WO2013155525A1

WO2013155525A1 - Ultra rapid blood culturing and susceptibility testing system

Info

Publication number: WO2013155525A1
Application number: PCT/US2013/036619
Authority: WO
Inventors: Gideon Eden
Original assignee: Biolumix, Inc
Priority date: 2012-04-13
Filing date: 2013-04-15
Publication date: 2013-10-17

Abstract

A method and device that significantly reduces diagnostic time associated with the detection of microorganism in a biological sample that combines (a) micro bacterial encapsulation with (b) ultraviolet light emitting diodes (UV LED) enhanced technology.

Description

ULTRA RAPID BLOOD CULTURING AND SUSCEPTIBILITY TESTING SYSTEM

BACKGROUND

[0001] The present disclosure pertains to methods and systems for blood culturing and susceptibility testing.

[0002] The state of art of bacterial blood infection detection and antimicrobial susceptibility testing dictates 4-7 testing days in which the patient may be in critical condition. Consequently the common practice is to treat potentially infected patients with wide spectrum of antibiotics, a practice which generates mutations of drug resisting bacteria that increasingly threaten the future welfare of the human race.

[0003] There are numerous medical diagnostic systems, mainly in hospitals, to detect the presence of microorganisms (bacteria, yeasts, and mold) in a patient's blood and provide anti microbial (antibiotic) susceptibility tests, in order to determine effective treatment for blood infection (Septicemia). The current state of art dictates that the results of these tests may not be available for days in which the patient may be in a critical clinical situation since no specific antibiotic treatment can be effectively administered. Consequently it is not uncommon to treat the patient with wide spectrum of antibiotics even before the test results are available. This practice can contribute to the development of drug resistant bacterial mutations. Such mutations that already affect global worldwide epidemics, and potentially present a threat to human welfare.

[0004] Faster testing technologies would be highly desirable. Faster new technology would enhance medical treatment, contribute to patient health and well-being and revolutionize the medical market place. In spite of this long-felt need, the older manual technologies as well as the (relatively) faster automated technologies cannot currently provide further time savings and result in shorter testing lag times. Without being bound to any theory, it is believed that these impediments are, at least partially due to measurement and analytical technologies that are dependent on current measurement principles which themselves are based upon blood culturing principles.

[0005] With existing fastest systems, it takes 2-5 days to determine the presence/absence of microorganisms in 10 milliliters of patient's blood. If the test is positive, at least one subsequent susceptibility test is typically performed on the cultured sample in order to determine the optimally responsive antibiotic. This may take additional 1-2 days for (a) growing colonies in Petri dish and (b) testing a matrix of antibiotics at various concentrations. Such susceptibility tests typically involve the use of a multi-well plate in which each well contains a mixture of specific antibiotics with the cultured microorganisms and growth media in which the material is further cultured to determine which antibiotic at which concentration effectively inhibits further organism growth.

SUMMARY

[0006] Disclosed herein is a method to detect the presence of living microorganism cells in a sample. The method includes the steps of enclosing at least one of the microorganism cells in a light transparent microdroplet. The microdroplet contains a mixture of liquid growth media and/or at least one fluorescent indicator dye capable of indicating the presence of at least one byproduct generated by growing microorganisms, wherein the indicator dye generates visible light when excited by ultraviolet light after exposure to the metabolic by-products. The method further includes the step of incubating the micro-droplet under conditions that promote rapid growth of the at least one microorganism and generation of metabolic by-products of said growth of the at least on microorganism and exposing the incubated microdroplet to a concentrated UV light emanating from a miniaturized UV light emitting diode. Visible light is generated from the at least one microdroplet and is indicative of the presence of at least one microorganism cell in the micro-droplet generating said metabolic by-products interacting with said fluorescent indicator.

[0007] Also disclosed is a device for detecting the presence of microorganisms in a sample. The device includes means for enclosing at least one microorganism cell in a light transparent micro-droplet. The micro-droplet also contains liquid growth media and at least one fluorescent dye capable of indicating the presence of by-products generated by growing microorganisms in which the indicator dye generates visible light when excited by ultraviolet light. The device also includes an incubator device configured to maintain the temperature of the micro-droplets at a value that promotes rapid microorganism growth and production of byproducts of metabolic growth together with at least one ultraviolet light emitting diode (UV- LED) paired with a detector configured to detect light in the visible spectrum. The UV-LED is configured to emit a concentrated beam of light in the UV spectrum. The device also includes means for introducing the at least one micro-droplet into the beam of concentrated UV light such that the UV light interacts with the fluorescent indicator to emit visible light.

DESCRIPTION OF THE DRAWING

[0008] In order to better understand the present disclosure the following drawing are included in which like reference numbers are used throughout the various views and figures, and in which: [0009] Figure 1 is a multi-stage diagram of an embodiment of the process and device as disclosed herein.

DETAILED DISCLOSURE

1. General Principles

[00010] In order to accelerate the entire culturing based detection followed by susceptibility tests from days to hours, several issues and limitations need to be considered and modified. These include but need not be necessarily limited to the following:

1. Decreasing the culturing/detection time of the organisms in the original blood

sample. With the current automated instruments (for example BacT- Alert, BacTek, VersaTREK), a threshold concentration of 10⁶ to 10⁷ CFU/ml is required in order to detect the presence of growing microorganisms. This threshold dictates culturing times of 1-2 days for fast growing organisms and up to 5 days for the slowest microorganisms.

2. Paralleling the culturing/detection process with the susceptibility tests. Currently, susceptibility tests can only start after the microorganism culturing process is complete and colonies are grown on a Petri dish to obtain sufficient concentration of the microorganisms. It is submitted that the incremental time can be reduced or eliminated with the new invention.

[00011] The new system is predicated on the unexpected discovery that two distinct technologies that have been developed for other distinct purposes and are demonstrated to be individually operational can be integrated into the method and device disclosed herein. The incorporation of the technologies in the device and method as disclosed herein will result in a new generation of rapid clinical tests that can save patients' life and reduce the growing risk of cultivating drug resisting bacteria. The one such technology is the technology related to gel micro-droplets. Variations on this technology have been developed and practiced since 1986. The additional technology pertains to miniaturization of Ultraviolet Light Emitting Diodes (UV LED). The unique combination of the two technologies in the device and method disclosed herein shall enable for the first time to break the severe delay limitation associated with the various current diagnostic procedures.

2. Gel Micro-droplets (GMDs)

[00012] Gel micro-droplets (GMDs) are spherical particles of gel that can be made by dispersing a liquid sample for example a polysaccharide such as agarose into an inert low dielectric fluid, such as mineral oil, and then cooling transiently. One such non-limiting example of a method for producing gel microdroplets is appended at the end of this disclosure and is incorporated by reference herein. Technology related to gel microdroplets is outlined in Weaver, et al., "Gel Microdroplets: Rapid Detection and Enumeration of Individual Microorganisms by Their Metabolic Activity" Pathology, Vol. 6, September 1986. The article was previously included as Attachment A and is incorporated by reference herein.

[00013] The article outlines a new, flexible method for rapid detection and enumeration of individual microorganisms using small (e.g. 10 to 100 micron diameter) gel particles surrounded by a non-aqueous liquid with low dielectric constant. Primary samples without prior cultivation can be used. In that study, gel microdroplets (GMDs) surrounded by an inert oil were statistically inoculated such that GMDs had a high probability of initially containing either zero or one acid-producing microorganism. Such GMDs retained dissociable metabolites produced by individual cells (or microcolonies) within the small GMD volume. The accumulated metabolic acids led to rapid changes in pH within GMDs initially occupied by one microorganism or colony forming unit (CFU), while GMDs with zero microorganisms had unchanged pH. The cumulative activity within individual GMDs was then determined using pH sensitive fluorescence indicators. This method was used to enumerate individual cell viability directly, without any prior culture, from clinically infected urine samples in about 1.5 hours for several rapidly growing pathogens, and was in agreement with much slower conventional culture methods. Because GMDs can be made readily in large numbers, and because many indicator systems can be used, GMDs used with automated measurement apparatus should have wide applicability.

[00014] Rapid detection and/or enumeration of viable microorganisms is of central importance to microbiological research, clinical microbiology, environmental science, food technology, toxicology and biotechnology. Two major classes or assays are used. The first class rapidly detects and identifies specific microorganisms directly from a primary sample, and is based on cell constituent assays, but does not determine cell viability. The most widely used in this class are specific ligand binding assays, particularly immunoassays and genetic probes ^5>8"10. However, these do not distinguish between dead and viable cells, do not provide an enumeration, but instead emphasize identification. For these reasons their microbiological use is restricted to determinations in which direct assessment of the physiological state of the microorganism is irrelevant.

[00015] The second class of assays is used for detection and/or enumeration of viable cells, either directly from the primary sample, or from a subculture of the primary sample. The most traditional and widely used method is the plate count, which allows determination of individual cell viability under many test conditions 7 ' 11. An important attribute of viable plate enumeration is that the count is independent of the concentration of the microorganism in the sample, because formation of each colony proceeds from an initial individual cell. The major disadvantage, which is addressed by the present gel microdroplet (GMD) method, is its slowness: typical determinations require one-half to several days, and are also labor and materials-intensive.

[00016] Instrumented technologies for rapidly determining cell or culture viability have been developed that partially address some of the limitations of the viable plate assay, but these instrumented methods generally require a prior culture and isolation from the primary sample, in order to first obtain a monoculture. These instrumented methods include optical techniques such as those that measure the light scattering properties of a culture¹² , metabolic activity based techniques such as those that measure overall changes in pH, carbon dioxide release, electrical impedance, chemiluminescence or fluorescence. A disadvantage of all of these metabolic activity methods is that they are based on the combined effects of a large, unknown number of cells, and therefore do not actually yield a count. A further disadvantage is that prior culture is generally required, which yields the isolates whose activity is actually measured. Finally, these total population methods exhibit detection times which become significantly longer as the sample's cell concentration decreases.

[00017] New methods using GMDs have the potential to rapidly yield an enumeration of individual cell viability directly from a primary sample with no prior cultivation. GMDs are approximately spherical particles of gel that can be made conveniently by dispersing a liquid agarose sample into an inert low dielectric constant fluid, such as mineral oil, and then cooling transiently. Here agarose was used, but other gel materials can also be used. The resulting GMDs typically have diameters ranging from 10 to 100 microns, with corresponding volumes of

which are exceedingly small. Microorganisms can be readily incorporated into the matrix of GMDs as they are made. Many of the GMDs contain initial individual microorganisms where encapsulated in gel or oil, they rapidly accumulate

extracellular products of individual viable cells, and are used with optical indicators for rapid determination of individual cell viability.

[00018] This method combines the rapidity of total population activity approaches with the ability to detect and enumerate viable cells independently of the sample cell concentration. Both the speed and independence of cell concentration result from the basic but simple principle that GMDs confine initial individual cells within a small volume, VGMD- By surrounding the GMDs with an inert oil, hydrophilic compounds such as pyruvate and other com-GMDs, containing metabolically active cells, and therefore accumulate metabolites, can be differentially detected (using a fluorescence indicator system) compared to GMDs that do not contain cells. A similar approach has allowed detection of individual β-galactosidase-containing bacteria²⁵'²⁶, or even of individual enzyme molecules using a fluorescence assay for this enzyme's activity. When the inoculum concentration, and therefore the average initial occupation of GMDs is low (e.g. less than 30%), there is a high probability of occupation by zero or one cell. This allows an enumeration based on individual cell viability counting the number of occupied and non- occupied GMDs.

[00019] There are two major advantages of using cells immobilized in GMDs. First, a cell entrapped within a GMD (or liquid microdroplet) surrounded by a low dielectric fluid

corresponds to an effective high cell density, which is inversely proportional to the GMD with VGMD- F°^r example, a 40 micron GMD with V_GMD = 3 x 10⁸ ml has an effective cell concentration of about 3 x 10⁷ cells/ml which initially occupied by an individual

microorganism. This effective high cell concentration makes determination within GMDs very rapid. Second, the gel matrix makes a microdroplet physically robust, increasing microdroplet stability and allowing physical manipulation.

[00020] In simplest terms, the use of GMDs consists of a rapid sub-division of a macroscopic sample containing cellso into many robust microscopic subvolumes (the GMDs). If the subvolumes are sufficiently small, many have a high probability of initially containing zero or one cell. For cells randomly distributed in a sample, the fraction P(n, n) of GMDs containing n cells depends on both the cell concentration and the volume of the GMDs, and is described by the Poisson distribution.

where n is the average number of cells initially entrapped in a particular size GMD. More specifically, if the cell concentration is the average number of cells initially entrapped in a particular size GMD. More specifically, if the cell concentration is p, the average number of cells in that Thus, the probability of unoccupied GMDs (zero initial

cells) is

and the probability of individually occupied GMDs (one initial cell) is

Further, the probability of initial multiple occupation (two or more initial cells) is

for small n and is small if V_GMD is somewhat smaller than 0.15/p. For budding and naturally aggregated microorganisms, "colony forming units" or CFUs, rather than individual cells, are the entities governed by Poisson statistics.

[00021] Detection of extracellular changes within GMDs due to individual cells or resulting microcolonies can be accomplished by use of fluorescent or colorimetric indicators. In general, extracellular assay of common biochemical parameters such as pH, metabolites or enzyme activity all should be suitable. This study demonstrated that the use of GMDs, a fluorescence pH indicator system and fluorescence microscopy allowed a viable count to be rapidly estimated by: (1) choosing GMDs in a particular size (V_GMD) range, (2) counting both the number of GMDs that exhibit a fluorescence color change and the number that does not change color in this size range, and (3) applying Poisson statistics. Although microscopy was used to illustrate basic principles, it is expected that automated measurements and analyses such as flow cytometry will be feasible, and more accurate and convenient.

[00022] The resulting GMDs as employed herein typically have diameters from 10 to 100 microns, with volumes of

It is envisioned that the targeted or tested microorganisms in the patient's sample can be incorporated into the matrix of the GMDs as the droplets are made. Alternately, the original patient sample can initially be mixed with growth media and pre-incubated for several hours in order to increase the bacterial population after which the mixture can be incorporated into GMDs. Initially, many of the generated GMDs will each contain a few individual microorganism cells surrounded by growth media. In addition to the gel forming material and microorganism cells, fluorescence indicating substrate can be incorporated in the mixture.

[00023] When the inoculums' concentration is low, the average initial occupation of GMDs is low, resulting in occupation by zero or perhaps one or a few cells in some of the microdroplets that are formed. It is understood that some of the micro-droplets formed will be unoccupied. This allows an enumeration based on individual cell viability that can be accomplished by counting the number of bacteria occupied and non-occupied GMDs after an additional short incubation time. One significant advantage of the gel micro-droplet entrapment of a microorganism cell or cells is the very high internal microorganism concentration in spite of the low actual numerical count. Microorganism concentration is inversely proportional to the

GMD volume. For example a GMD with 3x10 -^"8 ml can have an effective microorganism concentration of 3x10 CFU/ml although the GMD is occupied by a single microorganism cell. This concentration is high enough to generate sufficient concentration of metabolites to change the fluorescing properties of the embedded substrate within a few additional generation times of further incubation. The incorporation of fluorescent dye substrates enables detection of cell- occupied GMD's by utilizing an external light source that will penetrate the transparent wall of the GMD. If UV light is employed, the UV light will interact with the fluorescent substrate present in the GMD to generate visible light in the cell-occupied GMD; while no visible light is generated in non-cell-occupied GMDs. Consequently, with ultraviolet light source illumination, only the "viable" (cell- occupied) GMDs "glow in the dark" (emit detectable light) and can be easily identified and counted after very short incubation period such as a period of a few hours.

3. The Role and Function of Ultraviolet Light Emitting Diodes (UVLED)

[00024] While the GMD technology outlined herein is capable of ultra-fast detection of growing microorganisms, it cannot by itself provide a complete and practical system.

Microscopic observations of the GMDs, as recited in the article at Attachment A, are not considered practical in many situations, especially when large matrices of samples are tested. As disclosed herein optical instrument with appropriate sample "cartridge" is required to provide rapid and reliable detection and enumeration of the cell occupied GMDs, and to determine the antimicrobial or antibiotic susceptibility of the tested microorganisms.

[00025] In the last decades the general technology of light emitting diodes has

significantly progressed, and in particular, that related to ultraviolet LED technology. First, the ultraviolet spectrum has been successfully incorporated in the long UV range which permitted replacement of traditional gas discharge lamps for the first time. These older devices had an inherent limitation of repeatability. Every time a device such as a gas discharge lamp is switched on, the light level transient is not sufficiently repeatable. Consequently, the light intensity measured after fixed predetermined time period after switching may not be identical. This renders gas discharge lamps inadequate for precise fluorescence measurements. Since LED's are not based upon gas discharge but upon solid state energy levels, they are very repeatable and stable. They have also inherent longer shelf life and are becoming price effective.

[00026] The second advantage of the LED technology is the possibility to manufacture multiple miniaturized units on a single solid state chip. The most dramatic development was the replacement of the TV screens from Cathode Ray Tube (CRT) to screens constructed from LED pixels. Several commercially available arrays of UV LED are illustrated in Attachment B, the disclosure of which is incorporated by reference herein. It is contemplated that such devices can be further customized to operate in conjunction with the new invention as disclosed herein.

[00027] To further illustrate the invention as disclosed and claimed, the following discussion is presented referring to Figure 1 which illustrates an embodiment of the device and methodology disclosed herein. It is illustrated in stages, each describing a serial step, associated with newly designed devices and methods.

[00028] Stage 1: In this step a sample is obtained by any suitable method. In situations where the sample to be cultured is a blood sample, the blood sample is drawn by any suitable method. For example, the blood sample may be drawn, in a manner similar to those currently employed for blood culturing. In specific applications of the method disclosed herein, the sample may be introduced into a suitable container that includes suitable microbial growth media. Where desired or required, the container can also include at least one fluorescing indicator substrate that is capable of interacting with metabolic products of growing

microorganisms to produce a detectable light emission upon exposure to ultra violet light.

Preferably the indicator substrate is activated by ultraviolet light and fluoresces in the visible spectrum. There are several such specific indicator substrates that each can indicate the presence of a specific known microorganism. [00029] In specific embodiments, a sample such as a blood sample can be directly drawn into a suitably configured container. The suitably configured container can be equipped with suitable material such as growth medium and/or indicator substrate(s) such as material(s) that reacts with by-products of metabolic processes of microorganisms that may be present in the introduced sample. This drawing bottle may have vacuum in its head space to allow a direct blood draw of predetermined volume if desired or required.

[00030] Stage 2: The drawing bottle or other suitable container containing the mixture of the blood sample, the growth media and/or the indicator substrate is placed into an incubator device that maintains the resulting mixture at an optimal temperature for bacterial growth. To accelerate the process of bacterial growth, the vessel may be shaken or otherwise stirred to enable efficient oxygenation of the liquid. It is understood that the incubation time may differ for different kinds of organisms that may be present in the sample. For very fast growing organisms (for example, microorganisms exhibiting 20-35 minute doubling times) relatively short incubation time (for example 2-3 hours) should provide a sufficient number of cells in the liquid. For slower growing organisms, longer incubation times may be required. Since it is unknown what types of organisms are present in many particular given situations, several mixtures can be incubated for distinctive durations of incubation time.

[00031] Stage 3: In this stage microorganisms in the sample are encapsulated in GMDs by a suitable method and the GMD-encapsulated microorganisms are introduced into a suitably configured reading cartridge.

[00032] Encapsulation can progress by a suitable method. In one such method, a portion of the previously incubated mixture is drawn from the bottle or other suitable container via a line that flows through a microdroplets generation station. The generated droplets can encapsulate microorganisms as they flow by. The flow rate and the generation rate of the droplets are set so that each individual droplet may encapsulate 0-3 microorganisms. It is understood that the majority of the droplets may remain empty depending upon the microbial concentration in the sample after the incubation stage.

[00033] One non-limiting example of a suitable reading cartridge is a device that comprises a flat container defining a well or suitable sampler receiving area capable of containing a thin layer of liquid (for example, a device that receives sample to a width or depth of 0.2-0.5). A suitable device will be one having a sample receiving area that is transparent to UV light. It is understood that at this stage, one or more reading cartridges can be prepared. For example, at this stage similar reading cartridges can be filled, with some of the cartridges containing droplets with additional anti microbial agents (antibiotics) of different chemical compounds at various concentrations. These agents can be introduced by the same

injector/mixer station during the sample's flow through the station in certain embodiments.

[00034] Stage 4: In this stage each reading cartridge is incubated for predetermined time to allow several new bacterial generations to form. Within a few generations of growth cycles, the microorganisms contained in a respective microdroplet will exceed the concentration detection threshold, and the metabolites produced as a result of microorganism activity will interact with the fluorescent indicator substrate.

[00035] Stage 5: After incubation has been completes\d, the specific reading cartridge is placed into an optical reader that can identify and enumerate fluorescing micro-droplets. The optical reader as disclosed herein incorporates a specially designed array of UV pixels that can be activated one at a time. This UV LED screen provides illumination, one pixel at a time, to the transparent section of the reading cartridge. A photo detector with a wide reception area is placed on the other side of the reading cartridge. Non-limiting examples of suitable photo detectors include either a wide area Photo Multiplying Tube (PMT) or a sensitive CCD camera. A UV filter can be placed between the cartridge and the photo detector to prevent direct UV light from activating the photo detector.

[00036] When a UV pixel generated light impinges upon a micro droplet containing living organisms, visible light is generated by the fluorescence substrate, passes through to UV filter and sensed by the photo detector. By activating each of the screen pixels individually, the system enumerates how many micro droplets contain living cells are present, indicating the microorganisms' concentration in the associated sample.

[00037] Stage 6: If no fluorescing droplets are detected, the sample is negative, and no further action is required at this point in time (excluding repeat of the same test for longer incubated samples of Stage 2). If this (reference) sample is positive, the corresponding cartridges containing the anti-microbial agents as discussed previously are tested as well.

[00038] During the second incubation outlined in Stage 4, at least a portion of the encapsulated organisms can be exposed to the inhibitory action of the antimicrobial agent introduced into the micro-droplet. Consequently if the antimicrobial agent inhibits bacterial growth, no micro-droplet will fluoresce. The reading cartridge exhibiting an absence of or the least number of detected cells contains the effective antimicrobial agent and provides data regarding the antibiotic material and concentration effective to treat the patient. This

determination can be made simultaneously with the detection of the presence of the

microorganisms. For fast growing microorganisms these results may be available in several hours. For slower growing microorganisms, results may be available during the second day. The original sample can be further incubated to provide identification (ID) of the organisms if further diagnostic steps are required.

While the invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not to be limited to the disclosed embodiments but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims, which scope is to be accorded the broadest interpretation so as to encompass all such modifications and equivalent structures as is permitted under the law.

Claims

What is claimed is:

1. A method for detecting the presence of living microorganisms in a sample comprising the steps of:

enclosing at least one microorganism cell in a light-transparent microdroplet, the microdroplet containing at least one growth media material, fluorescent indicator dye, or a mixture of at least one growth media material and at least one fluorescent indicator dye, the indictor dye capable of indicating the presence of metabolic by-products generated by growth of the at least one microorganism cell enclosed in the microdroplet, wherein the indicator dye generates visible light when excited by an external light source;

incubating the microdroplet at a specific temperature selected to promote rapid growth of the at least one microorganism cell and generation of metabolic byproducts of said growth;

exposing the incubated microdroplet to a concentrated UV light emanating from a miniaturized UV light emitting diode; and

detecting visible light generated from said at least one microdroplet, the visible light indicating the presence of at least one microorganism cell in the micro-droplet generating said metabolic by-products interacting with said fluorescent indicator.

2. The method of claim 1 further comprising the steps of

generating at least one second micro-droplet that further contains at least one antimicrobial agent;

incubating the second microdroplet under conditions similar to the incubation of the first microdroplet; exposing the incubated second microdroplet to the concentrated UV light emanating from a miniaturized UV light emitting diode; and

noting an absence or presence of visible light emanating from the second micro- droplet, indicating the effectiveness of the anti-microbial agent to inhibit the growth of the microorganisms

3. The process of claim 1 or 2 wherein the sample is a biological fluid.

4. The process of claim 1 or 2 wherein the biological fluid is blood.

5. The process of claim 1 or 2 wherein the biological fluid is blood and wherein a plurality of microdroplets are introduced to at least one cartridge prior to incubation.

6. The process of claim 1 or 2 wherein the biological fluid is blood and wherein a plurality of microdroplets are introduced to at least one cartridge prior to incubation. The process further comprising the step of introducing the incubated cartridge to a cartridge reader. The cartridge reader having a photo detector and a UV-LED screen illuminating single pixels in a fast illumination sequence.

7. The process of claim 6 wherein the photo receptor is one of a wide-area photo detector or a photo multiplying tube.

8. A device for detecting the presence of microorganisms in a sample comprising: means for enclosing at least on microorganism cell present in the sample in a light transparent microdroplet, wherein the microdroplet also contains at least one of growth media, at least one fluorescent indicator dye, or a mixture of at least one growth media and at least one fluorescent indicator dye, the fluorescent indicator dye capable of indicating the presence of by-products generated by growing microorganisms in which the indicator dye generates visible light when excited by ultraviolet light; an incubator device configured to maintain the temperature of the microdroplets at a value that promotes rapid microorganism growth and production of by-products of metabolic growth;

at least one ultraviolet light emitting diode (UV-LED), configured to emit a concentrated beam of light in the UV spectrum;

means for introducing the at least one microdroplet into the beam of concentrated UV light such that the UV light interacts with the fluorescent indicator to emit visible light; and

a detector configured to detect light in the visible spectrum paired with the ultraviolet light emitting diode.

9. The device of claim 8 wherein the means for introducing the at least one microdroplet into the beam of said concentrated light is a reader cartridge.

10. The device of claim 9 wherein the cartridge reader has at least two layers configured to overlay the reader cartridge:

a first later that includes a photoreceptor and a second layer that includes a UV- LED screen. The UV-LED screen configured to illuminate individual pixels in a fast sequence.