoculated.
  • the lid assembly is a composite unit, housing several features, which could be a ‘sandwich’ construction where the ‘filling’ is a moulded rubber membrane showing different features in different areas.
  • the outer casework can be coded in various ways to identify contents and application. This can include colour coding with or without labelling, possibly bar coding as a computer compatible identification code.
  • the smaller components are assembled into the lid casing prior to closure and bonding into the vessel, followed by gamma irradiation to sterilise the complete assembly.
  • An aspect of the culture vessel design is the selection of appropriate materials. Avoidance of “sharps” and widely diverse materials is intended to simplify final disposal after use. For safety reasons terminal autoclaving is advisable; this would be followed by combustion using methods normally employed for disposables. Conventional culture containers create difficulties in disposal; the use of appropriate materials is intended to improve the environmental impact of large scale usage and disposal.
  • the durable part of the culture system takes the form of a compact, dedicated instrument.
  • the instrument In typical applications (eg Blood culture) the instrument has a capacity of two culture units (usually one aerobic/one anaerobic). The culture units are each housed in a incubation “silo”.
  • the pressure sensing system will register the effect of temperature change; indeed the progression from room temperature to incubation temperature (37°) produces a marked rise in pressure. This is a useful indication that the instrument is working well and that there are no leaks at the connection between disposable and instrument.
  • the silo wall is close to the disposable vessel and almost all heat transfers by radiation. The air layer slows heat transfer but this also applies to losses and thus has a stabilising effect. Particular attention is applied to extensive insulation using eg. NomexTM card.
  • a double ended rotor has a motor on both shafts with high power Neodymium Boron magnets mounted in each rotor.
  • the two “silos” are arranged to align with the two drive couplings, one on each side of the central drive module.
  • Data from the sensor starts to accumulate for further processing.
  • the electronics within a unit will monitor changes, decide the significance of events and, where necessary, operate the venting mechanism.
  • Essentially the ways in which data is handled depends on the configuration of test units. A basic unit, in small numbers, should be capable of individually reporting operational status. Where large numbers are in use then a centralised facility may be more appropriate.
  • the software is able to make decisions about prevailing pressure and any requirement to vent. There are criteria for this and the activator is motor driven on demand. Use of a small electric motor and a small lead screw is sufficient to apply adequate force. The action can be monitored and the effect confirmed by the pressure sensor itself.
  • the system of the present invention can match materials, characteristics and responses, to the biological application. It can operate at, or about atmospheric pressure, respond to small, subtle changes and provide a containment barrier (at the interface between disposable and durable parts of the system). It is not a direct pro rata pressure transfer system. Commercially it is very low cost, single use system, but includes a critical safety feature.
  • FIG. 1 Graph comparing primary pressure sensor reading with those from a secondary pressure sensor, over a range of known pressure values.
  • transfer of pressure was via a 30 mm diameter/0.8 mm thickness Butyl Rubber.
  • Sensor readings are recorded in millivolts, with actual pressure valves in mbar (using a fully calibrated Druck meter).
  • the traces of primary values are marked with circles, and the corresponding secondary pressures marked as triangles.
  • FIG. 2 General Base, side and front views of the single use disposable. These show the paddle wheel drive magnets on the RHS and the injection port closed with a tamper evident, detachable lid. It should be noted that the lid and closure have deliberate asymmetrical features, this ensures correct orientation on loading and hence fail/safe magnetic coupling of the drive system.
  • FIG. 3 The injection port, for addition of samples and removal of subcultures using syringes.
  • the port is expected to protect against needle-stick issues by virtue of the large diameter “chimney-like” structure.
  • FIG. 4 A single action valve for connection permanently between headspace and the sensor system. Operation of the valve is indirect, via a membrane barrier, using manual pressure to perforate foil seal.
  • FIG. 5 A multiple action valve, again indirect via a membrane, but only open when activated and thus providing momentary connection of headspace to atmosphere via a filter.
  • FIG. 6 This illustrates some of the many options for functional features on shafts through the paddle wheel centre axis.
  • FIG. 7 In a similar way there are options for refining the role and functions of paddles or other mixing structures.
  • FIG. 8 The instrument in front view, with a cutaway section to illustrate silo construction and operation of the central magnetic drive. Also a side view to illustrate scale and proportions.

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US10/508,081 2002-03-16 2003-03-14 Method and apparatus for the non-invasive monitoring of gas exchange by biological material Abandoned US20050170497A1 (en)

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GBGB0206275.0A GB0206275D0 (en) 2002-03-16 2002-03-16 Method and apparatus for the non-invasive monitoring of gas exchange by biological material
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US13/102,445 Expired - Fee Related US8389274B2 (en) 2002-03-16 2011-05-06 Method and apparatus for the non-invasive monitoring of gas exchange by biological material
US13/749,975 Expired - Fee Related US8795982B2 (en) 2002-03-16 2013-01-25 Method and apparatus for the non-invasive monitoring of gas exchange by biological material

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US13/749,975 Expired - Fee Related US8795982B2 (en) 2002-03-16 2013-01-25 Method and apparatus for the non-invasive monitoring of gas exchange by biological material

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US8389274B2 (en) 2002-03-16 2013-03-05 Bactest Limited Method and apparatus for the non-invasive monitoring of gas exchange by biological material
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AU2003216839A8 (en) 2003-09-29
US8795982B2 (en) 2014-08-05
EP1487962B1 (de) 2014-08-20
WO2003078563A2 (en) 2003-09-25
WO2003078563A3 (en) 2003-12-18
US8389274B2 (en) 2013-03-05
EP1487962A2 (de) 2004-12-22

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