GB2494202A

GB2494202A - Microorganism imaging and incubating apparatus able to maintain a temperature gradient

Info

Publication number: GB2494202A
Application number: GB1115301.2A
Authority: GB
Inventors: Frank Prior
Original assignee: BACTROSCAN Ltd
Current assignee: BACTROSCAN Ltd
Priority date: 2011-09-05
Filing date: 2011-09-05
Publication date: 2013-03-06
Also published as: GB201115301D0

Abstract

An apparatus for incubating and imaging microorganism growths comprising: a chamber for incubating a culture plate; a heated optically transparent platen for placing a culture plate adjacent thereto, said chamber and said platen being arranged so that a reducing temperature gradient, from higher to lower temperature, is formed in a direction moving away from the platen, further comprising an imaging device arranged to view through said platen for capturing an image of the culture plate. Also described is an apparatus and method of incubating and imaging a culture plate comprising incubating said culture plate by placing a culture plate on the heated optically transparent platen so the lid of the culture plate is adjacent the heated optically transparent platen, so that a reducing temperature gradient is formed and the temperature of growth media in a base of a culture plate is less than the temperature of the lid of the culture plate.

Description

Imaging AQparatus and Methods

Field of the Invention

The invention relates to imaging apparatus for incubating and imaging growths such as plant or animal tissue, cell cultures or micro-organisms (for example, bacterial growths) and methods for use in such apparatus. In particular the invention relates to an imaging apparatus comprising an imaging device and an incubator. In some erribodiments, the invent!on relates to a method and device for the incubation, detection and identification of growths such as micro-organisms.

Background

Present microbiological testing methods have changed little since their original invention by Robert Koch and Louis Pasteur in the 1850-60's. Micro-organisms are plated out on culture media, incubated and the number of organisms counted. Most plates are incubated for 2-7 days. After this they are observed by a microbiologist, the number of growths counted, and samples taken for identification through either colour chemistry or DNA sequencing. As a result bacteriology is a slow and expensive process. However, there are problems inherent in present microbiological testing methods.

Batches of "sterilized pharmaceuticals" or other sterilised products have to be kept in quarantine until the sterility of the batch is confirmed before they are released. This usually takes 5-7 days and is an added cost to sterilising services. A faster method of sterility testing is required. To comply with such requirements, as laid out in the US or UK Pharmacopoeias, plates have to be examined at specific time intervals and colony counts performed. This frequently results in the microbiologist having to perform colony counting out of hours, which is expensive. Apparatus and a method of viewing images without being in the laboratory are desirable.

Potentially contaminated areas in hospitals have to be kept empty for several days until sterility confirmation is obtained from the bacteriological lab. Taking these areas out of service slows down surgery and results in a slowing of patient throughput. A faster method of bacterial identification is required. New patients may be placed in isolation unnecessarily or may be placed on wards prematurely, if no prompt method and apparatus is available for testing such patients for bacteria such as MRSA or C. difficile in a cost effective, relatively quick manner.

Microbiological culture plates are destroyed after the colony counting and identification. As a result there is no visible record retained. This lack of QC record can cause problems when there is a retrospective query on the sterility of a particular sample or product. Apparatus and a method of recording images of the growth are desirable.

Furthermore, microbiological testing for microorganisms such as bacteria is expensive. A cheaper, less labour intensive method is required.

Identification of bacteria in samples from infected biological tissues using colonial morphology, biochemical tests or molecular methods requires initial culture and sub-culture of each sample to provide individual discrete colonies. This process may take several days. In animals or humans with infections, therapy should be started as soon as possible after diagnosis. Delay in starting therapy allows the bacteria more time to multiply. The more bacteria in the host the more difficult it is to eradicate the infection. Since bacterial identification takes so long, most infections are treated blindly with antibiotics until the results from microbiological testing are obtained. With the development of antibiotic resistant strains of Staphylococcus Aureus (S. aureus), Clostridium Difficile (C. difficile) and other antibiotic resistant organisms, treatment by blind therapy becomes even more problematic. To improve treatment and bacteriological eradication there is a need for a faster method of bacterial identification. Preferably, this should take less than 24 hours, or less preferably, less than 36 or 48 hours.

Identification of bacteria in samples from the health, veterinary, pharmaceutical, food or environmental sectors, traditionally requires that pure cultures of the bacteria present are obtained before a full identification can be carried out. This may involve a number of steps, including 1.

inoculation of a liquid medium with the sample. 2. incubation to promote bacterial growth; 3.

spreading of a sample of the liquid culture (or the direct sample) on a solid medium plate in a way which is likely to produce single isolated colonies; 4. incubation of the plate to promote bacterial growth; 5. picking off (or selection) of isolated colonies for identification using selective or differential growth media, or a range of biochemical, nucleic acid based or other types of tests. Such tests frequently take up to a week before a result is established. Various selective and differential growth media can be used for determining which type of micro-organism is present. Selective growth media are useful when testing for specific general or species of micro-organisms and act by inhibiting all (or nearly all) microorganisms except the microorganism of interest. Differential growth media are used to distinguish between certain species of bacteria based on a particular trait.

Such conventional plating techniques require either a large time commitment for preparation of the appropriate growth media, or the expense of purchasing pre-poured culture plates of selective media, significant laboratory space using traditional equipment and expertise to ensure that correct identification is made. These tests can take from 2-24 days to complete depending on the organism.

Such a delay is problematic when antibiotics should be started as early as possible.

Many researchers are attempting to develop methods of bacterial identification which are easier, simpler, faster, require less expensive equipment and/or are available at point of care. Many developments in this area have been aimed at reducing the time taken for bacterial identification so that remedial action can be taken as early as possible.

Patents disclosing such inventions involve the use of bacteriophages (SANDERS US6660470), (MILLER US2002127547), centrifugation with fluorescence quenching (ANDERSON US2002137026), phosphorence quenching (WILSON 0A2291476) and nucleic acid analysis (OLIVE D et a! US6372424), (PRESTON et 81 GB2367359), (ANTHONY at a! W00052203). Bacteriophage methods involve having the equipment and expertise necessary to culture specific bacteriophages. In addition the SANDERS technique requires the equipment and expertise to perform DNA amplification and sequencing. Both bacteriophage methods involve considerable expense in the required analytical equipment and expertise. Light quenching methods also involve considerable expense in both the required analytical equipment and expertise. The development of new nucleic acid based methods, including PCR technology, has enabled more rapid bacterial identification techniques to be developed, however these tend to be very expensive and require considerable expertise to perform the tests. PCR tests by their nature are highly strain specific. There is an ongoing need for manufacturers to work to ensure that all relevant strains are being detected by the PCR kits available. PCR technology requires significant financial investment and staff training which excludes many testing laboratories where such resources are not available. In addition, although commercial PCR kits recommend that a sample of material is retained for further use if necessary, in practice this tends not to happen. This means that there is no sample available which can be used for follow-up investigations.

A number of automated instruments exist for the identification of bacteria which rely on the metabolic activity of the bacteria. These exploit differences in metabolism of bacteria. By inoculating a culture of the bacteria of interest into an array of mini incubation chambers, each one containing a different substrate, the pattern of substrate metabolism is used to identify the organism present (see THACKER CA230621 1 and MARTIN eta! US20090221061, AXFORD eta! US4252897) MATSMUR.A at a/ (W00109371, US6251624, CA2380027) describe a system for detection and/or screening of microorganisms, and concurrently or consecutively on the same instrument determining the susceptibility of microorganisms to various antibiotics and/or identifying microorganisms. The instrument employs image acquisition technology, several image processing algorithms and a variety of specialised disposable plates to perform these functions.

GREEN et al (US20040101189, US2008064089, US20100330610) describe a device for imaging culture plates which have been incubated in a separate incubator. Green's device reads a bar code printed on the culture plate. Interpretation of the bar code enables the type of growth medium on the plate to be identified. This enables a predetermined set of decision criteria to be selected and applied to the illumination or analysis of the culture plate placed in the device. These criteria enable background illumination, viewing light colour or other variables to be selected depending on the type of media.

Hara et al (W09940176, EP1061127) describe a system for identification of microorganisms which comprises a culturing device, an image grabbing device, a corriputer processing device and a device for disposal of microorganisms. The method enables identification of microorganisms grown under specific conditions (time and temperature) by performing colour analysis of an image of culture plate growth at a specific single time point and comparing image data with a previously generated database. Analysis is based on determination of the colour, hue and colour density of the growth.

Further patent documents describing imaging devices suitable for imaging, typically, microorganism or tissue culture growths include: US6243486 (WEISS), W096/18720 (BRAIER). Examples of colony counting in CA71 7287 (MAIN), US3811036 (PERRY) and W09859314 (OLSTYN). Vessel body light transmisivity is described in JP2006230304 (TAKESHI) and a controlled environment incubator for light microscopy is described in US4301252 (BAKER), and a mounting platform in US200401 01951 (VENT).

Other imaging devices include JP9187270 (IKEDA), RU2385494 (NIKATAEV), US20100221765 (GONZALEZ), CN2602030 (KANZHUN). Examples of image analysis include GB2430026 (DUCKSBURY) and GB2344419 (WALL). Other imaging devices include US20100232660 (G RAE SS L E).

The methods described above require expensive analytical equipment and considerable expertise to perform the analysis.

During incubation, in a prior art incubator, condensation may form on the lid of the culture plate, adversely affecting viewing or on removal of a culture plate from an incubator for viewing, the change in temperature of the culture plate to the ambient temperature of the lab, frequently results in the formation of condensation and water droplets on the lid of the plate. This is problematic as the condensation makes it difficult to view the surface of growth media within the culture plate. Therefore, viewing of early growths can be difficult.

Thus, the formation of condensation on the transparent lid or the culture plate makes observation and identification of areas of growth difficult.

Furthermore accurate identification of the type of growths can be difficult. Early identification of certain problematic organisms e.g. MRSA, is problematic. Little identification can be done before a growth is visible.

Another common disadvantage with present incubation techniques is that the plates have to be removed from the incubator in order to visually inspect them.

The present invention seeks to alleviate one or more of the above problems.

Statements of the Invention

In a first aspect of the invention there is provided an apparatus for incubating and imaging growths.

In a second aspect of the invention there is provided a rriethod of analysing a sequence of collected images of growths over time to provide one or more colour fingerprints over time, for example in the form of colour fingerprint data or plots of colour finger print data in colour fingerprint graphs.

In a third aspect of the invention there is provided a method of manufacturing an apparatus according to the first aspect, In a fourth aspect of the invention, there is provided a method of analysing data collected using the apparatus of the first aspect to identify growth.

In a fifth aspect of the invention, there is provided a method of analysing data collected to identify growth, for example, using data from a series of images in a sequence to provide a colour fingerprint (data and/or graph) over time.

In a sixth aspect of the invention there is provided an apparatus and rriethod of identifying selected growths (such as MRSA) promptly using a selective culture plate and the apparatus of the first aspect of the invention.

In a seventh aspect of the invention there is provided a culture plate for use in an apparatus or a method according to any aspect of the invention.

Several embodiments of the invention are described and any one or more features of any one or more embodiments may be used in any one or more aspects of the invention as described above.

In a first aspect of the invention there is further provided an incubating and imaging apparatus for incubating and imaging growths, such as microorqanism qrowths, comprising: an incubating chamber for incubating a culture plate; a heated optically transparent platen for placing a culture plate against thereto; the incubating chamber and heated optically transparent platen being arranged so that a reducing temperature gradient, from a higher temperature to a lower temperature, is formed in a direction moving away from the platen; further comprising an imaging device arranged to view through the heated optically transparent platen into the incubating chamber for capturing an image of a growth media, and/or any growth, on a culture plate when placed against the heated optically transparent platen.

The incubating chamber and heated optically transparent platen may be arranged so that if a culture plate is placed against the platen, a reducing temperature gradient is formed from a first side of a culture plate adjacent the platen to a second side opposing the first side not against the platen. Thus, a reducing temperature gradient is formed across the major surfaces of a culture plate when placed against the heated optically transparent platen.

Preferably, against means being in thermal contact or in direct contact. The heated optically transparent platen may be substantially optically flat so as not to distort images captured by the imaging device through the platen. The platen may be colourless. The platen may be optically transparent in the visible range of frequencies. The platen may comprise glass or plastic. The platen may be temperature controlled so as to control the reducing temperature gradient. The platen may be substantially planar. The reducing temperature gradient may be in a direction substantially perpendicular to the plane of the platen. The platen may be the sole heat source within the incubating chamber. The platen may form a side wall, other than a roof or a floor, or a roof of the incubating chamber. The platen may form the floor of the incubating chamber or a shelf within the incubating chamber. The incubating chamber may be arranged so that minimal or no circulation of air occurs within it so facilitating the establishment of the reducing temperature gradient.

The platen may be heated substantially uniformly over its area or at least over an area of its surface for receiving culture plates thereon. The apparatus may comprise a heating mechanism arranged to heat the platen and in which the heating mechanism comprises at least one heating element. The heating element may be surface mounted. The heating element may comprise heating tape and/or conductive film. The heating element may be embedded within the platen.

The heating mechanism may be situated beneath the platen.

The heating element may be generally evenly distributed over an area of substantially the same size and/or shape as that of the heating platen, or as that of an area of the heating platen for receiving culture plates thereon, so that platen is heated substantially uniformly over its area, or over the area for receiving culture plates thereon.

The apparatus may comprise a fan for circulating heated air adjacent a surface of the platen not facing the incubating chamber. An optically transparent, second platen may be provided substantially parallel to the first heated optically transparent platen and heated air may be circulated in between the two platens so as to controllably heat the first heated optically transparent platen. The imaging device may be arranged to view through the first and second platens into the incubating chamber.

The first and second platens may be about 5 to 25mm apart (± 1mm), or about 10 to 15mm apart (± 1mm).

At least one temperature sensor may be provided on the heated optically transparent platen so as to control the reducing temperature gradient.

The platen may be substantially horizontal in use so that culture plates can be placed upon it and held in place by gravity.

The imaging device may comprise a flatbed scanner, the flatbed scanner having a scanning platen and in which the scanning platen may be the heated optically transparent platen. The heating mechanism may be provided inside the flatbed scanner. A fan may be provided in the flatbed scanner for circulating air on an underside of the flatbed platen. A heater box may be provided beneath the flatbed scanner for heating the flatbed scanner so that the scanning platen may be heated over substantially its whole surface. A heater box may be provided for encasing the flatbed scanner. In such an embodiment, the heater must be arranged to ensure a heat differential across the culture plate to avoid condensation.

The incubating chamber may comprise thermal insulation in one or more walls. The type and/or amount and/or thickness and/or location of thermal insulation may be selected to provide a suitable reducing temperature gradient from the heated optically transparent platen into the incubating chamber. The temperature gradient, emanating from the heated optically transparent platen into the incubating chamber, may be selected and/or controlled by suitable selection of thermal insulation in one or more side walls or roof of the insulation chamber, and/or distance of the roof of the insulation chamber away from the heated optically transparent platen and/or temperature of the heated optically transparent platen.

The temperature gradient may be selected from 0.5 to 6°C or 1 to 4°C or 1 to 3°C or 0.5 to 2°C or 1 to 2°C. A temperature of the incubating chamber may be 0 to 60°C (±0.5 to 1°C) or 30-45°C (±0.5 to 1°C) and/or the temperature of the heated optically transparent platen may be 0 to 60°C (±0.5 to 1 °C) or 30-45°C (±0.5 to 1°C).

The heated optically transparent platen may comprise a locating mechanism for locating a culture plate thereon. The locating mechanism may comprise a mark on the heated optically transparent platen. The locating mechanism may comprise an upwardly extending feature located on the platen such as a nodule or wall, or comprises one or more walls of the incubating chamber. The apparatus may comprise a culture plate and the culture plate may comprise a recess or notch for accommodating said upwardly extending feature.

The imaging device may comprise a digital imaging device. The imaging device may comprise a digital camera or a digital scanner or a digital camera-on-a-chip. The imaging device may be arranged to capture two or more images overtime. The imaging device may be arranged to capture images from an incubation start time t0 to an incubation end time t at time intervals At.

An image captured first may be set as a background image l.

A first condensation check time t1 may be defined between the incubation start time t0 and incubation end time t8 and a first condensation check image l may be captured at time t01 and checked for condensation and, if the condensation check is negative for condensation the first condensation check image I is set as a background image l = Id.

If a first condensation check is positive for condensation then a second condensation check time t2 may be defined between the first condensation check time and incubation end time t and a second condensation check image l2 may be captured at time t02 and checked for condensation and, if the condensation check is negative for condensation, then the second condensation check image l2 may

be set as a background image l = l2.

A condensation check may be carried out by selecting a region comprising a selected pixel or group of pixels in a first condensation check image l1, determining colour values of one or more or each colour components (C1, C2 such as RGB, CMYK) of the selected pixel or group of pixels in the first condensation check image l1, optionally, determining colour values one or more or each colour components (C1, C2 such as RGB, CMYK) of the corresponding pixel or group of pixels in a control image, comparing the determined colour values of one or more or each colour components of the first condensation check image l1, with expected colour values of a control image, or with determined corresponding colours values of a control image, to determine if condensation is present.

Further condensation checks may be carried out as required until a background image substantially free from condensation has been established.

A first growth check time tg may be defined between an incubation start time t0 and an incubation end time L and a first growth check image 1g1 is captured at time t91 and a first growth check is carried out.

A first growth check may be carried out by selecting a first growth region comprising a selected pixel or group of pixels in a first growth check image 1g1. determining colour values of one or more or each colour components (C, C2 such as RGB, CMYK) of the selected pixel or group of pixels in the first growth check image l1, determining colour values one or more or each colour components (C1, C2 such as RGB, CMYK) of the corresponding pixel or group of pixels in the background image l, comparing the determined colour values of one or more or each colour component of the first growth check image 1g1 with the corresponding colours values of the background image l to determine if there has been a change of sufficient difference in one or more colour values so as to indicate growth.

A threshold difference in colour values of one or more colour components may be set to indicate growth.

Colour values may be measured in unit values from 0 to 255 and the threshold difference may be 5 units in colour value of one or more or each colour components.

A colour value of a colour component in a geographical region in an image comprising one or more pixels or a block of pixels, may be the mathematical mean colour value for that colour component. A colour value of a colour component in a geographical region in an image comprising one or more pixels or a block of pixels, may be the mathematical mode, median or total colour value for that colour component.

Two or more images may be collected over time and colour values of one or more or each colour component of the same corresponding first growth regions A1 comprising the same corresponding pixel or a group of pixels in three or more or each captured image may be determined over time.

Colour values of one or more or each colour component of the first growth region A1 of the captured images It may be plotted against time in a graph as a first colour fingerprint graph. Colour values of one or more or each colour components of corresponding first growth regions A1 of the first or second or subsequent growth check image (l1 or l2 or l) may be included in the plotted graph against time.

Colour values of one or more or each colour component of a first growth region A1of a background image l may be first subtracted from the colour value of one or more or each colour component of the corresponding first growth legion Ai in the time images (li), and if also plotted, the corresponding colour values from the growth check image (Ig) and the resulting colour values may be plotted against time in a graph as a first colour fingerprint graph.

One or mole time images l may be captured at a time prior to the time of a first or second or subsequent growth check.

Colour values derived from time images captured prior to, and colour values derived from time images captured post a time of a first or second or subsequent growth check may be plotted against time.

A second growth region A2 may be selected and colour values of one or more or each colour component of one pixel or group of pixels forming the second growth region A2 may be determined from each of the timed images, l, and optionally from the selected growth check image l, and plotted in a graph over time to provide a second colour fingerprint graph.

A plot of colour value of one or more or each colour component against time may be compared to a library of comparable images of colour value against time of known growths, such as known microorganisms, so as to determine a likely identity of the growth.

The colour components may be red (R), green (G), and blue (B) or cyan (C), magenta (M), yellow (Y) and black (K). One or more or each of red (R), green (G), and blue (B), or one or more or each of cyan (C), magenta (M), yellow (Y) and black (K), components of images may be plotted in a graph over time to provide a colour fingerprint graph. Colour values of the colour components may be background corrected by subtracting corresponding colour values of a background image therefrom.

Means to detect differences in colour values of corresponding growth regions in a sequence of images may be provided. The difference in colour values may be determined as between each timed image and a selected background image. The differences in colour values may be determined between any two time images in the sequence or between neighbouring time images in the sequence.

The time interval between time images may be selected from the group of 1, 2, 3, 5, 10, 20, 30, 45, 60, 90 and 120 minutes.

The height of the incubating chamber may be 5 -10mm (± 1mm) above the height of a standard culture plate when in position on the heated optically transparent platen.

One or more standard calibration colour marks within the field of view of the imaging device may be used to correct variations in colour values of one or more colour components of an image. A 3 or 4 colour component standard calibration colour mark may be provided within the field of view on the heated optically transparent platen.

A microprocessor may be provided for carrying out image capture and analysis. Memory may be provided for storing images taken between an incubation start time t,,and an incubation end time t.

A specific microorganism selective culture plate selective to a specific microorganism may be provided and an indicator device may be provided for indicating a positive result if growth may be detected at a first growth check time and/or at any subsequent growth check time. A selective culture plate may be provided that is selective for MRSA or C. difficile. The indicator device may be an alarm and/or a print out and/or display. The alarm may be visible such as a light, flashing lights and/or audible such as an intermittent beep or continuous beep.

There may be provided an apparatus for imaging bacterial growth on a culture plate comprising an incubator having an incubating chamber for incubating at least one culture plate, an imaging device for imaging one or more culture plates in the imaging chamber, a microprocessor, a heated transparent platen for locating at least one culture plate thereon and imaging a culture plate therethrough by the imaging device, whereby when a culture plate is located on the heated transparent platen in an inverted manner so a lid of the culture plate is adjacent the heated transparent platen, the temperature of a growth medium in a culture plate is less than the temperature of the lid.

A method of manufacturing an incubating and imaging apparatus may be provided comprising providing an apparatus including any one or more of the above features.

A method of incubating and imaging a culture plate may be provided comprising: incubating a culture plate by placing a culture plate against the heated optically transparent platen so the lid of the culture plate is against the heated optically transparent platen, arranging so that a reducing temperature gradient, from a higher temperature to a lower temperature, is formed in a direction moving away from the heated optically transparent platen, capturing images of growth media and/ol any growth on one or more culture plates through the heated optically transparent platen, so that the temperature of growth media in a base of a culture plate is less than the temperature of the lid of the culture plate.

The method may comprise circulating heated air adjacent a surface of the heated optically transparent platen not facing the incubating chamber so as to transfer heat to the incubating chamber and to any culture plate placed in the incubating chamber via the heated optically transparent platen.

The method may comprise monitoring at least one temperature sensor provided on the heated optically transparent platen so as to control the reducing temperature gradient.

The method may comprise selecting thermal insulation in one or more walls of the incubating chamber so as to control, the reducing temperature gradient. The method may comprise selecting the type and/or amount and/or thickness and/or location of thermal insulation to provide a suitable temperature gradient from the heated optically transparent platen into the incubating chamber. The method may comprise selecting a height of the insulation chamber above from the heated optically transparent platen and/or temperature of the heated optically transparent platen and/or temperature of the incubating chamber so as to determine the reducing temperature gradient.

The method may comprise locating a culture plate on the heated optically transparent platen using a locating mechanism such as a locating mark on the heated optically transparent platen or an upstanding feature on the platen, such a nodule or wall, or one or more walls of the incubating chamber.

The method may comprise capturing two or more digital images over time. The method may comprise setting the first image as a background image l. The method may comprise capturing images from an incubation start time t0 to an incubation end time t at time intervals At.

The method may comprise a step in which a first condensation check time t may be defined between the incubation start time t0 and incubation end time t and a first condensation check image l is captured at time t and checked for condensation and, if the condensation check is negative for condensation the first condensation check image I is set as a background image l = l1.

The method may comprise a step in which if a first condensation check is positive for condensation then a second condensation check time t2 is defined between the first condensation check time and incubation end time t2, and a second condensation check image l2 is captured at time t2 and checked for condensation and, if the condensation check is negative for condensation, then the second condensation check image l2 is set as a background image l = l2.

The method may comprise a step in which a condensation check may be carried out by: selecting a region comprising a selected pixel or group of pixels in a first condensation check image L1, determining colour values one or more or each colour components (C1, C2 such as RGB, CMYK) of the selected pixel or group of pixels in the first condensation check image Id, optionally, determining colour values one or more or each colour components (C, C2 such as ROB, CMYK) of the corresponding pixel or group of pixels in a control image, comparing the determined colour values of one or more colour components of the first condensation check image k1 with expected colour values of a control image, or with determined corresponding colours values of control image, to determine if condensation is present.

The method may comprise repeating the step of checking for condensation until a background image substantially free from condensation has been established.

The method may comprise a step in which a first growth check time t1 may be defined between an incubation start time t0 and an incubation end time t and a first growth check image l1 is captured at time t91 and a first growth check is carried out. The method may comprise a step in which a first growth check may be carried out by: selecting a first growth region comprising a selected pixel or group of pixels in a first growth check irriage lg1, determining colour values one or more or each colour components (C1, C2 such as RGB, CMYK) of the selected pixel or group of pixels in the first growth check image 1g1 determining colour values one or more or each colour components (C, C2 such as RGB, CMYK) of the corresponding pixel or group of pixels in the background image l, comparing the determined colour values of one or more colour components of the first growth check image 1g1 with the corresponding colours values of background image l to determine if there has been a change of sufficient difference in one or more colour values so as to indicate growth.

The method may comprise a step in which a threshold difference in colour values of one or more colour components may be set to indicate growth.

The method may comprise a step in which colour values are measured in unit values from 0 to 255 and the threshold difference may be 5 units (or 2-6 units) in colour value of one or more or each colour components.

The method may comprise a step in which a colour value of a colour component in a geographical region comprising one or more pixels or a block of pixels, may be the mathematical mode, median or total colour value for that colour component.

The method may comprise a step in which two or more images are collected over time and in which colour values of one or more or each colour component of the same corresponding first growth region A1in each image comprising one pixel or a group of pixels are determined as a colour fingerprint over time. The method may comprise a step in which colour values of one or more or each colour component of the first growth region A1of the captured images l are plotted against time in a graph to provide a first colour fingerprint graph. The method may comprise a step in which the colour values of one or more or each colour components of the corresponding first growth region A1 of the first or second or subsequent growth check image (Id or l2 or 10fl) are also plotted against time in the graph to provide a first colour fingerprint graph. The method may comprise a step in which the colour value of one or more or each colour component of the corresponding first growth region A1of a background image l are first subtracted from the colour value of one or more colour components of the time images (l) and if also plotted, from the growth check image (l) and the resulting colour values are plotted against time in a graph to provide a first colour fingerprint graph.

The method may comprise a step in which one or more time irriages I are captured at a time prior to the time of the first or second or subsequent growth check. The method may comprise a step in which colour values derived from time images captured prior to and colour values derived from time images captured post a time of a first or second or subsequent growth check are plotted against time.

The method may comprise a step in which a second growth region A2 may be selected and colour values of one or more or each colour components of the one pixel or group of pixels forming the second growth region A2 are determined from each of the timed images, It, and optionally the selected growth check image l, and plotted against time in a graph to provide a second colour fingerprint graph. The method may comprise a step in which a second, or further, colour fingerprint graph may be developed from selection of second, or further growth, regions within the sequence of images, to provide additional identification information.

The method may comprise a step in which a plot of colour value of one or more colour components against time may be compared to a library of comparable images of colour value against time of known growths, such as known microorganisms, so as to determine a likely identity of the growth.

The method may comprise a step in which the colour components are red (R), green (G), and blue (B) or cyan (C), magenta (M), yellow (Y) and black (K). The method may comprise a step in which one or more or each of red (R), green (G), and blue (B), or one or more or each of cyan (C), magenta (M), yellow (Y) and black (K), components of images are plotted over time to provide a colour fingerprint graph.

The method may comprise a step in which the colour values of the colour components are background corrected by subtracting corresponding colour values of a background image therefrom.

The method may comprise a step in which means to detect differences in colour values of corresponding growth regions in a sequence of images may be provided. The method may comprise a step in which the difference in colour values are determined as between each timed image and a

selected background image.

The method may comprise a step in which the differences in colour values are determined between any two time images in the sequence or between neighbouring time images in the sequence.

The method may comprise a step in which the time interval between time images may be selected from the group of 1, 2, 3, 5, 10, 20, 30, 45, 60, 90 and 120 minutes.

The method may comprise a step in which one or more standard calibration colour marks within the field of view of the imaging device are used to correct variations in colour values of one or more colour components of an image.

The method may comprise a step in which a 3 or 4 colour component standard calibration colour mark is provided within the field of view on the heated optically transparent platen.

The method may comprise a step in which a microprocessor may be provided for carrying out image capture and analysis. The method may comprise a step in which memory may be provided for storing images taken between an incubation start time t0and on incubation end time t.

The method may comprise a step in which a specific microorganism selective culture plate may be provided and an indicator device may be provided for indicating a positive result if growth is detected at a first growth check time or at any subsequent growth check time. The method may comprise a step in which a selective culture plate may be provided and an indicator device may be activated upon first detection of any growth. The method may comprise a step in which the indicator device may be an alarm and upon first detection of a growth an alarm may be activated. The method may comprise a step in which a selective culture plate may be provided that is selective for MRSA or C. difficile.

Brief Description of the Invention

The present invention will now be described, by way of example only, with reference to the following figures in which like reference numerals refer to like reference features.

Figure 1 shows a schematic, perspective view of an apparatus according to a first embodiment of the invention.

Figure 2A shows a schematic, perspective view of apparatus according to a second embodiment of the invention.

Figure 2B shows a schematic, perspective cutaway view of the apparatus of Figure 2A.

Figure 3A shows a schematic, perspective view of an apparatus according to a third embodiment of the invention.

Figure 3B shows a schematic, perspective, cutaway view of the apparatus of Figure 3A.

Figures 4A and 4B illustrate a method of operating the apparatus of Figures SA and 3B according to a fourth embodiment of the invention, for example, at a point-of-care location such as a hospital ward or veterinary laboratory. The method may provide, for example, a growth alarm indicative of growth early on in the incubation period and continuing collection of images for use later in the confirmation of the identification of the growth.

Figure 5A shows a method of obtaining colour component values of a growth media according to a fifth embodiment of the invention, optionally for use as background colour component values. Steps for performing an optional condensation check are shown.

Figure 5B shows a method of analysing image data for identifying the occurrence of bacterial growth according to a sixth embodiment of the invention. Optional steps for counting and reporting colony numbers are shown.

Figure 6 shows a schematic view of a colony at three different times and example first and second pixel blocks for use in generating multiple colony/growth colour fingerprints of the same colony according to a seventh embodiment of the invention.

Figure 7 shows three methods for generating microorganism growth colour fingerprints (data and/or graphs) for comparison with reference microorganism fingerprints according to eight, ninth and tenth embodiments of the invention respectively.

Figure 8 shows two plots of example colour fingerprint graphs for Methicillin Resistant Staphylococcus Aureus (MRSA). Graph 1 shows red, green and blue colour values and luminosity values (in lumins) for a selected pixel block in an MRSA colony measured over time with no background subtracted. Graph 2 shows red, green and blue colour values and luminosity values as measured over time for the same pixel block in the same MRSA colony after subtracting background media colour values (optionally such as those measured in method 140 in Figure 5A).

Figure 9 shows a schematic, perspective view of a modular system comprising modular apparatus according to an eleventh embodiment of the invention and a cutaway, schematic, perspective view of one modular apparatus of the modular system.

Figure 10 shows a method of utilising a modular system such as that in Figure 9 according to a twelfth embodiment of the invention which may be suitable for, for example, a veterinary application where multiple animal patients may each require testing e.g. for MRSA, at around the same time prior to, for example, admission into veterinary hospital.

Figure hA shows a schematic, perspective view of a further apparatus according to a thirteenth embodiment of the invention which is suitable for batch use.

Figure 11 B shows a schematic, perspective, cutaway view of the further apparatus of Figure 11 A. Figure 1 1C shows a cross-sectional view through the apparatus of Figures hA and 1 lB.

Figure 12A shows a schematic, perspective view of yet a further apparatus according to a fourteenth embodiment of the invention which is suitable for batch use.

Figure 12B shows a schematic, perspective view of the apparatus in Figure l2Awith the lid removed.

Figure 13A and 13B show methods of operating a batch apparatus, such as those shown in Figures hA, 11B, and 11C and Figures 12A and 12B, according to a fifteenth embodiment of the invention.

Figure 14 shows a schematic, perspective view of an early prototype apparatus according to a sixteenth embodiment of the invention.

Figures 15A, 15B and 1SC each show scans of four culture plates at times, t = 0 hours, t = 18 hours, and t = equals 28 hours, respectively taken using the apparatus of Figures 2A and 2B..

Figures 16A and 16B show respectively, full size and zoomed views of a single culture plate of Staphylococcus Aureas (S. Aureas) on TSA.

Figure 17 shows complete colour fingerprint images of miscellaneous fungal and bacterial growths on TSA (Tryptone Soy Agar) culture plate agar, after image subtraction according to a seventeenth embodiment of the invention. Here a complete colour fingerprint image of the plate at 0 hours has been subtracted from a complete colour image of the plate at 18 hours. Colour values for slight changes in colour of contraol TSA plates at Oh have been subtracted from the colour values of each point in the fingerprint at 18h.

Figures 18A and 18B show, respectively, several different species of MRSA on Oxoid chroniogenic oxycillin MRSA agar media at, respectively, time t = 0 hours and t = 15 hours.

Figures 19A, 19B, 190, and 19D show growth colour fingerprint graphs of change in colour values of red, green, blue and luminosity values, according to an eighteenth embodiment of the invention, for E. Coli (Esherichia Ccli), salmonella, S. Aureas (Staphylococcus Aureas) and penicillium respectively, over 12 hours.

Figure 20 shows a colour fingerprint graph for a growth of B atrophaeus over 24 hours following

background correction.

Figure 21A shows growth colour fingerprint graphs of change in colour values of red, green, blue and luminosity value, according to a nineteenth embodiment of the invention, for E. Coli on TSA following background correction. The summed change in mean colour values for selected pixel block areas on each of six colonies on a single plate are shown.

Figure 21B shows growth colour fingerprint graphs of change in colour values of red, green, blue and luminosity value, according to the invention, for F. Coli on TSA following background correction for six colonies. The summed change in mean colour value for selected pixel block areas for each of six colonies each on separate plates are shown.

Figure 22A shows growth colour fingerprint graphs of change in colour values of red, green, blue and luminosity value according to the invention, for S Uberis (Steptococcus Uberis) on TSA following background correction for six colonies each on a single plate. The summed change in mean colour values for selected pixel block areas on each of six colonies on a single plate are shown.

Figure 22B shows growth colour fingerprint graphs of change in colour values of red, green, blue and luminosity value, according to the invention, for S Uberis on TSA following background correction for six colonies each on separate plates. The summed change in mean colour value for selected pixel block areas for each of six colonies each on separate plates are shown.

Figure 23A shows growth colour fingerprint graphs of change in colour values of red, green, blue and luminosity value, according to the invention, for Pasturella Multocida type A (PMA) on a TSA single plate following background correction for six colonies on a single plate. The summed change in mean colour values for selected pixel block areas on each on six colonies on a single plate are shown.

Figure 23B shows growth colour fingerprint graphs of change in colour values of red, green, blue and luminosity value, according to the invention, or Pasturella Multocida type A (PMA) on TSA following background correction for six colonies each on separate plates. The summed change in mean colour value for selected pixel block areas for each of six colonies each on separate plates are shown.

Figure 24 shows complete (including RGB components) images (following background subtraction) of standard organisms (E Coil, S Uberis and Pasteurella Multocida (PMA) growing on TSA agar after 24 hours.

Figure 25 shows a complete (including RGB components) image (following background subtraction) of a mixed culture growing on TSA agar after 24 hours.

Figure 26A shows an example plot of intensity of one or more or all colour components of bacteria over time showing lag phase, log or exponential growth phase and resting phase.

Figure 26B shows steps involved in a condensation check, according to a twentieth embodiment of the invention.

Figure 26C shows a method of collecting images according to a twenty-first embodiment of the invention, and optional steps for use in a method of activating a growth alarm according to a twenty-second embodiment of the invention.

Figure 26D shows further steps for use in the method of Figure 26C, according to the twenty-first embodiment of the invention.

Figure 27A shows plan and perspective views of plates for use in a batch apparatus according to a twenty-third embodiment of the invention.

Figure 27 B shows a heated optically transparent platen having an upstanding feature for engaging with a culture plate having a corresponding notch according to a twenty-third embodiment of the invention.

Figure 28A and 28B show, respectively, plan and perspective views of batch apparatus for use with a number of modified culture plates according to a twenty-fourth embodiment of the invention.

Detailed Description of the Invention

In Figure 1, apparatus 10 of a first example embodiment of the invention comprises a housing 1, having a lower imaging chamber 2, an upper incubating chamber 3, and an optically transparent platen 4 serving as a shelf for locating one or more culture plates 5 against (typically placed on an uppermost surface of platen 4). Culture plate 5 has an uppermost surface or lid 5A, and a lowermost surface or base SB typically having a culture media therein. Lower imaging chamber 2 and upper incubating chamber 4 may be completed or partially isolated from one another by optically transparent platen 4. Optically transparent platen 4 forms a support for culture plates 5 and is typically formed from a plate of optically transparent material such as glass or Perspex. Typically platen 4 is colourless. Typically platen 4 extends to the inner walls of housing 1, but it need not do so.

Where provided, this arrangement ensures there is little or no movement of air within the incubating chamber especially during an incubating and imaging sequence.

A light source 6, in the form of two tubular daylight light tubes, is located beneath optically transparent platen 4 to illuminate culture plates 5 for image capture. The light source may be above platen 4. An imaging device in the form of a camera on a chip 7 is typically located beneath optically transparent platen 4. A computer and display 9 is shown connected to imaging device 7 and is arranged to control and capture images from imaging device 7.

A heating plate 8 is shown. Heating plate 8 extends beneath and across a central area of platen 4 between light tubes 2 and underneath a central region for locating culture plates 5 on optically transparent platen 4. Thus, optically transparent platen 4 is heated from below. Preferably optically transparent platen 4 is heated substantially uniformly over its surface and is typically the only the heat source into incubating chamber 3. Typically, heated optically transparent platen forms one of the innerwalls (here a floor) of incubating chamber 3.

The desired incubation temperatures for incubating chamber 3 and for the platen 4 can be set on a control panel (not shown) with a temperature control and feedback system (not shown). Temperature sensors such as one or more thermisters or thermometers are provided (not shown) on platen 4 and/or in incubating chamber 3 to give feedback and enhance temperature control. Circuitry in a control panel (not shown) turns the heater plate 8 off if the temperature becomes too hot. The heater plate 8 is switched back on again, if the temperature is too low. This feedback is used to maintain the temperature of the incubating chamber 3.

In preferred embodiments the relative temperatures of opposing major surfaces 5A and SB of culture plate 5 are controlled carefully. In this embodiment, the culture plate 5 is placed on the platen (shelf) in an inverted manner, lid 5A being lowermost during an incubating and imaging sequence. As discussed elsewhere, it is preferred to establish a temperature gradient across the culture plate 5 from the lid 5A of culture plate 5 which is placed in contact with the optically transparent heated platen 4 to an opposite base SB that is somewhat distant (by the height of the culture dish) from the heated optically transparent platen 4. Preferably, this temperature gradient will be 1-5°c and more preferably 1-2°c so that, here, the lid 5A (the lid) of culture plate 5 against the platen 4 is hotter than the base of culture plate 5 not in contact with the heated optically transparent platen 4. Thus, in this embodiment, the culture plate 5 is placed on heated optically transparent platen 4 upside down in an inverted fashion so that the surface 5A (the lid) is lowermost immediately against and in contact with heated optically transparent platen 4.

The upper incubating chamber 3 may comprise insulation in sufficient quantity and/or position in its outer walls, and/or may be of a suitably selected height and/or shape and/or volume, to facilitate the establishment of a suitable reducing temperature gradient across opposing major surfaces 5A and SB of culture plate 5. Thus the lid 5A of the culture plate is kept slightly warmer than abase SB containing the growth media. This helps avoid condensation.

Thus one advantageous feature of one aspect of the present invention is that the apparatus is arranged so that a temperature gradient may be created across a culture plate 5 when it is placed upside down within incubating chamber 3, in other words in an inverted position. The incubator is arranged so that a lid of culture plate 5, forming culture plate surface 5A against heated optically transparent shelf 4 is slightly warmer than the base of culture plate 5, forming culture plate surface SB. By maintaining this temperature gradient, condensation on the lid of the culture plate 5 is reduced and may be substantially or completely eliminated.

The heated optically transparent platen 4 is preferably substantially planar and in the form of a plate and it may be formed from optically transparent (in the visible range) material such as glass or plastic.

Here, a heated optically transparent platen 4 is provided for placing one or more culture plates 5 thereon (typically held in place in a fixed location by gravity). Alternative arrangements can be envisaged, for example, a heated optically transparent panel may be provided in incubating chamber 3 wherein culture plates are held by suitable means (such as clips, mounts, supports or the like) against the platen (in direct contact or in thermal contact close to). Thus the heated optically transparent platen may not be horizontal but may be vertical or sloped. These, and other arrangements, are embodiments within the present invention. For example, an arrangement can be envisaged in which the heated optically transparent platen 4 forms a roof of the incubating chamber and a shelf is provided to hold the culture plates (right way up this time) against the heated optically transparent roof of the incubating chamber. The imaging device may view the culture plate from above.

Whilst both upper and lower chambers 2 and 3 are heated it is the temperature of the upper incubating chamber 3 and indeed the temperature of the heated optically transparent platen, here in the form of a shelf, and/or optional insulation and/or height of the incubating chamber that are controlled so as to provide a reducing temperature gradient across culture plates as described herein.

By placing culture plate 5 against the platen 4, it means being in thermal contact and/or in direct contact. The heated optically transparent platen 4 may be substantially optically flat so as not to distort images captured by the imaging device through the platen and may be colourless. The platen may be optically transparent in the visible range of frequencies. The platen rriay comprise glass or plastic. The platen may be temperature controlled so as to control the reducing temperature gradient.

The platen may be substantially planar and the reducing temperature gradient may be in a direction substantially perpendicular to the plane of the platen. The platen may be the sole heat source within the incubating chamber. The platen may form a side wall other than a roof or a floor, or a roof of the incubating chamber. The platen may form the floor of the incubating chamber or a shelf within the incubating chamber. The incubating chamber may be arranged so that minimal or no circulation of air occurs within it so facilitating the establishment of the reducing temperature gradient.

Culture plates may be placed in the incubator of the present invention with little concern as to x,y positioning. This is because once in position, the culture plates are not moved with respect to the heated optically transparent platen 4. In this embodiment, this is because the one or more culture plates 5 are held in position by gravity. The incubating chamber 3 may, optionally, be provided with a lock so that no access, which could result in movement of the culture plates 5 in an x-y direction across the heated optically transparent platen 4, is possible during an incubation and imaging run.

As described above incubating chamber 3 may have a manual or automatic lock (not shown), to prevent access to, and/or movement of, any culture plates located on the transparent shelf 4 during incubation and imaging. Thus, once a scanning and imaging sequence has begun following closure of the door lock, access to incubating chamber 3 is not desirable. Access may be controlled by computer 9.

In the present invention, a sequence of images of growth media of culture plates 5 are taken over time by imaging device 7, through the optically transparent platen 4 and through a lower most surface 5A (typically a lid) of culture plate 5. In one embodiment due to the extent of heated optically transparent platen 4, and the wide field of view of imaging device 7, one or a multiple of culture plates Scan be captured in a single image.

One or more culture plates are inserted through a door in the chamber (not shown). Inoculated culture plates 5 are placed lid side down on the optically transparent platen 4. The imaging device (camera on a chip) 7 and a computer 9 may be connected together by a cable or may be wirelessly connected.

Software in computer 9 enables the culture dish insertion time, incubation start time, optional first (and subsequent) condensation check times, first imaqe collection time, and first (and subsequent) growth check times as well as the time interval between images to be set on the computer 9. The camera on a chip 7 captures images as prescribed by the software. The time of capture of an image is stored with the captured image on the hard disk of the computer 9. At the end of a first incubation period (e.g. at a first growth check time Ti), the software analyses the images and may output a count number of regions on the culture plate that demonstrates growth. These regions can be observed individually or as a whole image of a plate by calling up stored digital images and viewing these on the screen of computer 9. Alternatively, these can be viewed remotely through local area network computers or over the internet.

Typically, the images are digital images and comprise, as is typical in digital images, a number of colour components such as colour components of primary colours red (R), green (G) and blue (B) separated into pixels. Other groups of colour components such as cyan (C), yellow (Y), magenta (M) and black (K) may be utilised within the present invention.

When required for bacterial identification, the software can generate colour fingerprint data and/or graphs of colour value (of one or more pixels or blocks of pixels) of one or more overall colour components against time for the regions on the culture plates that demonstrated growth. Plots of the data as colour fingerprint graphs can be visually observed by calling up any of the stored digital images and viewing these on the screen of computer 9. Alternatively, images can be viewed remotely through local area network computers or over the internet. The software may compare a plot of colour value of one or more regions of growth over time against a reference library of growth curves for known bacteria. Software may identify the bacteria on the plates, for example, by comparing colour value plots over time with comparable colour value plots over time from a library of reference growth colour value plots over time. The curve comparison may comprise least squares fitting, threshold setting, human visual inspection, etc. In use, the apparatus is connected to the computer and the power turned on. The desired temperature of the incubating chamber 3 and/or heated optically transparent platen 4 and/or reducing temperature gradient across a standard sized culture plate 5 are set by a control panel or via the computer 9 and the incubator chamber 3 is allowed to warm to the selected temperature. This, therefore, provides a desirable temperature gradient across a culture plate 5 due to selected parameters of dimensions such as height, volume, shape etc of incubating chamber 3, heating chamber 2, heating plate 8, heated optically transparent platen 4 and type and thickness and location of insulation inside walls and roof of incubating chamber 3.

One or more culture plates 5 are inoculated with the sample to be tested. The lid of the culture plates is then closed. One or more culture plates 5 are placed inside the device, inverted on the optically transparent platen, so that the lid of the one or more culture plates 5 is immediately against the optically transparent platen 4 to form surface 5k The desired start, end and interval times for the image capturing to be performed are entered into the computer either by being preset and selecting such a preset programme or manually entered, together with the maximum incubation time t and, optionally, a first (and subsequent) condensation check time and a first (and subsequent) growth check time t1.

Digital image It of the culture plates are automatically recorded at the selected times. During and/or at the end of the incubation period, the software is triggered to analyse the images It, (pixel by pixel or group of pixels by a group of pixels known as pixel blocks). A pixel block comprises a group of two or more neighbouring pixels.

The number and geographical location of regions in which colour value of one or more colour components of one or more pixels changes on each of the culture plates 5 is reported. Copies of the images can be printed out, saved to CD or DVD and or viewed remotely via a secondary computer link (not shown). When required for bacterial identification, the software will generate colour fingerprints (data and/or graphs) of colour value for each selected colour component against time for regions (such as selected pixel blocks) on the culture plates where growth occurred. Colour fingerprints can be viewed visually by calling up any of the stored digital images and viewing these on the screen. The images of the culture plates or the fingerprint graphs can be printed out, saved to CD or DVD and/or viewed remotely through local area network computers or over the internet. Software in the computer 9 will review colour fingerprints generated as above, and compare these against a reference library of growth curves for known bacteria. Best fits may be identified to deduce a likely identity for microorganism growth.

The apparatus 10 may be encapsulated in plastic or formed within a plastic housing I so that it may be easily cleaned. The incubation imaging apparatus 10 may be modular, so that banks of individual incubating and imaging apparatus can be stacked, for example, five wide and five high (see Figure 9). These may be designed so that these can be run and controlled by a single computer 9.

Figures 2A and 2B show scanning apparatus according to a second embodiment of the present invention comprising an imaging device here in the form of a flatbed scanner 12. The scanner has an optically transparent scanning platen 46 (see Figure 2B) typically made of glass for scanning an item placed thereupon. Thus, the scanning platen 46 of the scanner is an optically transparent shelf for placing culture plates 5 thereon. An insulated housing 14 forming an incubating chamber 3 is provided on the top of flatbed scanner 12. In this embodiment the insulated housing 14 completely covers the optically transparent scanning platen 46. The insulated housing 14 comprises a number of insulated walls of suitable size and shape to be mounted on scanning platen of scanner 12. A heater box 16 comprises a heater wire 18, a flue 20, a control panel 22 with controls 24, a display 26 and on/off switch 28. A main intake fan 29 is provided in one of the walls of heater box 16.

In use, flue 20 draws in air via fan 29 past heater wire 18 and circulates this upwards, typically in a distributed manner, to an underside (not facing the incubating chamber) of the platen 46 of the flatbed scanner 12. Thus, heated air is circulated on an underside of platen 46. A second platen (not shown) may be provided parallel to the first for circulating air in between so heating the first platen 46. The first and second platens may be about 5 to 25mm apart (± 1mm), or about 10 to 15mm apart (± 1mm).

Flatbed scanner 12 rests on heater box 16 so that, typically, the entire scanner 12 is overlapped by the heater box 16. Thus, in this example embodiment, the periphery of scanner 12 rests within the periphery of heater box 19 and no portion of the periphery of the scanner 12 is not in heated by heater box 16. Furthermore, heater wire 18 is distributed within heater box 16 to generally or substantially mirror optically transparent platen 46 of the scanner 12. Therefore, the heater wire 18 is more or less the same size and/or shape as that of optically transparent platen 46 of scanner 12.

Whilst only one flue 20 is shown, and that being somewhat in the centre of scanner 12, it will be appreciated by those skilled in the art that further flues may be provided in a distributed manner over the underside of scanner 12 to facilitate the even distribution of heat from the heater wire 18 to the underneath of scanner 12 particularly in the region of the optically transparent platen 46. Therefore, optically transparent platen 46 is heated substantially evenly over its surface facing the incubating chamber and forms a heated, optically transparent platen according to the present invention.

Scanner 12 is connected to a computer 32 via a computer cable 30. A data cable 34 connects the computer 32 to a display 36. A temperature sensor 38 is provided on the optically transparent platen 46 of scanner 12.

Several temperature sensors 38 may be provided although only one is shown here. Therefore, the temperature of the optically transparent platen 46 can be carefully controlled by feedback to the control panel 22 which controls heater box 16 and heater wire 18. Furthermore whilst a single heater wire 18 is shown, a distribution and/or layout of one or more heater wires may be arranged so that optically transparent platen 46 can be heated in a controlled manner through a combination of multiple heater wires 18 and multiple temperature sensors 38. This facilitates control of the developed reducing temperature gradient from the platen 46 into the incubation chamber 3.

Insulated housing 14 comprises an insulated housing wall 40 and an insulated housing lid 42. One or more culture plates 5 suitable for growing one or more microorganisms therein, are located within insulated housing 14 on optically transparent platen 46 in an inverted manner. A temperature gradient of 0.5°c, or 1°-3°c or 1°-2°c is established between a lid of the culture plate 44 in contact with the heated platen 46 and an opposing surface (a base) at a distance from heated platen 46.

Figures 3A and 3B shows schematic external and cutaway views respectively, of a single unit apparatus 60 according to a third embodiment of the invention typically designed to be used at a point of care, for example near a patient on a ward or in a surgery such as a doctor's or veterinarian's surgery.

Here apparatus 60 provides incubating and scanning functions within a single housing 61. Apparatus 60 is provided with a control panel 62, and an aperture 64 for control plate insertion and/or extraction.

Aperture 64 is in the shape of a slot typically of just larger size and similar shape as culture plates to be inserted therethrough and used with the apparatus 60. Housing 61 of apparatus 60 has a display 66 and a printer front panel 68 located thereon. A pair of indicator lights 70 is also shown; typically a red indicator light and a green indicator light are provided. For example, a red indicator light could indicate bacterial growth and a green indicator light could indicate no bacterial growth, for example, at an early stage of growth when a selective culture plate is used, as will be described later. A processor (not shown) is provided within housing 61 of apparatus 60 for providing control signals to an imaging device 72 and for capturing images to a storage media such as a hard disk or CD etc and for carrying out analysis on the captured images. An optional barcode (or other identification indicia) reader 71 is provided for reading a barcode or other identification indicia prior to insertion of a culture plate 82 into housing 61 via aperture 64. The processor may store this identification information along with stored images/data.

Turning now to Figure 3B, a cutaway view of apparatus 60 of Figure 3A is shown. Here a heated optically transparent generally planar platen 74 is provided in the form of an optically transparent shelf for supporting culture plates 82 thereon. Culture plates 5 are supported in an inverted manner on optically transparent generally planar platen 74 with culture media, such as agar, uppermost and the lid of the culture plate lowermost. Typically, the lid of the culture plate will lie against and in direct contact with an uppermost surface of the optically transparent generally planar platen 74. A heating device 76 is provided in the form of a heating plate of, in this example embodiment substantially the same size and shape as optically transparent platen 74. The heating device 76 is located beneath the optically transparent platen 74. A pair of daylight neon tubes 78 provides a lighting device for apparatus 60. These are located spaced apart each at one side of and underneath of optically transparent platen 74. Daylight neon tubes 78 may be replaced by one or more other suitable lighting devices which, if more than one, may also be distributed spaced part within the housing 61. A processor 80 is provided for controlling apparatus 60.

Selective media allow certain types of organisms to grow, and inhibit the growth of other organisms.

Differential media are used to differentiate closely related organisms or groups of organisms. Types of growth media which may suitable for culture plates in this and other embodiments of the invention include mannitol salt agar (MSA), eosin methylene blue agar (EMB agar), macconkey's agar.

Suitable commercially available products include: Oxoid Chromogenic MRSA Agar, Oxoid Brilliance MRSA, CLED agar, Brazier's Clostridium Difficile selective agar from Oxoid; chromlDTM MRSA, chromlDTM C. Difficile, Legionella, GVPC agar from Biomeriux; and HiCrome'TM EC 0157 from Sigma lAid rich A camera on a chip 72 forms an imaging device within the apparatus 60. Alternative imaging devices such as digital cameras, scanning devices, CCD devices and the like may be used. Typically the imaging device, here camera 72, is located underneath optically transparent platen 74 and arranged so that its field of view is directed so as to capture an image of a culture plate 5 located on platen 74.

Platen 74 may have dedicated regions (marked out on it for example) for placing a culture plate thereon and may be suitable for placing a number of culture plates thereon at one time. Camera 72 may be movable if its field of view is not sufficient to take in images of the number of culture plates required, or it may have a field of view sufficiently wide so that the required number of culture plates can be viewed at one time without movement of camera 72.

In this and other embodiments, movement of the culture plates is avoided, thus no complex moving or alignment apparatus is required, nor registration of images in software, simplifying the requirements of the system and reducing costs.

The lighting device in the form of neon daylight tubes 78 is illuminated when an image is required to be taken but otherwise may be switched off. Alternatively, the lighting device may be illuminated at all times.

Processor 80 is provided within apparatus 60 for controlling the functions of the incubator and scanning apparatus and for storing and analysing captured images for later viewing and/or analysis.

Once the growth is detected indicator lights 70 may be illuminated to indicate that growth is present as soon as growth is detected. This can be particularly important if a selective or semi-selective growth plate is used, such as an MRSA selective plate to indicate that a growth (in this case a selected growth, such as MRSA) is seen so as to give an early indication to an operator of a growth being detected. This will be described in more detail later.

Figures 4A and 4B show a method 100 of operating a single apparatus at a point of care, such as the apparatus shown in Figures 3A and 3B for detecting specific bacterial growths and issuing an alarm if detected, according to a fourth embodiment of the invention. At step 102 parameters for controlling incubation and scanning of images are selected. Typical parameters include a culture plate insertion time t2 incubation start time t1 a time zero for image capture to (this may well be the same as the incubation start time of team L1), optionally, a first condensation check time t1, and optionally a subsequent condensation check time a first growth check time t, optionally, a second growth check time t02, an image interval time delta At, or optionally a number of different length of image interval times At1 At2 an image capture end time t and an incubation end time t8°. These may be selected according to preset criteria or may be manually selected via control panel 62 and display 66.

In optional step 104 a barcode of a growth plates is scanned by barcode scanner 71. The barcode may indicate that the plate is non-selective or selective for certain bacteria. In this fourth embodiment of the invention a selective plate (e.g. MRSA) is used. The barcode may indicate other criteria for the culture plate, such as agar type, agar and/or plate production, incubation date and/or batch number, and/or imaging sequence and so on. At step 106 at time t-2, a culture plate is inserted and the time noted. In step 108, incubation is started at time t-1. In step 110 at time t0 a time zero image l is collected. This may occur following an optional condensation check step (not shown). In step 112, images are captured over time at time intervals of At. Thus at time, t = t3+At a first image l is captured. At time, t = t0+2 At, a second image 12 is captured. At time, t = t0+nAt an nth image is captured. In step 114, at a preselected time t-1 a first growth check image is captured. Thus, in step 114 after a predetermined time, such as number of hours (e.g. 4 to 6, typically 4, 5, or 6 hours) 1g1 the image analysis for the detection of growth is begun by capturing a first growth check image II. In step 116, the first growth check image is analysed for detection of bacterial growth. A preferred method of carrying out this growth check is shown in Figure 26C in a twenty-first example embodiment of the present invention. In step 118 bacterial growth is noted (positive growth) or no growth (negative growth) is noted. In step 120 an alarm output is given such as a beep or printout or by an illumination of say a red indicator light 70 or flashing of light 70 if positive growth is indicated.

Positive or negative growth information may be displayed on display panel 66.

Whilst the incubation and imaging sequence may stop after an alarm is output preferably the sequence continues. In step 122, the incubation continues. In step 124 image capture continues at time intervals of At. In step 126, at an end time of 12 hours incubation and image collection is stopped. Typically, incubation and image collection stop simultaneously but this need not be the case. For example, image collection may continue post-incubation, or vice versa. In step 128 image analysis for bacterial identification and/or confirmation of bacterial identification and/or confirmation of the negative result is carried out. In step 130 an output (such as entry in a data file and/or a beep and/or a light illumination and/or a light flash and/or printout) is provided to indicate the nature (positive or negative) of growth and any further information required such as the likely identify of the growth (e.g. MRSA, E. Coli, S Aureus, S Uberis).

If the growth is positive at the first growth check time, or, optionally, at any time thereafter, then the images already captured and later captured, are analysed to provide colour fingerprint data and optionally plotted over time to provided colour fingerprint graph(s) (optionally in real time).

Typically the images from one or more pixels or groups of pixels are broken down into colour components for plotting over time, the luminosity may also be plotted overtime. The colour values of pixels from images before, at and after the first growth check time are plotted over time. Thus data that has hitherto not been thought to be of relevance to identification of bacteria (prior to its visibility on growth plates to the naked eye) is captured in this embodiment of the present invention and utilised to provide a colour fingerprint(s) of growth both before, at and after a first growth check time.

Figure 5A shows a method 140 for obtaining a series of images to establish a background image and colour values of such a background image according to a fifth embodiment of the invention using an apparatus such as apparatus 10 in Figures 2A and 2B or apparatus 60 in Figures 3A and 3B. In this method an optional condensation check and/or optional colour analysis may be performed. In step 142, a time interval At between images is set on the apparatus. This can range from 1 or 2 minutes to 10, 15, 20. 30,60 minutes or more or even up to 24 hours. In step 143, images of a growth plate are taken at the set given intervals selected in step 142. Whilst typically the intervals are of equal duration, it will be appreciated by those skilled in the art, that the duration may be varied if desired. In step 144, the images are stored in hardware. In step 146, software is run on, for example, computer 32 in apparatus 10, or on processor 80 in apparatus 60 to select a first image. The first image may be at or shortly after time t2 or t1 or at or at t + At. In step 148, a software check is carried out on this first captured image to evaluate the image for condensation. This first condensation check may be carried out by manual visual inspection of the first captured image. Alternatively, this first condensation check may be carried out by comparing the colours values of colour components of the first image (such as 1R, G l) with expected reference colour components for the culture plate used (and in particular for the specific type of growth media for the culture plate used (e.g., ISA agar) If the condensation check is positive, then in step 150 the software selects the next image in the sequence of images collected in step 143 and stored in step 144 for analysis. A further condensation check is carried out on that image in step 148. This is repeated until a negative result is found for the condensation check in step 148 meaning minimal or no condensation. In step 152, the image for which a negative condensation check has been established is set as the background image l i.e. the

image of background growth media.

In step 154, the colour values e.g. 10R, 00 0B of a selected region (e.g. a pixel block) of the growth media in the culture plate are derived from the background image I. The colour values are expressed as components of colour such as red, qreen, blue (RGB) or cyan, magenta, yellow, black (CYMK) or other system of characterising digital colour into colour components. Typically, as other colour values in the present invention, these are expressed in one of 256 value units from 0-255 i.e. an 8 bit digital colour system. Other, types of digital colour value may be used, but this is a common type often used, and is preferable in the present invention.

In step 156, the background colour values in RGB or other suitable colour components for the background growth media are established as background colour values for colour components in the selected region R 0G 0B In an alternative preferred method of the invention, the corresponding pixel or group of pixels in a pixel block that are subsequently selected as a region of growth, and therefore are known to be suitable for later image analysis, are subsequently selected in this earlier collected first background image of the culture media to derive colour values for that pixel or group of pixels 10R, 0G 0B Thus the same geographic region of the later image, where growth is seen, is compared to the same geographic region of this earlier background image. The use of a background image assists in standardising changes in the performance of the imaging device and variables within the incubating and scanning apparatus, and in particular, the affect of variations, from plate to plate, in the background colour of the growth media in a culture plate.

The platen which here functions as a shelf provides a means of immobilising the culture plate by virtue of, in one embodiment, gravity holding the plate in place, so that movement of the plate between images in an incubating and imaging sequence is prevented. Friction enhanced regions, or one or more small upstanding features or walls of the incubating chamber may also be used. Thus, the culture plates are held fixed in position within the imaging device's field of view and do not move with respect to the platen during an incubation and imaging sequence.

Referring briefly to Figures 27A, 27B, 28A and 28B, two embodiments (for single and multiple plates, respectively) are seen in which, small locators in the form of notches 430 are provided, typically in the lid, in specially modified culture plates 5 for engaging with upstanding nodules 432 provided on the surface of the platen 46. Notches 430 and small upstanding nodules 432 are sized and shaped to fit together, perhaps by friction fit, so as to hold the culture plates in position on platen 46.

Alternatively, notch 430 and nodule 432 may loosely engage together to simply guide placement of the plates in the field of view of the imaging device (not shown) rather than hold the culture plates fixedly in position and provide some resistance to sideways movement of platen 46 (perhaps if the apparatus is knocked). Other interengaging features may be used, for example, an upstanding nodule may be provided on a lid of a culture plate for engaging with a small locating recess or notch on the platen Further, notches (or recesses) and nodules may be provided spaced around the periphery of the culture plates and/or on the platen. For example, 3 or 4 interengaging notches and nodules may be substantially spaced about a culture plate to hold the culture plate 5 in an imaging position on the platen 46.

In one embodiment, the present invention provides an apparatus in which the plates are incubated in the same place (and indeed the culture plates are typically not moved at all) as the image is captured without the need to transfer the plates to a secondary instrument. In known systems the colour of growths is compared in one image with the colours of a known organism in a previously captured image in identical growth media. In the present invention rather than comparing one image with a standard image, the present apparatus collects a sequence of images over time. The colour values of selected geographical regions (a pixel or group of pixels) of the growths over time are plotted to produce colour fingerprint data and/or graph(s) of colour values changes over time. Colour fingerprints can themselves be summed or averaged to provide a better signal to noise ratio. The colour fingerprint over time may reflect the lag, log growth, stationary and death phase of the organism. Each of these phases is characteristic of the organism and can be used for their identification. For example, some typical times are as shown.

The length of lag phase (hours) and length of exponential phase (hrs) respectively are as follows for the following organisms grown on TSA agar: E coil on TSA agar Lag 1 hour, Exponential Growth phase B hours S uberis on TSA, Lag 5 hours, Exponential Growth phase 15 hours P multocida on TSA, LaglO hours, Exponential Growth phase 24-30 hours B atropheus on TSA, Lag 6-B hours, Exponential Growth phase 25 hours Salmonella on TSA Lag<1 hours, Exponential Growth phase 4 hours S aureus on TSA, Lag 1-2 hours, Exponential Growth phase 12-16 hours Figure 6 shows selection of a pixel block comprising a group of pixels and subsequent selection of an alternative pixel block comprising a different group of pixels in a seventh embodiment of the present invention. Three images of the same growth at three different (succeeding) times t1, tg2 and t93 are shown as growth outlines 420, 422 and 424 respectively, indicating growth from time t1 to time t2 and from time t92 to time tg3. A first pixel block 426 comprises pixels 190 (xl to x2, yl to y2) in, in this example embodiment, a lOxlO pixel array 192.

The first pixel block 426 comprising the lOxlO pixel array 192 is selected in the software, typically shortly after capture of a first growth check image 420 at a first growth check time t91. Second pixel block 428 comprising a lOxlO pixel array 194 may be selected in software later as will be explained below. The average colour value of one or more or each colour component of the first pixel block 426 are determined. Typically the average colour values (for one colour component) will be a mathematical mean colour value, but may be a mode or a median colour value. Alternatively, total colour value may be used over pixels 190 of pixel array 192 of first pixel block 426.

This same pixel block 426 is used to define a corresponding pixel array 192 and corresponding pixels 190 (xl to x2, yl to y2) in one more and preferably a sequence of subsequent images of the same growth (422 at time tg2 and 423 at time tg3). The corresponding average colour value over the pixel block (typically the mathematical mean), or total colour value over the pixel block, of each colour component of the first pixel block 426 in the later images 422 and 423, of the same growth are determined. A plot of the average (mean, mode or median, or a total) colour value over pixels 190 of the first pixel block 426 as determined in later images 422 and 423 then forms a first colour fingerprint graph of colour over time of the selected growth (see Figures 19A to 19D for example).

Further images captured over time may be similarly analysed to extend the colour fingerprint plot over time. This can be done (and displayed or stored in real time so that a user can see the plots every time these are updated to include colour value data at a new time point.

If the first colour fingerprint graph corresponding to the colour values of the first pixel block 426 is not sufficient to enable identification or sufficiently sure identification of the growth on the culture plate, further analysis may be carried out. In this further analysis a second pixel block 428 is selected (perhaps based on a second image 422 or third image 423). The second pixel block 428 may be selected, for example, to provide further pixels in an area of strong growth (seen in the increase in extent of bulge 429 on the side of the growth). The second pixel block 428 may overlap the first block 426 but it need not do so.

A plot of average (mean, mode or median, or total) colour value over pixels 190 in second pixel array 194 (x3 to x4, y3 to y4) of one or more or each colour component (such as RGB, CMYK) over time forms a second colour fingerprint graph of the selected growth. Thus, further opportunities for developing a second or even a third or further colour fingerprints of a selected growth are provided by the incubating and imaging apparatus in a further embodiment of the present invention by its capability for collection, storage and analysis of captured images over time.

Whereas here pixel arrays 192 and 194 both comprise lOxlO groups of pixels, the pixel arrays may have different numbers of pixels and/or be of differing shape (circular, rectangular, triangular, polygonal etc). Indeed, one or more pixel arrays may be replaced by a single pixel to enable pixel by pixel comparison (although this is not currently preferred) Figure 7 shows three methods for generating microorganism growth fingerprints for comparison with reference microorganism fingerprints according to eight, ninth and tenth embodiments of the invention respectively. The three methods, identified collectively as process 200, have a common first step 202 of collecting and analysing data to determine colour values of colour components (such as RGB) from corresponding geographical locations (x, y), i.e. from selected pixel blocks, in background (control) image l and time images In step 204 of Method 1, a plot of the colour value of the colour components is plotted over time as a colour fingerprint graph. In step 206, the plot of colour value of one or more or each colour component versus time is printed out or displayed on a computer. In step 208, comparing the plot from step 206 with reference growth colour fingerprint for a know organism on a standard (or the same) culture media under similar conditions in similar or the same apparatus is carried out. Thus, standard growth fingerprints under the same or very similar conditions may be provided in advance, using, for example one or more or each of the following: the same or same design of incubating and imaging apparatus 10, 60, the same or similar temperature in the incubating chamber 3, the same or similar reducing temperature gradient emanating from the heated, optically transparent platen, the same or similar growth media, the same batch of culture plates. Following the comparison, identifying the micro-organism is carried out in step 210; this may be carried out by eye. Alternatively or in addition, fitting, such as least squares fitting, of colour value of one or more or each colour component over time to standard colour fingerprints may be carried out to identify automatically or facilitate manual identification of the growth.

In Method 2, step 212, removing the background colour is carried out, typically, by subtracting the background colour of, for example, a lOxlO pixel block in a background image (e.g. of a control plate or of a comparable geographical region of the same (inoculated) culture plate but prior to any growth thereon. This may follow a condensation check. Step 214 comprises correcting the colour values of each collected image over time (110R, 1G_10G, and l?_I0B) by subtracting the colour values of the background image for the corresponding geographical areas (pixel blocks) in the timed images of the same (inoculated) culture plate, as described in Figure 6. However, colour values from a correspondingly sized pixel block in a control (not inoculated) plate, preferably undergoing the same incubating and imaging sequence, may be used as background. In step 216, the background corrected colour values are plotted against the elapsed time at which the image was recorded. In step 218, the plot of background corrected colour value of one or more or each colour component versus time is printed out or displayed on a computer.

In step 220, comparing the plot from step 218 with reference growth (such as micro-organism) colour fingerprint on standard growth (typically the same) media under similar conditions in the apparatus is carried out. Following the comparison, identifying the micro-organism is carried out in step 222; this may be carried out by eye. Alternatively or in addition, fitting, such as least squares fitting, of colour value of one or more or each colour component over time to standard colour fingerprints may be carried out to identify automatically or facilitate manual identification of the growth.

In Method 3, step 224 comprises calculating a % change of the colour value of one or more colour components (such as RGB) from a colour value at zero time for each image in a sequence. Thus, for each image at step 226, determining the data associated with the colour values, for corresponding pixels or pixel blocks comprising groups of pixels, is carried out, for example: For Red (11R_10R,) xl 00110R, For Green (1G1G) xl 00/10° For Blue (11B_10B,) xlOO/IOB where 11R is the colour value of the red component for the selected pixel block at time t, I0 is the colour value of the green component for the selected pixel block at time t, l8 is the colour value of the blue component for the selected pixel block at time t, lj is the colour value of the red component for the selected pixel block at time 0, 10° is the colour value of the green component for the selected pixel block at time 0.

10B is the colour value of the blue component for the selected pixel block at time 0.

Alternatively a first timed image in the sequence may be used to provide reference colour values (instead of a background image l) against which a percentage change in the colour values of later times images can be determined. In step 228 the determined percentage changes in colour values are plotted against the elapsed time at which the image was recorded to provide a colour fingerprint plot. Step 230 comprises displaying and/or printing out the percentage change plot. In step 232, comparing the plot from step 230 with reference (such as micro-organism) colour fingerprint(s) on standard culture media (e.g. the same media) under similar conditions is carried out. Following the comparison, identifying the micro-organism is carried out in step 234; this may be carried out by eye.

Alternatively or in addition, fitting, such as least squares fitting, of colour value of one or more or each colour component over time to standard colour fingerprints may be carried out to identify automatically or facilitate manual identification of the growth.

Figure 8 shows two plots 236 246 of example colour fingerprint graphs for Methicillin Resistant Staphylococcus Aureus (MRSA). Graph 1 (236) shows red, green and blue colour values and luminosity values (in lumins) for a selected pixel block in an MRSA colony measured over time with no background subtracted. Line 238 (diamonds) shows mean luminosity colour values, line 240 (squares) shows mean red colour values, line 242 (triangles) shows mean green colour values, line 244 (crosses) shows mean blue colour values for first given geographical area (such as pixel or group of pixels in a pixel block) without any background correction such as background subtraction having been carried out.

Graph 2 (246) shows red, green and blue colour values and luminosity values as measured over time for the same pixel block in the same MRSA colony after subtracting background media colour values (such as those measured in method 140 in Figure 5A). Line 248 (diamonds) shows mean luminosity colour values, line 250 (squares) shows mean red colour values, line 252 (triangles) shows mean green colour values, line 254 (crosses) shows mean blue colour values for first given geographical area (such as a pixel or group of pixels in a pixel block) after background correction such as background subtraction of colour values of the corresponding pixel block having been carried out.

The colour values are typically in the range from 0 to 255 in an 8 bit digital imaging system. The initial colour values are in the range 120 to 160 when no subtraction has been carried out, and nearer zero following subtraction of background. Further the corrected plot graph 2 (246) shows a clear lag phase (when the colour value is close to zero and an exponential growth phase as expected in micro organism growth.

Figure 9 shows a schematic, perspective view of a modular system 260 comprising modular apparatus according to an eleventh embodiment of the invention and a cutaway, schematic, perspective view of one modular apparatus of the modular system.

Here, individual incubating and imaging apparatus 60 are combined to form a modular system 260 according to an eleventh embodiment of the invention. Typically each modular unit apparatus 60 has a culture plate insertion slot 64 suitable for receiving one culture plate at a time. Indicator lights 66 and a display 66 along with control buttons (not shown) are provided. A camera on a chip 72 is placed beneath a heated optically transparent platen 74. A heated plate 76 of substantially the same size and shape as platen 74 is situated beneath platen 74 to heat platen 74 substantially evenly over its whole area (or at least in the area for receiving culture plate(s)). No other heating is provided in the incubating chamber. One or more lights 78 are provided to illuminate a culture plate 82 placed on platen 74.

Indicator lights 66 may function as alarm lights, particularly if a plate selective for certain growth (such as MRSA or C. difficile) are used, and, according to a preset incubation and imaging programme, if growth is seen for example at a first growth check time t91 (typically between 4 to 8 hours or at around 4, 5, 6 hours) then one or more lights are illuminated when growth is seen, since any growth on a selective growth plate for MRSA at that time could indicate MRSA is present.

The modular apparatus can be used for the simultaneous detection and identification of microorganism on up to n culture plates where n= number of modules attached to the apparatus.

Figure 10 shows a method 262 of utilising a modular system such as that in Figure 9 according to a twelfth embodiment of the invention which may be suitable for, for example, a veterinary application where multiple animal patients may each require testing e.g. for MRSA, at around the same time prior to, for example, admission into veterinary hospital. In method 262, step 264 comprises selecting parameters for incubating and/or scanning or selecting a pro-set programme (such as a programme for use of a selective growth plate e.g. selective for MRSA or C. difficile). Step 266 comprises scanning a culture plate barcode (or other identification indicia) of a culture plate that has been inoculated with sample. Step 268 comprises inserting the culture plate and starting the incubating and imaging sequence. Step 270 comprises beginning incubation. Step 272 comprises collecting a time zero (e.g. a background) image. This may occur after an optional condensation check has been carried out (not shown). Step 274 comprises starting image capture and capturing images at time intervals t. Step 276 comprises analysing images for the detection of growth such as bacterial growth as described elsewhere herein (for example in connection with Figure 7). Step 278 comprises ending image capture and incubation at a stop time. This may be around 12, 18, 24, 36 or 48 hours after the start time. Step 280 comprises printing out and/or storing data from the incubation and imaging sequence.

The sequence may be repeated (typically overlapping or at substantially the same time) with several modules in a modular system such as that shown in Figure 9. One computing device may be used to control, analyse and display results from the modules used in the modular system. Typically each module will be inaccessible (for example locked) during its incubating and imaging sequence. If the sequence is stopped early this may be noted in a data file associated with that incubating and imaging sequence.

If growth is detected in one or more modules of a modular system, an alarm may be activated (such as light and/or sound), as being indicative of likely growth of the suspected organism. The incubating and imaging sequence is then typically continued, preferably without access to the culture plate at this time, to collect a sequence of images for later analysis and eventual confirmation (or not) of the identification of the suspected microorganism using the analysis techniques of the invention.

Figure 1 1A shows a schematic, perspective view of a further apparatus 290 according to a thirteenth embodiment of the invention suitable for batch processing of multiple culture plates at one time in an incubating chamber using a single incubating and imaging sequence and a single incubating chamber. Figure 11 B shows a schematic, perspective, cutaway view of the further apparatus of Figure hA. Figure 11C shows a cross-sectional view through the apparatus 290 of Figures hA and 11 B. Apparatus 290 comprises an integral computing device (not shown), an insulated casing 291, slots for inserting culture plates 292, a control panel 294 and a display 296. A heated, optically transparent platen 302 is provided with a number of optional recesses 295 for receiving culture plates thereon. Other holding means for plates could be used instead of recesses 298, such as one or more upstanding walls or nodules or the plates could simply be held in place by gravity and marks to show their preferred location on the platen provided on the platen. Typically recesses 298 are of the same size and shape as the culture plates except that the depth of the recesses is slightly less than the height of the culture plates to facilitate easy removal of culture plates from recesses 298. Illumination for image capture is provided in the form of lights 300 which here are tubular neon lights. Although a single imaging device may be used (such as a flatbed scanner that moves in fixed, known relation to the platen 302 or camera with a wide field of view) here each culture plate recess is provided with its own imaging device 306 here in the form of camera on a chip. The imaging devices 306 are fixed in relation to the platen. Thus separate image processing for each culture plate is facilitated. Turning now to Figure 11C, a housing base 312 is shown. An inverted culture plate 308 is shown resting on platen 302. A camera 306 is located beneath platen 302 having culture plate 308 in its field of view.

Two alternative heating devices are shown. A first is heating pad 304 which extends underneath the platen 302 and heats platen 302 over substantially all its surface. Typically this heating is relatively evenly distributed over the platen 302. The heating device 304 heats the platen 302 which then heats the underside of the culture plate 308 and provides a reducing temperature gradient across culture plate 308 emanating from the platen from its side in contact with the platen to its opposing side not in contact with the platen.

Referring to Figures 12A and 12B here apparatus 261 comprises an imaging device in the form of a single scanner 12, a heated optically transparent platen 74, provided with ultrafine "invisible" heating elements 310 that heat underneath and around culture plates (not shown) held in recesses 298 in optically transparent platen 74. The scanner 12 has a fixed, pre-defined path of movement with respect to the platen 74. A computer and keyboard 36, 32 are shown. The apparatus 260 has an insulated housing 292, a handle 314 and two hinges. During use a single image is taken by scanner 12 and used by computer 36 to analyse the images of each culture plate in recesses 298 separately.

Referring to Figures 13A and 13B, these show process flows 320 for batch apparatuses, such as those shown in Figures 1 1A, 11 B, and 11 C and Figures 12A and 12B. Batch process flows 320 show a sequence of common steps for the batch apparatus 260 seen in Figures hA, 11B and 110 and apparatus 261 seen in Figures 1 2A and 1 2B and several bespoke steps.

Step 322 comprises selecting parameters on an integral processor or computer and inserting a plate into each slot in the apparatus into a scanning position on the platen. Step 324 comprises selecting parameters on an external computer and opening the lid to place a number of culture plates on the scanning platen 74. Step 326 comprises selecting parameters using, in apparatus 260, an integral computing device or, in apparatus 261, an external computer 36 and 32. Common step optional 328 comprises scanning a barcode or other identification indicia, or entering the identification details manually. Step 330 comprises inserting the culture plates and beginning the incubation and imaging sequence, by, in apparatus 260, inserting the plates though a slot or other opening in the casing 291, whilst, in apparatus 261, this is carried out by opening the lid and placing culture plates in the culture plates holders (such as recesses). Step 332 comprises starting the incubation. Step 334 comprises collecting a time zero image (typically following a condensation check as described in connection with Figure 2GB and elsewhere herein).

Step 336 comprises capturing time images overtime at time intervals of At. Typically time intervals At are equally spaced but may not be particularly if extra data is required during a period of rapid change. Time intervals At may be a few minutes or hours in length but are typically 10, 15, 20, 30, 45 or 60 minutes in length. To reduce the amount of data storage required, images may be spaced apart in time even more for example, up to 2 hours in between time images for a long expected overall time sequence (>24-36 hours). Step 338 comprises commencing analysis of collected time images after a first growth check time t1. First growth check time, t1, may be 2-8 hours long and is typically 4, 5 or 6 hours long. Thus once a first growth check time has passed (set by the user or in a pre-set programme) image analysis of time images commences. Common step 340 comprises analysing one or more time images for growth. Common step 342 comprises determining, for example with reference to a threshold value on one or more colour values of one or more colour components, that a growth is seen (positive) or a growth is not seen (negative). An output is issued with the result.

Typically an alarm is activated if the one or more results are positive for growth (particularly of the culture plate or plates used are selective for one or more particular growths). Incubation continues in common step 343, and imaging continues according to the set schedule in the incubating and imaging sequence in step 345. At an end time, t5 (of start time plus Z hours) incubation and image collection stops.

Step 346 comprises analysing the time images to confirm the growth identification and/or the confirmation of the negative result. Step 348 comprises indentifying the growths using a colour finger print analysis technique according to one aspect of the present invention (such as that shown in Figure 7). Step 349 is identical to step 348 except that in addition it comprises locating individual plate images within the overall image of a number of plates. Common optional step 351 comprises outputting for display and/or printout and/or storage a data file for each plate within a batch. Thus both apparatus 260 and 261 can incubate and image a number of plates as a batch over one time period. Each has a single heated transparent platen but apparatus 260 has several discrete cameras, one for each culture plate imaging position Figure 14 shows a schematic, perspective view of an early prototype apparatus 350 according to a sixteenth embodiment of the invention. An incubation device in the form of a heater box 16 having a control panel 22 is located beneath a flatbed scanner 12. On top of scanner 12 is a scanner lid forming an insulating housing above heated, optically transparent platen (not shown) of flatbed scanner 12. A computer analyses the results and displays images on the screen 36. An edge of a culture plate 352 can be seen in the image alongside growths 356 on growth media 358.

Figures 15A, 15B and 15C demonstrate the images obtained of the growth of S aureus on TSA plates at 37°C at 0, 18 and 28 hours. Culture plates 352 are placed on heated optically transparent platen 354. The plates 352 include a growth media 358. Growths 356 are seen on the growth media 358 (and in Figures 16A and 16 B). A sterile control culture plate (seen top left) may also be used, for example to provide a background image (using a pixel block of a comparable size to provided colour values).

Figure 1 6B shows an image of S. Aureus on TSA agar plate after 8 hours, zoomed to show the whole plate. Figure 16B is a close up of the plate of Figure 16A zoomed to show individual growths.

Zooming enables significant niagnification of the growths without having to use a microscope. It aids in the counting of colonies that are growing into each other. Images can also be digitally subtracted and coloured to highlight changes.

Figure 17 shows miscellaneous fungal and bacterial growths on TSA agar. This image has been formed by taking an 18 hour image and subtracting a 0 hour image. The process works with all the normal culture media. Culture plates 352 show background corrected images of growths following subtraction, 360.

Figures 18A and 18B shows growth images of three different types of MRSA on the Oxoid oxycillin MRSA testing plates (Oxoid chromogenic agar plates) at time 0 and at time 15 hours. These plates are selective for MRSA but the oxycillin inhibits growth of normal S. Aureus. The blue areas (seen as lines in Figure 18B) on the 15 hour image show growth of MRSA not inhibited by the oxycillin in the growth media. This figure demonstrates that this embodiment of the present invention can identify MRSA in less than 24 hours.

This and other embodiments of the present invention are advantageous in enabling a sequence of growth images to be recorded. Analysis of these images enables change in red, green and blue (RGB) (or cyan, magenta, yellow, black (CMYK) or other colour components) in each growth area to be measured and to be plotted against time. These change in colour/ time values enable a colour fingerprint of growth to be plotted. Our results suggest that this colour fingerprint is specific for each microbiological species.

Figure 19A shows a growth fingerprint for E Coli on TSA agar (12 hour). Figure 19B shows a growth fingerprint of Salmonella on TSA agar (12 hour). Figure 19C shows a growth fingerplint of S aureus on TSA agar (12 hour). Figure 19D shows a growth fingerprint of Penicillium (12 hour). Red component values 364, 374, 384 and 394, green component values 366, 376, 386, and 396, and blue component values 368, 378, 388 and 398 are plotted over time to from colour fingerprints in this eighteenth embodiment of the invention. As can be seen from the figures all of these fingerprints are very different. After 24 hours incubation the fingerprints are even more characteristic.

Whilst colour fingerprints according to one or more embodiments of the invention may comprise one or more colour components, a colour fingerprint preferably comprises all the colour components in the system, such as all 3 in a 3 colour system such as RGB, or all 4 in a 4 colour component system such as CMYK. In figures 19A to 19D, intensity values in lumins 362, 372, 382, 392 are also plotted overtime. These may or may not form part of the colour fingerprint.

Reducing Quarantine time Figure 20 gives the 24 hour fingerprint of bacillus atrophaeus, the organism that is generally used as a control for ethylene oxide sterilisation. Early identification of the growth of this organism may be useful in routine monitoring of ethylene oxide sterilisation processes. After 12 hours there are large changes in colour values which again appear to be characteristic of the organism. Such plots may enable the quarantine time of ethylene oxide sterilised products to be significantly reduced from their present 7 day quarantine period.

Some examples of images of growths taken using apparatus according to the invention are seen in Figures 24 and 25. Figure 24 shows complete (including R,G,B components) images (following background subtraction) of standard organisms (E Coli, S Uberis and Pasteurella Multocida (PMA) growing on TSA agar after 24 hours. Two plates of each organism are shown. Figure 25 shows a complete (including R,G,B components) image (following background subtraction) of a mixed culture growing on TSA agar after 24 hours. A number of different types of growths can be seen.

Figure 26A shows an example plot (following background subtraction) of colour value of one or more or all colour components of a bacteria over time showing lag phase, log or exponential growth phase and resting phase. Figure 26B shows steps involved in a condensation check. Figure 26C shows a method of collecting images and optional steps for use in a method of activating a growth alarm.

Figure 26D shows further steps for use in the method of Figure 26C. During a lag phase, minimal growth is seen (see Figure 26A) during a log or exponential phase rapid growth is seen, somewhat later the organism enters a resting phase and the growth slows indicated by the flattening off of the colour values of the colour components of the growth.

Referring to Figures 26A and 26B, step 410 comprises at a first condensation check time t,,1, checking for condensation either by eye or automatically using known colours values of one or more colour components of the background media absent condensation and absent growth and comparing these with the image of the culture plate after a short period of time (i.e. pre any significant growth) say after 5, 10, 15, 20, 25, 30 minutes. Step 412 comprises an optional step of at a second condensation check time t2 checking for condensation, for example, in the same manner as in step 410. Step 414 comprises repeating the check until no or minimal condensation is observed. Step 416 comprises setting the image at which no or minimal condensation is seen as the background image l.

Referring to Figures 26A, 26C and 26D, step 440 comprises collecting images from immediately after the plate is inserted into the apparatus (t0) or from a time tt0 when an incubation and imaging sequence is begun. Step 442 comprises at a first growth check time tg1, commencing a check for growth. Step 444 comprises taking an image It = l1 at time t01. Step 446 comprises selecting a pixel or a pixel block in image l1. Step 448 comprises selecting the same pixel or a pixel block in background image l. Step 450 comprises separating It and 10 into colour components for the selected pixel or pixel block. Step 452 comprises checking if the colour value of any one or more colour components or luminosity (ALL) in the captured time image I is greater than the corresponding colour value in the background image 10 Therefore if any one or more of i ALL ALL It >0 or l?>loR or l2>1o0 or l?>I0B is true, this indicate that a growth is seen (ALL R+G+B, all colour components together i.e. a complete image).

Steps 454 and 456 comprise the optional steps of noting that growth is detected and activating an alarm if growth is seen, particularly if a selective or semi-selective plate is used.

If no growth is seen, optionally, a second (or further) growth check may be conducted (repeating steps 442, 444, 446, 448, 450 and 452 at later growth check times) until growth is detected.

Step 458 comprises, if growth is detected, then for previous images (before the growth check time at which growth was detected), determining form the image data the colour values of one or more or each colour component for the selected pixel or pixel block for previous images (direction B in Figure 26A). Step 460 comprises capturing further images over time (following the growth check time (direction F in Figure 26A). Step 462 comprises plottinq one or more of the colour values of one or more or each colour component over time ltR, ltG, lB as a colour fingerprint graph of the growth or first subtracting the corresponding colour values and then plotting the background corrected colour values over time..

Thus, the present invention, using digital imaging, can utilise standard culture plates, or modified culture plates, and current methods of inoculating test samples. The culture plate is inoculated and then incubated in the device (incubator). High resolution digital images of the culture plates are taken at set times during the incubation period. The images are stored on the hard disk of a computer or other digital computer storage device. Software enables pixel by pixel comparison of the digital images stored on the computer and reports, in certain embodiments, the geographical location and number of areas of colour change occurring during the incubation period. These can then be converted into a plot of colour value over time and this colour fingerprint used for the identification of the organism.

The present invention alleviates the problem of condensation by producing a temperature gradient between the top and the bottom of the culture plate in such a way that the transparent lid is held at a slightly higher temperature than the culture medium. By creating this temperature gradient, condensation is reduced and in most circumstances is substantially prevented, and the scanner obtains a clear view of the growth surface of the culture plate.

The present invention incorporates an integral heater so that the device functions not only as an imaging device but also as an incubator. The present invention records multiple digital images of the culture plate over the incubation period. Pixel by pixel comparison of changes between the images during the progression of the incubation enables early growth detection. Colour separation and analysis of images provides fingerprint growth curves which enables automated identification by comparing growth curves against a library of reference growth curves.

Unlike the prior art the present invention provides that (1) the image capture system is an integral part of the incubation chamber, (2) that there is no requirement to remove the lid of the culture plate, (3) that there is no requirement to move the culture plate for image capture and (4) in the process of image capture and analysis. Images are captured at several time points starting at time zero, image analysis for each time point is performed by generating histograms (such as for example mean, mode, median or total of two or more pixels or a pixel block) of red (R), green (0) blue (B) and luminosity (L) and calculating mean values (or mode median or total values) to generate kinetic plots for one or more or each colour component and, optionally, luminosity. Kinetic plots are compared with a library of known kinetic plots referred to as fingerprints which are unique to each micro-organism under pre-determined conditions.

The present invention describes a device which enables early visual detection and subsequent identification of micro-organisms. The device consists of a heated insulated chamber which encloses a digital image capture device connected to a digital processor or computer. Within the chamber, an optically transparent plate supports an inverted culture plate which is illuminated from below using a typically daylight lighting device. The image capture device is typically located below the optically transparent plate to record images of the surface of the culture plate. Computer software is designed to perform automatic image capturing of plates at time intervals selected by the operator. Early identification of growth is achieved by using software that identifies any areas of the image where there is developing colour change in a particular geographical location compared to the background colourofthe media. The x,ygeographical location of these changes is recorded. The colourvalues of all the images in the sequence corresponding to this geographical location (pixel or pixel block) are then measured. These colour values are then plotted as a colour/time colour fingerprint graph.

Identification of colour changes relative to the background colour of the media enables growth to be detected before it is visible to the naked eye. The use of high resolution image capturing equipment mimics the observation of the plate with a microscope. However, by using digital imaging comparison techniques, the plates can be examined more accurately and faster than a human observer.

Identification of the micro-organisms present is achieved by generating colour separated (R,G,B) time plots of colony growth and comparing the resulting time plots with a library of reference curves.

One common problem with incubating culture plates is the formation of condensation on the transparent lid. The present invention overcomes this problem by creating a temperature gradient across the culture plate with the lid being slightly warmer than the growth medium.

The present invention alleviates the need to remove the culture plates from the incubator as an image of the culture plates can be observed in situ by directly capturing a high resolution image with an imaging device such as a camera or scanner. Plates do not need to be removed from the incubator for this purpose. Recorded images can be viewed locally or remotely via a second computer and appropriate computer connections such as a network, radio transmission or the internet. The present invention may automatically store digital images of the growth on each plate during the incubation process. This provides the bacteriologist with a useful quality control record.

The device provides automated identification of micro-organisms and a quality control record is retained.

The present invention provides a quick result without the need for sub culturing. Operation of the device requires less skill and less experience than many of the more sophisticated methods described.

The apparatus of the present invention may comprise a number of components such as an incubation chamber, an optically transparent platen (typically for use as a shelf), an imaging device such as a digital camera or a scanner, a computer processor, an output device, a light source, a heating device, a temperature control system, and analysis software.

Features of the incubation chamber may include that: the chamber is heat insulated such that it allows the formation of a temperature gradient. The apparatus may have an opening and/or door to allow insertion and removal of culture plates. The apparatus may have connectors and/or ports which allow for connection to a computer or external processor by wire or wirelessly. The apparatus may have a power connector for a power supply to be connected to the internal electrical components, and/or an internal power supply. The apparatus may have one or more heat ports to allow heated air input and/or output for heating purposes. The apparatus may have a means of supporting one or more optically transparent shelves. Supports for the one or more optically transparent shelves may be provided in walls of a housing of the apparatus. The apparatus may be light proof (for example by provision of a housing and/or incubating chamber that is sealed so that no or minimal light can get in or out). The apparatus may have features to enable it to be stacked on top of or beneath another identical apparatus. The apparatus may have a background surface, for example, arranged to be viewed behind the culture plates by the imaging device when these are placed on the optically transparent platen, against which the culture plates can be viewed. The apparatus may have a means for the attaching of the imaging device securely, for example securely to a housing or to a part of the apparatus so it does not move in relation to the platen during an incubation and imaging sequence.

The heated, optically transparent platen may be optically transparent, for example, optically transparent in the relevant colour range, such as the visible range, so as not to unduly affect the colour of images taken by the imaging device if these are taken through the transparent platen. The range may include the infra red and/or ultraviolet or other regions of the spectrum. The platen may be optically transparent in the range or substantially optically transparent over the range, or sufficiently optically transparent and of fixed optical transparency so that the affect it has on images is substantially constant. The platen is typically rigid. The platen may be planar, for example in the form of a plate having two major substantially planar surfaces substantially parallel to one another so as reduce optical complexity in the imaging. The platen is typically colourless, but may be coloured. The platen may be removable for cleaning or replacement. The platen may comprise reference colours.

For example, one or more reference colours may be attached to one or more regions of the optically transparent platen which can be used as a standard, against which changes in the performance of the imaging device may be monitored and/or accounted for in image analysis. The heated, optically transparent platen may comprise a means of immobilising the culture plate so that movement of the plate is prevented. For example, the heated, optically transparent platen may comprise one or more recesses sized and shaped to receive a culture plate and/or the optically transparent platen may comprises one or more high friction regions and/or the optically transparent platen may comprise one or more low upstanding walls arranged so as to engage with the outer periphery of one or more culture plate. Interengaging features such as notches and nodules described in relation to Figure 27A to 28B may alternatively be used. Preferably any such location features are arranged so as not to impede or affect the view of the culture plate by the imaging device.

The imaging device may have a resolution which will meet the requirements of the data analysis of the images, namely 300 to 900 dpi (dots per inch)and preferably at least 300 dpi. Example of imaging devices that may be suitable include flatbed scanners, digital camers and high resolution cameras on a chip.

The heating device may be enabled so that the apparatus, and in particular the incubation chamber of the apparatus, is warmed to an incubation temperature in the range of 0 to 60°C preferably 30- 45°C.. The heating device may incorporate a feedback temperature control device which keeps the set temperature constant. For example, one or more temperature sensors such as thermistors may be placed in the apparatus such as on the heated, optically transparent platen and/or near the roof of the incubation chamber so as to monitor the temperature and/or the temperature gradient within the incubation chamber.

A culture plate is preferably placed upside down (inverted) in relation to the platen (on top of it if the platen is substantially horizontal so as to function as a shelf)) to take advantage of the reducing temperature gradient arranged within the incubating chamber and so reduce condensation. Thus, the heated platen enables a reducing temperature gradient of typically 0.5 to 6°C or ito 4°C or ito 3°C or 0.5/1 to 2°C to be established in a direction moving away from the platen (such as generally perpendicular away from the platen). The side walls of the culture plate are typically approximately parallel to this direction as the culture plate is placed in contact with the platen and therefore a reducing temperature gradient is set up across the culture plate from a lid of a culture plate to a base of the culture plate when the lid is placed in contact with the platen. Thus, the lid of the culture plate, and typically also the adjacent optically transparent platen, is hotter than the growth medium in the culture plate. This avoids the formation of condensation on the lid of the culture plate (or on a region of the optically transparent platen adjacent the culture plate). The lid of the culture plate is typically colourless and optically transparent so that images of the growths on the growth media within the culture platen can be captured through the lid of the culture plate and the heated, optically transparent platen by an imaging device. When the platen serves as a shelf, the imaging device is typically underneath the platen and its field of view is upwards.

Methods of heating the incubation chamber to create a temperature gradient across the plate may include one or more of the following: forcing a flow of hot air through the incubation chamber; forcing a flow of air through a gap between the optically transparent platen and a secondary platen; heating the optically transparent platen directly with embedded heater wires; heating the optically transparent platen directly with surface mounted heating devices such as heating tape or conductive film; incorporating a heater inside the chamber with or without forced air circulation, any other method of creating a temperature differential and/or arrangement of the apparatus in which the lid of the culture plate is kept hotter than the base of the culture plate.

The processor and/or external computer (if provided) may be connected to the imaging device by direct cabling, local area network or wireless network connection to enable two way flow of digital information. The processor and/or computer may enable storage, archival and retrieval of the digital images e.g. from the imaging device and/ or from memory devices media. The processor and/or computer may enable storage, archival and retrieval of other relevant information such as date and time at which the digital images were taken on the imaging device. The processor and/or computer may enable hard copy of the images and/or associated information to be printed out on a device such as a computer printer. The processor and/or computer may enable the recorded digital images to be viewed locally or remotely via a secondary computer. The secondary computer may be connected via a computer network, direct cabling, local area network or wireless network or any other form of electronic transmission.

By the term computer" any computing processing device is meant including but not limited to a personal computer, a main frame computer, a laptop computer, a smart phone with computing capabilities, a portable computing appliance such as a computing pad (e.g. Apple® ipad, Blackberry® playpad) and the like.

Software may be arranged to take a series of high resolution digital images at preset times, the times being entered into or preset on the computer. The software may be arranged to automatically store images on the computer or a secondary digital storage device connected to the computer. The software may be arranged to carry out a method of identifying any change in position of the culture plate during a run.

The software may be arranged to carry out a method of colour value calibration which may correct variations in colour output from the imaging device and/or from variations in illumination (e.g. if a bulb has to be changed in a light source). For example, this may be achieved by comparing the colours of one or more test features of one or more known standard colours within the incubation chamber in view of the optical imaging device. The one or more test features of one or more known standard colours may be located on the heated, optically transparent platen in view of the imaging device and/or on the culture plate (preferably on the upperside or underside of the lid). The software may comprise a method of comparing the high resolution digital images pixel by pixel or by groups of pixels with groups of pixels across all the imaqes in the sequence. For example, groups of pixels two or more pixels (such as a lOxlO pixel block) may, for each colour component, have the colour value summed over that group in the manner of a histogram, so that the summed colour value for that colour component for that pixel block at that geographical location can be plotted over time.

Alternatively, mathematical mean, mode or median colour value over the pixel block may be plotted over time for one or more or each colour components. The colour values of several growths on the same or different plates may be added or averaged together to provide an enhanced signal.

The software may be arranged to identify changes in colour value of growth areas compared to those of the original growth media as the initial colonies grow and identifying their x,y geographical locations on the image. The software may be arranged to identify the changes in colour as changes in the colour component value of red, green, blue (RGB)", cyan, magenta, yellow and black (CMYK)" or any other method of characterising colours or overall light intensity between the digital images. The software may be arranged to count the number of areas of change in colour value or intensity occurring during the incubation period and their geographical locations (x,y) on each plate.

The software may be arranged to report the total number of colonies identified to a data file and/or to a display and/or to a printout.

The software may be arranged to plot one or more colour component values and/or total colour values and/or luminosity values against the time across the sequence of images to give a colour fingerprint graph of the growth of the organism(s) at one or more geographical locations. The software may be arranged to sum and/or average the colour values across several growth areas i.e. across several geographical locations at each time point to enhance the signal to background ratio.

The software may be arranged to compare the colour fingerprints of one or more colour components of one or more geographical areas over time against a reference library of colour fingerprints over time for known bacteria.

The software may be arranged to analyse identification indicia (such as barcodes) on the culture plates denoting information on the type and composition of the growth media and optional numbering of the culture plates (for example to be captured by the processor or computer along with image data).

Example 1 -The Bug Scanner-an early prototype The initial concept was developed by growing a range of bacteriological samples on tryptone soya agar (TSA) plates in a standard bench top incubator. These were taken out of the incubator every 4 hours, placed on the platen of a computer scanner and scanned at 4 hourly intervals. Observation of these images in Photoshop demonstrated that most growths could be observed within about 12-24 hours. The images were diqitised and therefore these could be observed, subtracted and zoomed to assist colony counting by human manipulation of the imaging software including identifying the correct geographical area. As the images were stored on the computer these can be interpreted at leisure and viewed either locally or remotely. Measurement of the red, green and blue values of the growth areas enabled a colour fingerprint graph of the growth of the organisms to be plotted against time. Results suggest that these fingerprints were characteristic of the type of organism growing.

Examr)le 2 -The Bug Scanner-a later jrototype (see Figures 2A and 2B) A prototype was then built in which a thermostatically controlled heating circuit was incorporated into a computer scanner. Thermocouples were attached to the glass platen of the scanner and attached to the thermostatic control. The control measured the actual temperature of the platen and enabled the target temperature to be entered. When the temperature had risen to the selected value inoculated TSA agar plates were placed on the platen and the scanner cover placed over them.

Software on the computer enables entry of the times between scans and the maximum length of the incubation process. Once set up and running the software triggered the scanner to scan at the selected time intervals and store the sequential images on the hard drive of the computer.

This prototype according to the invention is a device for incubating, recording and early identification of microbiological growth. Standaid culture plates are inoculated as normal and placed in the incubation chamber of the scanner. The desired temperatule, the time interval between scans and the length of incubation are entered into the computer. The image sequence is automatically recorded onto the hard disk. The images can be viewed at any point during the incubation period.

This can be performed either locally or remotely over the Internet. The digital images can be subtracted to aid in the early identification of bacterial growth. They can be zoomed to aid colony counting. The type of organism growing can be identified by constructing a growth fingerprint of the change in colours overtime. The growth fingerprint is characteristic of the organism.

Features of the one embodiment:- 1. Provides a permanent sequential record of microbiological growth 2. Incubates and records sequential images automatically 3. Images can be viewed at any point during the incubation 4. Images can be viewed either locally or remotely via the internet 5. Early identification of microbiological growth by image subtraction 6. Enables colony counting to be performed retrospectively 7. Zoom facility and sequential imaging assists colony counting 8. Colour analysis of growth areas enables a colour fingerprint of the growth to be plotted. These fingerprints are specific for microbiological species enabling the type of microorganism to be identified 9. Compact size enables bacteriology to be performed in a small lab.

10. In house operation enables considerable cost saving over commercial microbiological testing Example 3-Proof of Principle A microbial incubating imaging device enables simultaneous incubation, viewing and recording of bacteriological culture plates. Digital images of the culture plates are taken at selected time intervals during the incubation. A temperature gradient is established between the top and bottom of each culture plate to remove and prevent condensation. Growths are identified as changes in colour (red, green blue (RGB) or other digital colour value systems) compared to the background colour of the media. Once a growth is identified in one of the sequential images, its x and y geographical location is noted: The colour values of all the previous and images in the image sequence corresponding to this geographical location are measured. The colour values are then plotted against the time at which the image was recorded. The colour/time graph of the growth, the colour fingerprint, can then be compared to the colour fingerprints of known organisms. The inventors suggest that each species of organism produce a colour fingerprint which is specific for that class of organism for the incubating conditions and time.

The prototype consists of a heating circuit inserted into a computer scanner. Inoculated culture plates are placed in the scanner and sequential growth images recorded onto the computer hard drive.

Images can be taken at preset time periods ranging from 5 minutes to once daily. Image sequences can be recorded over time periods ranging from hour to thirty days.

Observation of image sequences of bacterial growths while using the embodiment of the present invention for sterility testing in a Sterile Services Unit, showed that growth could be observed as a change in colour at a particular geographical location in each image in the sequence. Measurement of the change in red, green, blue and luminosity values over time of an individual colony in the image sequence showed that each organism appeared to have a specific colour fingerprint".

This study further investigated if colour fingerprinting could be used to identify different organisms growing in a mixed culture. As this was the case, this method enabled organisms to be identified in the initial culture of the bacterial identification sequence, thereby speeding up the process of identification. The process described only requires standard culture plates, (or in some alternative embodiments modified culture plates having positioning features such as a notch and/or colour standards thereon) and should therefore be considerably cheaper than DNA testing methods.

The aim of the study was to investigate if similar coloured organisms growing in mixed culture could be identified using colour fingerprinting using image sequences produced from the incubating and imaging and apparatus of the present invention. Such a finding should enable faster and more economical identification of bacteria.

Three organisms were selected that were known to produce colonies with a similar grey colour, namely Esherichia coil, Streptococcus uberis and Pasteure//a multocicia. Each were grown on three different rriedia; Tryptone Soya Agar (TSA), blood agar and TSA with 7% blood. All the media were manufactured by E&O laboratories.

The incubating and imaging apparatus of the present invention was run with the incubator temperature set at 37 DC The incubating chamber is arranged so that a temperature gradient is established emanating from the heated, optically transparent platen into the incubating chamber, The exact temperature gradient is not known, but is thought to be of the order of ito 2 or ito 3 °C from a position adjacent the platen to a distance around 8-10 rum from the platen (in other words to a distance roughly coincident with the expected position of a base of a culture plate, when one is placed on the platen lid side down).

A loop full (such as a Quadloops from Arben Bioscience, Rochester, NY, USA) each organism was taken from standard cultures and streaked onto TSA agar using standard microbiological techniques.

This process was repeated with all three organisms. Plates were labelled with sample details. A maximum of six plates were then placed top down on the platen and covered with an insulated lid and the imaging device (here a scanner) set to record images hourly for a time period of 48 hours.

Image sequences were taken of each culture. Further plates were then streaked and incubated so that six image sequences were available for each organism on six different plates. Mixed colonies were then prepared by mixing 20 microlitres of a stock solution of all three colonies into Sml of aqueous sodium chloride 0.9%. This was then vortexed and a loopful streaked on two TSA plates and incubated at 37degC for 48h.

The images from each run were opened in Adobe Photoshop. Distinct colonies were identified in the and 30 hour images. The XY coordinates of the top left hand edge of the colony were recorded. A by 10 pixel histogram was then drawn using this location as the top left hand location of the square. The red( R), green (G), blue (B) and luminosity (L) values of each histogram area were recorded. This process was repeated on every fifth image in the sequence. The colour values and corresponding times were entered into an Excel spreadsheet and plots of colour against time drawn.

This process was repeated so that colour fingerprints were obtained for six individual colonies of each organism on a single plate, and for six colonies each on six different plates of TSA. The process was then repeated for twelve mixed cultures on TSA. The RGBL results at 5 hours were used as baseline correction for the background colour of the media for all the subsequent colour values recorded in a growth sequence. Means and coefficients of variation (CV) were calculated for six growths on a single plate (Single plates, Tables 1-3) and for six growths on six different plates of the same media (Multiple plates, Tables 4-6).

This process was repeated so that growth fingerprints were obtained for six individual colonies of each organism on a single plate, and for six colonies on six different plates of each of the three media. The process was then repeated for twelve growths on the mixed cultures on TSA. The RGBL results at 5 hours were used as the background media colours. These were subtracted from the results of all the subsequent colour values recorded in the growth sequence. This removed the contribution of the background colour of the media. Means, and coefficient of variances were calculated for six growths on a single plate (Single plates tables 1-3) and for six growths on six different plates of the same media (multiple plates tables 4-6).

Results Table 1 Single plate (within plate variation) RGB values over time -E co/ion TSA, baseline corrected data for 6 individual colonies on one plate Incubation C.V Image time (h) Red Green C.V blue C.V lumin C.V 4 0.0 0.0 0.0 0.0 9 6.6 0.23 6.2 0.24 5.0 0.20 6.3 0.21 14 25.7 0.21 24.3 0.16 18.4 0.11 24.1 0.17 19 42.1 0.14 38.9 0.13 28.4 0.16 38.8 0.13 24 51.4 0.10 49.8 0.08 38.4 0.11 49.1 0.08 29 45.1 0.02 49.6 0.07 44.3 0.11 47.7 0.06 34 47.6 0.07 47.5 0.05 39.5 0.11 46.7 0.05 39 51.9 0.09 47.3 0.07 33.3 0.13 47.2 0.08 44 53.0 0.15 48.1 0.11 33.1 0.13 48.0 0.12 Table 2 Single plate (within plate variation) RGB values over time -S.uberis on TSA, baseline corrected data for 6 individual colonies on one plate Incubation Image time (h) Red C.V Green C.V blue C.V lumin C.V 4 0.0 0.0 0.0 0.0 9 1.2 0.39 0.9 1.31 1.2 1.15 1.0 0.68 14 5.0 0.16 5.5 0.33 3.9 0.22 5.1 0.25 19 9.5 0.15 10.2 0.18 8.2 0.21 9.7 0.17 24 14.5 0.15 16.2 0.12 12.1 0.19 15.3 0.13 29 17.6 0.27 18.0 0.22 14.8 023 17.6 0.23 34 23.6 0.31 25.9 0.27 21.7 0.24 24.7 0.27 39 22.1 0.31 26.3 0.32 23.5 0.35 25.0 0.31 44 22.2 0.25 26.7 0.28 27.4 0.29 26.3 0.26 Table 3 Single plate (within plate variation) RGB values over time -P.multocida on TSA, baseline corrected data for 6 individual colonies on one plate Incubation Image tine (h) Red C.V Green C.V blue C.V lumin C.V 4 0.0 0.0 0.0 0.0 9 0.9 0.61 0.8 0.11 1.3 0.50 0.9 0.46 14 1.5 0.41 1.7 0.45 0.6 1.48 1.5 0.38 19 3.0 0.57 4.6 0.47 32 0.70 3.9 0.50 24 10.3 0.56 11.0 0.55 8.5 0.53 10.5 0.55 29 22.3 0.27 22.0 0.30 17.3 0.30 21.5 0.29 34 34.1 0.13 32.0 0.17 24.6 020 31.6 0.16 39 37.4 0.20 35.9 0.16 27.8 0.20 35.5 0.17 44 39.8 0.21 39.8 0.20 31.7 0.24 38.9 0.20 Table 4 Multiple plate (between plate variation). RGB values over time -E co/i on TSA, baseline corrected data for six growths on six plates Incubation Image time (h) Red C.V Green C.V blue C.V lumin C.V 4 0.0 0.0 0.0 0.0 9 5.7 0.29 5.7 0.47 4.1 0.40 5.5 0.39 14 24.2 0.20 22.3 -0.21 15.5 022 22.1 0.20 19 38.7 0.14 364 0.16 25.4 0.22 35.9 0.15 24 45.1 0.15 46.1 0.11 36.1 0.18 44.7 0.11 29 43.0 0.05 45.0 0.09 39.1 0.12 43.8 0.07 34 43.7 0.07 412 0.06 33.8 0.10 41.1 0.04 39 46.8 0.10 41.7 0.07 28.1 0.14 41.7 0.06 44 49.7 0.10 42.4 0.09 26.7 0.13 0.08 Table 6 Multiple plate (between plate variation). RGB values over time -S uberis on TSA, baseline corrected data for six orowths on six plates CoV Sample time hrs Red green Coy Blue CoV lumin CoV 4 0.0 #DIV/0! 0.0 #DIV/0! 0.0 #DIV/0! 0.0 #DIV/0! 9 0.7 3.07 0.9 2.69 1.4 1.46 0.9 2.40 14 52 0.66 5.1 0.62 4.7 0.74 5.1 0.63 19 10.2 0.43 9.8 0.52 8.4 0.55 9.8 -0.49 24 15.4 0.39 15.8 047 13.7 0.41 15.5 0.44 29 15.0 0.28 16.5 0.39 15.1 0.40 15.9 0.36 34 15.2 0.49 18.8 047 19.6 0.47 17.8 0.47 39 15.3 0.44 18.8 0.44 19.7 0.45 17.9 0.44 44 14.2 0.41 18.7 0.47 20.3 0.50 17.5 0.45 Table 6 plate (between plate variation). RGB values over time -P. Multocida on TSA, baseline corrected data for six cirowths on six p a CoV Sample time hrs Red green Coy Blue CoV lumin CoV 4 0.0 0.0 0.0 0.0 9 -0.5 -4.16 -0.2 -5.82 -0.1 -24.66 -0.2 -5.16 14 1.1 140 12 044 1.9 0.78 1.2 0.43 19 7.0 0.75 7.8 0.55 6.5 0.61 7.4 0.57 24 17.7 0.49 172 0.44 13.6 0.44 16.9 0.44 29 27.5 0.31 25.6 0.34 20.9 0.29 25.7 0.32 34 33.5 0.25 33.0 0.25 26.6 0.22 32.5 0.24 39 38.9 0.25 36.3 0.25 28.3 0.27 36.2 0.24 44 40.7 0.28 38.0 0.26 29.1 0.28 37.8 0.26 The colour fingerprints of the mean results from the six organisms on one plate (single plate (5)) and six organisms on six plates (Multiplate (M)) are shown in Figures 21A, 21B, 22A, 22B, 23A, 23B. The fingerprints of the E coIl, S Uberis and PMA are very similar. The coefficients of variation are given in

tables 1-6.

At low colour values the Coy are as expected very high. At higher values (>20) the Coy values for E coli are 10-17% for both single and multiple plates. The Coy for S uberis is higher between 20-40%.

The Coy for PMA is 20-30%.

The aim of this study was to investigate if these fingerprints can be used to identify organisms in multiculture. The single and multiple plate results give us three standards with an estimate of the variation of the colour values over time for each of the organisms.

Examples 3 Discussion In these embodiments of the present invention, an incubating and imaging apparatus was used to record sequential hourly images of growths of E co/i, S. uberis and P. mu/too/cia on TSA. Analysis of 1 OxlO pixel areas of the growths enabled colour fingerprints of red, green, blue and luminosity values over time to be plotted for six separate organisms growing on one plate (single plate) and one organism growing on six separate plates (multiplate). The fingerprints for the single plate growths and the multiplate growths were very similar all having coefficient of variation of 10-20%. Multicultures were prepared in which all three organisms were mixed in saline and spread onto a TSA plate. These were incubated in the apparatus for 48 hours and images recorded at one hourly intervals. Twelve colonies were selected and colour fingerprints constructed for each organism. These fingerprints were then compared with the fingerprints of the standard organisms.

Five hourly images were taken in this study to reduce the amount of visual analysis required to extract the data for each fingerprint. However, analysing hourly rather than 5 hourly images should enable much better identification of features in the growth fingerprints. Measurement of changes in RGB at more frequent intervals will enable us to investigate the duration and changes in the lag phase, to more clearly define the exponential phase and hopefully identify the organism even earlier.

For slow growing organisms such as tuberculosis there can be up to 6 to 24 hours between readings.

Preferably, the culture plates are not moved from their initial position within the incubator between readings. This provides simple identification and registration of culture plates.

Methods of fitting such as best fit or least square fitting of one or more colour components forming the colour fingerprints to standard colour fingerprints could be used.

This pilot study suggests that for the three organisms chosen, individual colonies in mixed culture can be identified by using the apparatus of the present invention and colour fingerprinting. Identification of P. rnu/tocida in the mixed culture was almost impossible by eye. However, colour fingerprinting enabled positive identification of this organism in the mixed culture plate. This was still possible even although there appeared to be interaction between the S uberis and P. multocida which raised the amplitude of the colour peaks in the P. multocida fingerprint. Further testing is required to see if fingerprinting can be used to differentiate between other organisms.

This study suggests that colour fingerprinting can successfully identify individual organism in a mixed culture on the first culture plate. Further investigation is required to establish if this can be used for a range of common pathogens. Early and economic identification of these organisms could have a large impact on the diagnosis and treatment of infections in medical and veterinary practice.

Example 4 Analysis Process -Steps in obtaining a microorganism fingerprint using the invention Aim: Detection of microbial growth and colony counting. Outcome: Identification of growth or no growth, and colony count.

Method of Recording background media colour values: example MRSA on tryptone soya agar (TSA) Image Recording 1. The time interval between images is set on the software. This can range from 10 minutes to 24 hours.

2. Images of the culture plate are taken at these time intervals and then stored on the hard drive of the device 3. Software then checks the first image for evidence of condensation. If none is detected this image is used as the control. If condensation is detected then the second image is taken as the control.

Occasionally the third image will need to be used as the control.

4. The colour values of the media in the control image expressed as red, green or blue (RGB) or other system of characterising digital colour are recorded. These colour values are used as the

background colour of the media used.

Method of identifying bacterial growth 1. Software scans the control image measuring the colour values in lOxlO pixel blocks together with their geographical locations (x,y) in the image. These values are recorded.

2. Software scans each of the images in the sequence and records the colour values in lOxlO blocks together with their geographical location and the elapsed time at which these images were taken.

3. The two data banks are then compared. Any 1 Oxl 0 pixel block that has colour values greater than, preferably, 2-4 units or in the range of 1-6 units, different from the control colour values are considered to be early bacterial growths.

4. The xy coordinates of the lOxlO squares with the growth are recorded.

5. Squares with colour change which are in contact with another square with identified growth correspond to larger or growing colonies.

6. The number of colonies is counted 7. This process is continued for each of the images in the sequence Generation of the colour fingerprint 1. Using the data generated above the colour values of the growth areas are plotted against the elapsed times at which the images were taken to give a graph of colour value against time. These give an RGB against time graph (colour fingerprint) 2. Another method is to subtract the background colour values for each 10x10 pixel block in the control plate image which removes the background colour values. The corrected colour values are then plotted against the elapsed time at which the image was recorded.

3 Another method is to calculate the % change of the RGB values from zero time at each image in the sequence.

4. The images are displayed on the screen of the computer or printed out.

5. These are then compared against standard reference fingerprints grown on standard media under identical conditions in the apparatus Further embodiments of the invention will be apparent to the skilled reader from the disclosure herein, and all such variations are to be considered within the scope of the present application.

Claims

<claim-text>Claims 1. An incubating and imaging apparatus for incubating and imaging growths, such as microorganism growths, comprising: -an incubating chamber for incubating a culture plate; -a heated optically transparent platen for placing a culture plate against, -the incubating chamber and heated optically transparent platen being arranged so that a reducing temperature gradient, from a higher temperature to a lower temperature, is formed in a direction moving away from the platen -further comprising -an imaging device arranged to view through the heated optically transparent platen into the incubating chamber for capturing an image of growth media, and/or any growth, on a culture plate.</claim-text> <claim-text>2. An apparatus according to claim 1 in which the incubating chamber and heated optically transparent platen are arranged so that if a culture plate is placed against the platen, a reducing temperature gradient is formed from a first side of a culture plate against the platen to a second side opposing the first side not against the platen.</claim-text> <claim-text>3. An apparatus according to claim 1 or 2 in which adjacent comprises being in thermal contact or in direct contact.</claim-text> <claim-text>4. An apparatus according to claim 1, 2, 3 in which the heated optically transparent platen is substantially optically flat so as not to distort images captured by the imaging device through the platen.</claim-text> <claim-text>5. An apparatus according to any preceding claim in which the platen is colourless.</claim-text> <claim-text>6. An apparatus according to any preceding claim in which the platen is optically transparent in the visible range of frequencies.</claim-text> <claim-text>7. An apparatus according to any preceding claim in which the platen comprises glass or plastic.</claim-text> <claim-text>8. An apparatus according to any preceding claim in which the platen is temperature controlled so as to control the reducing temperature gradient.</claim-text> <claim-text>9. An apparatus according to any preceding claim in which platen is substantially planar and the reducing temperature gradient is in a direction substantially perpendicular to the plane of the platen.</claim-text> <claim-text>10. An apparatus according to any preceding claim in which the platen is the sole heat source within the incubating chamber.</claim-text> <claim-text>11. An apparatus according to any preceding claim in which the platen forms a side wall other than a roof or a floor, or a roof of the incubating chamber.</claim-text> <claim-text>12.An apparatus according to any of claims 1 to 10 in which the platen forms the floor of the incubating chamber or a shelf within the incubating chamber.</claim-text> <claim-text>13. An apparatus according to any preceding claim in which the incubating chamber is arranged so that minimal or no circulation of air occurs within it so facilitating the establishment of the reducing temperature gradient.</claim-text> <claim-text>14. An apparatus according to any preceding claim in which the platen is heated substantially uniformly over its area or at least over an area of its surface for receiving culture plates thereon 15. An apparatus according to any of claims 1 to 14 comprising a heating mechanism arranged to heat the platen and in which the heating mechanism comprises at least one heating element.16. Apparatus according to claim 15 in which the heating element is surface mounted.17. Apparatus according to claim 16 in which the heating element comprises heating tape and/or conductive film.18. Apparatus according to claim 15 in which the heating element is embedded within the platen.19. Apparatus according to claim 15 in which the heating mechanism is situated beneath the platen.20. Apparatus according to any of claims 15 to 19 in which the heating element is generally evenly distributed over an area of substantially the same size and/or shape as that of the heating platen, or as that of an area of the heating platen for receiving culture plates thereon, so that platen is heated substantially uniformly over its area, or over the area for receiving culture plates thereon.21. Apparatus according to preceding claim comprising a fan for circulating heated air adjacent a surface of the platen not facing the incubating chamber.22. Apparatus according to claim 21 in which an optically transparent, second platen is provided substantially parallel to the first heated optically transparent platen and heated air is circulated in between the two platens so as to controllably heat the first heated optically transparent platen.23. Apparatus according to claim 22 in which the imaging device is arranged to view through the first and second platens into the incubating chamber.24. Apparatus according to claim 22 or 23 in which the first and second platens are about 5 to 25mm apart (± 1mm), or about 10 to 15mm apart (± 1mm).25. An apparatus according to any preceding claim in which at least one temperature sensor is provided on the heated optically transparent platen so as to control the reducing temperature gradient.26. An apparatus according to any preceding claim in which the platen is substantially horizontal in use so that culture plates can be placed upon it and held in place by gravity.27. Apparatus according to any preceding claim in which the imaging device comprises a flatbed scanner, the flatbed scanner having a scanning platen and in which the scanning platen is the heated optically transparent platen.28. Apparatus according to claim 27 in which the heating mechanism is provided inside the flatbed scanner.29. Apparatus according to claim 28 in which a fan is provided in the flatbed scanner for circulating air on an underside of the flatbed platen.30. Apparatus according to claim 27 in which a heater box is provided beneath and/or encasing the flatbed scanner for heating the flatbed scanner so that the scanning platen is heated over substantially its whole surface.31. Apparatus according to any preceding claim in which the incubating chamber comprises thermal insulation in one or more walls.32. Apparatus according to claim 31 in which the type and/or amount and/or thickness and/or location of thermal insulation is selected to provide a suitable reducing temperature gradient from the heated optically transparent platen into the incubating chamber.33. Apparatus according to any preceding claim in which the temperature gradient emanating from the heated optically transparent platen into the incubating chamber is selected and/or controlled by suitable selection of thermal insulation in one or more side walls or roof of the insulation chamber, and/or distance of the roof of the insulation chamber away from the heated optically transparent platen and/or temperature of the heated optically transparent platen.34. Apparatus according to any preceding claim in which the temperature gradient is selected from 0.5 to 6°C or ito 4°C or ito 3°C or 0.5 to 2°C or ito 2°C.35. Apparatus according to any preceding claim in which a median temperature of the incubating chamber is 0 to 60°C (±0.5 to 1°C) or 30-45°C (±0.5 to 1°C) and/or in which the temperature of the heated optically transparent platen is 0 to 60°C (±0.5 to 1°C) or 30-45°C (±0.5 to 1°C).36. An apparatus according to any preceding claim in which the heated optically transparent platen comprises a locating mechanism for locating a culture plate thereon.37. Apparatus according to claim 36 in which the locating mechanism comprises a mark on the first platen.38. Apparatus according to claim 37 in which the locating mechanism comprises an upwardly extending feature located on the platen such as a nodule or wall, or comprises one or more walls of the incubating chamber.39. Apparatus according to claim 38 further comprising a culture plate and the culture plate comprises a recess or notch for accommodating said upwardly extending feature.40. Apparatus according to any preceding claim in which imaging device comprises a digital imaging device.41. Apparatus according to claim 40 in which the imaging device comprises a digital camera or a digital scanner or a digital camera-on-a-chip.42. Apparatus according to any preceding claim in which the imaging device is arranged to capture two or more images over time.43. Apparatus according to claim 42 in which the imaging device is arranged to capture images from an incubation start time t to an incubation end time t8 at time intervals At.44. Apparatus according to claim 43 in which an image captured first is set as a background image 10.45. Apparatus according to claim 43 in which a first condensation check time t01 is defined between the incubation start time t0 and incubation end time t5 and a first condensation check image l, is captured at time t01 and checked for condensation and, if the condensation check is negative for condensation the first condensation check image l is set as a background image l = l1.46. Apparatus according to claim 44 or 45 in which if a first condensation check is positive for condensation then a second condensation check time t02 is defined between the first condensation check time and incubation end time t8, and a second condensation check image l2 is captured at time t02 and checked for condensation and, if the condensation check is negative for condensation, then the second condensation check image l2 is set as a background image l _i2IC -47. Apparatus according to claim 45 or46 in which a condensation check is carried out by -selecting a region comprising a selected pixel or group of pixels in a first condensation check image ci, -determining colour values of one or more or each colour components (Cl, C2 such as RGB, CMYK) of the selected pixel or group of pixels in the first condensation check image Id -optionally, determining colour values one or more or each colour components (C1, C2 such as RGB, CMYK) of the corresponding pixel or group of pixels in a control image, -comparing the determined colour values of one or more or each colour components of the first condensation check image ll, with expected colour values of a control image, or with determined corresponding colours values of a control image, to determine if condensation is present.48. Apparatus according to any of claims 45 to 48 in which further condensation checks are carried out as required until a background image substantially free from condensation has been established.49. Apparatus according to any preceding claim in which a first growth check time t is defined between an incubation start time t0 and an incubation end time t and a first growth check image l1 is captured at time t91 and a first growth check is carried out.50. Apparatus according to claim 49, when dependent on claims 44 to 46, in which a first growth check is carried out by -selecting a first growth region comprising a selected pixel or group of pixels in a first growth check image 1g1, -determining colour values of one or more or each colour components (0, 02 such as RGB, CMYK) of the selected pixel or group of pixels in the first growth check image ll -determining colour values one or more or each colour components (C1, 02 such as kGB, CMYK) of the corresponding pixel or group of pixels in the background image l, -comparing the determined colour values of one or more or each colour component of the first growth check image 1g1 with the corresponding colours values of the background image l to determine if there has been a change of sufficient difference in one or more colour values so as to indicate growth.51. Apparatus according to claim 50 in which a threshold difference in colour values of one or more colour components is set to indicate growth.52. Apparatus according to claim 47, 50, 51 in which colour values are measured in unit values from 0 to 255 and the threshold difference is 5 units in colour value of one or more or each colour components.53. Apparatus according to any of claims 47, 50, 51 and 52 in which a colour value of a colour component in a geographical region in an image comprising one or more pixels or a block of pixels, is the mathematical mean colour value for that colour component.54. Apparatus according to any of claims 47, 50, 51 and 52 in which a colour value of a colour component in a geographical region in an image comprising one or more pixels or a block of pixels, is the mathematical mode, median or total colour value for that colour component.55. Apparatus according to any preceding claims in which two or more images are collected over time and in which colour values of one or more or each colour component of the same corresponding first growth regions Ai comprising the same corresponding pixel or a group of pixels in three or more or each captured image are determined over time.56. Apparatus according to claim 55 in which colour values of one or more or each colour component of the first growth region A1 of the captured images It are plotted against time in a graph as a first colour fingerprint.57. Apparatus according to claim 56 in which the colour values of one or more or each colour components of corresponding first growth regions A1 of the first or second or subsequent growth check image (l1 or l2 or lg) are included in the plotted graph against time.58. Apparatus according to claim 55, 56, or 57 in which the colour values of one or more or each colour component of a first growth region A1of a background image l are first subtracted from the colour value of one or more or each colour component of the corresponding first growth region A1 in the time images (li), and if also plotted, the corresponding colour values from the growth check image (Ig) and the resulting colour values are plotted against time in a graph as a first colour fingerprint graph.59-Apparatus according to any of claims 55 to 56 in which one or more time images I are captured at a time prior to the time of a first or second or subsequent growth check.60. Apparatus according to claim 59 in which colour values derived from time images captured prior to, and colour values derived from time images captured post a time of a first or second or subsequent growth check are plotted against time.61. Apparatus according to any of claims 53 to 60 in which a second growth region A2 is selected and colour values of one or more or each colour component of one pixel or group of pixels forming the second growth region A2 are determined from each of the timed images, l, and optionally from the selected growth check image l, and plotted in a graph over time to provide a second colour fingerprint graph.62. Apparatus according to any of claims 53 to 61 in which a plot of colour value of one or more or each colour component against time is compared to a library of comparable images of colour value against time of known growths, such as known microorganisms, so as to determine a likely identity of the growth.63. Apparatus according to any preceding claim in which the colour components are red (R), green (G), and blue (B) or cyan (C), magenta (M), yellow (Y) and black (K).64. Apparatus according to claim 63 in which one or more or each of red (R), green (G), and blue (B), or one or more or each of cyan (C), magenta (M), yellow (Y) and black (K), components of images are plotted in a graph over time to provide a colour fingerprint graph.65. Apparatus according to any preceding claim in which the colour values of the colour components are background corrected by subtracting corresponding colour values of a background image therefrom.66. Apparatus according to any of claims 42 to 65 in which means to detect differences in colour values of corresponding growth regions in a sequence of images is provided.67. Apparatus according to claim 66 in which the difference in colour values are determined as between each timed image and a selected background image.68. Apparatus according to clairri 66 in which the differences in colour values are determined between any two time images in the sequence or between neighbouring time images in the sequence.69. Apparatus according to any of claims 42 to 71 in which the time interval between time images is selected from the group of 1, 2, 3, 5, 10, 20, 30, 45, 60, 90, 120, 240, 360, 720 and 1440 minutes.70. Apparatus according to any preceding claim in which the height of the roof of the incubating chamber is 5 -10mm above the height of a standard culture plate.71. Apparatus according to any preceding claim in which one or more standard calibration colour marks within the field of view of the imaging device are used to correct variations in colour values of one or more colour components of an image.72. Apparatus according to claim 71 in which a 3 or 4 colour component standard calibration colour mark is provided within the field of view on the heated optically transparent platen.73. Apparatus according to any preceding claim in which a microprocessor is provided for carrying out image capture and analysis.74. Apparatus according to any preceding claim in which memory is provided for storing images taken between an incubation start time t0and an incubation end time t.75. Apparatus according to any preceding claim in which a specific microorganism selective culture plate selective to a specific microorganism is provided and an indicator device is provided for indicating a positive result if growth is detected at a first growth check time and/or at any subsequent growth check time.76. Apparatus according to claim 75 in which a selective culture plate is provided that is selective for MRSA.77. Apparatus according to claims 75 or 76 in which the indicator device is an alarm and/or a print out and/or display.78. Apparatus according to claim 81 in which the alarm is visible such as a light, flashing lights and/or audible such as an intermittent beep or continuous beep.79. Apparatus for imaging bacterial growth on a culture plate comprising -an incubator having an incubating chamber for incubating at least one culture plate -an imaging device for imaging one or more culture plates in the imaging chamber -a microprocessor -a heated transparent platen for locating at least one culture plate thereon and imaging a culture plate therethrough by the imaging device whereby when a culture plate is located on the heated transparent platen in an inverted manner so a lid of the culture plate is adjacent the heated transparent platen, the temperature of a growth medium in a culture plate is less than the temperature of the lid 80. A method of manufacturing an incubating and imaging apparatus comprising providing an apparatus according to any one of claims ito 79.81. A method of incubating and imaging comprising providing an apparatus according to any one of claims ito 79.82. A method of incubating and imaging a culture plate according to claim 81 comprising -incubating a culture plate by placing a culture plate adjacent the heated optically transparent platen so the lid of the culture plate is adjacent the heated optically transparent platen -arranging so that a reducing temperature gradient, from a higher temperature to a lower temperature, is formed in a direction moving away from the heated optically transparent platen -capturing images of the one or more culture plates through the heated optically transparent platen so that the temperature of growth media in a base of a culture plate is less than the temperature of the lid of the culture plate.83. A method according to claim 81 or 82 comprising circulating heated air adjacent a surface of the heated optically transparent platen not facing the incubating chamber so as to transfer heat to the incubating chamber and to any culture plate placed in the incubating chamber via the heated optically transparent platen.84. A method according to claim 81, 82 or 83 comprising monitoring at least one temperature sensor provided on the heated optically transparent platen so as to control the reducing temperature gradient.85.A method according to any of claims 80 to 84 comprising selecting thermal insulation in one or more walls of the incubating chamber so as to control, the reducing temperature gradient 86. A method according to claim 85 comprising selecting the type and/or amount and/or thickness and/or location of thermal insulation is selected to provide a suitable temperature gradient from the heated optically transparent platen into the incubating chamber.87. A method according to any of claims 80 to 86 comprising selecting a height of the insulation chamber above from the heated optically transparent platen and/or temperature of the heated optically transparent platen and/or temperature of the incubating chamber so as to control the reducing temperature gradient.88. A method according to any of claims 80 to 87 comprising locating a culture plate on the heated optically transparent platen using a locating mechanism such as a locating mark on the heated optically transparent platen or an upstanding feature on the platen, such a nodule or wall, or one or more walls of the incubating chamber.89. A method according to any of claims 81 to 88 comprising capturing two or more digital images over time.90. A method according to any of claims 81 to 89 comprising setting the first image as a background image l.91. A method according to any of claims 81 to 90 comprising capturing images from an incubation start time t0 to an incubation end time L at time intervals &.92.A method according to any of claims 81 to 91 in which a first condensation check time t is defined between the incubation start time t0 and incubation end time t and a first condensation check image l is captured at time t and checked for condensation and, if the condensation check is negative for condensation the first condensation check image I is set as a background image 1 = I1.93. A method according to any of claims 81 to 92 in which if a first condensation check is positive for condensation then a second condensation check time t2 is defined between the first condensation check time and incubation end time t8, and a second condensation check image l2 is captured at time t2 and checked for condensation and, if the condensation check is negative for condensation, then the second condensation check image l2 is set as a background image I,C94. A method according to any of claims 81 to 93 in which a condensation check is carried out by -selecting a region comprising a selected pixel or group of pixels in a first condensation check image Id, -determining colour values one or more or each colour components (C1, C2 such as RGB, CMYK) of the selected pixel or group of pixels in the first condensation check image Id -optionally, determining colour values one or more or each colour components (Cl, 02 such as RGB, CMYK) of the corresponding pixel or group of pixels in a control image, -comparing the determined colour values of one or more colour components of the first condensation check image 11, with expected colour values of a control image, or with determined corresponding colours values of control image, to determine if condensation is present.95. A method according to any of claims 84 to 97 comprising repeating the step of checking for condensation until a background image substantially free from condensation has been established.96. A method according to any of claims 81 to 95 in which a first growth check time t is defined between an incubation start time t0 and an incubation end time t and a first growth check image l1 is captured at time tg1 and a first growth check is carried out.97. A method according to any of claims 81 to 96 in which a first growth check is carried out by -selecting a first growth region comprising a selected pixel or group of pixels in a first growth check image l1, -determining colour values one or more or each colour components (Cl, 02 such as kGB, CMYK) of the selected pixel or group of pixels in the first growth check image 1g1 -determining colour values one or more or each colour components (Ci, 02 such as RGB, CMYK) of the corresponding pixel or group of pixels in the background image l, -comparing the determined colour values of one or more colour components of the first growth check image 1g1 with the corresponding colours values of background image 1 to determine if there has been a change of sufficient difference in one or more colour values so as to indicate growth.98. A method according to any of claims 81 to 97 in which a threshold difference in colour values of one or more colour components is set to indicate growth.99. A method according to any of claims 81 to 98 in which colour values are measured in unit values from 0 to 255 and the threshold difference is 5 units in colour value of one or more or each colour components.100. A method according to any of claims 81 to 99 in which a colour value of a colour component in a geographical region comprising one or more pixels or a block of pixels, is the mathematical mode, median or total colour value for that colour component.101. A method according to any of claims 81 to 100 in which two or more images are collected over time and in which colour values of one or more or each colour component of the same corresponding first growth region A1in each image comprising one pixel or a group of pixels are determined as a colour fingerprint over time 102. A method according to any of claims 81 to 101 in which colour values of one or more or each colour component of the first growth region A1of the captured images l are plotted against time in a graph to provide a first colour fingerprint graph.103. A method according to any of claims 81 to 102 in which the colour values of one or more or each colour components of the corresponding first growth region A1 of the first or second or subsequent growth check image (l1 or 1g2 or l) are also plotted against time in the graph to provide a first colour fingerprint graph.104. A method according to any of claims 81 to 103 in which the colour value of one or more or each colour component of the corresponding first growth region A1of a background image l are first subtracted from the colour value of one or more colour components of the time images (l) and if also plotted, from the growth check image (lu) and the resulting colour values are plotted against time in a graph to provide a first colour fingerprint graph.105. A method according to any of claims 81 to 104 in which one or more time images l are captured at a time prior to the time of the first or second or subsequent growth check.106. A method according to any of claims 81 to 105 in which colour values derived from time images captured prior to and colour values derived from time images captured post a time of a first or second or subsequent growth check are plotted against time.107. A method according to any of claims 81 to 106 in which a second growth region A2 is selected and colour values of one or more or each colour components of the one pixel or group of pixels forming the second growth region A2 are determined from each of the timed images, It, and optionally the selected growth check image l, and plotted against time in a graph to provide a second colour fingerprint graph.108. A method according to claim 107 in which a second, or further, colour fingerprint graph is developed from selection of second, or further growth, regions within the sequence of images, to provide additional identification information.109. A method according to any of claims 81 to 108 in which a plot of colour value of one or more colour components against time is compared to a library of comparable images of colour value against time of known growths, such as known microorganisms, so as to determine a likely identity of the growth.110. A method according to any of claims 81 to 109 in which the colour components are red (R), green (G), and blue (B) or cyan (C), magenta (M), yellow (Y) and black (K).111. A method according to any of claims 81 to 110 in which one or more or each of red (R), green (G), and blue (B), or one or more or each of cyan (C), magenta (M), yellow (Y) and black (K), components of images are plotted over time to provide a colour fingerprint graph.112. A method according to any of claims 81 to 111 in which the colour values of the colour components are background corrected by subtracting corresponding colour values of abackground image therefrom.113. A method according to any of claims 81 to 112 in which means to detect differences in colour values of corresponding growth regions in a sequence of images is provided.114. A method according to any of claims 81 to 113 in which the difference in colour values are determined as between each timed image and a selected background image.115. A method according to any of claims 81 to 114 in which the differences in colour values are determined between any two time images in the sequence or between neighbouring time images in the sequence.116. A method according to any of claims 81 to 115 in which the time interval between time images is selected from the group of 1, 2, 3, 5, 10, 20, 30, 45, 60, 90, 120, 240, 360, 720 and 1440 minutes.117. A method according to any of claims 8 to 116 in which one or more standard calibration colour marks within the field of view of the imaging device are used to correct variations in colour values of one or more colour components of an image.118. A method according to claim 117 in which a 3 or 4 colour component standard calibration colour mark is provided within the field of view on the heated optically transparent platen.119. A method according to any of claims 81 to 118 in which a microprocessor is provided for carrying out image capture and analysis.120. A method according to any of claims 81 to 1119 in which memory is provided for storing images taken between an incubation start time t0 and on incubation end time t.121. A method according to any of claims 81 to 1120 in which a specific microorganism selective culture plate is provided and an indicator device is provided for indicating a positive result if growth is detected at a first growth check time or at any subsequent growth check time.122. A method according to any of claims 81 to 121 in which a selective culture plate is provided and an indicator device is activated upon first detection of any growth.123. A method according to any of claim 122 in which the indicator device is an alarm and upon first detection of a growth an alarm is activated.124. A method according to any of claims 81 to 123 in which a selective culture plate is provided that is selective for a single organism, or is selective for MRSA or is selective for Clostridium Diffici I e.125. A culture plate for use in an apparatus according o anyone of claims 1 to 79 and/or for use a method according to any one of claims 81 to 124.126. An apparatus and a method for incubating and imaging substantially as described herein with reference to and/or as illustrated in the accompanying drawings.</claim-text>