Identifying bacteria and other organisms by determining growth profiles

Abstract

Some aspects of the present disclosure are generally related to methods of identifying cells based on a growth profile, e.g., growth rates obtain in respective growth media. Monitoring and determining respective growth rates of unknown cells as a function of growth media may facilitate identifying a cell. In some embodiments, identifying cells in a high throughput manner based on identifying growth rates in a relatively low number of growth media, compared to typical methods, is disclosed. In some embodiments, exposure of cells to different cell growth media is performed serially for at least some of a plurality of cells, which may decrease a time to identification. Additionally, in some embodiments, the growth rates of multiple unknown cells may be monitored in parallel by utilizing a microfluidic device comprising one or more cell growth trenches. Still other aspects of the present disclosure are generally directed to systems for monitoring and determining growth rates of cells, kits containing the systems, or the like.

Description

[0001] IDENTIFYING BACTERIA AND OTHER ORGANISMS BY DETERMINING GROWTH PROFILES

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 646,534, filed May 13, 2024, entitled “Systems and Methods for Identifying Cells Using Hyperspectral Imaging and Other Techniques,” by Paulsson, et al., U.S. Provisional Patent Application Serial No. 63 / 646,582, filed May 13, 2024, entitled “Identifying Bacteria and other Organisms by Determining Growth Profiles,” by Paulsson, et al., and U.S. Provisional Patent Application Serial No. 63 / 646,599, filed May 13, 2024, entitled “Microscopy Systems and Methods for Identifying Bacteria and Other Organisms in Microfluidic Devices,” by Paulsson, et al. Each of these is incorporated herein by reference in its entirety.

[0004] GOVERNMENT FUNDING

[0005] This invention was made with government support under 1AY2AX000005-01 awarded by U.S. Department of Defense / Defense Advanced Research Projects Agency (DOD / DARPA) and under W91 INF-19-2-0018 awarded by U.S. Army Research Office (ARO). The government has certain rights in this invention.

[0006] FIELD

[0007] Certain aspects of the present disclosure are generally directed to systems and methods for identifying bacteria and other organisms by determining growth profiles, e.g., in microfluidic devices.

[0008] BACKGROUND

[0009] Cell screenings play a fundamental role in biology and make it possible to identify one or more cells of interest based on a desired phenotype and / or genotype associated with the target cells. Current cell screening techniques only provide endpoint low-resolution snapshots, and offer low throughput when identifying multiple types of cells from within a sample. In some cases, current techniques are often limited by scalability, screening errors due to phenotypic mischaracterization, long time to identification, etc. Thus, more effective systems and methods for cell screening are needed.

[0010] SUMMARY

[0011] Certain aspects of the present disclosure are generally directed to systems and methods for identifying bacteria and other organisms by determining growth profiles, e.g., in microfluidic devices. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0012] Some aspects are related to methods. In some embodiments, the method comprises determining a first and a second growth rate in respective first and second growth media of at least some of a plurality of cells within a plurality of cell growth trenches of a microfluidic device.

[0013] In some embodiments, the method comprises exposing a plurality of cells within a plurality of cell growth trenches to a first growth medium; determining a first growth rate of the plurality of cells exposed to the first growth medium; exposing the plurality of cells to a second growth medium; and determining a second growth rate of the plurality of cells exposed to the second growth medium.

[0014] In some embodiments, the method comprises determining a first and a second growth rate in respective first and second growth media of at least some of a plurality of cells from a sample containing two or more unknown species of unicellular organisms.

[0015] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

[0018] FIGS. 1A-1B are schematic representations of a system, according to some embodiments;

[0019] FIG. 2A is a schematic representation of a top view of one embodiment of a microfluidic device, according to some embodiments;

[0020] FIG. 2B is a schematic representation of a perspective view of the microfluidic device of FIG. 2A, according to some embodiments;

[0021] FIG. 2C is a schematic representation of a partially exploded view of the microfluidic device of FIG. 2B, according to some embodiments; FIG. 3A is a schematic representation of a top view of a section of the microfluidic device of FIG. 2A in an unactuated state, according to some embodiments;

[0022] FIG. 3B is a schematic representation of a top view of a section of the microfluidic device of FIG. 2A in an actuated state, according to some embodiments;

[0023] FIG. 4A is a schematic representation illustrating the step of cell injection into the growth channel of FIG. 3 A, according to some embodiments;

[0024] FIG. 4B is a schematic representation illustrating the step of flushing the growth channel of FIG. 3A, according to some embodiments;

[0025] FIG. 5 is a clustered growth correlation matrix where the X and Y aces represent the same vector corresponding to a 96 Biolog media, according to some embodiments;

[0026] FIG. 6 shows the growth profiles of 26 NIST strains, categorized 0 to 2 for six cell growth media, according to some embodiments;

[0027] FIG. 7 shows the bacterial growth profiles in the post-incubated mixed sample within a microfluidic device, according to some embodiments;

[0028] FIG. 8 shows the bacterial growth profiles from FIG. 7 with the most similar growth profiles combined, according to some embodiments;

[0029] FIG. 9A shows the NIST strains detected in the blinded sample, according to some embodiments; and

[0030] FIG. 9B shows the growth profiles found in the blinded sample, according to some embodiments.

[0031] DETAILED DESCRIPTION

[0032] Some aspects of the present disclosure are generally related to methods of identifying cells based on a growth profile, e.g., growth rates obtain in respective growth media. Monitoring and determining respective growth rates of unknown cells as a function of growth media may facilitate identifying a cell. In some embodiments, identifying cells in a high throughput manner based on identifying growth rates in a relatively low number of growth media, compared to typical methods, is disclosed. In some embodiments, exposure of cells to different cell growth media is performed serially for at least some of a plurality of cells, which may decrease a time to identification. Additionally, in some embodiments, the growth rates of multiple unknown cells may be monitored in parallel by utilizing a microfluidic device comprising one or more cell growth trenches. Still other aspects of the present disclosure are generally directed to systems for monitoring and determining growth rates of cells, kits containing the systems, or the like. Cell screening plays a fundamental role in biology and makes it possible to identify one or more cells of interest based on a phenotype and / or genotype. Cell screening may be desirable in any of a variety of applications, for example, determining an illness in a human or other organism, in determining food spoilage, and / or determining contamination in the environment. Various cell screening methods have been developed over time. For example, a conventional technique for genotyping cells is sequencing, where at least a portion of the genome of a cell is amplified and sequenced to identify the cell. Phenotypic identification typically includes one or more of any of a variety of tests, including biochemical reactions, serologic tests, growth requirements, gram stain, bacterial morphology, and information about from where the cell was isolated. In either case of genotypic or phenotypic cell screening, there are a variety of challenges, including low screening throughput, slow identification times, limited potential for scalability and automation, limited screening capabilities for complex cell populations, and phenotypic mischaracterizations caused by screening errors.

[0033] One typical cell screening method that utilizes growth rates in various growth media leverages a 96 well plate loaded with the one or more of the unknown cells in each of the 96 wells. In such cases, each of the 96 wells contains a different growth medium, and a growth rate associated with each growth medium may be determined for the unknown cells, which may then be used to determine a cell identity. It will be appreciated that such a method has various drawbacks. For example, only a single cell type (i.e., loaded into each well of the plate) may be identified at a single time using such a method, resulting in low throughput and limiting the potential for screening complex cell populations and / or scaling the method. Further to this point regarding complex cell populations, the unknown cell must first be isolated and then amplified in order to load each well of the 96 well plate for monitoring the cell growth rate therein. Such restrictions limit the throughput of such a method and detract from the utility for determining unknown cells in time sensitive cases.

[0034] Accordingly, some aspects of the present disclosure are generally directed to methods of improved cell screening, e.g., by determining cell growth rates in various cell growth media and / or determining other growth patterns of cells in various cell growth media. It will be understood that while much of the disclosure described herein is in the context of growth rates, in some embodiments, other growth patterns (e.g., septum position, division patterns, etc.) or phenotypic identifiers (e.g., biochemical reactions, serologic tests, gram stain, bacterial morphology) may be similarly considered and / or considered in combination with the cell growth rates. In some embodiments, relatively few cell growth media, and corresponding cell growth rates, may be used to identify a cell species, compared to typical methods utilizing cell growth rates associated with various cell growth media. The identification may be qualitative and / or quantitative. For instance, some embodiments are generally directed to systems and methods for determining a species of a cell based on less than or equal to six cell growth rates associated with a respective six different cell growth media. In some embodiments, more or fewer cell growth media may be used based on the strain-discriminating potential of the cell growth media. Utilizing fewer cell growth media to obtain cell growth rates, as opposed to a typical 96 well plate experiment using 96 cell growth media, may accelerate the experiment. For instance, the time needed to grow and replicate the cells to a sufficient amount to populate the 96 wells of the 96 well plate is greatly reduced to a corresponding amount of cells needed for the reduced number of cell growth media.

[0035] For instance, some embodiments are generally directed to systems and methods for determining a species of a cell based on less than or equal to 50 cell growth rates associated with a respective 50 different cell growth media, less than or equal to 45 cell growth rates associated with a respective 45 different cell growth media, less than or equal to 40 cell growth rates associated with a respective 40 different cell growth media, less than or equal to 35 cell growth rates associated with a respective 35 different cell growth media, less than or equal to 30 cell growth rates associated with a respective 30 different cell growth media, less than or equal to 25 cell growth rates associated with a respective 25 different cell growth media, less than or equal to 20 cell growth rates associated with a respective 20 different cell growth media, less than or equal to 15 cell growth rates associated with a respective 15 different cell growth media, less than or equal to 10 cell growth rates associated with a respective 10 different cell growth media, less than or equal to 5 cell growth rates associated with a respective 5 different cell growth media, less than or equal to 3 cell growth rates associated with a respective 3 different cell growth media, less than or equal to 2 cell growth rates associated with a respective 2 different cell growth media, or a single cell growth rates associated with a respective cell growth medium. In addition, in some cases, at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, etc. growth rates may be determined. Combinations of any of these ranges are also possible in other embodiments, e.g., between 3 and 50 growth media and growth rates, between 10 and 30 growth media and growth rates, between 15 and 40 growth media and growth rates, etc.

[0036] In some embodiments, cell growth rates in different cell growth media may be determined in parallel, e.g., in the same device. In some embodiments, the device is a microfluidic device as described elsewhere herein. For example, a growth rate of a first population of cells in a first growth medium may be determined while a growth rate for a second population of cells in a second growth medium is simultaneously determined. In some instances, a third population of cells in a third cell growth medium, a fourth population of cells in a fourth cell growth medium, and so forth may be analyzed in parallel, e.g., within the same device, or within different devices.

[0037] Any suitable number of cell populations may be analyzed in different media in parallel, in accordance with some embodiments. According to some embodiments, this may reduce the time needed to grow and replicate cells at the analysis outset (e.g., before determining growth rates thereof) because only a first set of cells is needed in each cell population to be exposed to each cell growth media, as opposed to a large number of cells necessary to populate multiple wells in a 96 well plate as in typical methods. In some cases, relatively large numbers of growth rates may be determined in parallel, e.g., at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50, and / or up to 100, up to 200, up to 500, or up to 1,000 growth rates may be determined in parallel in accordance with certain embodiments.

[0038] In some embodiments, cell growth rates may be determined serially rather than in parallel. For example, a first growth rate associated with a first growth medium may be determined for an unknown cell, whereafter a second growth rate in a second growth media for the unknown cell may subsequently be determined by introducing the second growth medium in place of the first growth medium. According to some such embodiments, this may further reduce the time needed to grow and replicate cells at the outset (e.g., before determining growth rates thereof) because only a first set of cells is needed to be serially exposed to each cell growth media, as opposed to the number of cells necessary to populate multiple wells in a 96 well plate as in typical methods. In some cases, relatively large numbers of growth rates may be determined serially, e.g., at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, etc. growth rates may be determined serially in accordance with certain embodiments. In some embodiments, no more than 1,000, no more than 500, or no more than 100 growth rates may be determined serially.

[0039] In some embodiments, growth rates may be determined by exposing the exact same cells, and in some cases, a single cell, to different growth media at different points in time. In contrast, many other techniques require the measurement of the growth of a large number of cells, with no ability to, for example, separate different cells having different growth profiles or rates. In addition, such techniques may be unable to differentiate between cells having slightly different rates of growth, or determine the changes in growth rates as a cell is exposed to different media.

[0040] According to some embodiments, the methods described herein may be performed within a microfluidic device, e.g., having one or more cell growth trenches. As described in more detail elsewhere herein, one or more cell growth trenches may facilitate separating and isolating cells from a sample containing a complex cell population (e.g., containing two or more, five or more, 10 or more, 50 or more, 100 or more, 1,000 or more, or more types of cells etc.). In some embodiments, the cell growth trenches may facilitate flowing different growth media throughout the one or more cell growth trenches, which may allow the separated and / or isolated cells therein to grow within different cell growth media to increase throughput of determining an identity of cells within such a microfluidic device.

[0041] Some aspects are generally related to methods of identifying cells, e.g., qualitatively and / or quantitatively. In some cases, the methods may include providing one or more cells. In some embodiments, the method includes providing a plurality of cells. In some embodiments, at least some of the cells of the plurality of cells are of an unknown species. In accordance with some embodiments, the cells comprise one or more different unknown species of unicellular organisms. In some embodiments, providing the cells may comprise acquiring the cells, e.g., by sampling cells from a source. According to some embodiments, the sample may be obtained from any of a variety of suitable sources, e.g., from which it may be desirable to know the identity of the unknown cells. For instance, in some embodiments, the sample may be obtained from the environment (e.g., a potentially contaminated body of water, soil, etc.), from a food source, and / or a subject suspected of having a disease. The subject may be human or a non-human animal, e.g., a non-human mammal. The unknown cells may be any of a variety of suitable cells suitable for identification by cell growth profiles. Non-limiting examples of cells include bacteria cells, mammalian cells, fungi, algae, protozoa, archaea, etc. In some embodiments, the method comprises providing a substrate, such as a microscope slide and coverslip, a petri dish, or a microfluidic device, as described elsewhere herein. In some embodiments, the substrate comprises a microfluidic device. As a nonlimiting example, as noted above with respect to FIGs. 1B-2C, the microfluidic device may comprise, in one embodiment, a substrate 8, a cell flow layer 10 coupled to the substrate 8, and a control layer 50 configured to control flow of the fluid in the cell flow layer 10. In certain embodiments, the cell flow layer may comprise a growth channel having an inlet portion, an outlet portion, an inlet valve portion associated with the inlet portion, a main portion, and an outlet valve portion associated with the outlet portion. The microfluidic device and associated components (e.g., cell flow layer, control layer, growth channels, control channels, cell growth trenches, etc.) may have any configurations and properties described previously. Non-limiting examples include those described with reference to FIGs. 1-3 elsewhere herein.

[0042] In some embodiments, the method comprises adding or injecting a fluid comprising a plurality of cells into the microfluidic device. In some embodiments, the cells and the growth media may be flowed into a main portion of the growth channel. For example, as shown in FIG. 4A, cells 74 and growth media may be injected into the growth channel 12 through the inlet portion 14A. In some cases, the cells and growth media can be injected via one or more of the inlet openings. For example, as shown in FIG. 4A, cells 74 and growth media can be injected via one or more inlet openings 15A and 15B and flow into the main portion 18 of the growth channel 12. Any of a variety of cells may be injected into the microfluidic device. Non-limiting examples of cells include bacteria cells, mammalian cells, fungi, algae, protozoa, archaea, etc. In some embodiments, the cells may be from the sample that was provided. In some embodiments, the cells that are introduced into the microfluidic device may include one or more cell having an unknown species.

[0043] In some embodiments, during the injection step, the inlet and / or outlet valve portions of the growth channels are in an open state (e.g., an unactuated state). As noted above, in some embodiments, the control channels in the control layer are unpressurized. The control channels, when unpressurized, may allow fluid to flow through the valve portions of the growth channels. For example, as shown in FIG. 4A, when the control channel 52A is unpressurized, the inlet valve portions 16A and 16B are in an open state. The cells and growth media may thus able to flow through the inlet valve portion 16A into the main portion In some embodiments, the cells injected into the main portion of growth channel may fill at least one of the plurality of cell growth trenches fluidically coupled to the growth channel. As shown in FIG. 4A, for example, the cells 74 injected into the main portion 18 may fill into at least one of the plurality of growth trenches 20. Any of a variety of methods may be employed to populate the cells from the main portion of the growth channel into the growth trenches. In some cases, for example, the cells may populate the cell growth trenches via diffusion. Additionally or alternatively, centrifugation, backports, and / or open-ended trenches may be employed to load the cell growth trenches.

[0044] In some embodiments, the method comprises providing cells contained with a plurality of single-entry, single-file cell growth trenches fluidically coupled to a growth channel. For example, after the injecting step, the plurality of single-entry and single-file cell growth trenches in the growth channel may be configured to contain the injected cells arranged in a linear, one- dimensional grouping (e.g., the cells are geometrically constrained to a single-file line). See, for example, growth trenches in FIG. 4B. In some cases, as cells that initially fill the cell growth trenches begin to divide, the cells grow into an isogenic lineage of cells within the plurality of cell growth trenches. These cells within a given trench may have the same genetic makeup, e.g., as they may have originated from the same original cell. In some embodiments, the cells may be arranged in a single one-dimensional line within a trench. For example, as shown in FIG. 4B, as the cells 74 in each of the cell growth trenches 20 begin to divide, each of the cell growth trenches 20 may eventually contain an isogenic lineage of cells. In some embodiments, if there is more than one cell type present within a cell growth trench (e.g., a plurality of cells having different genetic lineages), then the cells may be allowed to expand and divide. The cell closest to the closed end of the channel may divide to produce additional cells, which “push” the other cells out of the channel (e.g., the ones having different genetic lineages) such that eventually, only an isogenic lineage of cells is present within the cell growth trench, originating from the cell closest to the closed end of the channel. In such a manner, according to some embodiments, each of a plurality of cell growth trenches initially containing cells may, after cell growth (e.g., replication, growth and division), contain and / or be filled by a single type of cell, e.g., after a cell initially closest to the closed end of the cell growth trench replicates and “pushes” any other cells out of the trench that were initial present. In this manner, according to some cases, cells injected into the microfluidic device may be separated and isolated (e.g., a cell type may be isolated after “pushing” out other cell types) within the cell growth trenches of the microfluidic devices.

[0045] In some embodiments, the method further comprises flushing the microfluidics device with a fluid, e.g., to remove the majority of cells from the growth channel while retaining one or more cells within at least one of the plurality of cell growth trenches. Referring again to FIG. 4B, as a non-limiting example, after the cells 74 populates the one or more of the cell growth trenches 20, a fluid may be injected through the main portion 18 of the growth channel 12 to flush out the majority of cells from the growth channels 12 while retaining the one or more cells 74 in the cell growth trenches 20.

[0046] Any appropriate fluids may be employed to flush one or more of the growth channels within the device, in various embodiments. Non-limiting examples of suitable fluids include cell media, buffer solutions, etc.

[0047] In some embodiments, the method comprises exposing a plurality of cells to a first growth medium. In some embodiments, the method comprises exposing a plurality of cells within a plurality of cell growth trenches to a first growth medium. Following the exposing to the first cell growth medium, the cells may grow. In accordance with some embodiments, the method may comprise determining a first growth rate of the plurality of cells exposed to the first growth medium. In some embodiments, the method may include exposing the plurality of cells to a second cell growth medium. In some such embodiments, the cells may be exposed to the second cell growth medium after exposure to the first cell growth medium and / or after determining a first growth rate of the plurality of cells. Such a process may be repeated for any of a number of cell growth media and / or respective growth rates, for example, a third growth medium and growth rate, a fourth growth medium and growth rate, a fifth growth medium and growth rate, a sixth growth medium and growth rate, and so forth. In various embodiments, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more growth media that are used, or other numbers such as any of those described herein.

[0048] The number of cell growth media may be selected based on the number of corresponding cell growth rates needed to distinguish different cell types, in accordance with some embodiments. As a non-limiting example, in some embodiments, at least 5 cell growth media and a corresponding 5 cell growth rates may be used to identify cells. In some embodiments, an early identification of cell identity may be made with a lower level of confidence to speed analysis. For instance, returning to the previous example where at least 5 cell growth media and a corresponding 5 cell growth rates are used to identify cells, in some such embodiments, an early identification of cell identity may be made before determining all 5 cell growth rates, for example, after obtaining 3 cell growth rates. This early identification may not be as specific or as confident as an identification made using the at least 5 cell growth rates, in some embodiments, but may speed analysis in certain cases. Moreover, following the initially early identification, the continual determination of further growth rates may be obtained to provide more confidence in the cell identification, in some embodiments.

[0049] In accordance with some embodiments, it may be advantageous to expose the cells within the cell growth trenches to another solution between consecutive applications of cell growth media, e.g., a recovery solution, so that the first and second cell media do not interact (e.g., or other sequential media such as third and fourth, fourth and fifth, and so forth do not interact). Alternatively, in some embodiments, cell growth media may be changed gradually such that there is a gradient in the cell growth media between a first and second cell growth media, between the second and third cell growth media, and so forth. In some embodiments, the gradient between cell growth media may provide a refined readout of cellular responses (e.g., growth rates and / or other physiological properties as a function of the gradient of cell growth media). In some embodiments, the cell growth media may be varied temporally. For instance, a first cell growth medium may be gradually introduced (e.g., from 0% cell growth medium and 100% buffer to 100% cell growth medium and 100% water over a certain amount of time such as at least 10 s, at least 1 min, or at least 10 min and / or up to 30 min or up to 1 hour). In some embodiments, the cell growth media may be varied spatially, for instance, across a width of a microfluidic device. The collection of cell growth rates in respective cell growth media associated with a cell type, in some embodiments, is a cell growth profile. In some embodiments, a cell growth profile includes at least two growth rates, at least three growth rates, at least four growth rates, at least five growth rates, at least six growth rates, etc., associated with respective cell growth media.

[0050] Any of a variety of cell growth media are suitable for exposing the cells and / or determining respective growth rates of the cells, in some embodiments. Non-limiting examples of cell media include cell media containing D-Galactose; P-Hydroxy-D,L-Butyric Acid; NaCl (e.g., greater than or equal to 2% by weight, greater than or equal to 2% by weight greater than or equal to 6% by weight greater than or equal to 8% by weight, etc., of the cell media), Niaproof 4, Potassium Tellurite, Sodium Lactate (e.g., in an amount of greater than or equal to 0.5% by weight, greater than or equal to 1% by weight, etc., of the cell media), and / or glucose. Other possible cell media are also possible. Other non-limiting examples of possible cell media may be found within a Biolog Gen III Microplate ® system. In some embodiments, the cell growth media may include chemicals capable of discriminating cell types, e.g., based on a growth profile of different cells present in the growth media. For instance, in some embodiments, cell growth media may contain one or more unique carbon sources, non-limiting examples of which include Glucose, D-Mannose, Sucrose, and Glycerol. Other carbon sources are also possible. In some embodiments, the cell growth media may include various other suitable components, including buffers (e.g., phosphate buffer), salts, detergents, gelling agents, and redox dyes. Other components of the cell growth media are possible. For instance, other cell growth media components may include pharmaceutical agents, antibiotics, osmotic agents, fluorescent dyes, specificity markers, or the like. Additionally, in some (but not all) embodiments where a microfluidic device is present, a gelling agent may be absent from the cell growth media.

[0051] Determining a growth rate of the cells may be done using any of a variety of suitable techniques. In some embodiments, a cell growth may be determined by time-lapse microscopy. The growth rates, in some embodiments, may be simplified by categorizing the growth of the associated cells in bins, e.g., as 0 - not growing, 1 - slight growth, 2 - important growth. According to some embodiments, the cell growth rate may be determined qualitatively or quantitatively. For instance, in some embodiments, the growth rate of cells may be determined by measuring a mass of cell growth exiting a microfluidic device in comparison the mass of cell growth entering the microfluidic channel. In some embodiments, images of cell growth may be obtained by other microscopic methods. A non-limiting example of microscopy that may be used to monitor the growth of cells is phase-contrast microscopy. In some embodiments, quantitative phase microscopy may be used to determine a cell growth rate. Other methods are also possible. In some embodiments, the images may be analyzed manually. In some embodiments, the images may be analyzed by computer assisted software. In some embodiments, a software program may be utilized to analyze images of cells over time and to quantify cell growth rates thereof. Various image analysis software programs may be obtained commercially.

[0052] It will be appreciated that, in some cases, certain cells may not grow substantially and / or at all within certain cell growth media. In some embodiments, some cells may be killed when exposed to certain cell growth media. In some such embodiments, it may be advantageous to flow a cell growth media in which certain cell types are susceptible to being killed later in a series of serially using different cell growth media. In such a manner, cell growth rates for cell growth media flowed before the cell growth media that kills the cells may be obtained, in accordance with some embodiments. This allows, in some cases, to obtain more information about a cell before a potential cell death. In some instances, cells may be serially exposed to a first growth medium and a second growth medium to determine respective growth rates. In some embodiments, cells may be serially exposed to a second growth medium and a first growth medium to determine respective growth rates. Other orders of exposing the growth media are possible, e.g., when more than two growth media are used.

[0053] In some embodiments, an alternative and / or additional method for addressing potential cell death is to take an initial sample containing one or more unknown cell types and dividing it, e.g., into two samples, three samples, four samples, or so forth. Assuming the initial sample has a relatively large amount of cells (e.g., greater than or equal to 105cells, greater than or equal to 106cells, greater than or equal to 107cells, greater than or equal to 108cells, greater than or equal to 109cells, etc.), dividing the sample into smaller subsets may still be a representative sample due to the large number of cells in each subset. Two or more of the samples may then be used to grow cells using a different cell growth media order.

[0054] As a non-limiting example, a sample containing a plurality of cells containing unknown species may be divided in three sets of cells, where a first set of cells may be introduced into a first microfluidic device having a plurality of cell growth trenches, a second set of cells may be introduced into a second microfluidic device having a plurality of cell growth trenches, and a third set of cells may be introduced into a third microfluidic device having a plurality of cell growth trenches. The cells in the first set may then serially be exposed to a first, second, and third cell growth media, in that order, to obtain respective growth rates of the cells. The cells in the second set may then serially be exposed to a second, third, and first cell growth media, in that order, to obtain respective growth rates of the cells. And the cells in the third set may then serially be exposed to a third, first, and second cell growth media, in that order, to obtain respective growth rates of the cells. In such a manner, in some embodiments, if any of the cells are susceptible to being killed by any of the three cell growth media, the other orders of exposing the cells to the cell growth media may determine respective growth rates.

[0055] In some embodiments, a method may further include identifying a cell based, at least in part, on the growth rates determined in various cell growth media (e.g., the cell growth profile). According to some embodiments, the growth rates of the cells may be indicative and / or may be correlated to a cell type. For example, cells having a known type may be exposed to various cell media to determine respective growth rates, as described above. In some such embodiments, the cell growth profile of the known cell may be obtained. Such a process may be repeated with other cell types to acquire corresponding cell growth profiles, in some embodiments. In this manner, a library of cell growth libraries that correspond to various types of known cells may be acquired. Accordingly, in some embodiments, the method may further include establishing a library of growth profiles of cells in a variety of cell growth media, e.g., as described above. The cell growth media used to acquire the cell growth profiles of the known cells to establish the library may be the same cell growth media in which the unknown cells may be grown. In some embodiments, establishing the library may occur before any steps with unknown cells. Identifying a cell based, at least in part, on the growth rates determined in various cell growth media may comprise using the library of growth profiles of cells in a variety of cell growth media, in accordance with some embodiments.

[0056] In some embodiments, determining one or more cell growth rates and / or identifying a cell species, at least in part, using the one or more cell growth rates may occur in a relatively fast manner. In some embodiments, the cell growth rates and / or identity of the cell species may be determined in, e.g., less than or equal to 48 hours, less than or equal to 24 hours, less than or equal to 12 hours, or less than or equal to 6 hours. Other timeframes are also possible in other embodiments. In some embodiments, a species of at least some of a plurality of cells may be determined in such a time period, e.g., by determining the cell growth rates of some of the plurality of cells in parallel using a microfluidic device. For example, a first growth rate of at least 2 (and / or at least 3, at least 5, at least 10, at least 100, at least 1,000, etc.) types of cells of the plurality of cells may be determined in parallel following exposure to a first growth media. In some embodiments, a second growth rate, third growth rate, fourth growth rate, and so forth may be determined (e.g., serially) of the at least 2 types of cells in parallel. The number of cells analyzed from a sample (e.g., a complex sample containing a plurality of bacterial cell species) may be dictated by the number of cell growth trenches present in a microfluidic device, which may be designed based on a size of the microfluidic device and the desired size of the cell growth channels.

[0057] According to some embodiments, as described above, the growth rates (e.g., the growth profiles) of the cells of an unknown species may be used to identify the species of the cell. For instance, in some embodiments, the method includes identifying the cells of the sample using the growth profile of the cells. In some embodiments, the method further comprises identifying one or more cells of interest in one or more cell growth channels based on a sensed property. In some such embodiments, the sensed property may be used in conjunction with the growth profile of the one or more cells. It should be noted the sensed property may include any property that can be directly or indirectly observed through various microscopy techniques, in accordance with various embodiments. For example, the sensed property may include, but are not limited to, a fluorescence property (e.g., a hyperspectral profile), a cell phenotype, a cell genotype, extracellular secretions, a marker or taggant associated with the cell (e.g., protein, antibody, etc.), etc. Specific examples of sensed properties include, but are not limited to, a cell morphology or physiology, gene expression, cellular localization patterns, enzymatic activity, DNA replication and modification, chromosome segregation patterns, metabolic state, cell envelopes, spatial distributions of organelles, intracellular structures, cell-cell interactions, cellular secretions, cell density, membrane permeability, membrane fluidity, membrane potential, ATP levels, physical changes, or any combinations thereof. The one or more other sensed properties, in some embodiments, may vary as a function of exposure to one or more cell growth media as described elsewhere herein. In some embodiments, the sensed property may be determined using methods described elsewhere herein and / or fluorescent reporters, which may be selected based on the desired sensed property.

[0058] Some aspects of the present disclosure are generally related to systems configured to monitor cell growth rates. In some embodiments, the system may include a light source, a substrate configured to contain one or more cells, and a detector. FIG. 1A shows a nonlimiting example of one such system configured for monitoring cell growth rates, including a light source 102, a substrate configured to contain cells 108, and a detector 110.

[0059] In some embodiments, a light source may be positioned and oriented to direct light toward a substrate. In some cases, a particularly beneficial type of light source having a certain intensity and / or wavelength of light may be employed in the system. In one set of embodiments, the light source may have a visible wavelength spectrum. In one set of embodiments, the light source (e.g., UV laser) may have a wavelength in the ultraviolet regime. Other ranges of light may also be used in other embodiments. Non-limiting examples of light source that may be employed include, but are not limited to, a laser, a lightemitting diode, an arc lamp, etc. In some cases, more than one light source may be used, and these may be the same or different, e.g., providing the same or different wavelengths of light. As described elsewhere herein in more detail, it may be desirable to illuminate only a portion of a sample (e.g., a portion of a microfluidic device). In some instances, this may be achieved using a mask in combination with the light source (e.g., a widefield light source). In other embodiments, the light source may be a collimated light source (e.g., a laser). In some embodiments, the light source comprises a structured light source such that light from the light source is directed to only a portion of a sample (e.g., a portion of a microfluidic device). In some embodiments, a structured light source illuminates only a portion of the microfluidic device, e.g., one or more cell growth trenches as described elsewhere herein in more detail. In some embodiments, the structured light source may provide light that is incident in any suitable shape such as a spot, a line, or the like. The shape of the structured light source, in some embodiments, may be selected based on a portion of a sample that is to be imaged.

[0060] It will be understood that in instances where the light source comprises a structured light source, the structured light source may be scanned to illuminate (e.g., an accordingly image) different portions of the microfluidic device. For instance, in some embodiments, the structured light source is configured with one or more components such that it is capable of separately illuminating and facilitating imaging a first portion and then a second portion of a sample (e.g., a microfluidic device). In some such embodiments, non-limiting components that may facilitate scanning different portions of a sample include a galvanometer scanner and acousto-optical devices. Other such components are possible, and may be selected based on the insight provided by this disclosure.

[0061] In some embodiments, the light source (e.g., a laser) is configured to produce light suitable to illuminate one or more cells for imaging. In some embodiments, the system may include one or multiple light sources. In some embodiments, a light source may be configured to produce light having an intensity of greater than or equal to 0.1 W / cm2, greater than or equal 0.5 W / cm2, greater than or equal 1 W / cm2, greater than or equal to 5 W / cm2, greater than or equal 10 W / cm2, greater than or equal to 50 W / cm2, greater than or equal 100 W / cm2, greater than or equal 500 W / cm2, greater than or equal to 1,000 W / cm2, greater than or equal 5,000 W / cm2, greater than or equal 10,000 W / cm2, or greater than or equal 50,000 W / cm2. In some embodiments, the light source may be configured to produce light having an intensity of less than or equal to 100,000 W / cm2, less than or equal 50,000 W / cm2, less than or equal 10,000 W / cm2, less than or equal 5,000 W / cm2, less than or equal 1,000 W / cm2, less than or equal to 500 W / cm2, less than or equal to 100 W / cm2, less than or equal to 50 W / cm2, less than or equal to 10 W / cm2, less than or equal to 5 W / cm2, less than or equal to 1 W / cm2, or less than or equal 0.5 W / cm2. Any of the above reference ranges are possible (e.g., greater than or equal to 0.1 W / cm2and less than or equal to 100,000 W / cm2, or greater than or equal to 100 W / cm2and less than or equal to 1,000 W / cm2). Other ranges are also possible.

[0062] In some embodiments, the light source (e.g., a laser) is configured to a produce light having any of a variety of appropriate wavelengths. In some cases, the light source is able to produce continuous spectral bands. In some embodiments, the light source (e.g., a laser) may have a wavelength of greater than or equal to 100 nm, greater than or equal to 125 nm, greater than or equal to 150 nm, greater than or equal to 180 nm, greater than or equal to 200 nm, greater than or equal to 225 nm, greater than or equal to 250 nm, greater than or equal to 275 nm, greater than or equal to 300 nm, greater than or equal to 325 nm, greater than or equal to 350 nm, greater than or equal to 375 nm, greater than or equal to 400 nm, greater than or equal to 420 nm, greater than or equal to 440 nm, greater than or equal to 450 nm, greater than or equal to 480 nm, greater than or equal to 500 nm, greater than or equal to 550 nm, greater than or equal to 600 nm, greater than or equal to 650 nm, greater than or equal to 700 nm, greater than or equal to 800 nm, or greater than or equal to 900 nm. In some embodiment, the light source (e.g., a laser) may have a wavelength of less than or equal to 1000 nm, less than or equal to 900 nm, less than or equal to 800 nm, less than or equal to 700 nm, less than or equal to 650 nm, less than or equal to 600 nm, less than or equal to 550 nm, less than or equal to 500 nm, less than or equal to 480 nm, less than or equal to 450 nm, less than or equal to 440 nm, less than or equal to 420 nm, less than or equal to 400 nm, less than or equal to 375 nm, less than or equal to 350 nm, less than or equal to 325 nm, less than or equal to 300 nm, less than or equal to 275 nm, less than or equal to 250 nm, less than or equal to 225 nm, less than or equal to 200 nm, or less than or equal to 180 nm, less than or equal to 150 nm, or less than or equal to 125 nm. The above-referenced values of wavelengths may have a deviation of + / - 5 nm, of + / - 10 nm, or + / - 15 nm. Any of the above-referenced ranges may be possible (e.g., greater than or equal to 100 nm + / - 5 nm and less than or equal to 1,000 nm + / - 5 nm, greater than or equal to 200 nm + / - 5 nm and less than or equal to 480 nm + / - 5 nm, or greater than or equal to 250 nm + / - 5 nm and less than or equal to 400 nm + / - 5 nm). Other ranges are also possible. In some embodiments, it may be desirable to use a light source that emits light corresponding to visible light wavelengths (e.g., greater than or equal to 400 nm and less than or equal to 800 nm).

[0063] Any of a variety of suitable substrates are possible in the systems described herein, in some embodiments. In some embodiments, the substrate may be transparent to wavelengths of light that may be used to excite cells and / or emitted from cells (e.g., as autofluorescence). For instance, in some cases, the substrate may comprise glass, optically transparent polymers (e.g., PDMS), or the like. Non-limiting examples of suitable substrates include a microscope slide and coverslip, a petri dish, and / or a microfluidic device. In accordance with some embodiments, and as described elsewhere herein, the substrate configured to contain cells may be a microfluidic device including one or more cell growth trenches. Other substrates are also possible. In some embodiments, the substrate configured to contain cells includes the cells. In some such embodiments, at least some of the cells are an unknown type of cell.

[0064] A detector, in some embodiments, is included in the system to image the cells. Any of a variety of suitable detectors are possible, including charge coupled devices (CCDs), complementary metal-oxide semiconductor (CMOS) sensors, photocathodes, photomultiplier tubes, or the like. One or more detectors may be integrated within a camera for imaging, according to some embodiments. A variety of different imaging techniques may be used in various embodiments, for example, quantitative phase microscopy, hyperspectral imaging, etc., e.g., as discussed in patent applications filed on May 13, 2024, entitled “Microscopy Systems and Methods for Identifying Bacteria and Other Organisms in Microfluidic Devices” (U.S. Pat. Apl. Ser. No. 63 / 646,599) and “Systems and Methods for Identifying Cells Using Hyperspectral Imaging and Other Techniques” (U.S. Pat. Apl. Ser. No. 63 / 646,534). Each of these is incorporated herein by reference in its entirety.

[0065] According to some embodiments, as mentioned above, the systems described herein may include a microfluidic device. In some embodiments, the microfluidic device may be configured to contain cells. In some embodiments, a system configured for individual cell screening is disclosed herein. In one set of embodiments, the system comprises a microfluidic device configured to contain a plurality of cells and a source of light (e.g., a laser) configured to induce autofluorescence in at least a portion of the plurality of cells (e.g., excite at least some of the molecules within at least a portion of the plurality of cells).

[0066] A non-limiting example of the systems described herein is shown in FIG. IB. As shown, system 1 comprises the microfluidic device 4, light source 2 (e.g., a laser) positioned to direct light at at least a portion of the microfluidic device, and mask 3 (e.g., an electronically reconfigurable mask) positioned to selectively shield a least a portion of the light directed at the portion of the microfluidic device 4. The mask, in some cases, may be configured to shield a portion 7 of the light originating from the light source 2, such that the portion of the microfluidic device underneath the mask is prevented from being exposed to light. As a result, cells residing within the portion of the microfluidic device may be shielded from light.

[0067] In some embodiments, the microfluidic device comprises two or more layers comprising microfluidic channels. For example, the microfluidic device may comprise a cell flow layer configured for flowing, receiving, and housing a plurality of cells. In some cases, the microfluidic device may further comprise a control layer positioned adjacent (e.g., coupled to) the cell flow layer. The control layer may be configured to control the flow of a fluid (e.g., cell media, a fluid comprising the plurality of cells, etc.) in the cell flow layer. In some embodiments, the microfluidic device further comprises a substrate (e.g., a coverslip) coupled to the cell flow layer, thereby forming a base layer of the microfluidic device. As shown in the non-limiting example of the microfluidic device in FIG. IB, the microfluidic device 4 may comprise a cell flow layer 10 and a control layer 50 positioned adjacent the cell flow layer 10. A substrate 8 may be coupled to the cell flow layer 10, forming the base layer of the microfluidic device 4.

[0068] While FIG. IB shows one embodiment in which the microfluidic device comprises three layers, it should be noted that not all embodiments described herein are so limiting, and in other embodiments, the microfluidic device may comprise any additional layers disposed adjacent (e.g., directly adjacent) and / or between the one of more layers described above. In other embodiments, more or fewer layers may be present.

[0069] As noted above, the microfluidic device may include a substrate (e.g., coverslip), a cell flow layer coupled to the substrate, and a control layer coupled to the cell flow layer, in one embodiment. In some embodiments, the cell flow layer may include a variety of different channels through which cells (and other fluids such as growth media and cleaning fluids or solutions) can flow during use. The control layer may include various channels that can be filled with a fluid in order to actuate various different portions (e.g., valve portions) of the cell flow layer. In some embodiments, the cell flow layer may comprise various portions (e.g., valve portions) that can be actuated by the control layer to selectively control the flow of cells and other fluids through the various channels of the cell flow layer. It should be understood, however, that flow layers, control layers, etc. in this example are but one method of confining or growing cells on a microfluidic device, e.g., for screening or other purposes, but that in other embodiments, other systems for confining or growing cells in a microfluidic device are also contemplated. In some embodiments, the microfluidic device may comprise various channels having any of a variety of configurations and arrangements described herein. A non-limiting example of one embodiment is shown in FIGs. 2A-2C. As shown, FIGs. 2A-2C respectively illustrate a top down view, perspective view, and a partially exploded perspective view of the microfluidic device 4 of FIG. IB.

[0070] In one set of embodiments, the microfluidic device includes a cell flow layer comprising one or more growth channels configured to receive, flow, and / or house a plurality of cells. As shown in FIGs. 2A-2C, the microfluid device 4 may comprise a cell flow layer 10 comprising one or more growth channels 12. In some embodiments, the control flow layer comprises one or more control channels configured to control flow of a fluid in the cell flow layer. For example, as shown in FIGs. 2A-2C, the control flow layer 50 may comprise one or more control channels 52A configured to control flow of a fluid in the one of more growth channels 12 within the cell flow layer 10.

[0071] While FIGs. 2A-2C show a set of embodiments in which the microfluidic device comprises growth channels and control channels, it should be noted that not all embodiments described herein are so limiting, and in other embodiments, the microfluidic device may comprise various other types of channels in the cell flow layer and / or control layer. For example, as described in more detail below, the cell flow layer may further comprise one or more collection channels in additional to the growth channels.

[0072] In some embodiments, the channels (e.g., growth channels) in the cell flow layer are separated from the channels (e.g., control channels) in the control layer by an upper wall of the cell flow layer. See also Int. Pat. Apl. Pub. No. WO 2020 / 257746. As shown in FIG. 2C, the various channels (e.g., growth channels 12) of the cell flow layer 10 may be defined on an underside of the cell flow layer 10. For instance, the cell flow layer 10 may include an upper wall 10A that forms the upper wall (e.g., the ceiling) of the various channels defined in the cell flow layer 10. When the cell flow layer 10 is bonded to the substrate 8, the substrate 8 forms a lower wall (e.g., a floor) of the various channels of the cell flow layer 10. Similarly, the various channels (e.g., control channels) of the control layer 50 may be defined on an underside of the control layer 50. An upper wall 50A of the control layer 50 may form an upper wall (e.g., a ceiling) of the various channels (e.g., control channels) of the control layer 50, and the upper wall 10A of the cell flow layer 10 may form a lower wall (e.g., a floor) of the various channels of the control layer 50. In some embodiments, the channels of the cell flow layer and the control layer may be fluidically coupled to the atmosphere via a plurality of vertical channels or ports. For example, as shown in FIGs. 2A-2C, the various channels (e.g., growth channel 12) of the cell flow layer 10 may be fluidically coupled to the atmosphere via a plurality of vertical channels extending upward through the cell flow layer 10 and the control layer 50 via one or more openings 15 and 17 defined in the upper wall 50A of the control layer 50. Similarly, the various channels (e.g., control channels 52A) of the control layer 50 may be fluidically coupled to the atmosphere via a plurality of vertical channels extending upward through the control layer 50 via one or more openings 19 defined in the upper wall 50A of the control layer 50. In some embodiments, the one or more openings may serve as inlets and / or outlet that allow for fluid flow into or out of the various channels in the control layer and cell flow layer.

[0073] In some embodiments, the cell flow layer comprises a growth channel comprising various portions, including an inlet portion, an outlet portion, and a main portion positioned between the inlet portion and the outlet portion. The growth channel may further comprise an inlet valve portion associated with the inlet portion and an outlet valve portion associated with the outlet portion. The various portions of the growth channel may be located at various positions along the length of the cell flow layer. For example, the inlet portion and the inlet valve portion may be located at a first end of the cell flow layer, while the outlet valve portion and the outlet portion may be located at a second end of the cell flow layer. The main portion may be located between the first end and the second end of the cell flow layer. Generally, each portion of the growth channel may function as a channel through which cells and fluids can flow.

[0074] A non-limiting example of one embodiment of a growth channel is illustrated in FIG. 3A. Specifically, FIG. 3A shows a top-down view of a section (e.g., section 4A) of the same microfluidic device illustrated in FIG. 2A. As shown, the growth channel 10 comprises various portions, including an inlet portion 14A at a first end of the cell flow layer, an outlet portion 14B at a second end of the cell flow layer, and a main portion 18 positioned between the inlet portion 14A and the outlet portion 14B. The growth channel 10 may further comprise an inlet valve portion 16A associated with the inlet portion 14A and an outlet valve portion 16B associated with the outlet portion 14B.

[0075] In some embodiments, the cell flow layer further comprises a plurality of cell growth trenches fluidically coupled to a main portion of the growth channel. In some embodiments, the plurality of cell growth trenches is configured to contain cells during use of the device. As shown in FIG. 3A, the cell flow layer may comprise a plurality of cell growth trenches 20 fluidically coupled to the main portion 18 of the growth channel 10. In some cases, the cell growth trenches may be positioned on a first side of the main portion of the growth channel, and may be configured to extend outward from the main portion of the growth channel in a direction that is perpendicular to the direction in which the main portion extends between a first end of and a second end of the cell flow layer. For example, as shown in FIG. 3A, the plurality of cells growth trenches 20 may be positioned on a first side of the main portion 18 of the growth channel and may extend outward from the main portion 18 in a direction perpendicular to a direction in which the main portion 18 extends. However, it should be understood that other arrangements of cell growth trenches are also possible in other embodiments. For example, the cell growth trenches may appear on both sides of growth channel, there may be more than one such growth channel, the cell growth trenches may independently be of the same or different sizes or lengths, the cell growth trenches may be symmetrically or asymmetrically arranged within the cell flow layer, or the like.

[0076] In some embodiments, the microfluidic device comprises a plurality of single-entry, single-file cell growth trenches fluidically coupled to the main portion of the growth channel. That is, some or all of the plurality of cell growth trenches may be sized so that individual cells are permitted to enter into the cell trenches one at a time in a linear, single-file fashion. In some cases, the cell growth trenches (e.g., growth trenches 20 in FIG. 3A) may have a width (e.g., width w in FIG. 3 A) that is generally equal to or slightly larger than the width of individual cells entering into the growth trenches. For example, as shown in FIG. 4B, the single-entry, single-file cell growth trenches 20 may be sized such that individual cells are configured to fill the trenches in a linear, single-file fashion. In addition, in other embodiments, some or all of the plurality of cell growth trenches may be sized to allow more than one individual cell to enter at a time.

[0077] In some embodiments, the microfluidic device may facilitate flowing different growth media throughout the one or more cell growth trenches. This may be desirable to allow the separated and / or isolated cells therein to grow within different cell growth media, which may be done serially with multiple cell growth media as disclosed elsewhere herein. Such an arrangement, in some embodiments, may increase throughput of determining an identity of cells within such a microfluidic device using the methods described herein. For instance, cell growth rates in serially introduce cell growth media may be determined for multiple unknown cells that are separated and isolated within the cell growth trenches of the microfluidic device.

[0078] In some cases, as noted above, the system may include a mask, which may be positioned to selectively shield light directed at at least a portion of the cell growth trenches in the cell flow layer of a microfluidic device. For example, light may be directed at the cells that may be sufficient to inhibit or kill the cells. However, the light may not necessarily reach all of the cells in the device due to the presence of the mask, which may be able to selectively shield the incoming light. By positioning or configuring the mask appropriately, in some embodiments, certain cells or portions of the device may be subjected to light, while other cells or portions of the device may be shielded from the light, e.g., such that those cells survive. Alternatively or additionally, in some embodiments, a structured light source may be used such that light is directed at only a portion of the microfluidic device (e.g., a portion of the cell growth trenches) in the cell flow layer of a microfluidic device. Accordingly, in some embodiments, a structured light source may be structured such that certain cells or portions of the device may be subjected to light, while other cells or portions of the device may be shielded from the light, e.g., such that those cells survive. In some embodiments, this may be advantageous as the light may inadvertently kill cells exposed thereto, but the cell growth within the cell growth trenches may facilitate the “pushing” out of dead cells, as described elsewhere herein. It should additionally be understood that, in some embodiments, the light incident on the cells may not kill the cells. Accordingly, in some such embodiments, the cell screening method may be non-destructive, and cells may then be separated and / or collected following identification.

[0079] As mentioned above, the system may include a mask positioned to shield at least a portion of the substrate from the light source. In some embodiments, the mask may be configured to allow light to be incident on only a portion of the substrate. For example, in some embodiments, a mask may be configured to allow light to be incident on a microfluidic device in a line that is perpendicular to a direction of one or more cell growth trenches of the microfluidic device, e.g., as shown as dotted line 51 in FIG. 3B. In this manner, a portion of cells from each of the cell growth trenches containing cells may be excited by the light source, such that they may autofluoresce and hyperspectral profiles of cells from each populated cell growth trench may be collected and analyzed in parallel by the detector and / or processor, in accordance with some embodiments. Such an arrangement, in some embodiments, may desirably facilitate high throughput screening of cells within a microfluidic device, e.g., in combination with determining cell growth rates in respective cell growth media.

[0080] As noted above, the growth channel (e.g., 10 in FIG. 3A) may include, in certain embodiments, various valve portions, e.g., such as an inlet valve portion (e.g., 16A in FIG. 3A) and an outlet valve portion (e.g., 16B in FIG. 3A). In some embodiments, these valve portions may be actuated to aid in selectively controlling the flow of cells and fluid through the growth channel in the cell flow layer. For instance, the inlet valve portion of the growth channel may allow for control of fluid flow between the inlet portion of the growth channel and the main portion of the growth channel. Similarly, the outlet valve portion of the growth channel may allow for control of fluid flow between the main portion of the growth channel and the outlet portion of the growth channel. For example, as shown in FIG. 3A, the inlet valve portion 16A of the growth channel 10 may allow for control of a flow 22A between the inlet portion 14A of the growth channel 10 and the main portion 18 of the growth channel 10. Similarly, the outlet valve portion 16B of the growth channel 10 may allow for control of a flow 22B between the main portion 18 of the growth channel 10 and the outlet portion 14B of the growth channel 10. See also Int. Pat. Apl. Pub. No. WO 2020 / 257746. However, it should also be noted that in other embodiments, there may be only a single valve portion present (e.g., an inlet valve portion or an outlet valve portion), and / or there may be no valve portions present.

[0081] In some embodiments, in the microfluidic device, the control layer may include one or more control channels configured to actuate the valve portions (e.g., inlet and / or outlet valve portions) within the growth channel in the cell flow layer. A variety of microfluidic valve configurations may be used in various embodiments.

[0082] A non-limiting example of one such embodiment is illustrated in FIG. 3A. As shown, the control layer (e.g., control layer 50 in FIG. 2B) may include one or more control channels 52A coupled to (e.g., overlaps with) the inlet valve portion 16A and outlet valve portion 16B of the growth channels 10 in the cell flow layer (e.g., cell flow layer 10 in FIG.2). The control channels 52A may be configured to aid in actuating the inlet valve portion 16A of the growth channel 10 and the outlet valve portion 16B of the growth channel 10.

[0083] The control channels may have any of a variety of appropriate configurations in the control layer. For example, in one set of embodiments, the control channel may have a U shape, as shown in FIGs. 2A-2C. As shown, the control layer may include one or more control channels 52A fluidically connected by a base channel 52B, thereby forming an overall U-shaped structure. In this embodiment, the control channels 52A may extend across (e.g., overlap with) the inlet valve portion 16A and outlet valve portion 16B of the growth channels 10. Specifically, the control channel 52A may overlap with portions of the upper wall 10A of the cell flow layer 10 that form the upper wall of the inlet valve portions 16A and outlet valve portion 16B.

[0084] While FIGs. 2A-2C illustrate an embodiment in which the control channels have an overall U shape structure, it should be noted that the control channels can have other shapes and / or configurations. In some embodiments, the control channels may extend across (e.g., overlaps with) all of the necessary valve portions of the cell flow layer. By having such a configuration, the control channel may serve as an on-off switch configured to close or open the valve portions of the cell flow layer. In addition, it should be understood that other valve configurations are also possible in other embodiments, for example, valve configurations that are able to control flow proportionally.

[0085] In some embodiments, one or more control channels in the control layer can be pressurized in order to actuate the valve portions (e.g., inlet and / or outlet valve portions) of the growth channels in the cell flow layer, for example, to serve as an on-off switch, or such that flow through the valve is proportional to the amount of pressure. As one non-limiting example, when the one or more control channels become pressurized, the inlet and outlet valve portions of the growth channels may be actuated to transition from an open state that allows for fluid flow through the valve portions to a closed state that prevents fluid from flowing through the valve portions. Conversely, when the one or more control channels become depressurized, the inlet and outlet valve portions of the growth channels may transition back from a closed state (i.e., actuated state) that prevents fluid from flowing through the valve portions to an open state (i.e., unactuated state) that allows for fluid flow through the valve portions.

[0086] FIGs. 3A-3B shows a non-limiting example of actuation of the inlet and outlet valve portions via the one or more control channels. As shown in FIG. 3A, when the one or more control channels 52A are not pressurized, the inlet and outlet valve portions 16A and 16B of the growth channels 12 remain in an open state that allows for fluids 22A and 22B to flow through the valve portions 16A and 16B. As shown in FIG. 3B, when the one or more control channels 52A become pressurized, the inlet and outlet valve portions 16A and 16B of the growth channels 12 become actuated and transition to a closed state that prevents fluid from flowing through the valve portions. Conversely, when the one or more channels become depressurized, the inlet and outlet valve portions 52A of the growth channels may revert from a closed state (as shown in FIG. 3B) to an open state (as shown in FIG. 3A).

[0087] In some embodiments, the cell flow layer and / or the control layer comprise polymers (e.g., polydimethylsolixane (PDMS)) and may be cast together, or from separate molds. In some embodiments, the substrate is made from glass. The various channels of the cell flow layer and the control layer can be formed using any suitable fabrication technique(s). In some embodiments, the cell flow layer and the control layer are fabricated using multilayer soft lithography. In some embodiments, molds are initially formed from silicon wafers using UV lithography techniques. The cell flow and / or control polymer layers may then cast by flowing liquid polymer into the silicon molds, and then subsequently cured so that the polymer hardens. The two polymer layers can be bonded together (for example via curing or partial curing), and bonded to the substrate (for example via plasma bonding), and then further baked. Thus, the negative space of the channels of the cell flow layer and the control layer may be imprinted from the positive silicon wafer molds.

[0088] In some embodiments, the cell flow layer may have a length (e.g., such as a distance between the inlet and outlet of each channel in the cell flow layer) of between about 5 mm and about 100 mm, or about 30 mm; the control layer may have a span between various control channels (e.g., such as a distance between the two control channels 52A in FIG. 2A) of between about 4 mm and about 99 mm, or about 29 mm. The cell flow layer 10 and the control layer 50 may independently have a width of between about 20 micrometers and about 500 micrometers, or about 100 micrometers; the cell flow layer 10 may have a height of between about 5 micrometers and about 80 micrometers, or about 15 micrometers; and the control layer 50 may have a height of between about 10 micrometers and about 100 micrometers, or about 50 micrometers. It will be understood that while FIG. 2A depicts that the distance and width of control channels and cell growth channels in the cell flow layer are generally uniform, it is also possible for the length, width, and / or spacing between cell growth trenches and cell growth channels to differ.

[0089] The cell growth trenches may have any of a variety of appropriate dimensions. In some embodiments, the length (e.g., 1 in FIG. 3 A) of the cell growth trenches (e.g., the distance that the cell growth trenches 20 extend outward from the main portion 18 of growth channel 10) may be at least 1 micrometer, at least 5 micrometers, at least 10 micrometers, at least 25 micrometers, at least 50 micrometers, at least 75 micrometers, at least 100 micrometers, at least 200 micrometers, at least 300 micrometers, or at least 400 micrometers. In some embodiments, the length of the cell growth trenches may be no more than 500 micrometers, no more than 400 micrometers, no more than 300 micrometers, no more than 200 micrometers, no more than 100 micrometers, no more than 75 micrometers, no more than 50 micrometers, no more than 25 micrometers, no more than 10 micrometers, or no more than 5 micrometers. Any of the above-referenced ranged are possible (e.g., between 1.0 micrometer and 500.0 micrometers). Other ranges are also possible. It will be understood that while FIG. 3A depicts the cell growth trenches as being similarly sized, it is also possible for the length, width, and / or spacing between cell growth trenches to vary.

[0090] As noted above, the width (e.g., w in FIG. 3A) of the cell growth trenches (e.g., 20 in FIG. 3A) may be comparable to the size of a single cell in certain embodiments. For example, the cell growth trench may have a width that is large enough fit a single cell. In some embodiments, the width of the cell growth trench may be at least 1 micrometer, at least 5 micrometers, at least 10 micrometers, at least 25 micrometers, at least 50 micrometers, or at least 75 micrometers. In some embodiments, the length of the cell growth trenches may be no more than 100 micrometers, no more than 75 micrometers, no more than 50 micrometers, no more than 25 micrometers, no more than 10 micrometers, or no more than 5 micrometers. Any of the above-referenced ranged are possible (e.g., between 1.0 micrometer and 100.0 micrometers). Other ranges are also possible.

[0091] In some embodiment, the height of the cell growth trenches may be at least 0.1 micrometers, at least 0.5 micrometers, at least 1 micrometer, at least 5 micrometers, at least 10 micrometers, at least 20 micrometers, or at least 40 micrometers. In some embodiments, the length of the cell growth trenches may be no more than 50 micrometers, no more than 40 micrometers, no more than 20 micrometers, no more than 10 micrometers, no more than 5 micrometers, no more than 1 micrometers, or no more than 0.5 micrometers. Any of the above-referenced ranged are possible (e.g., between 0.1 micrometer and 50.0 micrometers). Other ranges are also possible.

[0092] In some embodiment, the distance between adjacent pair of cell growth trenches (e.g., 20 in FIG. 3A) may be at least 0.1 micrometers, at least 0.5 micrometers, at least 1 micrometer, at least 2 micrometers, at least 4 micrometers, at least 6 micrometers, at least 8 micrometers, at least 10 micrometers, at least 20 micrometers, at least 30 micrometers, or at least 40 micrometers. In some embodiment, the distance between adjacent pair of cell growth trenches may be no more than 50 micrometers, no more than 40 micrometers, no more than 30 micrometers, no more than 20 micrometers, no more than 10 micrometers, no more 8 micrometers, no more 6 micrometers, no more 4 micrometers, no more 2 micrometers, no more 1 micrometers, or no more 0.5 micrometers. Any of the above-referenced ranged are possible (e.g., between 0.1 micrometer and 50.0 micrometers). Other ranges are also possible.

[0093] The cell growth trenches may have any of a variety of appropriate aspect ratios. For example, in one set of embodiments, the cell growth trenches may have a length (e.g., 1 in FIG. 3A) to width (e.g., w in FIG. 3A) aspect ratio of at least 2, at least 5, at least 10, at least 25, at least 50, or at least 75. In some embodiments, the cell growth trenches may have a length to width aspect ratio of no more than 100, no more than 75, no more than 50, no more than 25, no more than 10, or no more than 5. Any of the above-referenced ranged are possible (e.g., between 2 and 100). Other ranges are also possible.

[0094] International Patent Application No. PCT / US2020 / 038867, filed on June 22, 2020, published as international Patent Publication No. WO 2020 / 257746 on December 24, 2020, and entitled “Isolating Live Cells After High-Throughput, Long-Term, Time-Lapse Microscopy,” by Luro, et al., is incorporated herein by reference in its entirety. U.S. Patent Application Serial No. 18 / 140320, filed April 27, 2023, entitled “Systems and Methods for Retrieving Cells from a Continuous Culture Microfluidic Device,” is incorporated herein by reference in its entirety.

[0095] In addition, the following are each incorporated by reference in their entireties: U.S. Provisional Patent Application Serial No. 63 / 646,534, filed May 13, 2024, entitled “Systems and Methods for Identifying Cells Using Hyperspectral Imaging and Other Techniques,” by Paulsson, et al., U.S. Provisional Patent Application Serial No. 63 / 646,582, filed May 13, 2024, entitled “Identifying Bacteria and other Organisms by Determining Growth Profiles,” by Paulsson, et al., and U.S. Provisional Patent Application Serial No. 63 / 646,599, filed May 13, 2024, entitled “Microscopy Systems and Methods for Identifying Bacteria and Other Organisms in Microfluidic Devices,” by Paulsson, et al.

[0096] The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the disclosure.

[0097] EXAMPLE 1

[0098] The following example describes identifying bacterial cells by determining respective growth profiles thereof.

[0099] Experimental outline

[0100] Sequential phenotypic tests were performed in order to profile and identify bacteria in the mixed sample. The Biolog Gen III Microplate ® system was used in combination with microfluidics to identify bacteria from their respective growth patterns on different microbiological media. The Biolog Gen III Microplate ® system contains 71 carbon source utilization assays, 23 chemical sensitivity tests and 2 colorimetric dosages. Although this 96- well plate is suitable for characterizing an isolated bacterial strain, it cannot be used as such to analyze a mixture of different strains. Rather than directly using a Biolog plate, a heterogeneous bacterial sample was loaded into a microfluidic device having a plurality of cell growth trenches in which the bacterial samples was separated. That is, in the microfluidic device, bacteria were trapped in dead-end microchannels, called growth trenches, where they can be monitored for many successive divisions. In addition, all growth trenches are connected to a main feeding channel in which fresh media circulates, thus ensuring a constant replenishment of nutrients. This device facilitated the study of the heterogenous sample, as individual bacterial cells were isolated in separate growth trenches. Individual cells may then be analyzed in each of the separate cell growth trenches. In addition to making the analysis at a single cell level possible, it also prevents any cell competition interaction (fast growing bacteria versus slow growing bacteria). The bacterial sample was then sequentially exposed to different Biolog media. By varying different Biolog media, specific growth profiles for each growth trench containing isogenic bacteria were established. In parallel, the growth profiles of 26 NIST strains were analyzed within a separate microfluidic device. Finally, by comparing the different growth profiles, the presence of some NIST strains was determined in the mixed sample.

[0101] Results

[0102] Establishment of growth profiles of known bacterial strains. The 96 media of the Biolog Gen III Microplate ® were combined with Microfluidics to identify bacteria in a mixed sample. In order to reduce the total number of media used in the microfluidic experiment and to increase the speed to identification, a combination of some non-redundant media with the highest strain-discriminating potential were selected. To do this, 26 NIST isolated strains were tested with the 96- well Biolog plates. Cell growth was measured under each condition by using a plate reader 20h after inoculation of the plates. Based on the growth phenotype of the 26 strains, the correlations between different cell media was calculated. After clustering, 6 redundant groups of media were identified. Within each group, the growth profile of each strain is similar (FIG. 5). In order to optimize the discriminating potential of the protocol in this Example, a single medium per group showing the highest variability between strains was selected. The 6 selected media were: 1) D-Galactose; 2) 0- Hydroxy-D,L-Butyric Acid; 3) 8% NaCl; 4) Niaproof 4; 5) Potassium Tellurite; 6) 1% Sodium Lactate.

[0103] Next, the growth profiles of the 26 NIST strains were established. To do this, the data of the Biolog plates by only keeping the 6 selected media were reevaluated. In order to simplify any further analysis, the growth rate was categorized as 0 - not growing, 1 - slight growth, 2 - important growth. This binning was also justified by the fact that growth rate distribution was trimodal over the 6 media. The simplified method to profile strains showed very satisfactory results as all 26 NIST strain were identified by a unique growth profile (FIG. 6). Furthermore, this method was found to be very consistent, as most of the closely related strains exhibited similar (but not identical) growth profiles - see, e.g., the classification tree on FIG. 6.

[0104] Estimation of the number of different strains in the blinded sample. A post incubation (pre-grown) mixed sample was loaded into a microfluidic device having a plurality of cell growth trenches. The sample was diluted before loading the microfluidic device to load the growth trenches with single cells. For this experiment, a device with relatively large trenches (2 pm wide) that can retain bacteria of varying size was used. The feeding media was switched successively during the experiment so that bacteria were exposed to the 6 previously selected discriminant media (3h of exposure to each media). The sequence was: 1) D-Galactose; 2) P-Hydroxy-D,L-Butyric Acid; 3) 8% NaCl; 4) Niaproof 4; 5) Potassium Tellurite; 6) 1% Sodium Lactate. To avoid any interaction between these media, bacteria in the microfluidic device were allowed to recover in glucose enriched media for i h before introducing each next successive media. The bacterial growth rate was monitored by timelapse microscopy and directly quantified on phase-contrast kymograph images. The growth rates were categorized by following the same rules we used for the Biolog plates: 0 - not growing, 1 - slight growth, 2 - important growth. In addition to simplifying the analysis, this also allows for the comparison between the 96-well experiments and the microfluidic experiments.

[0105] Of the growth trenches, 140 out of 1000 growth trenches from the microfluidic device were quantified. The reason for this low N-value was due to a biofilm formation. To avoid biofilm formation, the flow rate in the feeding channel was increased, which resulted in some of the cell growth being unloaded.

[0106] There were 44 different growth profiles found, suggesting the presence of 44 different bacterial strains (FIG. 7). However, the number of different strains in the mixed sample may be lower than 44 if there is variability in the growth profile within a strain (e.g., experimental variability, such as how the growth rate is measured in different growth trenches). Therefore, it is possible that the most similar growth profiles correspond to the same strain. By grouping such similar growth profiles together, only 30 different strains were obtained (FIG. 8). This places the estimated total number of strains in the mixed sample between 30 and 44.

[0107] Identification of bacteria in the blinded sample. To identify the bacteria in the mixed sample, their growth profiles (e.g., in 6 different media) were compared with the profiles of the 26 NIST strains (e.g., in the same 6 media). Because these profiles were established using completely different approaches (microfluidics versus 96-well plates), a 100% correlation was not the considered, but instead a similarity score was calculated (tolerating some level of difference). This was done by first calculating the distances between the growth profiles quantified in each microfluidic trench (e.g., the mixed simple) and the profiles of each of the 26 NIST strains. As the growth categories are the same (e.g., 0, 1, or 2) for microfluidic and for 96-well plate experiments, this was simply the absolute difference between two growth profiles. Next, each microfluidic trench was associated with one of the NIST strain with the closest growth profile (e.g., having the smallest distance, smallest difference). Finally, for each NIST strain, the number of associated microfluidic trenches (e.g., the abundance) was scored and what was the average growth profile similarity between them and the strain (see FIG. 9 and Table 1). Assuming the blinded sample contains some of the NIST strains, a higher similarity score suggested higher likelihood that a particular strain is present in the sample.

[0108] Table 1. Detailed relative abundance and similarity score for 26 NIST strains

[0109] Discussion

[0110] In this Example, a Microfluidics and Growth Profile based method to identify bacteria in a mixed sample is described. The accuracy of this method was satisfactory, but could greatly be improved by optimizing the experimental protocol. One of the most important limitations of the approach is that growth profiles established under different conditions were compared -e.g., growth rates obtained in microfluidics were compared with growth rates obtained from bulk experiments. In order to make this experiment more coherent, it would be advantageous to characterize the growth profile of known strains also in a microfluidic environment to compare growth profiles of unknown bacterial cells obtained from a microfluidic environment thereto. Another limitation of this experiment was due to the formation of biofilms in the microfluidic device, leading to the unloading of many bacteria and an associated relatively low N-value for the number of cell growth trenches analyzed. Coating the microfluidic device with anti-adherents and using biofilm inhibiting drugs may alleviate this problem.

[0111] While several embodiments of the present disclosure have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present disclosure is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the scope of the present disclosure.

[0112] In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and / or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

[0113] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0114] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0115] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0116] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0117] When the word “about” is used herein in reference to a number, it should be understood that still another embodiment of the disclosure includes that number not modified by the presence of the word “about.”

[0118] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0119] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

CLAIMSWhat is claimed is:

1. A method, comprising: determining a first and a second growth rate in respective first and second growth media of at least some of a plurality of cells within a plurality of cell growth trenches of a microfluidic device.

2. A method, comprising: exposing a plurality of cells within a plurality of cell growth trenches of a microfluidic device to a first growth medium; determining a first growth rate of the plurality of cells exposed to the first growth medium; exposing the plurality of cells to a second growth medium; and determining a second growth rate of the plurality of cells exposed to the second growth medium.

3. A method, comprising: determining a first and a second growth rate in respective first and second growth media of at least some of a plurality of cells from a sample containing two or more unknown species of unicellular organisms.

4. The method of any one of claims 2-3, wherein the plurality of cells is contained within a microfluidic device comprising a plurality of cell growth trenches.

5. The method of any one of claims 1-4, further comprising identifying a species of at least some of the plurality of cells based on the first and second growth rates.

6. The method of any one of claims 1-5, wherein the cells are serially exposed to the first growth medium and the second growth medium.

7. The method of any one of claims 1-6, wherein the cells are serially exposed to the second growth medium and the first growth medium.

8. The method of any one of claims 1-7, further comprising determining a third, fourth, fifth, and sixth growth rate in respective third, fourth, fifth, and sixth growth media of at least some of the plurality of cells from the sample of two or more unknown species of unicellular organisms and identifying the species of at least some of the plurality of cells based on the first, second, third, fourth, fifth, and sixth growth rates.

9. The method of any one of claims 1-8, wherein the cells are bacteria cells.

10. The method of any once of claims 1-9, further comprising exposing the cells to a recovery solution between the first and second cell growth media.

11. The method of claim 10, wherein the recovery solution comprises glucose enriched media.

12. The method of any one of claims 5-11, wherein the identifying the cell species of at least some of the plurality of cells occurs in less than or equal to 48 hours.

13. The method of any one of claims 5-11, wherein identifying the cell species of at least some of the plurality of cells is based on the first and second growth rates and one or more other sensed properties.

14. The method of claim 13, wherein the one or more other sensed properties comprises a cell morphology.

15. The method of claim 13 or 14, wherein the one or more other sensed properties comprises a fluorescence property.

16. The method of any one of claims 1-15, wherein determining the first growth rate comprising monitoring a growth of cells using phase-contrast microscopy.

17. The method of any one of claims 1-16, wherein determining the first growth rate comprising monitoring a growth of cells using time-lapse microscopy.

18. The method of any one of claims 1-17, further comprising providing the plurality of cells.

19. The method of claim 18, wherein providing the plurality of cells comprising acquiring the cells by sampling cells from a source.

20. The method of claim 19, wherein the source comprises a body of water.

21. The method of claim 19, wherein the source comprises soil.

22. The method of claim 19, wherein the source comprises a food source.

23. The method of claim 19, wherein the source comprises a subject having a disease.

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