Abstract: The present invention provides a method of preparing a suspension of intact microorganisms from a sample containing microorganisms and mammalian cells, comprising contacting the sample with a buffer solution with a pH of at least pH 6 and less than pH 9, a detergent and one or more proteases to allow lysis of the mammalian cells; filtering the mixture through a filter suitable for retaining microorganisms to remove the lysed mammalian cells; resuspending the microorganisms retained by the filter in a liquid to provide a suspension comprising the recovered intact microorganisms; and determining the concentration of microorganisms in the suspension by: i. heating and/or contacting an aliquot of the suspension with an alcohol; ii. optionally diluting one or more aliquots of the suspension to provide one or more diluted aliquots before, during and/or after step (i); iii. contacting at least a portion of an aliquot of step (i) or (ii) with a single fluorescent stain capable of binding to DNA; iv.

Abstract: A method for analysis of a digital image in order to determine a value relating to an amount of a substance shown in the image includes using a threshold to identify all pixels of the digital image above or below a certain intensity value in order to produce a first binary image. Texture filtering is used to identify regions of high texture in the digital image to produce a second binary image in which all regions of high texture are pixels with a first state. The first and second binary images are overlayed to produce a combined binary image. A value relating to the amount of the substance is determined based on the area of the combined binary image with pixels having the first state.

Abstract: The present invention provides methods for determining the antimicrobial susceptibility of a microorganism in a clinical sample said method comprising removing a test aliquot from a clinical sample culture before the culture reaches 0.5 McFarland units, isolating the microbial cells and transferring the cells into a suitable culture medium for microbial growth, and performing an AST assay using the isolated microbes, wherein the concentration of microbial cells in the microbial cells used to set up the AST assay is measured before the degree of microbial growth in the different growth conditions of the AST assay is measured. Devices for determining the antimicrobial susceptibility of a microorganism in a clinical sample are also provided.

Abstract: A method for performing microscopy-based imaging of samples comprises: loading a sample holder (100) onto a support (50) configured to receive the sample holder (100); moving the sample holder (100) in a first direction, from a starting position on a first strip of the sample holder (100), to move the sample holder (100) relative to an imaging line of a line camera (10), to capture an image of the first strip of the sample holder (100); monitoring a focal plane using an autofocus system (15) as the sample holder (100) is moved in the first direction; in response to a signal from the autofocus system (15), moving an objective lens (25) along the optical axis to adjust the focal plane; and moving the sample holder (100) in a second direction, to align the imaging line of the line camera (10) with a position on a second strip of the sample holder (100).

Abstract: A sample holder 100 for use in a device for image-based analysis of multiple samples, the sample holder comprising: an optically flat surface 10; multiple locations of interest 16, 18 dispersed across the optically flat surface 10 that each, in use, correspond to locations of a sample of the multiple samples; and multiple focal structures 12 associated with the multiple locations of interest 16, 18; wherein the focal structures 12 each comprise at least one pyramid shaped indentation 30 in the optically flat surface 10.

Abstract: A method for determining the antimicrobial susceptibility of a microorganism in a clinical sample includes removing a test aliquot from a clinical sample culture before the culture reaches 0.5 McFarland units and isolating the microbial cells. The cells are transferred into a suitable culture medium for microbial growth and an AST assay is performed using the isolated microbes. The concentration of microbial cells in the microbial cells used to set up the AST assay is measured before measuring the degree of microbial growth in different conditions of the AST assay. Devices for determining the anti-microbial susceptibility of a microorganism in a clinical sample are also disclosed.

Abstract: A sample holder (10) comprises: an upper layer (20); a lower layer (40); a middle layer (30) between the upper and lower layers; and a sample chamber (33) formed by a through-hole in the middle layer (30), covered at its upper extent by a portion of the bottom surface of the upper layer (20), and at its lower extent by a portion of the top surface of the lower layer (40), wherein at least part of the bottom surface of the upper layer (20) overlapping a portion of a top periphery of the sample chamber (33) comprises a hydrophobic surface, wherein the hydrophobic surface is sufficiently hydrophobic that a contact angle of a water droplet on the hydrophobic surface would exceed 110°.

Abstract: A method for detecting and characterizing a microorganism in a clinical sample includes introducing a clinical sample to a first culture vessel containing the culture medium; removing a test aliquot; separating DNA from the test aliquot; and performing nucleic acid tests on the DNA to identify the microorganism and to detect the presence or absence of one or more genetic antimicrobial resistance markers in the microorganism. If a microorganism is identified, an antimicrobial susceptibility test is performed wherein microbial growth in the antimicrobial susceptibility test is monitored by accessing growth or markers for growth and wherein the type and concentration of antimicrobial agents used in the antimicrobial susceptibility test is determined by the identity of the microorganism and the antimicrobial resistance markers detected. A device for performing the method is also provided.

Abstract: The present invention relates to a method of determining the concentration of intact microorganisms in a sample comprising optionally diluting an aliquot of the sample to provide a diluted aliquot at a dilution value; contacting at least a portion of an aliquot or of a diluted aliquot of the sample with first and second stains capable of binding to DNA, wherein the first stain is a fluorescent stain, is cell-permeable, and has a first emission wavelength, and the second stain is cell-impermeable, and is capable of acting as an acceptor molecule in a FRET pair with the first stain acting as a donor molecule; imaging the aliquot-stain mixture at the first emission wavelength and determining an image analysis value for the number of objects corresponding to intact microorganisms in the imaged mixture; and comparing the image analysis value for said aliquot to a pre-determined calibration curve, as well as an apparatus, a consumable and a kit therefor.

Abstract: A method for detecting and characterizing a microorganism in a clinical sample includes introducing a clinical sample to a first culture vessel containing the culture medium; removing a test aliquot; separating DNA from the test aliquot; and performing nucleic acid tests on the DNA to identify the microorganism and to detect the presence or absence of one or more genetic antimicrobial resistance markers in the microorganism. If a microorganism is identified, an antimicrobial susceptibility test is performed wherein microbial growth in the antimicrobial susceptibility test is monitored by accessing growth or markers for growth and wherein the type and concentration of antimicrobial agents used in the antimicrobial susceptibility test is determined by the identity of the microorganism and the antimicrobial resistance markers detected. A device for performing the method is also provided.

Abstract: Multiplexed methods of detecting an analyte in a sample using two or more padlock probes each specific to a different target sequence are described. The target sequence is either part of an analyte or indicative of the presence of an analyte in the sample. Each padlock probe includes an analyte-specific reporter sequence, and either a restriction cleavage site located 3? of the analyte-specific reporter sequence, and/or a first amplification primer binding site for an amplification reaction. Where the padlock probe includes a restriction cleavage site, cleavage at the restriction cleavage site occurs 3? of the analyte-specific reporter sequence. Where the padlock probe includes a first amplification primer binding site for the further amplification reaction, it does not contain a second amplification primer binding site 5? of the analyte-specific reporter sequence. Panels of probes and kits for the same are also described.

Abstract: A sample holder 100 for use in a device for image-based analysis of multiple samples, the sample holder comprising: an optically flat surface 10; multiple locations of interest 16, 18 dispersed across the optically flat surface 10 that each, in use, correspond to locations of a sample of the multiple samples; and multiple focal structures 12 associated with the multiple locations of interest 16, 18; wherein the focal structures 12 each comprise at least one pyramid shaped indentation 30 in the optically flat surface 10.

Abstract: A method for analysis of a digital image in order to determine a value relating to an amount of a substance shown in the image includes using a threshold to identify all pixels of the digital image above or below a certain intensity value in order to produce a first binary image. Texture filtering is used to identify regions of high texture in the digital image to produce a second binary image in which all regions of high texture are pixels with a first state. The first and second binary images are overlayed to produce a combined binary image. A value relating to the amount of the substance is determined based on the area of the combined binary image with pixels having the first state.

Abstract: The present invention provides a method of recovering viable microbial cells from a complex sample, said method comprising: a) providing a sample having a volume of at least 1 ml; b) contacting said sample with a buffer solution and one or more proteases, wherein said buffer solution has a pH of at least pH 6 and less than pH 11, wherein said buffer solution and said one more proteases do not comprise a detergent or a chaotrope, and wherein the buffer solution/protease/sample mixture is non-hypotonic; c) filtering the mixture obtained in step (b) through a filter suitable for retaining microbial cells; and d) recovering the microbial cells retained by the filter in step (c), wherein the recovered microbial cells are viable, and a microbial recovery device for the same.

Abstract: A method for detecting and characterising a microorganism in a clinical sample includes introducing a clinical sample to a first culture vessel and then simultaneously transporting and incubating the clinical sample in a thermally insulated compartment of a sealable container. A test aliquot is removed from the sample and the remainder of the sample is cultured further. The method further includes removing DNA from said test aliquot and performing nucleic acid tests on said DNA to identify the microorganism and to detect the presence or absence of one or more genetic antimicrobial resistance markers in said microorganism.

Abstract: A method for performing a rolling circle amplification (RCA) reaction includes at least two rounds of RCA. The method includes providing a concatemeric first RCA product having a multiplicity of monomer repeats, each repeat representing a complementary copy of a first RCA template, wherein the nucleic acid molecule to be detected and/or analyzed, or its compliment, is contained in the first RCA template. The first RCA product is cleaved into monomer units which are reduced in size compared to the monomer repeat of the first RCA template. Monomer units resulting from the cleavage are circularized to form second RCA templates, which are smaller than the first RCA template. A second RCA reaction is performed on the second RCA template and a primer for the second RCA to form a second RCA product. The second RCA product, or a monomer unit derived from it, is detected or analyzed.

Abstract: A substrate for use in manufacture of a production master plate for production of a detection disc for carrying samples in an apparatus for detection of microscopic objects in a fluid, the substrate having a channel and separate focus structure, wherein the focus structure is a groove.

Abstract: A substrate for use in manufacture of a production master plate for production of a detection disc for carrying samples in an apparatus for detection of microscopic objects in a fluid, the substrate having a channel and separate focus structure, wherein the focus structure is a groove.

Abstract: The present invention provides methods for determining the antimicrobial susceptibility of a microorganism in a clinical sample said method comprising removing a test aliquot from a clinical sample culture before the culture reaches 0.5 McFarland units, isolating the microbial cells and transferring the cells into a suitable culture medium for microbial growth, and performing an AST assay using the isolated microbes, wherein the concentration of microbial cells in the microbial cells used to set up the AST assay is measured before the degree of microbial growth in the different growth conditions of the AST assay is measured. Devices for determining the antimicrobial susceptibility of a microorganism in a clinical sample are also provided.