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Publication Number
US-10487349-B2
Publication Date
2019-11-26
Expiration Date
Abstract
A fluidic device (1) has a culture chamber (10) configured to house a 3D culture matrix (2) comprising a culture of microorganisms (6). A concentration gradient of a test substance is established over the 3D culture matrix (2) by providing respective fluid flows at different end portions (12, 14) of the culture chamber (10) and comprising different concentrations of the test substance. The response of the microorganisms (6) to the test substance is determined based on the position of a border zone (5) in the 3D culture matrix (2).
Core Innovation
The invention provides a method of determining a response of a microorganism to a test substance using a fluidic device. A culture of the microorganism is provided in a three-dimensional, 3D, culture matrix arranged in a culture chamber of the fluidic device, and the culture chamber has a first fluid channel flanking a first end portion and a second fluid channel flanking a second, different end portion. A first fluid flow comprising the test substance at a first concentration is connected to the first fluid channel, while a second fluid flow lacking the test substance or comprising the test substance at a second concentration lower than the first concentration is connected to the second fluid channel.
This arrangement forms a concentration gradient of the test substance over at least a portion of the 3D culture matrix. The method takes at least one computer-processable image of the 3D culture matrix and identifies a border zone between a growth zone of growing microorganisms and a non-growth zone lacking growth of the microorganism. The border zone is determined relative to the first end portion and/or the second, different end portion by computer processing of the image.
A minimal inhibitory concentration, MIC, is determined based on the position by correlating the position to a concentration or concentration range of the test substance based on gradient information defining concentration versus position in the 3D culture matrix for the concentration gradient. A resistance of the microorganism to the test substance is determined based on the width of the border zone.
Claims Coverage
Independent claim clm-00001 defines four inventive features covering 3D culture gradient formation, computer-processable image analysis of a border zone, MIC determination from border-zone position, and resistance determination from border-zone width.
3D culture matrix in a flanking two-channel fluidic device
providing a culture of said microorganism in a three-dimensional, 3D, culture matrix arranged in a culture chamber of a fluidic device having a first fluid channel flanking a first end portion of said culture chamber and a second fluid channel flanking a second, different end portion of said culture chamber
Concentration gradient across the 3D culture matrix using first and second fluid flows
connecting an input of said first fluid channel to a first fluid flow comprising said test substance at a first concentration; connecting an input of said second fluid channel to a second fluid flow lacking said test substance or comprising said test substance at a second concentration that is lower than said first concentration to form a concentration gradient of said test substance over at least a portion of said 3D culture matrix
Border-zone identification between growth and non-growth by computer image processing
taking at least one computer-processable image of said 3D culture matrix; identifying, in said at least one image, any border zone in said 3D culture matrix between a growth zone of growing microorganism, and non-growth zone lacking growth of said microorganism by processing said at least one image by a computer configured to identify said border zone
MIC from border-zone position correlated to concentration-versus-position gradient information
determining a position, relative to said first end portion and/or said second, different end portion, of said any border zone by processing said at least one image by said computer; determining a MIC of said microorganism to said test substance based on said position of said border zone by correlating said position to a concentration or a concentration range of said test substance based on gradient information defining concentration versus position in said 3D culture matrix for said concentration gradient
Resistance from border-zone width
determining a resistance of said microorganism to said test substance based on said width of said border zone
The claim coverage links a 3D culture matrix in a fluidic device with a test-substance concentration gradient to image-based border-zone identification, then derives MIC from border-zone position and resistance from border-zone width.
Stated Advantages
Faster readout (hours vs overnight)
Documented Applications
Determining MIC and resistance of E. coli K12 MG1655 using ampicillin and spectinomycin gradients in the CELLDIRECTOR® Ruby microfluidic device.
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