WO2017147427A1 - System and method for microscopic observation of microbial fitness - Google Patents

Abstract

The disclosure provides an optically acceptable culture chamber, and related methods, systems, and kits, effective to observe one or more cells of interest in a stable culture environment. The chamber, and related methods, systems, and kits, allows for observation effects of controllable factors on the growth and development from a single, identifiable cell deposition, or from a plurality of single cell depositions in parallel, over extended periods of time.

Description

SYSTEM AND METHOD FOR MICROSCOPIC OBSERVATION OF MICROBIAL

FITNESS

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Provisional Application No.

62/300,672, filed February 26, 2016, the disclosure of which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under Grant No. 1R21AI111181-01A1 awarded by the National Institute of Health. The Government has certain rights in the invention.

BACKGROUND

Traditional and contemporary methods for assessing growth of microorganisms involve inoculating liquid cultures with millions or billions of cells and calculating population average responses. Other methods involve depositing colony forming units on the dorsal surface of solid growth medium and waiting for the visible appearance of colonies to assess growth rate. A common application of these methods is to use microorganism growth rate as a measure of drug susceptibility, in which case the liquid or solid growth medium is adjusted to contain the desired drug, microorganisms are introduced, and growth is assessed by using macroscopic measurements and population averages as described above. From these, data susceptibility profiles are then derived, with slower or no growth of drug-treated cells compared to untreated cells indicating drug susceptibility. Despite widespread use, these approaches are outdated and provide only limited resolution that reflects aggregate cell-responses. Thus, the approaches obscure responses of single cells on biologically-relevant time scales and resolution. These challenges are particularly problematic for slow-growing organisms like Mycobacterium tuberculosis, for which time-to-visualization takes at least seven days, and drug susceptibility testing routinely requires >3-5 weeks. Being able to track microbe growth on a microscopic level would allow for much faster assessment of growth kinetics and drug susceptibility profiles. Culturing cells using solid growth medium on a microscope stage has been proposed, but these efforts are hindered by the practical challenges of maintaining microscopically-smooth, optically-clear, solid medium without Z-plane drift caused by desiccation or other factors.

Accordingly, there remains a need for improved strategies to provide stable culture environments for microscopic observation of individual cells over time. The present disclosure addresses these and related needs.

SUMMARY

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

In one aspect, the disclosure provides a culture chamber for microscopic observation of one or more cells of interest maintained in a stable environment. The culture chamber comprises a solid housing comprising a base substrate that permits microscopic observation through the substrate; and a solid or semi-solid growth medium layer disposed proximal to the base substrate to provide a stable culture space between the base substrate and growth medium layer. The chamber is configured to receive one or more cells in the stable culture space and subsequently seal the stable culture space to provide a stable environment for maintenance of the cells.

In one embodiment, the culture chamber further comprising a sealable channel or valve configured to deliver cells into the stable culture space. In one embodiment, the solid substrate is re-sealably detachable from the housing and growth medium layer to permit delivery of the one or more cells and subsequent reattachment and sealing to provide the stable environment for maintenance of the cells. In one embodiment, the chamber is configured to receive an applicator to inject the one or more cells in the stable culture space. In one embodiment, the base substrate is an optically acceptable barrier. In one embodiment, the base substrate is plastic or glass. In one embodiment, the solid or semi-solid growth medium permits growth and/or development of the one or more cells of interest. In one embodiment, the culture chamber comprises, or is configured to receive, a coverslip over the solid or semi-solid growth medium layer on a side opposite from the solid substrate. In one embodiment, the culture chamber comprises, or is configured to receive, a liquid medium over the coverslip on a side opposite from the growth medium layer. In another aspect, the disclosure provides a method for microscopically observing one or more cells in a stable environment. The method comprises providing the one or more cells in a culture space disposed between an optically acceptable base substrate and solid or semi-solid growth medium layer; sealing the stable culture space to provide a stable environment for maintenance of the cells; and observing the one or more cells with a microscope through the optically acceptable base substrate.

In one embodiment, the method further comprises placing a coverslip over the growth medium layer on a side opposite from the one or more cells. In one embodiment, the method further comprises placing liquid medium over the coverslip on a side opposite from the growth medium layer. In one embodiment, the one or more cells are observed two or more times to assess development and/or viability of the cells over time. In one embodiment, the one or more cells are bacteria, such as Mycobacterium tuberculosis. In one embodiment, the method further comprises providing the one or more cells with an experimental environmental condition. In one embodiment, the experimental environmental condition is the presence of one or more potential antibiotics, such as isoniazid, ethambutol, rifampin, pyrazinamide, and the like.

In another aspect, the disclosure provides a method of assessing viability and/or development of a cell in a stable environment. The method comprises providing the one or more cells in a culture space disposed between an optically acceptable base substrate and solid or semi-solid growth medium layer; sealing the stable culture space to provide a stable environment for maintenance of the cells; and observing the one or more cells with a microscope through the optically acceptable base substrate.

In one embodiment, the one or more cells are bacteria, such as Mycobacterium tuberculosis. In one embodiment, the method further comprises providing the one or more cells with an experimental environmental condition. In one embodiment, the experimental environmental condition comprises an amount of one or more potential antibiotics, such as isoniazid, ethambutol, rifampin, pyrazinamide, and the like. In one embodiment, the method further comprises assessing the susceptibility or resistance of the cell to the potential antibiotic or combination of antibiotics.

In another aspect, the disclosure provides a system for microscopic observation of a cell. The system comprises a solid housing comprising a base substrate that permits microscopic observation through the substrate; a solid or semi-solid growth medium layer disposed proximal to the base substrate to provide a stable culture space between the base substrate and growth medium layer; a microscope; and a microscopy enabled imaging device. In one embodiment, the system is adapted to receive one or more cells of interest into the stable culture space and subsequently be sealed to provide a stable environment for maintenance of the cells.

In another aspect, the disclosure provides a kit, comprising a solid housing comprising a base substrate that permits microscopic observation through the substrate; a solid or semi-solid growth medium layer adapted to be placed proximal to the base substrate to provide a stable culture space between the base substrate and growth medium layer, wherein the stable culture space is capable of receiving one or more cells to provide a stable environment for maintenance of the cells; and instructions for use with one or more cells of interest. In one embodiment, the kit further comprises a coverslip configured to be placed over the growth medium layer. In one embodiment, the kit further comprises liquid medium.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIGURE 1 is a schematic illustration of a culture well indicating the inverted growth assembly to permit microscopic observation during long-term culture.

FIGURE 2 is a schematic illustration of the platform design in a multi-well format for microscopic observation of microbial fitness in response to experimental conditions.

FIGURE 3 is a series of forty photomicrographs taken of the same field of view of a culture of Mycobacterium tuberculosis covering 117 hours from the initial plating of the individual bacteria.

FIGURES 4A-4C are graphical illustrations demonstrating that M. tuberculosis strain H37Rv in rich culture conditions exhibit the expected growth parameters. Specifically, 264 microcolonies were tracked over four+ days of culture with repeated imaging. FIGURE 4 A illustrates the log₂ of growth over time (hours). FIGURE 4B illustrates a histogram of doubling times as frequency vs. time (hours). FIGURE 4C illustrates the range of derived population doubling times, indicating that the population median doubling time was 23.9h with 1st and 3rd quartile range of 20.6-28.2h, in keeping with standard replication rates of M. tuberculosis.

FIGURES 5A-5E graphically illustrate the comparison of growth doubling times for different M tuberculosis from diverse evolutionary clades. Specifically, the different M. tuberculosis isolates exhibited variable and idiosyncratic growth characteristics. Clinical isolates of M. tuberculosis representing diverse evolutionary clades were cultured on the disclosed multi-well platform and tracked over a 96 hour experiment with image acquisition at three hour intervals. FIGURES 5A-5D illustrate the object size quantification yields growth curves for each strain with the y-axis representing object size on a log2 scale. FIGURE 5E illustrates the median doubling times +/- the 1st and 3rd quartile from all objects. The illustrated growth rates vary in a strain-specific manner and corroborate with batch culture measurements (data not shown). Lineage and strain identifiers detailed in Table 1.

FIGURES 6A-6U graphically illustrate the growth rate of M. tuberculosis strain H37Rv is differentially affected by drug concentration and type. FIGURE 6A schematically illustrates a standard experimental setup where cells are exposed in individual experiment wells to a defined drug concentration. FIGURES 6B-6K provide an aggregate (FIGURE 6B) and individual graphical representations (FIGURES 6C-K) of stepwise drug titration for the minimal inhibitory concentration (MIC) of different concentrations of rifampin on M tuberculosis as observed in the microscopic platform of the present disclosure. FIGURES 6L-6U provide an aggregate (FIGURE 6L) and individual graphical representations (FIGURES 6M-6U) of stepwise drug titration for the minimal inhibitory concentration (MIC) of different concentrations of isoniazid on M tuberculosis as observed in the microscopic platform of the present disclosure. As observed, the growth rate of cells is idiosyncratic to drug type and drug concentration.

FIGURES 7A and 7B graphically illustrate that the endpoint of M. tuberculosis

H37Rv microcolony size varies by drug concentration and type. FIGURE 7 A illustrates the median microcolony size at TO and T90h for cells exposed to rifampin (left) or isoniazid (right) over drug exposures ranging from untreated to 8. Ox the batch culture- defined MIC99. FIGURE 7B illustrates the T90:T0 ratio of microcolony size for each drug treatment can be used to derive the "Growth Inhibitory Concentration" (GIC) by deriving the nonlinear regression best fit line and determining the intersection for "x" of the maximum. Here the GIC90 (90% inhibition of maximal ratio) was calculated for rifampin (left, 16 ng/mL) and isoniazid (right, 1235 ng/mL).

FIGURES 8A-8F illustrate that the uptake of propidium iodide provides rate of bacterial death and defines killing dose vs. inhibitory dose of antibiotics. FIGURES 8A-8C illustrates that the vital dye propidium iodide (PI) is excluded from viable cells but is incorporated into nucleic acids and detected as a red fluorescent signal in cells that have lost membrane integrity in untreated (FIGURE 8A), and cells treated with 0.5x the batch culture-defined MIC99 for isoniazid (FIGURE 8B) and rifampin (FIGURE 8C). FIGURES 8D-8F illustrate that the quantifying rate of PI uptake and percentage of dead microcolonies distinguishes the time-to-death and bactericidal concentrations for antibiotics with unique mechanisms of action.

FIGURES 9A-9D graphically illustrate the determination of phenotypic drug susceptibility against all frontline M. tuberculosis antibiotics occurs 48 hours or less. The median slope of microcolony area over time for populations of individual objects is demonstrated for all frontline TB antibiotics tested. Cell growth response is shown over a range of drug concentrations for (FIGURE 9A) isoniazid, (FIGURE 9B) ethambutol, (FIGURE 9C) rifampin, and (FIGURE 9D) pyrazinamide. The growth rate response corresponding to each drug concentration was calculated by constraining the analysis window from 0 - 48 hours. By 48 hours of drug exposure, significant (*** = p<0.0001, Kruskal-Wallis test with Dunn's correction) differences in growth rate are apparent, and persist for the remainder of the exposure. In all cases, the "untreated" population is positioned at the far left. The vertical dashed line on each graph indicates where the batch culture-derived " l .Ox MIC99" drug concentration was tested. Only two supra-killing drug concentrations were tested for pyrazinamide (250 and 500 μg/mL).

FIGURES 10A and 10B illustrate low-dose isoniazid disrupts ATP homeostasis and induces increased cell growth. FIGURE 10A: M. tuberculosis strain H37Rv was grown for 99 hours in the presence of isoniazid concentrations ranging in 2-fold steps from 2. Ox - 0.03x the batch culture-defined MIC₉₉. Bars reflect the ratio of the median microcolony area of the population (99h:3h) as measured by the PMDF live cell imaging method. FIGURE 10B: M. tuberculosis strain H37Rv was grown for 96 hours in the presence of isoniazid concentrations ranging in 1.5-fold steps from 1.5x - 0.03x the batch culture-defined Μ ½9· Bars reflect the mean ATP levels of the cell population from 4 replicate wells on 4 replicate plates for each treatment condition.

DETAILED DESCRIPTION

This disclosure describes the inventors' development of a new approach for observing individual cells in a stable culture that facilitates reliable long-term microscopic observations of the cells for an extended period of time, for example over a period of days or weeks.

In one aspect, the disclosure provides a culture chamber for the microscopic observation of one or more cells of interest maintained in a stable environment. The culture chamber comprises a solid housing comprising a base substrate that permits microscopic observation through the substrate, and a solid or semi-solid growth medium layer disposed proximal to the base substrate to provide a stable culture space between the base substrate and growth medium layer. The chamber is configured to receive one or more cells in the stable culture space and subsequently seal the stable culture space to provide a stable environment for maintenance of the cells.

The term "one or more cells of interest" encompasses any type of cell that is intended to be observed. Cells can include, for example, cells isolated or derived from multicellular organisms. For example, the cells can be mammalian (e.g., human or mouse) cells from tissue culture lines or as isolated from a subject (e.g., via biopsy). Cells can also include single cellular organisms, such as bacteria, archaea, protozoa, fungi, algae. The "one or more" language encompasses multiple cells of the same type, or, alternatively, encompasses one or more cells of different types (e.g., that live commensally or are in host-parasite or predator prey relationships). For example, as described in more detail below, the cell can be a pathogenic bacterium, such as M tuberculosis, which can be studied for the effects of an antibiotic or suspected antibiotic composition.

The term "stable environment" indicates that the environment is conducive to a controlled maintenance of environmental factors conducive to growth and survival of the cell(s), including factors such as nutrients and hydration, without undue risk of contamination or entry of detrimental factors (except for controlled application of experimental conditions). The term also encompasses the stability of the target cell(s) and offspring thereof as positioned in a single location (e.g., a stable and identifiable position on defined x, y, and z, planes) such that horizontal, vertical, or otherwise movement is minimized. This aspect facilitates the repeated observation of the cell(s) over time reliably at the initially established coordinates within the chamber.

The term "solid-housing" refers to structural aspects of the culture chamber, which can include walls, lid, and base substrate. Exemplary, non-limiting examples can include typical tissue or cell culture plates commonly used in laboratories. The housing is typically composed of suitable polymers/plastics that can be sterilized and can receive cells and medium components to maintain viability and sterility of a cell environment. The "base substrate" permits microscopic observation therethrough. Thus, the base substrate is composed of clear, transparent materials, or other materials that allow passage of light. The base substrate can thus provide an optically acceptable barrier to permit observation, while contributing to the stability of the culture environment. Many appropriate materials for the base substrate are known in the art. For example, the base substrate can be glass, plastic, polydimethylsiloxane (PDMS), and the like. The base substrate can be considered to be the "floor" of the chamber, but does not necessarily need to be on the bottom of the chamber space. In many embodiments, the base substrate can receive the cell(s) of interest, which is/are deposited thereon. The base substrate can also be coated with any known composition to facilitate attachment or growth of the cell(s), such as poly-L-lysine, a polyphenolic protein solution (CellTak), etc. However, such additional composition(s) preferably do not significantly reduce the optical access to the cell(s) through the base substrate.

The term "solid or semi-solid growth medium" refers to any appropriate medium that facilitates the growth of the one or more cells of interest. Formulations of solid or semi-solid growth media are well-known and can be appropriately selected for any particular cell of interest by persons of ordinary skill in the art. The solid or semi-solid growth medium can be shaped such that it is positioned within the solid housing proximal to the base substrate. The term "proximal" is meant to indicate close positioning such that a "stable culture space" exists between the base substrate and the growth medium layer. The solid or semi-solid growth medium does not necessarily need to be in contact with the base substrate, but can maintain points of contact. The growth medium layer can be considered a "puck" or "plug" (see FIGURE 1) that is positioned sufficiently close to permit cell(s) deposited on, or attached to the base substrate to be able to also derive nutrients and possibly even physical support/contact from the solid or semi-solid growth medium. Thus, the stable culture space is typically narrow, encompassing a gap between the base substrate and the growth medium layer that is on the scale of the size of a cell. The term "stable" as used in "stable culture space" thus refers in part to the constricted gap that provides sufficient physical contact or support to prevent lateral or vertical migration of the cell(s) within the particular coordinates within the chamber (or within a field of view, thereof).

One example of a culture chamber includes glass borosilicate base substrate with arrangement of alginate or PDMS pucks (as the medium layer) to provide a reinforcing base support and localization medium for cells.

The chamber is configured to receive the one or more cells in the stable culture space, such that the cell(s) are ultimately stably positioned between the base substrate the growth medium layer (see FIGURE 1). This can be performed by depositing the cell(s) on the base substrate followed by placing an appropriately shaped medium puck over the cells such that the cells occupy the formed stable culture space. However, numerous other configurations are encompassed by the present disclosure, which will be apparent to persons of ordinary skill in the art.

For example, non-limiting examples of configurations include having a removable base substrate such that the cell(s) can first be deposited on the medium puck which is first or thereafter placed within the housing. The removable base substrate can then be (re)attached to the solid housing in a manner where it is positioned proximal to the growth medium layer (puck) with the cells deposited thereon. Upon reattachment of the removable base substrate forms the stable culture space with the cell(s) therein.

Another exemplary configuration includes a sealable channel or valve that permits the delivery or deposition of the cell(s) into the stable culture space. The valve or channel can be integrated into any aspect of the solid housing, including the base substrate. The valves or channels can serve as access ports with or without dedicated lines to facilitate cell inoculation/deposition or media perfusion/exchange. These same lines could also be functionalized to provide vacuum suction to facilitate, for example, rapid deposition and immobilization of cell(s) on the base substrate. These same lines could also be functionalized to deliver and remove processing reagents for the liberation of bacteria from clinical isolates, e.g., Mycobacterium tuberculosis from sputum or bronchoalveolar lavage samples, etc.

In another example, the chamber is configured to receive a removable applicator that delivers the cell(s) into the stable culture space. Such an applicator could resealably breach the medium puck layer. For example a needle could be inserted through a pre- placed puck to inject the cells into the stable culture space, and upon removal of the needle, the puck closes and seals the hole resulting from the needle.

In some embodiments, the chamber further comprises markers, such as on the puck or the base substrate, to provide landmarks to facilitate navigation during microscopic analysis.

In some embodiments, the culture chamber comprises, or is configured to receive, a coverslip over the medium layer (puck). In some embodiments, the culture chamber comprises or is configured to receive, a liquid layer over the coverslip. See FIGURE 1.

It will be appreciated that the culture chamber can be replicated in a single device, to provide a multi-chamber format in a single device, such as a plate. One example is the 24^_well platform, described in more detail below, used to observe M. tuberculosis strains in parallel under various experimental conditions. The housing, of a single chamber or a multi-chamber device, can be outfitted with seals, gaskets, and locking mechanisms on a per-chamber or per-plate basis to provide reversible biosafety containment for imaging and maintenance of the potential bio-hazardous cell(s) within culture chambers. The geometry of a single chamber or a multi-chamber device can be outfitted or functionalized with additional features (e.g., tracks, channels, etc.) that assist plate manipulation on, around, and off the imaging space. Such features could be appropriately designed to facilitate automated handling to permit high-throughput imagine analysis of the chamber(s) and the cell(s) therein. The single chamber or a multi-chamber device can be functionalized to be compatible with any available microscope (e.g., upright or inverted microscope configurations) or other imaging device setup (e.g., with film or digital cameras, tablet or cell phone cameras, etc.).

The culture chamber is configured to be sealable after receipt of the cell(s) in the stable culture space. As used herein, the term "seal," or variations thereof, refers to a substantial, but not necessarily complete, isolation of the stable culture space from the environment outside the chamber. This can be sufficient isolation to prevent significant evaporation/desiccation, etc., of the medium layer, which prevents the movement of cell(s) over and in the surface of the medium layer puck. In some embodiments, the isolation is sufficient for biocontainment of biosafety-level 2, 3, or 4 microorganisms.

In another aspect, the disclosure provides a method for microscopically observing one or more cells in a stable environment. The method comprises providing the one or more cells in a culture space disposed between an optically acceptable base substrate and solid or semi-solid growth medium layer; sealing the stable culture space to provide a stable environment for maintenance of the cells; and observing the one or more cells with a microscope through the optically acceptable base substrate.

The method can comprise placing a coverslip over the growth medium layer on a side opposite from the one or more cells and/or placing liquid medium over the coverslip on a side opposite from the growth medium layer. For analysis with a temporal component, the one or more cells are observed two or more times to assess development and/or viability of the cells over time.

The present methods are not limited to any target cell, but rather are amendable for application with any cell capable of in vitro culture. In some embodiments, the cell or more cells is a bacterium relevant to human or other animal health.

The method can incorporate the application of any of a number of controlled conditions to test the effect(s) on the one or more cells in the stable environment. The controlled conditions can be the application of one or more compounds, at one or more determined concentrations. An advantage of the platform method is the ability to scale up the experiments and run several in parallel, thus permitting the testing a variety of conditions simultaneously (e.g., multiple potential antibiotics each at a plurality of concentrations). Other conditions can include differing media conditions to optimize growth and replication, culturing temperature, light exposure, and the like. The platform method is flexible and can be suitably modified and applied by persons of ordinary skill for myriad assay designs.

In one illustrative, non-limiting embodiment, the one or more cells are bacteria, such as M. tuberculosis. The one or more cells are provided in an experimental environmental condition, such as a medium comprising a potential or known antibiotic at a concentration. Exemplary applications of the method, as described in more detail below, include the exposure of M. tuberculosis to varying concentrations of antibiotics such as isoniazid, ethambutol, rifampin, and pyrazinamide. However, it will be understood that any potential antibiotic or other composition can be utilized as the experimental condition to be tested on the cultured cell(s). The effects of the antibiotics can be assessed at multiple concentrations over time in parallel, with effects observable in 48 hours or less.

It will be understood by persons of ordinary skill in the art that a wide variety of parameters can be observed and subject to empirical analysis using the disclosed method and culture chamber platform. The empiric determination of GIC₉₀ is an exemplary determination of the effects of imposed conditions on the cultured cells (e.g., to determine drug susceptibility of cells at different concentrations). However, applications of the method are not so limited. Due to its modular nature, the disclosed platform method encompasses other known empiric determinations. To facilitate observation of the cultured cells and the effects of an imposed variable, the cells can incorporate detectable tags to indicate the presence, levels, or absence of any appropriate known marker that is indicative of a relevant biological state of the cell. The detectable tags can be recombinantly expressed in the cell, or can be added to the culture via a detectable reagent that specifically binds to a biomarker expressed or displayed by the cell(s). Such tags can include cell-specific fluorescent stains, dyes, immune-derived tags that bind to cell biomarkers, recombinantly expressed fluorescent proteins, and the like. In some embodiments, a cell stain that distinguishes between live:dead state of cells, known as vital dye, can be applied to the culture along with the variable conditions (e.g., antibiotic). A non-limiting example of a vital dye used in the description below is propidium iodide (PI), although numerous other such vital dyes can be readily incorporated into this platform method by persons of ordinary skill in the art.

In one very specific embodiment, the disclosed method comprises exposing one or more clinically relevant M. tuberculosis strains to varying doses of pyrazinamide, which is an important first-line component of many anti-M tuberculosis therapies. In this specific embodiment, the method can comprise adjusting the growth medium to pH 6.0 by dropwise addition of phosphoric acid or similar acid supplement. This media formulation has been demonstrated to both support M tuberculosis growth in the absence of additional media supplement and enhance the activity of pyrazinamide when this antibiotic is added to the culture medium. Adjusting pH does not preclude additional modification to media formulation including but not limited to alternate nutrient sources or antibiotic supplementation. This specific embodiment overcomes serious limitations in existing tests of this antibiotic's efficacy in vitro against genetic variants of M. tuberculosis because the pH often required to assess the efficacy of pyrazinamide also impedes growth of cultured M. tuberculosis in standard assays, thus obscuring the results. However, as described below in more detail, the present method was surprisingly able to achieve test culture conditions that were simultaneously conducive for M. tuberculosis (without the antibiotic) and permissive of the antibiotic activity, thus allowing for a reliable and direct inference of the antibiotic's effect when applied to the culture.

As indicated below, the disclosed chamber and related methods can be applied to any temporal analysis of cell(s), such as the effect of an imposed condition on the cell(s) over time. Such analyses could include, for example, the testing of any antibiotic of interest on the growth and development of a bacterium, or even to compare the differential effects of an antibiotic of interest on distinct variants of a bacterium species. For example, as described below in more detail, the disclosed chamber, system, and methods, as described herein can be applied to observe the growth of target bacteria under (potentially variable) antibiotic conditions, determine the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and/or growth inhibitory concentration (GIC), which serve as useful metrics to ascertain the effect of particular antibiotics on the cell(s).

The present disclosure also encompasses related systems and kits encompassing one or more elements of the culture chamber described herein.

To establish proof of the design, the inventors developed a high-content imaging platform for the parallel microscopic determination of fitness of M. tuberculosis at single cell resolution over long periods. By implementing automated time lapse transmitted light microscopy on an environment-controlled stage, the inventors quantitated the growth rate from thousands of individual colony forming units (CFU) under dozens of conditions in a single experiment. Fluorescent imaging of vital stains was used to provide a concurrent enumeration of dying and dead cells. These data provide a quantitative measure of growth rate per CFU as well as a measure of population heterogeneity under rich and defined-stress conditions. By exposing viable M. tuberculosis to dilution series of antibiotics, the drug-induced alteration of growth rate was translated into a functional "real time minimum inhibitory concentration" (RT-MIC) and "real time minimum bactericidal concentration" (RT-MBC) values. These values were obtained simultaneously and in under four days. Remarkably, in some cases measurable effects were even observed in less than 48 hours. This represents a significant reduction in time-to-results for standard/batch culture MIC enumeration from seven days, and the MBC from -35 days. Furthermore, because this approach does not require tagged or fluorescently-labeled bacteria, it is suitable for the rapid quantitation of antibiotic susceptibility profiles of laboratory-or clinically-isolated bacteria with unknown resistance. However, while this platform does not require modified cell strains, it will be apparent that the modular nature of the approach accommodates any appropriate for microscopic observation of a signal, such as the incorporation of dyes, stains, immune-derived binding agents coupled to markers, and the like (generically referred to as "markers"). Appropriately applied, these markers could contribute to speed and sensitivity of the approach for myriad applications.

Specifically, FIGURE 1 provides a schematic illustration of a representative individual culture chamber of the present disclosure. As illustrated, one or more cells can be deposited over a base substrate and are overlaid with a layer of appropriate solid or semi-solid culture medium (indicated as a "puck"). Additionally, an optional coverslip and liquid medium can be further added over the puck within the chamber. These additional layers can assist the preservation of the culture conditions by providing additional sealing, nutrient or drug delivery, and/or resistance to evaporation or drying of the one or more cells' immediate culture environment.

In this demonstration, a multi-well format was used, as represented in FIGURE 2. This format permitted the deposit of cells in distinct wells to facilitate simultaneous testing and comparison of multiple variables, such as differing drug doses and combinations, while otherwise maintaining uniformity for constant conditions such as temperature and ambient gas, etc. Each well can provide a sufficient surface area to maintain distinct coordinates for multiple sustained observations, thus providing robust sample sizes for each experimental condition. The images can be acquired through the base substrate, in this case the optically transparent floor of the chamber, over set intervals on a scale of days or even weeks or more. To demonstrate that M. tuberculosis cells can grow and multiply within the present culture setup, and that such development could be microscopically observed over a relevant time period, each of 24 wells in a 24-well plate was assembled as represented in FIGURE 1 with approximately 100,000 cells of H37Rv strain of M tuberculosis per well. Pucks of approximately 1x14 mm (height x diameter) were placed over the deposited cells in each well. The pucks were composed of M. tuberculosis rich growth medium 7H9 supplemented with glycerol, albumin, dextrose, catalase, and tween-80 (7H9+GAT), 50 nanomolar propidium iodide, +/- variable concentrations of antibiotic and rendered semi-solid with the addition of alginate (Sigma) powder to a final concentration of 4% and cross-linking with 50 millimolar calcium chloride (Sigma). Finally, a 15mm diameter circular #2 micro coverglass and 1.8 milliliters 7H9+GAT+prodium iodide +/- antibiotic of liquid medium were added over the puck. Images were obtained by a Nikon Eclipse Ti-E Inverted Fluorescent Microscope with automated stage and In Vivo Scientific incubated enclosure. A series of forty representative micrographs representing a subsection of a single well at every three hours over a five day period are illustrated in FIGURE 3. Timing of image acquisition can be varied as needs of application dictate, ranging from multiple acquisitions per minute, to a frequency on the order of days. This illustrates that the deposited CFUs were able to establish themselves and grow in the novel configuration. Moreover, this establishes that the format provides stability for the growing cell cultures and, thus, facilitates reliable imaging over time without lateral or vertical migration of the cells over the substrate. Stated otherwise, repeated images of a developing cell and colony could be obtained at a single coordinate set without having to compensate for movement. Thus, data on individual cells can be collected from such a culture format for a significant period of time.

The stability of the cultured cells in this environment permitted the derivation of doubling times for the individual CFUs. Cells of the H37Rv strain of Mycobacterium tuberculosis were cultured and observed as described for FIGURE 3 above. Specifically, the individual bacterial microcolonies were identified algorithmically as objects based on differential signaknoise (mi crocolony: background substrate) pixel intensities in photomicrographs. Outlines of cell perimeters were then determined using these same signal :noise parameters and colony size parameters such as area and volume were calculated. Implementing a combination of fixed coordinates and nearest-neighbor location parameters allowed for the faithful identification of microcolonies at time, T, and T+x, where x=successive time points. Object area over time was translated into slope of object size and used to calculate the doubling time for individual microcolonies Specifically, FIGURE 4A illustrates the log of growth over time (hours). FIGURE 4B illustrates a histogram of doubling times as frequency vs. time (hours). FIGURE 4C illustrates the range of derived population doubling times, indicating that the population median doubling time was 23.9h with 1st and 3rd quartile range of 20.6-28.2h. These data indicate that the H37Rv cells exhibited the expected doubling times for this strain given the medium used. Accordingly, the present format permits normal culture growth and development of M. tuberculosis.

Next, the comparative growth rates of different M. tuberculosis isolates were compared. Using the general culture format described above, multiple wells of four clinical M. tuberculosis isolates representing diverse evolutionary clades were established and growth rates were observed over 96 hours. As illustrated in FIGURES 5A-5E, the rates vary in a strain-specific manner and corroborate with batch culture measurements previously observed. Lineage and strain identifiers referred to in FIGURES 5A-5E are detailed in Table 1. This provides further support that the present culture format permits normal culture of the target bacteria and does not significantly skew doubling or other developmental processes of the bacteria.

TABLE 1 : Linea e and strain identifiers indicated in FIGURES 5A-5E.

The broad range of replication rates described above suggested a cell-intrinsic variability in behavior from diverse M tuberculosis isolates. To assess the ability of this system to measure the impact of external factors that affect growth and viability, strain

H37Rv was exposed different frontline anti -tubercular antibiotics. Cells were treated with different antibiotics applied in multiple concentrations from 0.0625x to 8. Ox the batch culture MIC99 dose over a 90-100 hour time course and compared to untreated controls using the described multi-well format. As illustrated in FIGURE 6A, the individual wells represent individual experiments that receive a particular drug concentration so as to determine the minimum concentration that elicits an inhibitory effect (i.e., the minimum inhibitory concentration, or MIC). FIGURES 6B-6K are a series of graphs showing the average object size, corrected for TO size, for the population of microcolonies over time in hours (i.e., the size of the observed microcolony as an indicator of doubling in absolute relation to microcolony size at time of deposition) in response to different treatments of the antibiotic rifampin. FIGURE 6B is an aggregate illustration that overlays the trends observed for all concentrations of the antibiotic rifampin (Rif). FIGURES 6C-6K illustrate the growth curve for each Rif titration individually. Compared to untreated controls (FIGURE 6B) exposure to rifampin leads to a nearly complete blockade of biomass expansion, even at the lowest doses tested (0.0625x the batch culture MIC99). This blockade persists throughout the treatment series (up to 8. Ox the batch culture MIC99), and reveals a strikingly different response pattern compared to isoniazid exposure.

FIGURES 6L-6U illustrate a similar experiment performed with a series of titrations of the antibiotic isoniazid (INH). Here, the methodology followed that described for rifampin, and the final critical Growth Inhibitory Concentration (GIC) compared favorably to values obtained through traditional/slower methods. By following the population average microcolony size over time in response to increasing doses of isoniazid, a steady diminution in growth rate with higher drug concentrations was observed. Compared to untreated cells (FIGURE 6L, slope = 0.054), the rate of biomass expansion decreases marginally at the 0.0625x the MIC99 dose (slope = 0.050), and this decrease continues more markedly at a l .Ox dose (slope = 0.01). At supra-MIC99 treatment concentrations, there is essentially a cessation of biomass expansion.

Specifically, it is noted that the Growth Inhibitory Concentration ("GIC") reported in FIGURES 6B-6U are within 2.5 fold, which is a close recapitulation of the value inferred from more traditional methods of observation.

FIGURES 7 A and 7B illustrate that the endpoint of M. tuberculosis strain H37Rv microcolony size varies by drug concentration and type. Specifically, FIGURE 7A illustrates the median microcolony size at TO and T90h for cells exposed to increasing concentrations of rifampin (left graph) or isoniazid (right graph). As demonstrated in

FIGURE 7B, by creating a ratio of colony sizes at TO and T90h, a metric of growth inhibition can be derived for each drug, thus permitting comparisons of different drugs and doses under otherwise constant conditions. Furthermore, these growth characteristics of cells exposed to different concentrations of diverse antibiotics can be used to calculate the critical Growth Inhibitory Concentration (GIC) for each drug - an analog of the traditionally-defined minimum inhibitory concentration. In this application, the T90:T0h microcolony area ratio was used to derive a nonlinear regression best fit line through the data. Solving for the intersection at point "x" provides the GIC_X value. GIC₉₀, as demonstrated here, is the antibiotic concentration required to confer 90% inhibition of bacterial growth.

To further asses the viability of cells over time, a vital dye (specifically propidium iodide (PI)) was applied to the cells facilitate live vs dead cell imaging. Free PI is undetectable by light- and fluorescent-microscopy modalities described here. Viable cells exclude PI and keep it in its "free" form, whereas PI infiltrates dead cells as a result of lack of membrane integrity and is detected as a red fluorescent signal upon incorporation into nucleic acids. Thus, PI uptake can provide a measure of the number of viable to dead cells within the field of view. FIGURES 8A-8F illustrate the ratio of Pi-negative to Pi-positive cells (indicating the ratio of viable to dead cells) for M. tuberculosis strain H37Rv cells that have been untreated by any antibiotic (FIGURE 8A), or exposed to 0.5x the batch culture-derived minimum inhibitory concentration of isoniazid (FIGURE 8B) and rifampin (FIGURE 8C). These brightfield and fluorescent object sizes were then quantified as described above. FIGURE 8D-8F depict the quantitative and condition-specific amount and rate of PI uptake for untreated cells (FIGURE 8D), and cells treated with isoniazid (FIGURE 8E) and rifampin (FIGURE 8F).

Single CFU exposed to 1.8μΜ isoniazid or 0.061 μΜ rifampin (microcolony micrographs in FIGURES 8B and 8C, respectively) both exhibited marked decrease in replication compared to untreated control cells (microcolony micrographs in FIGURE 8A). Quantifying biomass expansion of these individual CFU demonstrated that untreated cells replicated with a doubling time of 16.8 hours. This single-CFU doubling time is faster than the H37Rv group median value reported above, and points to the spectrum of phenotypes present in a population of M. tuberculosis. Both isoniazid and rifampin exposure leads to a marked decrease in replication from the earliest points of drug exposure (FIGURES 8D-8F, left Y-axis), with doubling times for isoniazid- and rifampin-exposed cells were 49.3 & 51.4 hours, respectively.

Because the vital stain PI is included in these experiments, quantification was possible for both rate and magnitude of cell death under rich- or drug-treatment conditions. For example, cells grown in the absence of any antibiotic demonstrate a standard replication rate, but exhibited a -5% cell death phenotype during the time course (FIGURE 8D, right Y-axis). This observation that replication in a clonal bacterial population is associated with a measurable death rate raises interesting questions about both basic bacterial physiology, as well as effective means of measuring of fitness, and demonstrates the surprising power and sensitivity of this platform to assess heterogeneous behaviors of cells in parallel. Considering the drug treatment conditions, the nearly- complete cessation of biomass expansion was observed in response to antibiotic treatment was accompanied by antibiotic-specific rates of PI uptake. In the case of isoniazid, after -50 hours of decreased cell division rates we observed a spike in PI uptake in the microcolony (FIGURE 8E, right Y-axis). The increased uptake of PI is in keeping with the primary mechanism of action of isoniazid toxicity through inhibition of cell wall biosynthesis. Conversely, cells exposed to rifampin exhibit an almost immediate diminution in growth rate, but in general do not demonstrate uptake of PI, possibly owing to the RNA polymerase-specific mechanism of action of this antibiotic (FIGURE 8F, right Y-axis). Combined, these results point to the ability to distinguish different readouts of drug activity using the PMDF platform. In the case of both isoniazid and rifampin drug treatment results in almost immediate diminution in growth rate. While detection of Pi-derived fluorescence can be a useful proxy for M. tuberculosis viability, the drug- specific signal kinetics suggest that quantifying biomass over time is a more sensitive measure of drug susceptibility.

The results described above assess growth of M. tuberculosis under rich conditions, and in response to the frontline anti-TB agents isoniazid and rifampin over periods of 90+ hours. Next, additional TB drugs were assessed to determine the minimum time-to-detection of differential growth in response to these drugs. Mycobacterium tuberculosis cells were cultured in the presence of different concentrations of isoniazid, rifampin, ethambutol, and pyrazinamide (see FIGURES 9A-9B). Untreated M. tuberculosis grown in rich medium was used as a control, and for all drugs except pyrazinamide bacteria were dosed in 2-fold increments bracketing the drug-specific batch culture MIC99. Pyrazinamide was assessed at two supra-MIC₉9 drug concentrations. In these experiments biomass expansion was quantified for dozens to hundreds of individual CFU over time. The median slope of the population is reported for each condition. The slope of biomass expansion was large for untreated cells (indicating a faster doubling time) and approached 0 for cells exposed to the highest concentrations of antibiotic (indicating a drug-induced decrease in cell growth). By constraining the analysis window in progressively shorter blocks (12-hour steps) of a 90+ hour time course, the minimum time frame required was defined to make statistically significant drug susceptibility determinations (i.e., the point at which the population behavior of the treatment group irreversibly differentiates from the untreated controls). For all frontline antitubercular antibiotics, the expected decrease in growth rate attendant with higher concentrations of drug was observed, and for each antibiotic an unequivocal determination of susceptibility was made within 48 hours (FIGURES 9A-9D).

A potential advantage of tracking individual M. tuberculosis CFU is the direct, rapid, and sensitive quantification of phenotypes. In this work, the impact of drug treatment on M. tuberculosis replication rate is described, with particular attention to a decrease in replication and the potential impact that increased sensitivity and decreased time-to-detection might have in phenotypic drug susceptibility testing; however, over the course of these experiments a modest, but statistically significant (Kruskal-Wallis test with Dunn's post test), increase in growth rate of cells exposed to very low doses of cell wall-active antibiotics was also observed. For example, cells exposed to 0.225 and 0.45μΜ (sub-MICgg) of isoniazid had doubling times of 17.0 & 18.6 hours, respectively, as compared to the untreated control cells that exhibited a doubling time of 21.5 hours (FIGURE 9A). This observation suggests the counter-intuitive possibility that persistent exposure to low levels of isoniazid may induce an increase in M. tuberculosis replication. Accordingly, microcolony growth of M. tuberculosis exposed to isoniazid was measured in a titration series ranging from 0.03 lx to 2. Ox the batch culture MIC99 (corresponding to a range of 0.113 - 7.2μΜ). Cell growth was quantified as a ratio of median microcolony size at late:early time points. By this measure, a ratio of 1 indicates that microcolony area was unchanged over the course of the experiment, a ratio of <1.0 corresponds to a decrease in microcolony size (for example, through cell lysis or fragmenting of microcolonies), and a ratio of >1.0 indicates that microcolony size increased over the duration of the experiment. M. tuberculosis grown in rich media with no antibiotic exhibited a nearly 20-fold increase in microcolony size over a 4 day time course, corresponding to just over 4 cell doublings (FIGURE 10A, leftmost bar). As before, at concentrations equal to or greater than the MIC₉₉ a substantial isoniazid- induced diminution ofM tuberculosis replication was observed (FIGURE 10B, rightmost bars). Exposure to low doses of isoniazid, however, led to increased growth compared untreated cells (see "0.03 lx" bar of FIGURE 10A). Considering whether detection of this phenomenon was limited to the particular to the PMDF platform, and to assess if low level INH treatment can lead to a paradoxical increase other measures of cell viability, an independent experimental system to quantify ATP levels in batch culture was used.

M. tuberculosis cultures in 96-well plates were treated to a titration series of isoniazid ranging from 0.03 lx to 1.5x the batch culture MIC₉₉ (corresponding to a range of 0.113 -

6.0μΜ). After 96 hours of drug exposure, cells were lysed and ATP levels were recorded by Bac Titer-Glo (BTG) assay. By this measure also, an increase in signal was observed for cells exposed to sub-MIC₉₉ doses of isoniazid as compared to untreated cells

(FIGURE 10B). These results in aggregate suggest that compared to untreated control cells persistent long-term exposure to sub-MIC₉₉ doses of isoniazid leads to an increase in at least two measures ofM tuberculosis cell viability: growth rate and ATP pool size. This again illustrates the benefits and advantage of the disclosed approach to culturing and observing cells.

Discussion: The above investigation illustrates the development of a platform that facilitates quantification of growth from thousands of M. tuberculosis CFU exposed dozens of conditions in parallel. Growth characteristics of diverse M. tuberculosis lineages were investigated, and the response of strain H37Rv exposed to a range of concentrations of all frontline TB antibiotics was assessed. The inventors established that using growth rate as a measure of drug susceptibility allows the determination of sensitivity to all frontline anti -tubercular antibiotics in 48 hours or less. The inventors also observed that M tuberculosis exposed to sub-killing doses of isoniazid exhibited the paradoxical phenotype of increased growth rate. Such cultures also exhibited elevated levels of ATP in independent experiments. This work provides a platform for rapid and sensitive simultaneous growth determination for thousands of M. tuberculosis microcolonies in each experiment that elucidates novel phenotypes with potentially far- reaching clinical implications, as well as having the potential to increase the number of antibiotics interrogated and accelerate drug susceptibility testing.

The platform described in the present work (see, e.g., FIGURES 1 and 2) adds several capacities to the existing experimental landscape for characterizing cell growth and response to environmental factors. The number of conditions able to be interrogated in parallel is well-beyond what has been described for microfluidics platforms (e.g., at least 24 versus 8 or less), and as an extension, in any one experiment one is able to assess the growth of substantially more microcolonies and/or strains of target cells (e.g., of M. tuberculosis). Further, the inventors determined that for any one uniform condition they are able to maintain viable cells on the microscope stage for at least 7 days. An additional important feature of the disclosed approach is the ability to quantify cell growth of hundreds-to-thousands of cell (e.g., M. tuberculosis) CFU. This reflects an improvement over existing multi-well/fixed medium approaches that use as endpoints either a qualitative assessment of growth (as for MODS) or require longer time frames to distinguish phenotypes.

For example, as described above, cells were treated with a range of concentrations of frontline antibiotics and observed a rapid and dose-dependent decrease in growth rate of M. tuberculosis compared to untreated controls, particularly at and above the batch culture-defined MIC99 (see, e.g., FIGURES 6B-6U). This rapid diminution in growth rate allowed determination of susceptibility of M. tuberculosis to multiple drugs in 48 hours (FIGURES 9A-9D). ForM tuberculosis, this approach is at least 4-fold faster than current phenotypic drug susceptibility tests, yielding diagnostic information days-to- weeks before comparable results could be generated by traditional culturing methods. In addition, unlike the Xpert M. tuber culosisi xi test, the disclosed method can be applied to any drug and is independent of bacterial genotype. This has important potential clinical applications all for first- and second-line anti -tubercular antibiotics, but is perhaps nowhere more immediately significant than for pyrazinamide DST. Inclusion of pyrazinamide in combination drug regimens reduced the treatment duration from 9 to 6 months, and it remains an important pillar in current M. tuberculosis therapies, though based on the current landscape improved DST of pyrazinamide has been identified as a research priority by the WHO. The genotypic correlates of pyrazinamide resistance are not well understood and provide poor prognostic accuracy, and batch culture phenotypic testing for pyrazinamide susceptibility requires careful control of inoculum size and an acidic pH that can independently impact M. tuberculosis viability. Given these challenges it is significant, then, that in the present work the inventors identified conditions that support M. tuberculosis growth and simultaneously accommodate microscopic determination of pyrazinamide susceptibility, and provide time-to-detection in 48 hours or less (FIGURE 9D).

For all drugs tested in this demonstration, a dramatic decrease in growth rate was noted when cells were exposed to antibiotic concentrations at or above their respective MIC99. This approach to cell culture and microscopic analysis permitted an intriguing observation of the apparent increase in growth rate attendant with very low doses of isoniazid exposure (FIGURE 9A). To test this observation independently with additional experiments, cell growth was quantified microscopically (FIGURE 9A) and also measured ATP levels in small batch culture (FIGURE 9B). Independent of the readout, exposure to sub-MIC₉₉ levels of isoniazid resulted in increased M. tuberculosis biomass (measured either as microcolony size or elevated ATP levels compared to untreated controls). It is worth noting that the concentration of isoniazid treatment at which we observe these increases in signal differs between the two assays we deployed. It is possible that these measures detect different manifestations of a mechanism that culminates in a general growth advantage at sub-killing drug doses. Other studies using M. smegmatis have noted that an increase in intracellular ATP levels during antibiotic exposure is associated with elevated levels of ATP following drug washout, and is consistent with metabolic activity during antibiotic insult. The batch culture increase in ATP levels are consistent with this, and can be further characterized as a perturbation (increased ATP levels) to metabolic homeostasis at a moderate isoniazid dose. Using cell growth as the readout of drug treatment, an increase in growth rate attendant with very low doses of antibiotic was observed. A synthesis of these observations suggests that the altered ATP levels attendant with drug exposure (consistent with maintained metabolic activity) represents a rapid and transient perturbation of metabolic homeostasis. It is therefore possible that at sub-MIC₉₉ doses of isoniazid ATP cycling is broadly affected, and that at very low doses of isoniazid this perturbation is resolved through an increase in biomass expansion; however, at moderate doses of drug growth rate is kept in check - perhaps by the cell-wall mechanism of isoniazid - and a disequilibrium of increased ATP pools is manifest in the population of cells. The clinical implications of this observation, which is enabled by the disclosed platform, are highly significant. Variable bioavailability and drug penetration in granulomas suggest that antibiotic treatment within a single person is not monolithic, and that bacteria at different sites may be exposed to different (sub-killing) doses of drug. The impact of low-dose or transient exposure to isoniazid could fall along multiple lines. In one scenario, an uptick in growth rate and perturbation of metabolic homeostasis could prime the bacteria to be more susceptible to other antibiotics - a "one-two punch" combination that results in more effective combination therapies. Alternatively, increased growth rate could contribute to a poorer prognosis or increased transmission rates - a numbers game in which more bacilli translate to more opportunities for colonization within and across hosts. The reality is likely to be a complex and heterogeneous combination based on multiple factors, which can now be more readily discerned using the microscopic observation platform disclosed herein.

After decades of research and punctuated progress, tuberculosis disease remains a tremendous global health burden. The standard antibiotic interventions are generally effective against drug-sensitive M. tuberculosis, however, in the face of emerging drug resistant strains the current suite of diagnostics and treatment options are over-matched. The disclosed novel approach allows a better focus at the disease-relevant level of the single CFU and provides important advances in the understanding of the basic physiology of the target bacterium. This increased sensitivity can be leverage to improve diagnostic drug susceptibility testing speed and breadth. The parallel microscopic determination of fitness of M. tuberculosis efforts described in this work contribute to this landscape and have the potential to speed drug susceptibility testing, as well as reveal critical novel insights about bacteria, such as M. tuberculosis, a deadly human pathogen on a global scale. Summary: The present data show that the novel culture configuration provides a stable culture environment for bacteria, such as M. tuberculosis, that permits sufficient stability that the individual bacteria can be repeatedly observed at stable, predictable coordinates in the medium over an extended period. This configuration is amenable to multi-complex format, such as a multi-well format to permit a significant scale-up to assess myriad experimental conditions in parallel. Furthermore, the ability to observe and quantify the development of numerous individual CFU's for each experimental condition permits the rapid acquisition of informative data, thus compressing the time required to assess conditions, such as efficacy of an antibiotic on cells.

This novel approach described herein can be further optimized to enhance imaging applications, to address any antibiotic of interest, test compound, or chemical and to facilitate the efficient deposition of cells within the physical chambers. Such a system can be readily applied to assessing the susceptibility of cells isolated from an infected individual (such as from sputum samples) to assess the susceptibility of the isolates to an antibiotic and, thus, predict an optimal antibiotic regimen. Additionally, this strategy can be applied to co-cultures of host and pathogen cells to ascertain aspects of infection strategies and host responses.

It is generally noted that the use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

Following long-standing patent law, the words "a" and "an," when used in conjunction with the word "comprising" in the claims or specification, denotes one or more, unless specifically noted.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise," "comprising," and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, such as in the sense of "including, but not limited to." Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words "herein," "above," and "below," and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application. Words such as "about" and "approximately" imply minor variation around the stated value, usually within a standard margin of error, such as within 10% or in some cases 5% of the stated value.

Disclosed are materials, compositions, and components that can be used for, in conjunction with, and in preparation for the disclosed methods and compositions. It is understood that when combinations, subsets, interactions, groups, etc., of these materials are disclosed each of various individual and collective combinations is specifically contemplated, even though specific reference to each and every single combination and permutation of these compounds may not be explicitly disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in the described methods and components of the described chamber or system. Thus, specific elements of any foregoing embodiments can be combined or substituted for elements in other embodiments. For example, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed. Additionally, it is understood that the embodiments described herein can be implemented using any suitable material such as those described elsewhere herein or as known in the art.

Publications cited herein and the subject matter for which they are cited are hereby specifically incorporated by reference in their entireties.

While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A culture chamber for microscopic observation of one or more cells of interest maintained in a stable environment, comprising:

a solid housing comprising a base substrate that permits microscopic observation through the substrate; and

a solid or semi-solid growth medium layer disposed proximal to the base substrate to provide a stable culture space between the base substrate and growth medium layer; wherein the chamber is configured to receive one or more cells in the stable culture space and subsequently seal the stable culture space to provide a stable environment for maintenance of the cells.

2. The culture chamber of Claim 1, further comprising a sealable channel or valve configured to deliver cells into the stable culture space.

3. The culture chamber of Claim 1, wherein the solid substrate is re-sealably detachable from the housing and growth medium layer to permit delivery of the one or more cells and subsequent reattachment and sealing to provide the stable environment for maintenance of the cells.

4. The culture chamber of Claim 1, wherein the chamber is configured to receive an applicator to inject the one or more cells in the stable culture space.

5. The culture chamber of Claim 1, wherein the base substrate is an optically acceptable barrier.

6. The culture chamber of Claim 5, wherein the base substrate is plastic or glass.

7. The culture chamber of Claim 1, wherein the solid or semi-solid growth medium permits growth and/or development of the one or more cells of interest.

8. The culture chamber of Claim 1, wherein the culture chamber comprises, or is configured to receive, a coverslip over the solid or semi-solid growth medium layer on a side opposite from the solid substrate.

9. The culture chamber of Claim 1, wherein the culture chamber comprises, or is configured to receive, a liquid medium over the coverslip on a side opposite from the growth medium layer.

10. A method for microscopically observing one or more cells in a stable environment, comprising:

providing the one or more cells in a culture space disposed between an optically acceptable base substrate and solid or semi-solid growth medium layer;

sealing the stable culture space to provide a stable environment for maintenance of the cells; and

observing the one or more cells with a microscope through the optically acceptable base substrate.

11. The method of Claim 10, further comprising placing a coverslip over the growth medium layer on a side opposite from the one or more cells.

12. The method of Claim 11, further comprising placing liquid medium over the coverslip on a side opposite from the growth medium layer.

13. The method of Claim 10, wherein the one or more cells are observed two or more times to assess development and/or viability of the cells over time.

14. The method of Claim 10, wherein the one or more cells are bacteria, such as Mycobacterium tuberculosis.

15. The method of Claim 14, further comprising providing the one or more cells with an experimental environmental condition.

16. The method of Claim 15, wherein the experimental environmental condition is the presence of one or more potential antibiotics, such as isoniazid, ethambutol, rifampin, pyrazinamide, and the like.

17. A method of assessing viability and/or development of a cell in a stable environment, comprising

providing the one or more cells in a culture space disposed between an optically acceptable base substrate and solid or semi-solid growth medium layer;

sealing the stable culture space to provide a stable environment for maintenance of the cells; and

observing the one or more cells with a microscope through the optically acceptable base substrate.

18. The method of Claim 17, wherein the one or more cells are bacteria, such as Mycobacterium tuberculosis.

19. The method of Claim 17, further comprising providing the one or more cells with an experimental environmental condition.

20. The method of Claim 19, wherein the experimental environmental condition comprises an amount of one or more potential antibiotics, such as isoniazid, ethambutol, rifampin, pyrazinamide, and the like.

21. The method of Claim 20, further comprising assessing the susceptibility or resistance of the cell to the potential antibiotic or combination of antibiotics.

22. A system for microscopic observation of a cell, comprising:

a solid housing comprising a base substrate that permits microscopic observation through the substrate;

a solid or semi-solid growth medium layer disposed proximal to the base substrate to provide a stable culture space between the base substrate and growth medium layer; a microscope; and

a microscopy enabled imaging device.

23. The system of Claim 22, wherein the system is adapted to receive one or more cells of interest into the stable culture space and subsequently be sealed to provide a stable environment for maintenance of the cells. kit comprising a solid housing comprising a base substrate that permits microscopic observation through the substrate;

a solid or semi-solid growth medium layer adapted to be placed proximal to the base substrate to provide a stable culture space between the base substrate and growth medium layer, wherein the stable culture space is capable of receiving one or more cells to provide a stable environment for maintenance of the cells; and

instructions for use with one or more cells of interest.

25. The kit of Claim 24, further comprising a coverslip configured to be placed over the growth medium layer.

26. The kit of Claim 25, further comprising liquid medium.