Automated methods and systems for obtaining and preparing microbiological samples for both identification and antibiotic susceptibility testing

Abstract

To provide efficient and automated methods and systems that obtain a sample of microorganism from a colony and subject the obtained sample to multiple tests.SOLUTION: A method comprises an automated step of locating and selecting a colony of microorganisms on a culture dish, an automated step of obtaining a sample of the selected colony of microorganisms, and an automated step of automatically preparing a suspension of a sample of microorganisms by submerging in the suspension a pick tool having the sample, subsequently, the pick tool being vibrated in at least the vertical direction to release in the suspension the sample from the pick tool. An apparatus and a system optionally enable downstream processing of samples prepared for antibiotic susceptibility testing (AST).SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] This application claims the benefit of the filing dates of U.S. Provisional Application No. 62 / 167,577, filed May 28, 2015, U.S. Provisional Application No. 62 / 318,494, filed April 5, 2016, U.S. Provisional Application No. 62 / 167,593, filed May 28, 2015, and U.S. Provisional Application No. 62 / 269,545, filed December 18, 2015, the disclosures of which are incorporated herein by reference. [Background technology]

[0002] Methods and systems for locating, selecting, and identifying microbial colonies using mass spectrometry, particularly Matrix Assisted Laser Desorption and Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS), and systems for implementing the same are known. Such systems and methods are described in WO 2013 / 147610 to Botma et al., the disclosure of which is incorporated herein by reference.

[0003] MALDI analysis is a useful tool for solving structural problems in biochemistry, immunology, genetics, and biology. Samples are ionized in the gas phase, and a time-of-flight (TOF) analyzer is used to measure the ion mass. TOF analysis begins with ions being formed and accelerated to a constant kinetic energy as they enter a drift region. Ions reach the detector after a flight time proportional to the square root of the ion mass. Because ions of different masses arrive at the detector at different times, a mass spectrum is produced.

[0004] Mass spectrometry in general can be a powerful tool in the fields of drug discovery and development, genotyping, and proteomic research. MALDI, a specific type of mass spectrometry, has already been used for the characterization and identification of bacteria and microorganisms. The current research trend is to analyze an ever-increasing number of samples using individual sample quantities ranging from micromolar to atomic mole levels. As a result, samples are also becoming smaller, and a need exists for efficient and reliable collection of the correct amount of microorganisms and precise deposition of the collected amount of sample onto the target plate used in the MALDI instrument.

[0005] In a typical MALDI TOF MS operation, the sample to be analyzed is spotted or deposited onto a MALDI target plate, which may be a metal or other material that will allow for ionization of the sample. A commonly accepted method for preparing a MALDI target plate is to spot or smear the sample suspected of containing microorganisms directly onto the target plate from a plate medium. After sample application, a matrix reagent is often added to support sample ionization. In some cases, an extraction reagent is added as well. In other cases, an offline extraction step may be required before the sample is applied to the target plate.

[0006] Once the target plate is prepared, it is positioned at a fixed location within the MALDI instrument. The target plate has multiple deposition spots (e.g., 24-384 deposition spots on a single target plate), which have a fixed orientation with respect to the edge of the target plate. The target plate is positioned on an XY stage so that acquired samples of microbial colonies can be deposited onto selected deposition spots. A high-voltage potential is maintained between the target plate and a metal grid. This voltage can be maintained or pulsed, depending on the desired result, and a vacuum is created within the chamber. A laser is fired into the sample / matrix, forming a plume of ions. The voltage difference is used to accelerate the ions into a flight tube so that they can be analyzed. Analysis directly relates the time-of-flight to the mass of the ionized components.

[0007] Several parameters can affect the quality of the results, including target flatness, amount and type of matrix, concentration of sample, conductivity of the sample target, placement accuracy on the deposition spot, and other variables.

[0008] Because the process requires picking colonies and depositing them directly onto a plate, the picked sample cannot be used as a sample source for other analyses. As a result, if it is desired to perform another test on the sample, another portion of the sample must be taken to perform the test. Because multiple colony picks are required for multiple tests, there is an increase in the processing time required and the potential for conflicting results due to differences between the two picked samples. Therefore, there continues to be a need for an automated and efficient method and system for obtaining microbial samples from colonies and subjecting the obtained samples to multiple tests. Summary of the Invention [Problem to be solved by the invention]

[0009] To solve at least one of the problems set forth above, the present invention provides an automated method and system for locating and selecting colonies of microorganisms on culture plates and identifying the microorganisms within the selected colonies using MALDI and at least one other test. [Means for solving the problem]

[0010] The method includes the automated steps of locating and selecting colonies of microorganisms on a culture dish, acquiring a sample of the selected colonies of microorganisms, preparing a suspension for the acquired sample, dispensing a portion of the acquired sample onto a target plate and placing the target plate in an apparatus for performing MALDI for identification of the microorganisms in the selected colonies of the sample, and using or transferring another portion of the suspension for another test. In one embodiment, the second test is an antibiotic susceptibility test (AST). The AST can be performed using an existing automated AST method (BD Phoenix or Vitek), or can be Kirby-Baur / disk diffusion, disk dilution, broth dilution and agar dilution, or other methods.

[0011] In one embodiment, a suspension is prepared in a cuvette. The suspension in the cuvette is tested using a nephelometer to determine whether the turbidity of the sample is within a predetermined range of values ​​determined to be suitable for MALDI testing. If not, the amount of sample or the amount of diluent in the suspension is adjusted to provide a suspension with a target turbidity value. Once an aliquot of the suspension is removed from the MALDI cuvette, the suspension is tested again, and the turbidity of the suspension is determined by the nephelometer. This time, the turbidity of the suspension is evaluated to determine whether the turbidity is within a range of turbidity values ​​suitable for using the sample in a second test (e.g., an AST test). If not, the amount of diluent in the suspension is adjusted to provide a suspension with the appropriate turbidity.

[0012] All steps are performed automatically, which, to a large extent, eliminates the problems mentioned above, because automation avoids unwanted variations and mistakes that result in inaccurate results from the MALDI instrument, additional costs, and lost time. By automating each of the steps, these problems can be overcome, at least to a large extent. It has been taken for granted in the art that at least some of the steps can be performed only manually. In contrast, however, the present invention offers, for the first time, the possibility of automating all of the steps necessary to locate and select microbial colonies and identify the microorganisms within the selected colonies using MALDI.

[0013] By fully automating the preparation of the suspension, the present invention provides an accurate and reproducible method for using the suspension for MALDI identification and AST or other testing. The method further includes an automated step of overlaying an aliquot of MALDI matrix solution onto the dispensed sample suspension on the target plate. In some embodiments, the dispensed sample suspension deposited on the target plate is allowed to dry before the aliquot of MALDI matrix solution is overlaid. Further embodiments will include spotting an extraction reagent, such as formic acid, prior to the matrix reagent for improved results.

[0014] This alternative method using a suspension is even more significantly useful when additional tests or analyses are to be performed on the microbial colony sample. Such additional analyses may be accomplished in a particularly reproducible and efficient manner in one embodiment of the method according to the present invention, which further includes the automated steps of obtaining a second aliquot of the sample suspension, depositing the second aliquot of the sample suspension into broth for AST testing, and transporting the inoculated AST broth tube to an instrument for performing susceptibility testing or another additional analysis. Consequently, the method of the present invention may be used to automatically obtain or pick samples that can be fed into available ID / AST instruments, including, but not limited to, BACTEC™, Phoenix, MGIT, VITEK, and BacT / Alert.

[0015] A fully integrated embodiment of the automated method includes the above-described steps combined into a single process flow. In particular, a stage for a culture dish carrying microorganisms is provided. The culture dish is positioned on the stage. An automated pick tool is provided having an automated positioning device with a pick tool holder for holding a pick tool (e.g., a pipette). The positioning devices are arranged to position the pick tool at a start position above the culture dish, automatically lower and lift the pick tool toward and away from the culture dish, and position the pick tool at a transfer position. The pick tool is positioned within the pick tool holder of the positioning device. The pick tool is placed at a start position above the culture dish and automatically lowered toward the culture dish in contact with the microorganisms to pick up a sample of the microorganisms. The pick tool, carrying the sample of the microorganisms, is automatically lifted from the culture dish to the transfer position. An automated suspension medium dispenser is provided for automatically dispensing suspension medium into a suspension tube held in the suspension tube holder. The automated dispenser automatically dispenses an initial amount of suspension medium into the suspension. The positioning device automatically moves the pick tool from above the culture dish to a position above the suspension. The positioning device lowers and lifts the pick tool toward and away from the suspension medium contained in the suspension tube, respectively, and optionally positions the pick tool at a waiting position above the suspension tube. The positioning device oscillates the pick tool in a linear vertical motion for a period of time while the pick tool with the microorganism sample is immersed in the suspension medium. After the period has elapsed, the pick tool is lifted away from the suspension medium contained in the suspension tube to a waiting position. A turbidity meter (also referred to herein as a nephelometer) is provided for performing measurements of the turbidity of the suspension medium contained in the suspension tube held in the suspension tube holder. After at least the period of time during which the pick tool oscillates has elapsed, the turbidity of the suspension medium contained in the suspension tube held in the suspension tube holder is measured by the turbidity meter, and a final measurement value indicative of the measured turbidity is provided.

[0016] A controller is communicatively connected to the positioning device, the transfer device, the automated suspension medium dispenser, and the turbidity meter to automatically control the movement of the positioning device, the movement of the transfer device, the operation of the automated suspension medium dispenser, and the operation of the turbidity meter, respectively. The controller operates to control and monitor the suspension, as described above, and to provide a suspension having a turbidity within specifications.

[0017] The present invention further relates to an apparatus for the automated preparation of a suspension of a sample of microorganisms for carrying out the above-described method of automatically selecting colonies of microorganisms on a culture dish, preparing a suspension of the sample of microorganisms, and using the suspension to test for at least both microbial identification and antibiotic susceptibility, the apparatus comprising: a stage for a culture dish carrying microorganisms; a positioning device having a pick tool and a pick tool holder for holding the pick tool, the positioning device being arranged to position the pick tool at a start position above the culture dish, to automatically lower and lift the pick tool towards and away from the culture dish, and to position the pick tool at a transfer position, respectively; a suspension tube station for holding a suspension tube; an automatic suspension medium dispenser for automatically dispensing suspension medium into suspension tubes held within the suspension tube station; a positioning device for automatically transferring the pick tool from a transfer position of the positioning device to a position above the suspension tube held in the suspension tube holder, for lowering and lifting the pick tool towards and away from the suspension medium contained in the suspension tube, and for positioning the pick tool at a standby position above the suspension tube held in the suspension tube holder, respectively, the transfer device being further arranged to oscillate the pick tool in a linear vertical motion for a period of time; a turbidity meter for performing measurements of the turbidity of the suspension medium contained in the suspension tube held in the suspension tube holder and for providing a final measurement value indicative of the measured turbidity; a controller communicatively connected to the positioning device, the transfer device, the automatic suspension medium dispenser, and the turbidity meter, and configured to automatically control movement of the positioning device, movement of the transfer device, operation of the automatic suspension medium dispenser, and operation of the turbidity meter, respectively; Equipped with.

[0018] The controller is a) determining whether the final turbidity measurement value is greater than a first threshold value (maximum value) pre-stored in the memory of the controller, and if yes, the controller is configured to perform step b) (dilution); or determining whether the final turbidity measurement value is less than or equal to the first threshold value and greater than or equal to a second threshold value (minimum value) pre-stored in the memory of the controller (the first threshold value being greater than the second threshold value), and if yes, the controller is configured to perform step c) (acceptable turbidity); or determining whether the final turbidity measurement value is less than the second threshold value, and if yes, the controller is configured to perform step d) (concentration); b) controlling the automatic suspension medium dispenser to supply a further amount of suspension medium into the suspension tube; c) providing a signal that the suspension tube with the suspension may be removed from the suspension tube holder for further processing; d) Positioning a further pick tool in the pick tool holder of the positioning device in the manner described for the first pick tool.

[0019] In a further embodiment of the apparatus according to the invention, the controller is arranged to control the turbidimeter such that measuring by the turbidimeter the turbidity of the suspension medium contained in the suspension tube held in the suspension tube holder is initiated before the pick tool is immersed in the suspension medium contained in the suspension tube.

[0020] In an advantageous embodiment of the apparatus according to the invention, in step d), the first pick tool is provided as a further pick tool, and the controller is arranged to control a transport device for positioning the further pick tool in the pick tool holder of the positioning device.

[0021] Preferably, the controller is arranged to determine a further amount of suspension medium based on the initial amount of suspension medium, the final measured value, and the first and / or second threshold values, In particular, the controller is arranged to control the automated suspension medium dispenser in the manner set out above.

[0022] In a fully automated device according to the invention, when the apparatus includes an automated culture dish positioning and unloading device for automatically positioning and unloading culture dishes containing microorganisms onto and from the stage, respectively, the controller is communicatively connected to the automated culture dish positioning and unloading device and is arranged to control the operation of the automated culture dish positioning and unloading device and to automatically position the culture dishes containing microorganisms onto the stage; when the apparatus includes an automated suspension container positioning and unloading device for automatically positioning and unloading suspension containers into and from the suspension container station, respectively, the controller is communicatively connected to the automated suspension container positioning and unloading device and is arranged to control the automated suspension container positioning and unloading device and to automatically position the suspension containers into the suspension container stations. In this case, it is preferred that the controller is arranged to subsequently allow the culture dishes to be automatically removed from the stage by the automated culture dish positioning and unloading device only after a signal has been provided that the suspension container with the suspension may be removed from the suspension tube container station for further processing. Furthermore, the controller is preferably arranged so that only after receiving a signal that the suspension container with the prepared suspension therein may be removed from the suspension container station, the controller automatically removes the suspension container from the suspension container station by the automatic suspension container positioning and removal device.

[0023] The present invention further relates to a method for automatically depositing droplets of a suspension containing a sample of a microbial colony onto a deposition spot of a MALDI target plate. In certain embodiments, the system and method are configured to use the suspension as a source for a sample for another test (e.g., AST).

[0024] The apparatus includes a positioning device having a pipetting tool and a pipetting tool holder for holding the pipetting tool. The positioning device is arranged to position the pipetting tool at a start position above a suspension tube holding a suspension containing a sample of microbial colonies. The pipetting tool automatically lowers and raises the pipetting tool into and out of the suspension, and positions the pipetting tool at a transfer position, respectively.

[0025] The pipetting tool picks up a volume of suspension and raises the pipetting tool with the volume of suspension to a transfer position. The pipetting tool has a pressurizable chamber, closed by a controlled valve, for containing the volume of suspension medium.

[0026] A target plate holder is provided for holding a target plate, the target plate having at least one deposition spot.

[0027] The apparatus positions the target plate in a target plate holder.

[0028] The apparatus includes a transfer device for automatically transferring the pipetting tool from a transfer position of the positioning device to a position above one of the deposition spots on the target plate, and for lowering the pick tool (e.g., pipette tip) to a predefined distance above the target plate, pressurizing the chamber (e.g., the pressure is in the range of about 0.5 bar to 1.1 bar, which is by way of example and not limitation), and opening the valve for a time period during which a droplet of the suspension having a volume in the range of about 0.5 μl to 3.0 μl is deposited on one of the deposition spots. Preferably, the shape of the pipetting tool is such that deposition of the droplet of the suspension on the target plate occurs in a splash-free manner.

[0029] The suspension tube is then moved to a second location, where the turbidity of the suspension is adjusted for a second test (e.g., an AST). The second location has a nephelometer to determine whether the turbidity of the suspension is suitable for the second test. A pipetting tool is then used to obtain additional suspension and use that suspension to inoculate a container for another test (e.g., an AST).

[0030] In one embodiment, an automated system for preparing a single sample suspension is described, from which an aliquot is removed for identification (ID) of a microorganism within the sample and secondary testing. In another embodiment, the automated system prepares a single sample suspension from which an aliquot is removed for identification (ID) of a microorganism within the sample and antibiotic susceptibility testing (AST) of the microorganism. The system includes at least a first section for performing an ID assay. The first section has a mechanism for receiving plates, either automatically or manually. The system includes or is in communication with an imaging device that optically inspects the culture plate, and recognizes colonies of interest from the image. In an alternative embodiment, colonies are selected before the image is acquired and the plates are received by the system. The system includes a mechanism for identifying the location of the colony of interest on the plate and designating the colony of interest to be picked for testing. The first section includes an automated robotic picking tool. The system also includes a controller in communication with the robotic pick tool that directs the robotic pick tool to capture the pipette and then bring the pipette to a location above a colony of interest. The top of the plate is removed to facilitate colony picking. The robotic pick tool then lowers the pipette so that the tip is in contact with the colony of interest.

[0031] After the colonies are picked, the controller commands the robotic pick tool to transport the picked sample to the first sample suspension preparation station. Optionally, the system captures a new image of the plate after the colonies are picked to verify that the pick is from the correct location. The first sample suspension station has a suspension dispenser that dispenses the sample suspension into a suspension tube, cuvette, or other suitable receptacle. The first sample suspension station has a nephelometer or other suitable device for measuring the turbidity of the liquid in the suspension tube or cuvette. The robotic pick tool releases the carried sample from the culture plate into the suspension. In some embodiments, the robotic pick tool vibrates the pick tool to facilitate the release of the sample into the suspension. The nephelometer measures the turbidity of the suspension, and the automated system adjusts the suspension to make it acceptably heavy (i.e., cloudy) for the ID assay in response to turbidity measurements that fall outside of a predetermined turbidity value.

[0032] The first section further includes a first robotic pipettor. The first robotic pipettor obtains a first aliquot of the suspension at a first station and inoculates a receptacle for use in an ID assay. The receptacle (e.g., a MALDI plate) is then removed from the system and transported to an apparatus for performing MALDI. The receptacle can be transported mechanically or manually. The suspension tube or cuvette is then transported to a location within the first section where the remaining portion of the suspension is prepared for use in a second assay (e.g., an AST assay). Transport is by automated means using a conveyor.

[0033] The first section has a second nephelometer at a second sample suspension station for measuring the turbidity of the suspension. The first robotic pipettor is further configured to adjust the concentration of the sample in the suspension tube or cuvette to a predetermined concentration for a second assay, obtain a second aliquot of the sample suspension having the adjusted concentration, and inoculate the second aliquot of the suspension into a sample tube for an AST assay. Such a sample tube is commonly referred to as an AST broth tube.

[0034] The system optionally has a second section for preparing panels for AST assays. The automated system has an automated mechanism for transporting inoculated sample tubes from the first section to the second section. In one embodiment, the inoculated sample tubes are lowered through the deck for the second sample suspension station, transported below the deck, and exit below the deck in the second section. The second section has a second robotic pipettor that obtains aliquots from the inoculated sample tubes and inoculates the obtained aliquots into the AST panel. The second section also has means for storing, dispensing, manipulating, and pushing caps 99 (see FIG. 26) into the cap holes in the inoculated panel. The second section also has a robot that loads the inoculated panel into the device in which the AST is performed, and the AST device is configured with at least two doors, the first door receiving the panel from the panel loading robot, and the second door for manual loading of the inoculated panel by the user. The AST device is not required to be located in the second section of the system and can be adjacent to the second section. The second section also has a controller communicatively connected to the AST equipment for requesting and scheduling access and opening to the first door of the AST equipment.

[0035] The invention will be further explained with reference to the following figures. [Brief explanation of the drawings]

[0036] [Figure 1] FIG. 1 is a front view of a system according to one embodiment of the present disclosure including a system housing. [Figure 2] 2 is a schematic diagram of a component layout within the system housing of FIG. 1 according to one embodiment of the present disclosure. [Figure 3] FIG. 2 is a block diagram of the architecture of the system of FIG. 1 according to one embodiment of the present disclosure, including exemplary components suitable for implementing the methods described herein. [Figure 4A] FIG. 1 is a perspective view of one embodiment of a low volume single cuvette nephelometer. [Figure 4B] 4B is a top cross-sectional view of the low-volume single-cuvette nephelometer of FIG. 4A taken along a horizontal plane extending through the nephelometer. FIG. [Figure 5A] FIG. 4B is a perspective view of a single cuvette according to an embodiment of the present disclosure for use with the low-volume single-cuvette nephelometer of FIG. 4A. [Figure 5B] FIG. 4B is a perspective view of a single cuvette according to another embodiment of the present disclosure for use with the single cuvette nephelometer of FIG. 4A. [Figure 6] FIG. 4B is a process flow diagram illustrating one process embodiment for preparing a sample using the nephelometer of FIG. 4A. [Figure 7A] FIG. 1 is a top cross-sectional view of a continuous cuvette nephelometer according to one embodiment of the present disclosure. [Figure 7B] 7B is a top cross-sectional view of the continuous cuvette nephelometer of FIG. 7A taken along a horizontal plane extending through the nephelometer. [Figure 8] FIG. 7B is a perspective view of a linear low-volume multi-cuvette array / strip according to an embodiment of the present disclosure for use with the continuous cuvette nephelometer of FIG. 7A. [Figure 9] FIG. 1 is a partially transparent perspective view of stacked cuvettes. [Figure 10] FIG. 10 is a perspective view of a nephelometer according to another embodiment of the present disclosure. [Figure 11] 11 is a cutaway view of the nephelometer of FIG. 10 showing the transmitted light detector path of the nephelometer. [Figure 12] 11 is a further cutaway view of the nephelometer of FIG. 10 showing the transmitted light detector path of FIG. 10, while also showing the light source and transmitted light detector. [Figure 13] 11 is another cutaway view of the nephelometer of FIG. 10 showing the scattered light detector path of the nephelometer. [Figure 14] FIG. 1 is a diagram of a sample preparation decision tree in which sample preparation is based on measured sample turbidity. [Figure 15] FIG. 1 shows a pipette removing a mucoid sample from a target plate, where strings begin to form in the pipette. [Figure 16] FIG. 16 shows the target plate of FIG. 15, where the pipette is further removed from the agar surface of the plate, further extending the string. [Figure 17A] 1 is a time graph showing the change in capacitance over time as the pipette picks a sample but no strings are formed. [Figure 17B] 1 is a time graph showing the change in capacitance over time as the pipette picks a sample and a string forms. [Figure 18] 1 is a flowchart illustrating an automated process according to one embodiment of the present invention. [Figure 19] 19 is a flowchart comparing a timeline of the automated process of FIG. 18 with a timeline of an equivalent process performed manually. [Figure 20] 2 is a schematic side view of the system of FIG. 1 together with a cartridge transfer device and a plurality of test devices. [Figure 21] FIG. 21 is a diagram of an exemplary system for automatically preparing, transporting, and testing samples, including the system, cartridge transport device, and testing device of FIG. 20, as well as the exemplary microbiology test cartridge and controller 30 of FIG. 3. [Figure 22] FIG. 10 is a rear perspective view of a cartridge gripper of a cartridge transfer device according to an embodiment of the present disclosure, as the cartridge gripper approaches a cartridge within a cartridge holding structure. [Figure 23]FIG. 22 is a side perspective view of the cartridge gripper of FIG. 21 highlighting the pivotable connection between the gripper plate and the arm of the automated cartridge transfer device. [Figure 24] FIG. 22 is a front perspective view of the cartridge gripper of FIG. 21. [Figure 25] FIG. 22 illustrates the exemplary microbiology test cartridge of FIG. 21. [Figure 26] FIG. 10 is a diagram showing a tray for temporarily storing cartridges. [Figure 27A] FIG. 21 is a front perspective view of one of the test instruments of FIG. 20 including a manual door. [Figure 27B] 27B and 27C are various perspective views of the testing equipment of FIG. 27A, including the automatic door of such equipment. [Figure 27C] 27B and 27C are various perspective views of the testing equipment of FIG. 27A, including the automatic door of such equipment. [Figure 27D] 27B and 27C are various perspective views of the testing equipment of FIG. 27A, including the automatic door of such equipment. [Figure 28] Figures 28A and B are diagrams of exemplary test equipment components that may be automatically controlled by the controller of Figure 3. Figure 28C is a diagram further illustrating an exemplary architecture of the controller of Figure 3. [Figure 29] 12 is a schematic diagram of a pick station according to another embodiment of the present disclosure. [Figure 30] 2 is a schematic diagram of a component layout within the system housing of FIG. 1 according to another embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0037] As used herein, a "cuvette" and / or "micro-cuvette" and / or "low volume cuvette" and / or "LVC" and / or "sample vessel" or "vessel" is a container suitable for receiving a liquid suspension. The container is preferably made of optically clear plastic or glass designed to hold a test sample in a specific space and orientation for testing or processing.

[0038] As used herein, an "algorithm" is one or more mathematical instructions that are used to manipulate multiple data values ​​to make decisions based on the mathematical values ​​and then generate corrected or more accurate data values ​​that represent a desired output.

[0039] As used herein, an "amplifier" is an electronic circuit used to take a smaller original electronic signal and increase its amplitude, thereby producing a proportionately larger new signal that represents the original signal. Suitable amplifiers are well known to those skilled in the art and will not be described in detail herein.

[0040] As used herein, an "analog to digital converter" or "A / D converter" is an electronic device that is capable of taking a variable electrical signal and converting it into a numerical value that represents the amplitude of the original signal.

[0041] As used herein, "dilution" means a solution or suspension produced by adding a liquid diluent to a concentrated solution or suspension, resulting in a new suspension or solution having a lower and more uniform concentration of sample within the solution or suspension compared to the original solution or suspension.

[0042] As used herein, a "laser" or "laser diode" is an electronic device that produces a concentrated and focused beam of light when an electric current is applied to it.

[0043] As used herein, a "light attenuation filter" is a device placed in a light path to absorb and reduce the amount of light as it passes through the filter, resulting in the filtered light having a proportionally lower intensity than the original light source.

[0044] As used herein, a "light emitting diode" or "LED" is an electronic device that emits light of a particular type and orientation when an electric current is applied to it.

[0045] As used herein, a "McFarland" is a unit of measure for the amount of solid particulate matter dispersed within a fluid or liquid suspension.

[0046] As used herein, a "nephelometer" is an instrument capable of measuring the amount of solid particles in a suspension. As used herein, "nephelometry" refers to a method by which the amount of suspended matter in a suspension can be measured.

[0047] As used herein, a "photo-diode" and / or "detector" is an electronic device used to measure the intensity of light in a given environment.

[0048] As used herein, "saturated" and / or "saturation" refers to the point at which a detector has reached the maximum amount of output signal it is capable of producing. For example, adding more light to a photodetector beyond saturation will not result in any further change in the detector output signal, which has reached its maximum operating capability.

[0049] As used herein, a "suspension" is a solution in which a solid is uniformly distributed in a liquid.

[0050] As used herein, "turbidity" is a measure of the amount of suspended solids in a solution (i.e., the cloudiness of a liquid sample).

[0051] Described herein are methods and systems for preparing a single suspension from a colony of a microorganism, which serves as a source for a sample, to determine both the identity and antibiotic susceptibility of the selected colony of the microorganism. Because samples used to characterize and identify microorganisms are typically obtained from a culture dish with multiple colonies growing on the medium, it is important that the sample be obtained from a colony of interest. If a sample is taken from a colony of no interest, time and efficient use of the MALDI instrument are reduced. The present invention contemplates an automated process for identifying and selecting a colony of interest from among multiple colonies present on the dish. The process of distinguishing colonies can be at least partially automated by providing a culture dish with several colonies of the microorganism, acquiring an initial image of the culture dish containing all of the colonies of the microorganism, displaying the initial image of the culture dish containing all of the colonies of the microorganism on a display, and selecting at least one colony of the microorganism from the initial image.

[0052] In this way, the researcher or analyst may select colonies of interest based on their education and knowledge. In certain embodiments, the culture dish is provided with individual identification information, such as a barcode, that identifies the culture dish, and the method further includes storing an initial image of the culture dish containing all colonies, storing information regarding at least one selected colony of microorganisms, and storing the identification information of the culture dish in the memory of the central control computer. In further embodiments, the researcher or analyst may manually enter treatment instructions regarding treatments to be received by the selected colonies of microorganisms in the culture dish. The treatment instructions are stored in the memory of the central control computer for later use.

[0053] In one embodiment, colonies on plates are imaged according to the methods described in Provisional Patent Application No. 62 / 151,681, entitled "Colony Contrast Gathering," filed April 23, 2015, and also filed as International Application No. PCT / US2016 / 028913, and also in International Application No. PCT / EP2015 / 052017, entitled "A System and Method for Image Acquisition Using Supervised High Quality Imaging," both of which are incorporated herein by reference. The contrast of different colonies relative to the medium provides the ability to differentiate colonies and facilitate automated colony picking. As described elsewhere, images of plates can be acquired in a separate device before being received by the systems described herein, or the systems herein can be integrated with modules in which such images are acquired.

[0054] After the initial image of the culture dish is acquired, the culture dish is incubated for a period of time to allow microorganisms on the plate to grow, if present. In a further embodiment of the invention, the method includes the automated steps of positioning the culture dish on a stage for the culture dish, acquiring an image of the culture dish positioned on the stage, acquiring identification information for the culture dish, and comparing the image acquired by the imaging device of the pick tool device with a stored initial image of the culture dish, thereby obtaining information regarding the location of a selected colony of microorganisms and, optionally, processing instructions regarding a process to be performed on the selected colony of microorganisms. By comparing the image of the culture dish as placed in the pick tool device with the initial image, the location of the selected colony can be obtained automatically, for example, by computerized image comparison.

[0055] In another embodiment, fiducial markings on the agar surface or culture dish can be used to relocate the colony. These fiducial markings can be embedded on the plate during manufacturing, applied by the user or organic growth, or incorporated into the dish or agar surface by any suitable means. Another reference point, such as the center of the dish, can be detected using a machine vision device, and from that reference point, dish coordinates can be determined. A barcode is an example of a fiducial. The location of the colony on the dish can be determined by reference to its relative distance from the center and angular offset to the barcode zero offset. Once the relative location of the colony is determined, the dish can be moved to another system, and the following two steps are performed: The dish is centered, for example, by mechanical means. The barcode zero offset is detected, for example, by rotating the dish while a fixed sensor detects the presence of the barcode label and a barcode scanner scans the barcode. At this point, the center of the dish is known, and the barcode zero offset is known; therefore, the location of the previously referenced colony can be easily calculated, since it is stored as the distance from the center of the dish and angular offset to the barcode label. The method, as described herein, does not require a camera or computer vision system in the second system (in this example, the colony picking system) or any other system where colony location information is needed. The zero offset used in this example is relative to the barcode label, but the barcode label could be any unique reference feature on the dish or could be applied to the dish as described above.

[0056] One automated method and apparatus for picking up microorganisms from the surface of a culture medium is described in U.S. Patent Application Publication No. 2014 / 0242570 (U.S. Serial No. 14 / 347,841) to Botma et al., entitled "Method For Picking Up Cell Material And Assembly For Performing Said Method," which publication is commonly owned and incorporated herein by reference.

[0057] As described in Botma et al., in an advantageous embodiment, the method further includes removing the pick tool a predetermined distance away from the contact position toward a check position, holding the pick tool in the check position, and measuring the capacitance of the system consisting of the pick tool and support at the check position. In some cases, the sample material to be picked up is very sticky or viscous. After contacting such sample material, when the pick tool is removed from the sample, a thin thread remains in contact between the pick tool and the sample material remaining in the culture dish. This thin thread may be severed and possibly contaminate the pick tool device. By measuring the capacitance of the pick tool at the check position and the pick tool support, which may be, for example, several millimeters above the culture dish, it is possible to detect the presence of such a thread so that appropriate action can be taken. In embodiments in which a pick tool holder adapted to grasp and release the pick tool is provided to removably hold the pick tool, an automated response to the detection of the remaining thread may be implemented. For example, if the electrical capacitance measured at the check position differs from the starting electrical capacitance at the start position, the pick tool may be released from the pick tool holder, causing the pick tool to fall into the culture dish, which may then be discarded. Because these steps can be easily performed in an automated manner, no time-consuming human intervention is required to discard the pick tool and culture dish.

[0058] In one embodiment, a pipette tip is used to pick a colony from the surface of the medium (e.g., agar) on which the colony is disposed. The pipette, in one embodiment, may use suction to draw the colony into the tip. In other embodiments, suction is not used to draw the colony into the pipette tip, and the contact force between the colony and the pipette tip alone forces the colony into the pipette tip.

[0059] In a still further embodiment of the method according to the invention, the method comprises the step of automatically preparing a suspension of a sample of microorganisms. In such a method, the following steps are carried out:

[0060] A first pick tool is provided with a positioning device having a pick tool holder for holding the pick tool (e.g., the pipette tip pick tool described above). The positioning device is arranged to position the pick tool at a starting position above the acquired location of the selected colony of the microorganism on the culture dish. The positioning device automatically lowers and lifts the pick tool toward and away from the culture dish, respectively, and positions the pick tool at a transfer position.

[0061] A first pick tool is positioned in a pick tool holder of a positioning device. The pick tool is then positioned at a starting position above the acquired location of a selected colony of microorganisms on the culture dish. The pick tool is then automatically lowered to contact the microorganism colony, thereby picking up a sample of the microorganisms. The pick tool is then automatically lifted from the culture dish to a transfer position together with the acquired sample of the microorganisms.

[0062] A suspension tube holder is provided for holding at least one suspension tube. The suspension tube is positioned within the suspension tube holder. Although referred to herein as a suspension tube, the container for the suspension may be a tube, vial, cuvette, or other container for holding a suspension solution.

[0063] An automatic suspension medium dispenser is provided for automatically dispensing suspension medium into suspension tubes held in the suspension tube holder. The automatic dispenser automatically dispenses an initial amount of suspension medium into the suspension tubes held in the suspension tube holder. A transfer device, which may be separate from or part of the positioning device, is similarly provided for automatically transferring a pick tool (with an already collected sample) to a position above the suspension tubes held in the suspension tube holder. The transfer device lowers and lifts the pick tool (and the sample carried by the pick tool) into and out of the suspension medium contained within the suspension tube. The transfer device similarly positions the pick tool in a waiting position above each suspension tube held in the suspension tube holder.

[0064] The transfer device vibrates the first pick tool with a sample of the microorganisms in a linear vertical motion for a period of time while the first pick tool is immersed in the suspension medium to release the sample into the suspension medium and mix the suspension. After the period has elapsed, the first pick tool is lifted away from the suspension medium contained in the suspension tube to a waiting position. Alternatively, instead of vibration to release the microorganism sample, repeated suction by the pipette-tip pick tool while partially immersed in the suspension medium can be used to release the microorganisms and mix the suspension.

[0065] In an automated method, a turbidity meter is provided that measures the turbidity of a suspension medium contained in a suspension tube held in a suspension tube holder. In one embodiment, the turbidity meter is described in U.S. Provisional Patent Application Serial No. 62 / 056,911, filed September 29, 2014, and International Application PCT / IB 2015 / 00272 (published as WO 2016 / 051267), which are assigned to the assignee of the present invention and are incorporated herein by reference in their entireties.

[0066] After a period of time during which the pick tool is vibrated has elapsed, the turbidity of the suspension medium contained within the suspension tube held within the suspension tube holder is measured by the turbidimeter to provide a final measurement value indicative of the measured turbidity.

[0067] In a further embodiment, a controller is provided that is communicatively connected to the positioning device, the transfer device, the automated suspension medium dispenser, and the turbidity meter, such controller automatically controlling the movement of the positioning device, the movement of the transfer device, the operation of the automated suspension medium dispenser, and the operation of the turbidity meter, respectively.

[0068] 6, in one embodiment, the controller determines whether the final turbidity measurement is greater than a first threshold (maximum value) pre-stored in the controller's memory. If yes, step b) (dilution, described below) is performed. If the final turbidity measurement is less than or equal to the first threshold and greater than or equal to a second threshold (the first threshold being greater than or equal to the second threshold) pre-stored in the controller's memory, step c) (acceptable turbidity, described below) is performed. If the final turbidity measurement is less than the second threshold, step d) (increase turbidity, described below) is performed.

[0069] In step b), the automatic suspension medium dispenser is automatically controlled to dispense an additional amount of suspension medium into the suspension tube, and in step c), a signal is provided that the suspension tube with the suspension is to be removed from the suspension tube holder for further processing.

[0070] According to step d), a further pick tool is obtained and positioned in the pick tool holder of the positioning device as described above. The positioning device positions the further pick tool in a start position above the culture dish, automatically lowers the further pick tool toward the culture dish into contact with the microorganisms to pick up a further sample of microorganisms, and automatically lifts the further pick tool with the sample of microorganisms away from the culture dish to a transfer position, all as described for the first pick tool. The pick tool described herein is a pipette, although other suitable pick tools are described in U.S. Provisional Application No. 62 / 144,574, filed April 8, 2015, entitled "Device And Apparatus For Collecting Microbial Growth From A Semi-Solid Surface," and International Application No. PCT / US2016 / 026625, filed April 8, 2016, both of which are incorporated herein by reference.

[0071] The transfer device automatically transfers a further pick tool having a further sample of microorganisms from the transfer position of the positioning device to a position above the suspension tube held in the suspension tube holder, lowers the further pick tool having the further sample of microorganisms into the suspension medium contained in the suspension tube, and oscillates the further pick tool in a linear vertical motion for a period of time while the further pick tool having the further sample of microorganisms is immersed in the suspension medium. After the period of oscillating the further pick tool has elapsed, the turbidity of the suspension medium contained in the suspension tube held in the suspension tube holder is measured by the turbidimeter to provide a further final measurement value indicative of the measured turbidity.

[0072] After the sample is collected, in yet another embodiment, the pipetting system may perform a series of rapid retractions and dispenses of the pipette tip in the liquid suspension. For example, the pipetting system may repeat the series of retractions up to approximately 24 times within a 20-second period to dispense approximately 250 μL to 300 μL of sample. The repetitive action generates high shear forces at the pipette tip. The high shear forces allow for the dispersion of clumps or mucoid strands in the sample, including microorganisms, to create a more uniform suspension.

[0073] It is thus possible to prepare suspensions of microbial samples in a highly automated manner, while the controller and turbidity meter provide suspension tubes containing a suspension medium that always contains a sufficient (reproducible) amount of microorganisms to perform a correct analysis of the microorganisms.

[0074] In a further embodiment of the method for automatically preparing a suspension of a microorganism sample according to the present invention, the controller is configured to further perform a step of measuring the turbidity of the suspension medium contained in the suspension tube held in the suspension tube holder with a turbidimeter during the period during which the pick tool is vibrating. The turbidimeter is configured to provide the controller with an online measurement value indicative of the measured turbidity during the period during which the pick tool is vibrating. In this way, a very fast automatic determination of the amount of microorganisms in the suspension can be obtained. In particular, if during the vibration the online measurement value of turbidity is below a first threshold value and above a second threshold value, the controller controls the movement of the transfer device so that the pick tool is lifted to a standby position, and the controller also provides a signal that the suspension tube with the suspension can be removed from the suspension tube holder for further processing. In this way, the method can be performed very time-efficiently, since the vibration of the pick tool is stopped when the suspension medium contains a sufficient amount of microorganisms.

[0075] The relative positioning of the pick tool and the sensor of the turbidity meter is such that the pick tool does not obstruct the path of the turbidity meter during vibration of the pick tool.

[0076] In a further embodiment of the method for automatically preparing a suspension of a microorganism sample according to an embodiment of the invention, the controller is arranged to, for example, control the turbidity meter so that a step of measuring the turbidity of the suspension medium contained in the suspension tube held in the suspension tube holder is initiated by the turbidity meter before the pick tool is immersed in the suspension medium contained in the suspension tube. In this way, it is possible, for example, to check whether the initial suspension medium used is not contaminated. Furthermore, this provides an indication of a starting value for the turbidity, which is useful when determining the final measurement value.

[0077] In yet a further embodiment of the method for automatically preparing a suspension of a microorganism sample, the method further includes providing a suspension tube holder for holding the suspension tube. The suspension tube holder may be adapted to rotate the suspension tube held within the rotatable suspension tube holder. In a further embodiment, a controller is arranged to be communicatively connected to the rotatable suspension tube holder for controlling the rotation of the suspension tube holder. The controller is further arranged to rotate the suspension tube during measurement of the turbidity of the suspension medium contained within the suspension tube. Such rotation of the suspension tube allows turbidity measurements at several positions within the suspension tube that are rotationally spaced apart from one another to provide a more accurate final measurement of the turbidity of the suspension. Such rotation is not necessary to eject the sample from the pick tool. The vibration of the pick tool described above is more than sufficient to eject the sample.

[0078] Although a further pick tool different from the first pick tool may be used, the method can be economically carried out when in step d) the first pick tool is provided as the further pick tool, and the positioning of the further pick tool in the pick tool holder of the positioning device is carried out by the device transport device under the control of the controller.

[0079] In yet a further embodiment of the method for automatically preparing a suspension of a microbial sample according to the present invention, the additional amount of suspension medium is determined by the controller based on the initial amount of suspension medium, the final measured value, and the value of the first threshold and / or the second threshold. This allows the amount of suspension medium used to be carefully controlled. Thus, suspension medium is conserved.

[0080] In some embodiments, because the pick tool vibrates in a vertical linear motion relative to the suspension tube, the horizontal cross section of the suspension tube can be relatively small. This allows for smaller suspension volumes to be used. In one embodiment, the controller dispenses an initial volume of suspension of about 0.1 ml to 5 ml, preferably less than about 1 ml (in one example, about 300 μl). In other embodiments, the volume of the suspension is about 0.5 ml to about 2 ml. In one embodiment, the dispensed volume is 300 μl. This relatively small amount of suspension medium is sufficient to prepare a proper suspension of the microbial sample.

[0081] In these methods for automated preparation of suspensions of microbial samples, relatively small tubes, vials, or cuvettes having a maximum cross-sectional diameter of about 2 mm to about 12 mm, preferably about 3 mm, can be used as suspension containers, compared to traditional suspension tubes having a diameter of about 16 mm. The tubes can have square, rectangular, or circular cross sections, with the actual cuvette shape being primarily a matter of design choice. In one embodiment, the tube is circular with a diameter of about 6 mm to about 12 mm. In one advantageous embodiment, the diameter is about 10 mm. With these relatively small suspension tubes, proper release of the sample from the pick tool is obtained when a controller is configured to control the vibration of the transfer device so that the pick tool vibrates at a frequency between about 5 Hz and about 250 Hz. The selection of a frequency within this range is primarily a matter of design choice and will depend on the components of the suspension being formed. For suspensions formed with samples and solutions that readily interdisperse, frequencies of 5 Hz to 12 Hz may be appropriate. For components that do not readily form suspensions, frequencies of about 100 Hz or higher may be required. Preferably, the controller is configured to control the vibration of the transfer device so that the pick tool vibrates at an amplitude of about 0.5 mm to about 4 mm, preferably about 2 mm to about 3 mm, and most preferably about 1 mm, which results in optimal release of the sample from the pick tool. In embodiments in which the controller is configured to control the vibration of the transfer device so that the period for which the pick tool vibrates is about 3 seconds to about 120 seconds, preferably about 30 seconds to 60 seconds, the complete sample can be released from the pick tool in substantially all cases. For efficiency and throughput, it is advantageous if vibration is only required for about 3 seconds to 10 seconds, with 6 seconds being approximately the average minimum vibration time.

[0082] The values ​​for frequency, amplitude, and duration depend on the characteristics of the particular microorganism, for example, its adherence to the pick tool. In one embodiment, imaging inspection can be used to infer whether the sample has, at least mostly, been released from the pick tool by first using the preferred values ​​mentioned above. If some material still remains on the pick tool, the vertical vibration is repeated at different values ​​within a given range.

[0083] The automated method for preparing a suspension of a microbial sample further includes providing an automated culture dish positioning and unloading device for automatically positioning and unloading a culture dish on and from the stage. A controller is communicatively connected to the automated culture dish positioning and unloading device to control the operation of the automated culture dish positioning and unloading device. Thus, positioning of the culture dish (carrying the target microorganism) on the stage can be performed automatically under the control of the controller. In another embodiment, an automated suspension tube positioning and unloading device is provided that automatically positions and unloads a suspension tube in and from the suspension tube holder, respectively. The controller is communicatively connected to the automated suspension tube positioning and unloading device to control the operation of the automated suspension tube positioning and unloading device, so that positioning of the suspension tube in the suspension tube holder can be performed automatically under the control of the controller. Advantageously, the controller is configured to subsequently allow the automated culture dish positioning and unloading device to unload the culture dish from the stage only after providing a signal that the suspension tube containing the suspension may be unloaded from the suspension tube holder for further processing. In yet a further embodiment, the controller is arranged such that the suspension tube is automatically removed from the suspension tube holder by the automatic suspension tube positioning and removal device only after a signal is provided that the suspension tube with the suspension may be removed from the suspension tube holder for further processing.

[0084] In yet a further embodiment of the method according to the invention, an identification mark is provided on the suspension tube. According to this method, the identification mark of the suspension tube is stored in the memory of the central control computer together with the characteristics of the suspension, with a link to the identity of the culture dish from which the selected colony of the microorganism was obtained. This ensures that not only can the method work automatically in a very efficient way, but also that a correct and fast processing of the obtained analytical results is achieved.

[0085] In a further embodiment of the methods described herein, a pipetting tool is provided (either separately or a pick tool device adapted to receive and use a pipette) for depositing an aliquot(s) of the suspension onto a MALDI plate, as well as for depositing aliquots of the suspension for other downstream analyses (e.g., AST). A positioning device is provided with a pipetting tool holder for holding the pipetting tool. The positioning device is arranged to position the pipetting tool at a start position above the suspension tube. The positioning device automatically lowers and lifts the pipetting tool into and out of the suspension, respectively, and positions the pipetting tool at a transfer position. The pipetting tool is received by the pipetting tool holder of the positioning device. The positioning device positions the pipetting tool at a start position above the suspension tube, lowers the pipetting tool into the suspension in the suspension tube, operates the pipetting tool to pick up a volume of the suspension, and lifts the pipetting tool with that volume of suspension to the transfer position. The pipetting tool has a pressurizable chamber, closed by a controlled valve, for containing the volume of suspension medium.

[0086] The method provides a target plate holder for holding a target plate having at least one deposition spot. The target plate is positioned within the target plate holder. A transfer device is provided, which automatically transfers a pipetting tool from a transfer position of the positioning device to a position above one of the deposition spots on the target plate and lowers the pipetting tool a predetermined distance above the target plate. The chamber is pressurized to a pressure within a range of approximately 0.5 bar to 1.1 bar, and the valve is then opened for a time period during which a droplet of suspension having a volume within a range of approximately 0.5 μl to 3.0 μl is deposited on the deposition spot, specifically, covering at most about half of one of the deposition spots on the target plate. The pipetting tool is then lifted from the target plate. The pressure and opening time are adjusted depending on the characteristics of the specific microorganism, such as its stickiness, to obtain a droplet of suspension, which can be reproducibly prepared and accurately deposited on the target plate as a result of the automated process.

[0087] A pipetting tool is used to obtain more suspension in the manner described above, which is then used to dispense the suspension into containers for other analyses (e.g., suitable containers for performing antibiotic susceptibility testing (AST)).

[0088] In order to avoid cross-contamination in a preferred embodiment of the method according to the invention, the shape of the pipetting tool, in particular its dispensing tip, is such that the deposition of the drops of suspension on the target plate or other container occurs in a splash-free manner. Depending on the type of microorganism used, in particular its viscosity, it has been found that in addition to choosing the correct pressure within the above-mentioned ranges and the opening time of the valve within the above-mentioned ranges, a suitable shape of the pipetting tool ensures that the drops of suspension can be deposited in a splash-free manner.

[0089] In a further embodiment of the method, identification marks are provided on the target plate and other container(s) for sample testing (e.g., AST), and optionally, identification marks are provided on the deposition spots of the target plate. According to the method, the identification marks of the target plate and the deposition spots are all stored in the memory of a central control computer, together with the characteristics of the suspension, with a link to the identity of the culture dish from which the selected colony of the microorganism was obtained. Not only can the method work automatically in a very efficient manner, but also a correct and fast processing of the analytical results obtained is achieved.

[0090] In a further embodiment of the method, a prepared container, such as a container supporting the performance of an additional test such as an AST, may be moved from a location where a tube is inoculated with a microbial suspension and other appropriate reagents to a second location where a further pipettor pipettes the mixture from the container and inoculates a cartridge to be used for the test. After inoculation, such a cartridge may be further positioned by the robot in a holding structure that holds the cartridge until it is removed by a cartridge transfer device. When available, the cartridge transfer device picks up or grasps the cartridge from the holding structure and transfers the cartridge to another holding structure located within a testing instrument, such as an AST testing instrument.

[0091] Mass spectrometry, performed by MALDI or MALDI-TOF-MS, is used to identify microorganisms. In MALDI-TOF-MS operations, samples of microbial colonies are spotted or deposited onto a target plate held in a fixed position within the MALDI instrument. Such target plates typically have multiple deposition spots (e.g., 24 to 384 deposition spots on a single target plate). These deposition spots have a fixed orientation relative to the edge of the target plate. The target plate is positioned on an XY stage so that acquired samples of microbial colonies can be deposited onto selected deposition spots. The location where a particular sample is deposited is indicated by XY coordinates / parameters and stored in the memory of a central control computer.

[0092] Although not shown in detail in Figure 2, a target plate 42 is shown positioned below the transfer track 18 at the position indicated at B. Samples can be transferred along the transfer track 18 from the culture dishes 3 and / or suspension tubes 11 to above the target plate at position B, where the samples are lowered and deposited onto a deposition spot on the target plate. Other transfer mechanisms than that shown in Figure 1 are contemplated. For example, a deck-mounted transfer mechanism can be provided.

[0093] The present invention is described in detail below with respect to preparing a suspension containing a sample and depositing the suspension onto a deposition spot on a target plate. Generally, colonies of microorganisms are automatically located and detected on a culture dish. Samples of selected colonies of microorganisms are obtained in an automated manner, for example, by a pick tool brought into contact with the colonies.

[0094] When characterizing and identifying microorganisms, multiple colonies typically grow on a culture dish. Furthermore, multiple different culture dishes are processed through the device. Therefore, the present invention provides the ability to separately identify each culture dish, for example, by barcode, and further, each colony of interest on a single culture dish is selected and given an identifying mark. In this regard, prior to the automated step of locating and selecting the microbial colonies on the culture dish, a culture dish determined to contain several microbial colonies is provided. An initial image of the culture dish is acquired. The image includes all of the microbial colonies. The device, or a device operating in communication with the device, displays the initial image of the culture dish including all of the microbial colonies on a display and selects at least one microbial colony within the initial image. Thus, a researcher or analyst may select colonies of interest based on sufficient education and knowledge. In one embodiment, the imaging information is processed to identify colonies for picking based on specifications. Since each culture dish is provided with individual identification information, such as a barcode, that identifies the culture dish, an initial image of the culture dish containing all colonies is stored, and information about at least one selected target colony of microorganisms is stored (preferably with a link provided in the (electronic) initial image). All information and identification information of the culture dishes is stored in the memory of a central control computer, which allows a high degree of accuracy and integrity of the process.

[0095] Thus, the only possible manual action in the methods and apparatus described herein is the act of selecting a colony of interest. All data regarding the sample is processed in an automated manner. Optionally, a researcher or analyst may manually enter processing instructions regarding the treatment to which the selected colony of microorganisms on the culture dish will be subjected. The processing instructions are likewise stored in the memory of the central control computer for later use. After this manual action, all further steps performed are automated in a reliable and efficient manner.

[0096] In this further automated process, the culture dish is automatically positioned on a culture dish stage of a pick tool device equipped with an imaging device. An image of the culture dish positioned in the pick tool device is acquired, and this image acquired by the imaging device of the pick tool device, along with the identification information of the culture dish, can be compared with a stored initial image of the culture dish, thereby deriving information regarding the location of the selected colony of the microorganism and, optionally, processing instructions regarding the process to be performed on the selected colony of the microorganism. By comparing the image of the culture dish as it is placed in the pick tool device with the initial image, the location of the selected colony can be automatically obtained, for example, by computerized image comparison. Furthermore, each target plate is provided with an identification mark, and optionally, each deposition spot on the target plate has an individual identification mark or location identifier. Containers used for AST similarly carry identification marks to associate results with the correct sample. After storing the identifying marks of the target plate and the deposition spots in a central control computer together with the properties of the suspension with a link to the identity of the culture dish from which the selected colony of the microorganism was obtained, correct linking of the obtained MALDI / AST results to the specific colony of the microorganism under test is possible in a correct and automated manner.

[0097] It has been discovered that when a sample covers at most about half of one of the deposition spots on a target plate, analytical results obtained from a MALDI instrument on the portion of the deposition spot not initially covered by the sample are unexpectedly significantly more accurate than analytical results obtained from a MALDI instrument on the portion of the deposition spot initially covered by the sample. It is hypothesized that crystallization, which occurs after a drop of matrix material is overlaid on the sample covering a portion of the deposition spot, causes the uncovered portion of the deposition spot to also contain a certain amount of sample material, and that this amount is highly suitable for providing superior analytical results. The physical or chemical process responsible for this effect is currently unknown, but may perhaps become clearer once the basic processes underlying MALDI are understood.

[0098] Sample preparation systems and methods Method embodiments of the present invention in which a suspension is made from a sample of microbial colonies picked from a culture dish will now be described, along with an embodiment of a sample preparation system 1000 for carrying out such methods.

[0099] FIG. 1 illustrates a system 1000 for implementing the methods described herein. The system 1000 includes a housing 1005 that provides an environment for implementing the methods and for the components that implement the described methods. In this regard, the components are distributed among multiple stations within the housing 1005. From left to right, the housing provides a receiving station 1010, a pick station 1020, a preparation station 1030, and a transfer station 1040. The receiving station 1010 receives one or more culture dishes suspected of carrying a microorganism of interest and automatically delivers the culture dishes to the pick station 1020. The pick station 1020 automatically detects colonies of interest and picks samples from the colonies. The preparation station 1030 automatically prepares samples for testing, such as identification (ID) and antibiotic susceptibility testing (AST). The transfer station 1040 automatically transfers the prepared AST samples to AST cartridges (also referred to herein as panels), which are then automatically transferred to the AST system.

[0100] In a typical method, an automated pick tool device 8 is provided to acquire a pick tool 6 and transfer the tool to a stage 2 supporting a culture dish 3 mounted on the stage 2. Prior to picking, a colony 4 of interest is identified on the culture dish 3 and its location on the culture dish 3 is determined. The pick tool 6, informed of its location via a controller 30, moves the pick tool 6 over the colony 4 of interest and picks the colony. Once picked, the picked sample 19 is transferred into one or more cuvettes or suspension tubes 11. Additionally, an aliquot of suspension liquid 14 is dispensed into the suspension tubes 11. This dispensing is preferably performed prior to transferring the picked colony sample 19 into the tube 11. The pick tool 6' is then positioned such that the portion of the pick tool 6' carrying the picked sample 19 is immersed in the suspension liquid 14. The pick tool 6' is vibrated to release the microorganisms. The turbidimeter 20 monitors the turbidity of the suspension and provides such information to the controller 30, which cross-references the measured turbidity with concentration specifications for tests to be performed on aliquots of the suspension, such as ID and AST. Target concentrations for both ID and AST are set forth herein.

[0101] Once the suspension reaches a desired turbidity, an aliquot of the suspension is pipetted from tube 11, and the suspension is inoculated into plate 42 for performing ID testing. Pipetting tool 46 then obtains another aliquot of the suspension for AST. In some embodiments, the suspension may require further dilution with a suspension before pipetting the suspension for AST. Once obtained, pipetting tool 46 then dispenses the suspension into containers 82 for AST testing or other assays, such as molecular diagnostic assays. Such containers may contain reagents utilized for such further testing and may be barcode scanned by tube gripper robot 50 prior to dispensing.

[0102] The container 82 is then transported by the movable device 80 to a secondary location. At this location, another pipetting tool 66 pipettes the suspension from the container 82 and inoculates the test cartridge 90. This cartridge 90 may be moved by the cartridge transfer robot 70 to a holding structure of the cartridge loading unit 78 prior to inoculation. The cartridge loading unit 78 may be operable to rotate the cartridge 90 to an optimal angle for inoculation. A decapper robot (not shown) may remove the cap from the cartridge 90 as needed for inoculation by the pipettor 60. After the cartridge 90 is inoculated, it may be transported by the transport instrument 2000 to the testing instrument 2050 (see FIG. 20 ). Once the test is performed, the system may then output a final specimen report showing quantification of specimen growth, as well as ID and AST results.

[0103] The method will now be described more particularly with respect to system 1000 and its components. Figure 2 shows a schematic of stations 1020, 1030, and 1040 disposed within housing 1005 of system 1000.

[0104] Pick station 1020 includes a stage 2 for a culture dish 3 containing microorganisms 4 on a nutrient layer 5, such as an agar gel layer. The culture dish 3 may be positioned on stage 2 by a movable arm (not shown) that transfers the dish 3 from receiving station 1010. Receiving station 1010 may automatically receive multiple culture dishes from other upstream laboratory equipment and arrange the dishes in a stacked arrangement before delivering the dishes 3 to station 1020.

[0105] When a dish 3 is received at pick station 1020, colony identification and colony picking are performed. Station 1020 includes a positioning device 8 with a pick tool holder 9 for releasably holding a pick tool, such as a disposable pipette tip. As shown, pick tool holder 9 holds a first pick tool 6. Positioning device 8 is arranged to position first pick tool 6 at a start position (shown in solid lines in FIG. 2 ) above culture dish 3 and to automatically lower and lift first pick tool 6 toward and away from culture dish 3 so that first pick tool 6 can be positioned (shown in dashed lines) to contact microorganisms 4 and pick up a sample 19 of microorganisms 4. After first pick tool 6 picks up sample 19 (first pick tool with held sample 19 is shown as 6′ in FIG. 2 ), positioning device 8 lifts and positions first pick tool 6′ at transfer position “A” above suspension tube 11. The positioning device 8 preferably lifts the pick tool 6' vertically to a start position before moving it horizontally along the transfer track 18 to the transfer location A. This may help prevent contamination from mucoid strings that may form during sample pick. However, in other embodiments, the positioning device 8 may move both vertically and horizontally simultaneously (as indicated by the arrows in FIG. 2 ) toward the transfer location A.

[0106] When picking colonies for further testing, some colonies have the sticky or slimy characteristic mentioned above. This is called a mucoid consistency, which makes it difficult to remove the colony from the agar surface. As mentioned above, after the colony is contacted by the picking device, a mucoid string will often form between the picking device and the colony on the agar surface (Figures 15 and 16). This string can be difficult to break in a controlled manner, presenting the problems mentioned above regarding possible contamination of other samples and surfaces within the instrument.

[0107] When manually picking colonies, the user will see the string forming and may take any number of manual actions to break the string. This may include rotating the picking device and / or rubbing the device on a clean portion of the plate. Because the string can be visually observed, the user will see when the string breaks and can proceed with testing. All these actions can be taken with little risk of cross-contamination.

[0108] In one embodiment of an automated process for addressing the presence of mucoid strings, the strings are detected optically (e.g., a camera may be used to monitor and detect such strings) or by monitoring changes in an electric field. Strings are conductive to the surrounding air. Once a string is detected, practitioners will appreciate that any number of mechanical devices may be used to sever the string. Referring to FIG. 15 , a culture dish 710 is shown disposed within an automated system 700. The culture dish 710 has agar 720 disposed thereon, on which many different colonies 730 have formed. A pipette tip 740 is lowered into contact with one colony, and as the pipette tip 740 is retracted, a string 750 forms. Referring to FIG. 16 , as the pipette tip 740 continues to be raised from the surface of the agar 720, the string 750 extends. Moving the pipette tip at this point will move the string to another location within the system 700. This can lead to cross-contamination with strings elsewhere in the system 700 .

[0109] In one embodiment, the presence of the string is detected by monitoring the capacitance of the pipette tip as the colony is picked and the pipette is retracted from the plate surface, transferring the picked sample into suspension. The string will cause a difference in capacitance charge as the pick tool retracts from the sample.

[0110] Capacitive level sensors can detect a variety of solids, aqueous liquids, and organic liquids. Capacitive detection relies on a radio frequency signal applied to a capacitance circuit. By monitoring the capacitance (pF = picofarads), the formation of a mucoid string can be detected. Figure 17A is a plot of the capacitance as the pick tool descends and contacts the agar surface. The capacitance drops rapidly as the pick tool is lifted away from the agar surface. Figure 17B shows the change in capacitance signal as the mucoid string forms as the pick tool is removed from the surface. The capacitance drops slowly as the string becomes thinner and thinner and eventually breaks.

[0111] The conductivity level sensor uses a low voltage level between two sensors. Because the mucoid string is conductive, the conductivity will remain high as long as the agar surface is connected to the pick tool by the mucoid string.

[0112] The strings may also be detected optically: an optical signal across the plate is diffracted by the strings formed between the plate and the pipette; this interruption of the signal can be detected by software, thereby indicating the presence of the strings.

[0113] Any number of mechanical devices can be used to remove the strings. Preferred solutions are cost-effective and do not generate aerosols or contaminate other plates in the system. Mucoid coatings on some bacteria make picking difficult in manual and automated systems. Mucoid biofilms protect the organism but make working with the organism difficult. In these instances, additional automated steps and features are provided after picking of the mucoid sample to eliminate the strings and prevent contamination of the system.

[0114] In one embodiment, a resistively heated hot wire or blade is provided to cut through the mucoid string. The wire or blade is heated to a temperature that is sufficient to sterilize the cutting device so that it can be reused continuously. A professional can select a suitable temperature that will decontaminate the wire or blade by killing microorganisms, but is not so hot as to induce rapid vaporization of the picked sample, which could result in aerosol release of organisms.

[0115] Very low temperatures can also be used to sever the string. Once the string is detected, a small spray of liquid nitrogen onto the pipette tip will harden the string, severing it. In an alternative embodiment, a cutting probe frozen to freezing point would be used to slice through the mucoid string, allowing for a clean severance.

[0116] In another embodiment, a rotating disposable rod is used to sever the string. Once the string is sensed, a cutting rod is brought into contact with the string. In an alternative embodiment, the cutting rod may rotate to wrap the mucoid string around the rod, thereby ensuring that the string is severed.

[0117] In another embodiment, a sonication device is provided that severs the strings as the pipette tip moves away from the agar plate and forms a string. For example, an ultrasonic horn is connected to the pipette tip adapter. When the pick device is pulled away from the agar surface, short pulses of high frequency easily shear the mucoid strands.

[0118] In another embodiment, the strings are allowed to dry, causing them to become brittle and break. Drying time is reduced by blowing air onto the strings through small nozzles positioned near the plate. Drying time is controlled so as not to significantly increase the time for any one pick.

[0119] In another embodiment, a strong electric current is passed through the string. The natural resistance of a thin mucoid string results in the greatest resistance at the thinnest (least conductive) portion of the string. This increased resistance causes the string to break. The electric current is selected to be strong enough to break the string, but not so strong as to induce rapid vaporization, which could result in aerosol release of biological matter.

[0120] After the string is detected, the tip is advanced across the agar surface (3-6 mm above the surface). As the string falls onto the agar and the tip continues to move, the string will be stretched to the point where it breaks off. However, because the string will break off on the agar, there is no risk of cross-contamination. In an alternative embodiment, a rapid zigzag pattern is used to break the string as the tip changes direction on the agar.

[0121] In an alternative embodiment, the tip is struck into the agar in a plate where there is no growth. This wipes the tip clean and removes the string. In another embodiment, the pipette tip is moved across the agar surface to the edge of the plate. The string can be effectively wiped away at the edge of the plate, eliminating the string.

[0122] In another embodiment, when mucoid strings are detected, a small vacuum device near the tip is used, which uses a HEPA filter system to suck up the strings and eliminate environmental contamination.

[0123] In an alternative embodiment, the pipette is treated or coated with a mucolytic agent capable of cleaving high molecular weight glycoproteins found in mucoid strings. One example of such an agent is n-acetyl-l-cysteine.

[0124] In another embodiment, a low-power laser is positioned off the side of the plate. As the pipettor leaves the plate area, the pipette tip moves directly above the laser beam. If a string is present, the mucoid string then moves through the beam. The mucoid string will be heated to the point where the string breaks.

[0125] In another embodiment, once a string is detected, the tip can be rotated 360 degrees. The rotation cuts off the mucoid string. In another embodiment, the pipette tip moves up and down, contacting the agar surface in the same location where the pick was made, thereby severing the string. With each touchdown, the pipette tip aspirates a partial volume. This dislodges the mucoid string, effectively drawing most or part of the string into the pipette tip.

[0126] The pick station 1020 further includes a suspension tube holder 10 for holding a suspension tube 11 that may contain suspension medium 14. In this embodiment, the suspension tube holder 10 is a rotatable suspension tube holder for rotating the suspension tube 11 about a vertical axis D. However, in some embodiments, the tube holder 10 may be stationary. The illustrated suspension medium 14 is dispensed from an automated suspension medium dispenser 12, which has a dispensing nozzle 13 for automatically dispensing the suspension medium 14 into the suspension tube 11 held in the suspension tube holder 10. However, in some embodiments, an automated pipettor, such as pipettor 40, may separately dispense the suspension medium 14 into the tube 11.

[0127] The positioning device 8 also includes a transfer device 15 incorporated therein for assisting in the automated transfer of the sample 19 to the suspension medium 14. The transfer device 15 is connected to the pick tool holder 9 and configured to vibrate the pick tool 6' in a linear vertical motion for a period sufficient for the sample 19 to be released from the pick tool 6'. In the method, once the suspension tube 11 is inoculated with the suspension medium 14 and the pick tool 6' is positioned at a start position at a transfer location A above the tube 11, the positioning device 8 lowers the pick tool 6' into the suspension medium 14. As shown schematically in FIG. 2 , with the sample 19 immersed, the transfer device 15 is activated to vibrate the pick tool 6', thereby releasing the sample 19 into the suspension medium 14. The positioning device 8 then positions the pick tool 6' at a waiting position above the suspension tube 11, which may be the same as the starting position. In other embodiments, the waiting position and the starting position may be different from each other.

[0128] System 1000 also includes a turbidity meter 20 for performing measurements of the turbidity of suspension medium 14 contained within suspension tube 11 held within suspension tube holder 10. As commonly known in the art, a turbidity meter may provide a measurement that is a measure of the concentration of a material, in this case, the concentration of microorganisms suspended within the suspension medium. As shown in FIG. 2 , turbidity meter 20 includes a laser 21 that transmits laser light toward and through suspension medium 14, and a sensor 22 that detects the amount of laser light transmitted through suspension medium 14. Preferably, a sensor (not shown) may be positioned perpendicular to the path of the laser light to detect the amount of laser light scattered by the suspension.

[0129] The operation of the system 1000 is controlled by a controller 30. The controller 30, shown schematically in FIG. 3 , includes a processor 32 and a memory 34. The controller 30 is communicatively connected to the positioning device 8, the transfer device 15, the automated suspension medium dispenser 12, and the turbidity meter 20 to automatically control the movement of the positioning device 8, the movement of the transfer device 15, the operation of the automated suspension medium dispenser 12, and the operation of the turbidity meter 20, respectively. Additionally, the controller 30 may be communicatively connected directly to other parts of the apparatus, such as the pick tool holder 9, the laser 21, and the sensor 22.

[0130] 2 and 3, the controller 30 is arranged to control the turbidity meter 20 so that the turbidity measurement of the suspension medium 14 is initiated before the pick tool 6' is immersed in the suspension medium 14. The controller 30 further controls the rotatable suspension tube holder 10 to initiate rotation of the suspension tube 11 held in the holder 10 before the pick tool 6' is immersed in the suspension medium 14, and to maintain the rotation of the suspension tube 11 during the measurement of the turbidity of the suspension medium 14. The controller 30 further controls the turbidity meter 20 so that turbidity measurements are performed during the entire period during which the pick tool 6' oscillates. Thus, the turbidity meter 20 provides the controller 30 with online measurements whose values ​​indicate the measured turbidity, and therefore the concentration of microorganisms, during the period during which the pick tool 6' oscillates.

[0131] As mentioned above, the controller 30 includes a memory 34 that stores a first threshold value and a second threshold value. The first threshold value is equal to or greater than the second threshold value. When the turbidity measurement provided by the turbidimeter is equal to or between the first and second threshold values, the concentration / amount of microorganisms in the suspension medium is sufficient to allow the suspension tube 11 containing the suspension 14 to be further processed. When the measured turbidity is between the first and second threshold values, the controller 30 provides a signal that the suspension in the suspension tube 11 may be further processed. Furthermore, in such a situation, the pick tool 6 may be discarded, for example, by moving a positioning device over a waste receptacle, activating the pick tool holder, and releasing the pick tool 6 into the waste receptacle.

[0132] If the final measurement value of turbidimeter 20 is greater than a first threshold value pre-stored in memory 34 of controller 30, it is determined that the concentration of microorganisms is too high to allow the suspension in suspension tube 11 to be further processed. In such a situation, controller 30 controls automatic suspension medium dispenser 12 or some other medium dispenser to supply an additional amount of suspension medium 14 into suspension tube 11. This additional amount of suspension medium 14 is based on the initial amount of suspension medium, the final measurement value, and the value of the first threshold value and / or the second threshold value, such that the addition of the additional amount of suspension medium to the suspension medium 14 already present in suspension tube 11 will result in a concentration of microorganisms in suspension medium 14 of tube 11 that meets the requirements for further processing, which can be confirmed by additional or further measurements of turbidity by turbidimeter 20.

[0133] If the final measurement value of the turbidity meter 20 is less than the second threshold value (indicating that the concentration of microorganisms in the suspension medium 14 is too low), the controller 30 controls the positioning device 8 so that an additional sample 19 of microorganisms 4 is picked up by the first pick tool 6 to further concentrate the suspension medium 14. Alternatively, the first pick tool 6 may be discarded, and the second pick tool may be used to pick up such additional sample. In this regard, upon determining that the final measurement value is less than the threshold value, the controller controls the first pick tool 6 in the pick tool holder 9 of the positioning device 8 to be lowered from its starting position above the culture dish 3 toward and into contact with the microorganisms 4 to pick up the additional sample 19 of microorganisms 4. The first pick tool 6' is then automatically lifted, along with the additional sample 19 of microorganisms 4, away from the culture dish to its starting position at transfer location A above the suspension tube 11. The pick tool 6' with the further sample of microorganisms is then lowered into the suspension medium 14 and vibrated by the transfer device 15 in a linear vertical motion for a period of time to release a further sample 19 of microorganisms 4 into the suspension medium 14. Again, turbidity is measured during the vibration and the measurements are compared with first and second threshold values ​​stored in the memory 34 of the controller 30. In this case, the controller 30 may be arranged to control the movement of the positioning device 8 so that the first pick tool 6 is raised to a standby position once the further sample has been at least partially removed from the first pick tool 6 and if, during the vibration, the online measurements of turbidity taken by the turbidimeter 20 are below the first threshold value and above the second threshold value.

[0134] If the measured turbidity is less than the threshold level, the concentration of microorganisms in the suspension medium 14 may be increased by multiple subsequent colony picks as just described, although other procedures may be implemented instead to account for the measured concentration being determined to be too low. In this regard, as described in more detail below, multiple dispenses of low concentration suspension may be deposited onto the same spot on the MALDI plate. This has the effect of concentrating the microorganisms 4 on the MALDI plate rather than in the suspension medium 14.

[0135] Suspension tubes 11, or alternatively vials or cuvettes particularly useful in the apparatus of the present invention, have a cross-section with a target maximum dimension of about 2 mm to about 12 mm, preferably about 3 mm. For these relatively small suspension tubes, controller 30 can control automated suspension medium dispenser 12 or other medium dispenser so that the initial volume of suspension medium dispensed is about 0.1 ml to 5 ml, preferably less than about 1 ml.

[0136] The vibration of the transfer device 15 is controlled by the controller 30 so that the pick tool 6' vibrates at a frequency between about 5 Hz and about 250 Hz, preferably about 100 Hz, with an amplitude of about 0.5 mm to about 4 mm, preferably about 2 mm to about 3 mm. The controller 30 is further arranged to control the vibration of the transfer device 15 so that the pick tool 6' vibrates for a period of about 3 seconds to about 120 seconds, preferably about 30 seconds to about 60 seconds.

[0137] Automated system and method for nephelometry Various nephelometer embodiments are described herein. It should be understood that one of the nephelometers described herein may comprise the nephelometer 20 described above. In one embodiment, the nephelometer used in the automated system 1000 may be the nephelometer described in U.S. Provisional Application No. 62 / 056,911, which is assigned to the assignee of the present invention and incorporated herein by reference. In this embodiment, the suspension is not vibrated when measuring turbidity.

[0138] Another nephelometer embodiment 100 is shown in Figures 4A and 4B. Nephelometer 100 is a low-volume nephelometer designed to accommodate a single suspension tube, shown as a cuvette 110, with suspension fluid 120 contained within nephelometer base 101 as shown in Figure 4A. Nephelometer 100 also includes a light source 130, a focusing lens 170, a side scatter detector 140, a transmitted light detector 150, and a light-attenuating filter 160 (best shown in Figure 4B). Cuvette 110 with sample 120 is positioned at the center of nephelometer 100 and within nephelometer base 100. Light source 130, scatter detector 140, and transmitted light detector 150 are positioned at 90-degree angles relative to each other around cuvette 110. Scatter detector 140 is positioned very close to cuvette 110 containing sample suspension 120 and parallel to the incident beam from light source 130. This minimizes the effects of diffraction, refraction, and reflection on the scattered light. The transmitted light detector 150 is positioned 180 degrees, i.e., opposite, from the light source 130. The detector 150 may also be oriented perpendicular to the incident light beam or at a different angle to reduce the effects of reflections from its surfaces. A light-attenuating filter 160 is positioned between the cuvette 110 and the transmitted light detector 150. In this configuration, the sample suspensions are individually processed inside the containers 110, and the nephelometer 100 detects scattered light and / or transmitted light that passes through the tested samples 120 at an angle.

[0139] The use of low-volume containers / cuvettes (or microcuvettes) designed for processing relatively small volumes of biological and fluid suspensions is contemplated for use with low-volume nephelometers such as nephelometer 100. FIGS. 5A and 5B illustrate alternative embodiments of such low-volume cuvettes. Cuvettes 110, 110′ are molded from optically clear plastic and have minimally tapered sides 430, 440 with optically smooth polishes for convenient orientation within nephelometer 100. Cuvettes 110, 110′ may be configured as individual units for single-use applications. However, in some embodiments, discussed further below, in which a series of cuvettes is used to prepare suspensions, cuvettes 110, 110′ may be configured for use with a linear array strip for such applications. Alternatively, cuvettes 110, 110′ may be configured for use with a matrix array designed to process multiple samples simultaneously. In matrix embodiments, multiple series of suspensions are prepared in parallel.

[0140] As shown, the cuvettes 110, 110′ have a lower portion 410 having a relatively small volume compared to the upper portion 400. A suspension is first prepared in the small-volume portion 410. Thus, the suspension is first disposed within the lower portion 410 of the cuvettes 110, 110′. A biological sample suspected of containing the target microorganism is added to and mixed with the fluid suspension to provide the test sample suspension 120. The turbidity of the suspension within the lower portion 410 is measured. In this regard, when the cuvette 110 or 110′ is coupled to the nephelometer 100, light generated by the light source 130 passes through the sample suspension 120 disposed within the lower portion 410. The nephelometer 100 detects the light scattered by the lower portion 410 with the detectors 140 and 150 and measures the turbidity of the sample within the lower portion 410 of the cuvette based on the detected light.

[0141] Beneath the lower portion 410 of each cuvette 110, 110′ is a “large particulate” collection area 420 designed to receive large particles that settle out of the sample suspension, which would otherwise adversely affect the accuracy of the turbidity measurements made by the nephelometer 100. Low-volume samples typically have insufficient volume to allow particulate contaminants to settle out of the portion of the suspension interrogated by the nephelometer. For example, light passing through a low-volume suspension containing particulate impurities may not distinguish between the sample and the impurities in the suspension and may result in an inaccurate McFarland value (i.e., a value indicative of turbidity) that causes the sample to be improperly processed. For example, an inaccurate McFarland value may signal an incorrect dilution. An inaccurate McFarland value may also cause the sample to be processed downstream (e.g., by either AST or MALDI) when the sample would not have been further processed if the true McFarland value had been known. That is, the true McFarland value would have informed the operator that the sample was unsuitable for MALDI or AST. Furthermore, the presence of impurities within the sample can interfere with accurate concentration measurements of the sample being tested. Therefore, the cuvette 110, 110' according to the illustrated embodiment provides this separate particulate collection area 420, which is outside the direct light path through the lower portion 410. Particulate contaminants settle within the collection area 420 and do not remain within the area under test of the sample suspension emerging in the lower portion 410. The cell length of the lower portion is in the range of approximately 5.5 mm and is designed to provide a cell length sufficient for low-volume samples to obtain adequate turbidity measurements. The lower portion is designed to provide a sufficient cell length when the test sample suspension is prepared for light to pass through the sample and be captured by the detectors 140 and 150. Preferably, the lower portion 410 is made of a highly polished optical material or a material having near optical clarity and other optically transmissive materials known to those skilled in the art. Such a material allows light to pass through the walls 440 of the lower portion of the cuvette without interference.

[0142] Those skilled in the art will recognize that there are three dimensions of design freedom for configuring the small-volume portion 410 of the cuvette 110, 110′. The dimensions of the small-volume portion 410 are primarily a matter of design choice. In one embodiment, the dimensions of the small-volume portion 410 are configured to receive a device (e.g., a pick tool) that will introduce a sample into the lower portion of the cuvette. For example, and not by way of limitation, the lower portion of the cuvette is sized to provide adequate space for a 3 mm diameter pick tool to be immersed and rotated within the lower portion without contacting the sides of the cuvette 110 or 110′ and creating scratches and surface aberrations that would reduce the optical transparency of the cuvette.

[0143] Of course, the dimensions of the lower portion 410 must accommodate optical inspection of the sample. In particular, the lower portion 410 of the cuvette 110, 110' is sized to work with the light source 130 and detectors 140, 150 of the nephelometer 100. Thus, the dimensional constraints on the cuvette design are determined by the configuration of the nephelometer 100.

[0144] Above the lower portion 410 is the upper portion 400, which is used to dilute the sample suspension contained within the vessel for further processing in downstream applications such as AST. The upper portion 400 has a wider width and a longer length than the lower portion 410. Preferably, the vessel's interior dimensions are designed to accommodate automated mixing of the biological sample with the suspension fluid to further dilute the test sample suspension directly within the vessel when needed. In operation, the hierarchical vessel design of the cuvettes 110, 110' allows the turbidity of the sample suspension within the cuvette to be measured, and if the target turbidity is not reached, the sample can be further diluted and the turbidity measurement repeated. This configuration allows for sample dilution in real time (i.e., as the sample is being optically interrogated). Furthermore, the hierarchical vessel design allows for measuring the turbidity of low-volume sample suspensions (e.g., suspensions having a volume of about 200 μL to about 500 μL) while still having the benefit of a large volume to accommodate sample dilution.

[0145] As shown, the upper level or upper portion 400 of each cuvette 110, 110′ has a generally square or rectangular perimeter. Essentially, the geometric configuration of the upper portion 400 is a matter of design choice. The lower level or lower portion 410 similarly has a generally square perimeter. In this regard, the cuvettes 110, 110′ “telescope” from top to bottom due to the larger cross-sectional dimensions of the upper portion 400 compared to the lower portion 410. Alternative shapes for the cuvettes 110, 110′ are also contemplated, so long as the walls 440 of the lower portion 410 are at an angle from each other (e.g., the cuvette is not cylindrical, elliptical, etc.). It has been found that positioning the walls 440 of the lower portion 410 (i.e., the portion received by the nephelometer) at an angle from each other allows for less aberration to the optical signal and better mixing of the test sample (compared to a circular tube). This is shown in the illustrated embodiments 110, 110', where the lower portion 410 has four sides 440 that are perpendicular to one another, thereby defining a square. Additionally, the upper portion 400 similarly has four sides 430 that are perpendicular to one another, except that the sides 430 are wider than the sides 440. The smaller lower portion 410 is configured to be received by the nephelometer base 101 and / or a linear cuvette array (described below). The top of each cuvette 110, 110' has an opening 450 for receiving the sample and diluent / suspension medium. The sidewalls 430 and 440 of the upper and lower portions 400, 410, respectively, are defined by planar surfaces. Without being bound to any particular theory, it is believed that the planar surfaces minimize diffraction and refraction of light passing through the cuvettes 110, 110'. Additionally, the square configuration of the cuvettes / vessels 110, 110' allows the light path to pass through and enter the sample suspension and vessels perpendicular to the planar surfaces of such vessels 110, 110'. This configuration also minimizes the potential for diffraction or refraction of the light source 130 as it enters and exits the cuvettes 110, 110'.

[0146] Various configurations of the cuvettes 110, 110' are contemplated. In the embodiment shown in Figure 5A, the upper portion 400 of the cuvette 110 is tapered relative to the lower portion 410. The corners of the upper portion 400 where the sidewalls 430 intersect are aligned with the corners of the lower portion 410, as seen by the straight edges 401. The tapered edges 401 define the transition between the wider upper portion 400 and the narrower lower portion 410.

[0147] In another embodiment 110′ shown in FIG. 5B , the corner of the upper portion 400 where the sidewalls 430 intersect is offset from the corner of the lower portion 410 where the sidewalls 440 intersect. Such offset occurs at an offset edge 402, as shown in FIG. 5B . In one particular example, the corner of the lower portion 410 is offset by 45 degrees from the corner of the upper portion 400. Advantageously, this configuration allows the light source 130 and detectors 140, 150 to be positioned on either side of the cuvette 110′ when the cuvette 110′ is installed inside the nephelometer base 101.

[0148] A method of using a nephelometer 100 and a cuvette 110 to measure turbidity, as shown in the flowchart of FIG. 6, is now described. The cuvette 110 is manually or automatically placed inside the nephelometer base 100. An initial suspension fluid (free of microorganisms) is placed inside the cuvette 100. The fluid volume is about 200 μL to about 500 μL. Preferably, the initial suspension fluid volume is about 300 μL. If dilution is required, additional fluid may be added to the cuvette 110 to obtain a specified McFarland value. A biological sample suspected of containing microorganisms is then added to the cuvette 110 and mixed with the suspension fluid to produce a test sample suspension 120. The nephelometer 100 measures the initial turbidity of the test sample 120, and the McFarland value is recorded in memory 34. The sample suspension is further diluted by adding additional suspension fluid if the initial turbidity reading is too high. Dilution is automated in one embodiment. The upper portion 400 of the cuvette 110 allows the volume of suspension fluid to exceed the volume of the lower portion 410. The nephelometer 100 measures the turbidity of the diluted suspension. Once a predetermined McFarland value is obtained, the suspension is processed for downstream testing, stored, or discarded. The suspension may be diluted as many times as necessary to obtain the predetermined McFarland value.

[0149] Light from the light source 130 interrogates the suspension 120 (e.g., the sample under test) disposed inside the cuvette 110. Light that strikes a surface (e.g., the flat sidewall 440 of the cuvette / container 110) is referred to herein as incident light. Light that is scattered from the particles of the suspension 120 is referred to herein as scattered light. A portion of the incident light is reflected by the cuvette surface. Refracted or transmitted light is the portion of the incident light that is transmitted through the surface (e.g., the flat sidewall 440 of the cuvette / container 110).

[0150] In operation, transmitted light is received by transmitted light detector 150. In an exemplary embodiment, transmitted light detector 150 is positioned on the incident light path to maximize detection of light transmitted through the suspension. In cases where the surface of detector 150 is highly reflective, detector 150 may be positioned so that the detector surface is positioned at a slight (non-90 degree) angle relative to the axis of the light path. Positioning detector 150 at an angle optimizes detection of transmitted light without reflecting light back into suspension 120 or directing light to other portions of nephelometer 100. The intensity of light collected by detector 150 is proportional to the turbidity of the suspension.

[0151] A light-attenuating filter 160 is positioned immediately in front of the transmitted light detector 150. The filter reduces the intensity of the light incident on the detector 150 by an amount proportional to the amount of incident beam. In an exemplary embodiment, the filter 160 allows the detector 150 to operate without saturating and provides a detector operating intensity bandwidth sufficient to detect small variations in the intensity of the transmitted light.

[0152] The nephelometer 100 also measures the amount of scattered light. The scatter detector 140 is positioned with its detection surface parallel to the incident light path and along one side of the cuvette 110. A portion of the light passing through the suspension sample 120 is scattered by particles in the suspension. The side scatter detector 140 collects a portion of the scattered light. The amount of scattered light collected by the detector 140 provides a signal proportional to the amount of particles in the suspension 120 under test. One method of measuring the turbidity of the suspension 120 is to process the amount of scattered light collected by the scatter detector 140 with various algorithms well known in the art. The data collected from the scatter detector 140 may be combined with the data collected from the transmittance detector 150 in various ways. For example, the signals may be physically combined, or the detector values ​​may be mathematically manipulated to combine the detector values ​​to further increase the accuracy and reliability of the initial signal. The signals or data values ​​may be combined additively, subtractively, differentially, etc. to provide a result signal representative of the combined signal. Such combining may be performed by processor 32. When detector value signals are combined in this manner, the resolution and accuracy of collected data for measuring turbidity can be increased. Advantageously, data collected from two separate detectors (scattered and transmitted data) may provide more accurate results for small volume samples. In embodiments where a scatter measurement is not sufficient, dual measurements are advantageous. Measuring both transmitted and scattered light may be more accurate due to the limited optical path length through small volume samples 120.

[0153] In the exemplary embodiment, the scatter detector 140 and the transmittance detector 150 are standard high-efficiency photodiode detectors. However, other detectors with similar characteristics may be used as well. Suitable detectors include detectors that operate across the visible light spectrum, from ultraviolet (UV) to infrared (IR). Suitable detectors may be selected based on their linear response curve, size, reproducibility of results, and ability to operate / detect light paths in low light conditions and detect subtle variations in light intensity with measurable resolution. Examples include photodiodes, photomultiplier tubes, avalanche detectors, solar cells, photoresistors, photosensors, etc. Such detectors are commercially available, well known to those skilled in the art, and will not be described in detail herein.

[0154] In an exemplary embodiment, the light source is a high-intensity light-emitting diode (LED) or diode laser. Preferably, the wavelength of the LED light is approximately 650 nm. Preferably, the wavelength of the detector light is within the red band (i.e., approximately 620 nm to 750 nm). However, those skilled in the art may use interrogation light at different frequencies of visible light. Optionally, a converging lens 170 (FIG. 4B) is used to focus the light into a narrow beam (e.g., a beam with a diameter of approximately 3 mm). The converging lens 170 is positioned in front of the light source 130. The use of the converging lens 170 focuses the light from the light source 130 into the sample area 410 of the container / cuvette, minimizing the amount of light that may be scattered from the test area. Those skilled in the art know that light scattered outside the test area (i.e., the lower portion 410 of the cuvette 110) will prevent the scattering from being used to measure sample turbidity due to a high background signal. The converging light then passes through a converging lens 170 (not shown) at an angle perpendicular to the plane of the cuvette 110 to enter the lower portion 410 of the cuvette 110. The perpendicular angle reduces undesirable diffraction and refraction that occur when a light beam passes from one medium (e.g., air) to another (e.g., the flat surface side of the cuvette). The path of the converging light beam is maintained as the light passes through the suspension toward the detectors 140 and 150. In embodiments in which the light source 130 is a diode laser, an additional lens 170 may not be needed to focus the light beam. This is due, in part, to the properties of lasers, which provide collimated and focused light for interrogating the suspension. The converging lens 170 is used in embodiments in which the light source 130 is an LED and collimation or focusing of light is desired or required.

[0155] 7A and 7B show another nephelometer embodiment 200 in which cuvettes are advanced serially through the nephelometer. The system is designed for use with a series of cuvettes (described below) advanced through the nephelometer in a continuous manner. Individual cuvettes 110 may be placed directly inside the nephelometer base 201 by placing the lower portion of the cuvette into the channel 220 shown in FIG. 7A. Alternatively, individual vessels 110 may be first placed inside a linear vessel array 300, and a linear array 300 (FIG. 8) containing multiple vessels may be placed inside the nephelometer via the pass-through channel 220. After the vessels are placed individually inside the nephelometer base or inside the linear array, suspensions are prepared in the cuvettes and turbidity is measured, as described above.

[0156] Nephelometer 200 similarly includes a light source 230, a focusing lens 270, a scatter detector 240, a transmitted light detector 250, and a light-attenuating filter 260, all of which are described above in connection with the nephelometer of FIG. 4B. The cuvette 110 containing the sample 120 is positioned at the center of nephelometer 200 and within nephelometer base 201. Light source 230, scatter detector 240, and transmitted light detector 250 are positioned at 90-degree angles from each other around the cuvette 110, as described above. Side scatter detector 240 is positioned parallel to the incident beam from light source 230. Positioning scatter detector 240 closely to the sample under test 120 and parallel to the incident light minimizes the effects of diffraction, refraction, and reflection on the scattered light. Transmitted light detector 250 is positioned opposite light source 230, and the incident light from the light source propagates toward the transmitted light detector. The detector 250 may similarly be positioned perpendicular to the incident light path or at a few degrees from perpendicular to reduce reflection effects from its surfaces. A light-attenuating filter 260 is positioned between the cuvette 110 and the transmitted light detector 250.

[0157] FIG. 8 illustrates a series cuvette array / receptacle for use with one embodiment of an apparatus of the present invention, such as nephelometer 200. This embodiment differs from the previously described embodiment in that the suspension tubes installed for turbidity measurement rotate. The series cuvette array 300 is a series cuvette strip that moves along a guided channel 220. An LED light source 230 is installed on one side of the guided channel 220, which guides the strip 300. The strip 300 slidably engages with the channel 220. The strip 300 may also include standoffs or other structures 530 ( FIG. 9 ) for convenient stacking, packaging, and shipping. The strip 300 is advanced through the nephelometer, and the cuvette wells 320 are positioned between the light source 230 and the detectors 240 and 250 for processing. After processing is completed, the linear strip 300 is indexed and advanced to the next cuvette, and processing may continue for subsequent samples using the same nephelometer. The cuvette strip 300 may be stored or discarded based on the needs of an individual user. In this embodiment, a single nephelometer is designed to efficiently process multiple samples without the need to remove individual cuvettes and replace them with new ones. The linear cuvette strip 300 may be designed with a variety of cuvette shapes, sizes, and configurations. For example, the wells 320 of the strip 300 may be designed to be more or less deep, wide, narrow, long, short, etc., depending on the cuvette design. Furthermore, the wells may be attached to each other before or after individual wells, or may be individually inserted into wells positioned next to each other. Placing multiple cuvettes 110′ with the rim 402 inside the linear array 300 allows for more efficient transport of the cuvettes 110′ through the nephelometer 200 because the cuvettes 110′ can be processed serially, received by the nephelometer, and measured without further manipulation.

[0158] In another single series cuvette embodiment shown in FIG. 9, the cuvette strip is stackable and can be separated into individual cuvettes or a linear strip of cuvettes, depending on the configuration of the nephelometer. In the illustrated embodiment, the cuvettes 500 are carried by a rack 510. The rack 510 has a flat surface from which the cuvettes are suspended. The flat surface is scored (not shown) to allow the cuvettes to be separated into individual cuvettes or a strip of cuvettes. The stackable cuvettes also have the standoffs 530 described above. Note that the lower portion 540 of the cuvette 500 is received by the wider upper portion 550 to facilitate stacking.

[0159] FIG. 10 is a perspective view of nephelometer 590 showing aperture 575 for light source 570, aperture 635 for the scattered light sensor, and aperture 605 for the transmitted light sensor.

[0160] 11 is a cutaway view of a nephelometer 590 showing the path for light transmitted through the lower portion 540 of the cuvette 500. A light source (570, FIG. 12) is received by an aperture 575 on one side of the cuvette receptacle 580 of the nephelometer 590. Aperture 575 receives the light source. A sensor 600 (FIG. 12) is positioned in an aperture 605 directly opposite aperture 575, with the lower portion 540 of the cuvette positioned between them. The nephelometer has a lid 620.

[0161] Figure 12 is a cutaway view of a nephelometer 590 showing the path for light scattered through the lower portion 540 of the cuvette 500. A light source 570 (Figure 12) is on one side of the cuvette receptacle 580 of the nephelometer 590. A sensor 630 (Figure 10) is positioned in an aperture 635 perpendicular to the light source 570, with the lower portion 540 of the cuvette positioned between them.

[0162] 13 is a cutaway view of a nephelometer 590 showing the path for light transmitted through the lower portion 540 of the cuvette 500. A light source 570 is received by an aperture 575 on one side of the cuvette receptacle 580 of the nephelometer 590. Between the sensor 600 and the cuvette 500 is a light-attenuating filter 640, which is placed before the transmittance detector to reduce the light intensity to a usable level so as not to saturate the sensor. Aperture 575 receives the light source 570 and a lens 650 for focusing the light signal. The sensor 600 is positioned in aperture 605 directly opposite aperture 575, with the lower portion 540 of the cuvette positioned between them.

[0163] In one embodiment, samples are placed inside cuvettes and processed individually when placed in the nephelometer. Once the sample is processed and a McFarland value is obtained, the cuvette is removed from the nephelometer and replaced with a new cuvette. In this embodiment, one or more nephelometers operate individually. In an alternative embodiment, the nephelometer is configured to deliver a continuous series of cuvettes to the nephelometer for measurement. The linear cuvette channel 220 receives a strip 300 of individual cuvette wells 320 (FIG. 4B). The strip is transported through the nephelometer, stopping for each cuvette to be optically interrogated for measurement, as described in detail elsewhere herein.

[0164] The method for measuring turbidity according to the present invention is automated. Data collected from the measurement may be further processed to produce meaningful results. In these embodiments, the signal from the detector is fed to a signal amplifier. The amplifier output is transmitted to an analog-to-digital converter circuit, which outputs a digital representation of the input signal. The digital representation of the input signal is then processed using various algorithms to determine whether the measured value is a target value. If the measured value is greater than the target value, the sample is diluted as described above, and the turbidity is remeasured. This remeasurement can be performed manually by an operator or in an automated manner, with the cuvette being transported out of the nephelometer for dilution and back into the nephelometer for further measurement. Methods have been developed to process the signal into usable outputs, using various dilutions of various biological and non-biological samples and relating McFarland values ​​to suspension concentrations. These data are then used to generate data sets that are further analyzed and compared to target values ​​using an algorithm that corrects for linearity and offset in the data curve to generate a representative output value for turbidity. This process is repeated until the target turbidity is obtained, as described elsewhere herein.

[0165] System 1000 may also include a conveyor, the end of which may form stage 2 for a culture dish, or a conveyor and stage 2 positioned relative to one another so that culture plates can be transported onto and removed from the stage by appropriate operation of the conveyor. The conveyor is controlled by controller 30 to automatically position and remove culture dishes containing microorganisms onto and from the stage, respectively. Note that in other embodiments not shown, different means for automatically positioning and removing culture dishes onto and from the stage, respectively, may be used. In particular, controller 30 is arranged to allow the culture dish to be automatically removed from the stage by the automated culture dish positioning and removal device only after a signal is provided that the suspension tube containing the suspension may be removed from the suspension tube holder for further processing. This ensures that it is always possible to pick up additional samples, if needed.

[0166] As shown in FIG. 2 , the apparatus 1000 of the present invention may also include an automatic suspension tube positioning and removal device (not shown) for automatically positioning and removing suspension tubes in and from the suspension tube holder, respectively. Such an automatic suspension tube positioning and removal device may include gripping means for releasably gripping the suspension tube 11. Again, a controller 30 may be communicatively connected to the automatic suspension tube positioning and removal device and configured to control the operation of the automatic suspension tube positioning and removal device and automatically position the suspension tube 11 in the suspension tube holder 10. The controller 30 is particularly configured to automatically remove the suspension tube from the suspension tube holder by the automatic suspension tube positioning and removal device only after a signal is provided that the suspension tube with the suspension may be removed from the suspension tube holder for further processing. The automatic suspension tube positioning and removal device may be movable along the rail 18 independently of the movement of the positioning device 8. The suspension tube 11 may be fetched, and the suspension tube with a suspension medium containing a sufficient concentration of microorganisms may be passed to equipment for further processing, such as a bacteriological incubator. It should be noted that a multi-track system may direct the suspension tube positioning and removal device and the positioning device 8 to different locations where different components may be present or where different processes may be performed.

[0167] The sample suspension thus prepared is used to perform characterization or identification of microorganisms using MALDI, and optionally for other analyses such as AST. To identify microorganisms using MALDI, an aliquot of the sample suspension is obtained using a pipetting or picking tool, and the aliquot is transferred onto the target plate 42. A drop can be obtained using such a tool 46, which is held by the gripping means 49 of the pipettor 40 and then automatically lowered into the suspension at position A. As the tool 46 is lifted out of the suspension, the drop of suspension will adhere to the tip of the tool 46, and the tool 46 can be transferred along a track to position B, where the tool 46 with the suspension is lowered until the drop of suspension contacts the deposition spot 44 on the target plate 42. At least a portion of the suspension will remain on the deposition spot 44 after the tool 46 is lifted away from the target plate 42. Alternatively, pick tool 6 may be used to pick up a quantity of suspension 14 from suspension tube 11, transfer this quantity to position B, and deposit a droplet of suspension onto target plate 42. After the droplet of suspension has been deposited on target plate 42, and in particular when this droplet has been allowed to dry, MALDI matrix solution is automatically overlaid onto the amount or portion of sample deposited on target plate 42. To perform other tests or other analyses, a second droplet of sample suspension may be obtained in a similar manner, and such droplet may be automatically transferred and deposited into, for example, a test culture dish, which may then be further transferred in an automated manner to perform a susceptibility test or other further analysis.

[0168] In one embodiment, the matrix solution is dispensed in multiple spots on the target plate 42. This improves throughput and reduces the cost of consumables such as pipettes. In this embodiment, a sufficient volume of matrix solution (i.e., for many target spots) is aspirated into a pipette, which is then used to sequentially dispense into multiple spots. Dispensing low volumes of fluid, typically in the range of 1 μL to 20 μL, requires contacting a droplet of fluid with the surface being dispensed, thereby allowing the surface tension of the fluid contacting the target plate to pluck the droplet from the pipette tip 46. During the process of "touching off" a droplet onto the target plate surface, the pipette tip 46 may unintentionally contact the surface of the target plate 42. If the pipette tip 46 contacts the target plate 42, there is a risk of carryover of sample material from one target plate spot to the next, resulting in cross-contamination. In an effort to prevent cross-contamination, capacitive liquid detection is integrated into the pipettor 40 to detect when a droplet contacts the target plate 42. Capacitive liquid detection is used to perform multiple dispenses from a single volume of matrix solution in a single pipette according to the following steps.

[0169] First, a new pipette tip is picked, then the dry tip is moved to the target plate 42, and the plate is contacted by the pipette tip 42 at a non-target location to determine and record the precise vertical (Z) position of the target plate location relative to the tip interface.

[0170] A sufficient volume of matrix solution is then aspirated from the matrix reagent container. The container has a septum that prevents evaporation of the matrix solution. After the matrix solution is aspirated and the tip is removed from the matrix container, the septum wipes away any residual matrix fluid that may have coated the tip 46. This ensures that a droplet will form at the end of the tip as the matrix solution is dispensed from the tip and will not travel up the side of the tip 46.

[0171] The pipette tip 46 is then moved to the target plate 42. A droplet forms on the end of the tip 46. The tip 46 moves down in the vertical (Z) direction until the droplet contacts a target plate spot 44. When the droplet contacts the plate, the capacitance sensing circuit provides a signal indicating that the droplet is contacting the plate 42. The vertical (Z) position of the tip 46 is checked to ensure that the tip 46 is not contacting the plate 42. If the tip 46 is not contacting the plate 42, the multi-dispense process continues. If the tip 46 is contacting the plate 42, the tip 46 is ejected into the waste, a new tip is captured, and the dry tip is moved into position to contact the target plate, establishing a vertical (Z) position for the new tip to contact the target plate. The process continues until all target spots 44 on the plate 42 are inoculated with matrix solution.

[0172] Obtain ID and AST samples from the same suspension Preparation of a suspension from a single colony pick used for both MALDI and AST testing is described in U.S. Patent No. 9,180,448, which is assigned to the same assignee as the present application and is incorporated herein in its entirety. While this disclosure may refer to a sample preparation device (hereinafter, sample preparation or preparation station) as a "Phoenix AP," an AST system as a BD Phoenix™, or a mass spectrometry system as a MALDI, it should be understood that the meaning of these terms is not limited to devices bearing these trademark names, but may include devices with substantially similar functionality. Devices with substantially similar functionality may include Vitek (bioMerieux) and MicroScan (Siemens Healthcare) ID / AST systems.

[0173] In one embodiment, the devices described herein integrate the microbial identification capabilities of a MALDI instrument with the AST and data processing capabilities of a laboratory analysis or processing system such as a Phoenix, Phoenix AP, BACTEC, or EpiCenter system.

[0174] As described above, a suspension is prepared from microorganisms picked from prepared plates 3 or collected from blood culture vials. In one embodiment, suspension tube 11 is over-inoculated with microorganisms 4. Tube 11 is advantageously used as the source for both ID and AST. This ensures that not only samples from the same patient, but also the same isolates, undergo ID and AST testing.

[0175] The suspension is prepared to a concentration suitable for MALDI. Suspensions suitable for MALDI typically have a McFarland value of about 2. An automated system is used to inoculate the suspension tube 11 using a pick tool 6, monitor the turbidity, and process the suspension to provide a suspension with a target turbidity. The automated process for providing a suspension with a target turbidity is described in detail herein. The suspension is then used to inoculate the MALDI plate 42 as described above. As described above, the system 1000 associates the culture dish 3 with the suspension tube 11 and the MALDI plate 42 through the use of machine-readable tags and codes. In one embodiment, the device scans the barcode on the MALDI plate 42 and writes the plate ID to an RFID tag on the suspension tube rack. The system 1000 uses an automated pipettor 40 to automatically add MALDI reagents (e.g., formic acid, matrix, etc.) to prepare the suspension to be dispensed onto the MALDI plate 42 for the analyses described herein.

[0176] System 1000 then uses automated pipettor 40 or dispensing nozzle 30 to dispense additional solution (e.g., deionized water) into tube 11, and nephelometer 20 monitors turbidity to provide a suspension with a turbidity suitable for AST or other diagnostic tests (e.g., molecular tests). Automated systems and methods for providing a suspension with a target turbidity are described in detail herein and will not be repeated. For AST, the target turbidity is approximately 0.5 McFarland, and typically is approximately 0.25 McFarland or higher. Pipettor 40 then transfers the aliquot to AST tube 82. RFID tags on the test tube rack are updated with the calibration results.

[0177] The AST tube 82 shown in FIG. 2 is held by an AST tube mover 80. The AST tube mover 80 is generally a robot disposed below the system deck 7 and configured to move in at least two dimensions, as indicated by the vertical and horizontal arrows in FIG. 2. In particular, the AST tube mover 80 is configured to hold the AST tube 82 by a receptacle, gripper, or the like, move the AST tube 82 below the deck 7, and move the AST tube 82 between predesignated positions located at the preparation station 1030 and the transfer station 1040, respectively. In this regard, the deck 7 may have openings through which the mover 80 can lift and lower the AST tube 82 at these predesignated positions. Of course, it is also contemplated that the suspended tube gripper robot 50 may move the AST tube 82 between the preparation station 1030 and the transfer station 1040 from a suspended position above the deck 7 rather than from below the deck 7.

[0178] AST tube 82 may be stored in station 1040. Prior to transferring the aliquot to AST tube 82, tube gripper robot 50 grips AST tube 82 with gripping means 59 and moves tube 82 from its storage position to a barcode scanner to register tube 82 with controller 30. The gripper robot 50 then hands tube 82 to mover robot 80. Robot 80 then shuttles tube 82 below deck 7 to position C located at preparation station 1030, where tube 82 is at least partially elevated above deck 7. Pipettor 40 then removes an aliquot of diluted suspension from tube 11, moves to position C, and then inoculates AST tube 82 with the aliquot at position C. Tube mover 80 then shuttles tube 82′ with the suspension therein back to transfer station 1040, as shown in FIG. 2 .

[0179] While at station 1040, another pipettor 60 removes an aliquot of suspension from the AST tube 82'. Prior to removing this aliquot, the cartridge transfer robot 70 grasps the empty AST cartridge 90 with a grasper means 79 and transports the cartridge 90 from storage to a cartridge loading unit 78, which includes a cartridge holding structure for holding the cartridge 90. The unit 78 may be movable to pivot the cartridge 90 from a vertical configuration to an inclined configuration, as shown, to facilitate inoculation. A decapper (not shown) may also remove the cap sealing the cartridge 90 prior to inoculation. The pipettor 60 then automatically inoculates the AST cartridge 90 with the diluted suspension. Both the AST suspension tube 82 and the AST cartridge 90 carry codes that allow the suspension undergoing AST analysis to be associated with the pick from which the suspension was prepared. The device has a data management system that associates cartridges with the suspension used to inoculate the cartridge 90. System 1000 reads the MALDI plate ID and plate location for each suspension and makes the necessary associations between the picked colonies from the identified culture plate 3 with the suspension prepared for the culture plate 3, the MALDI plate 42, the location on the plate 42 where the associated suspension will be inoculated, the AST suspension tube 82, and the AST cartridge 90 where the AST suspension will be inoculated. Automation is provided for inoculating the AST cartridges and transporting the inoculated cartridges to a testing instrument that will perform an AST on the inoculated cartridge. An exemplary cartridge transport instrument for automatically moving the inoculated cartridge 90 into the AST testing instrument and removing the tested cartridge from the AST testing instrument is described below.

[0180] Preparing MALDI plates using layering techniques In one embodiment of the present invention, the suspension is automatically deposited onto the MALDI plate 42 using a dispense / layer method. This method is described in commonly assigned U.S. Provisional Application No. 62 / 038,509, entitled "Method Of Sample Preparation For Maldi," filed August 18, 2014, which was filed as International Application No. PCT / US21015 / 45506, published as WO 2016028684. U.S. Provisional Application No. 62 / 038,509 and International Application No. PCT / US21015 / 45506 are incorporated herein by reference in their entireties.

[0181] In the solution dispensing / layering method described herein, the bacterial suspension to be dispensed is first evaluated to determine its turbidity, as described elsewhere herein.

[0182] A bacterial suspension is prepared as described elsewhere herein. The solution dispense / layering method, as its name suggests, requires the formation of two or more layers of solution for identification by MALDI. A selected volume of sample is dispensed onto a MALDI plate 42 and dried. Subsequently, at least a second aliquot of the suspension (preferably the same volume) is dispensed onto the dried suspension. Dispensing is accomplished using the automated method described above. The second dispensed aliquot is dried. Optionally, more layers of suspension can be deposited and dried. After the last of the two or more layers is dried, the sample is processed for MALDI (e.g., by adding formic acid and then applying a matrix over the sample, as described herein). The sample is then evaluated by MALDI. The solution dispense / layering method has been determined to provide acceptable MALDI results for liquid samples with McFarland turbidity values ​​significantly below 2.0 for both Gram-positive and Gram-negative bacteria.

[0183] For example, in one embodiment, if a liquid bacterial suspension (prepared from bacterial colonies picked from an agar plate as referenced above and suspended in water (mass spectrometry grade)) has a 0.5 McFarland value, which is significantly less than a 2.0 McFarland value, this is an indication that solution dispense / layer sample preparation should be used to prepare this sample for MALDI.

[0184] After deciding to use solution dispensing / layering to prepare the sample for MALDI, the amount of suspension per layer is selected. In the above example for a sample with a 0.5 McFarland value, a volume per layer of at least about 3 μl but not more than about 4 μl is selected. The number of layers depends on the turbidity value and sample volume. Once the layer volume is selected and deposited on the MALDI plate, the sample is dried. The exact drying conditions are a matter of design choice and are selected to obtain rapid drying while maintaining the integrity of the sample for MALDI testing. Suitable drying conditions are easily determined by one of ordinary skill in the art. For example, the drying step can be completed at ambient temperature or with the aid of a hot plate (illustratively, about 40°C to about 45°C). After drying, a second layer of suspension is deposited on top of the first layer. The second layer has the same volume as the first layer. If needed, additional layers are added and dried. Because layering requires additional time and resources, the number of layers is limited to the number needed to obtain accurate results from MALDI.

[0185] Following solution dispensing / layering sample deposition, the sample target wells are processed using typical MALDI procedures (addition of 70% formic acid and matrix).

[0186] It has been determined that the sample preparation process for MALDI depends on a variety of factors, most notably i) the concentration of microorganisms in the suspension, ii) the volume of the suspension, and iii) the number of aliquots, if applicable. The microbial concentration is reflected in the turbidity of the sample. Roughly speaking, the higher the turbidity, the higher the microbial concentration.

[0187] Turbidity is measured by a nephelometer as described elsewhere herein. Once the turbidity of the suspension has been assessed as described above, a decision is made on how to prepare the sample for MALDI. Such a decision is made by evaluating the turbidity information and the sample volume. In these embodiments, the sample information is entered into a database. The database (pre-programmed with information on the sample preparation best suited for a particular sample) outputs a recommended method for MALDI sample preparation.

[0188] In an automated system, a processor controls the MALDI preparation protocol in response to information received about the sample. The system processor compares the measured turbidity to a predetermined turbidity threshold, as described herein. If the processor determines that the sample turbidity is within a predetermined range of turbidity values, the processor provides instructions to transfer a predetermined volume of diluted sample to the MALDI plate 42. The automated system prepares the sample for MALDI based on instructions from the processor (i.e., adding formic acid to immobilize the sample prior to MALDI, followed by application of a MALDI matrix solution over the sample, as described elsewhere herein). If the processor determines that the turbidity exceeds the predetermined range, the processor provides instructions to prepare the MALDI sample using a volume less than typical (i.e., if 0.5 μl is normally deposited on the MALDI plate 42, only 0.25 μl is deposited on the MALDI plate 42 instead of the highly turbid sample). If the processor determines that the turbidity is below a predetermined range, the sample is deposited in multiple layers onto the MALDI plate 42, with the sample drying between depositions. As noted above, the methods and apparatus described herein are fully automated embodiments, such that the system, based on instructions from the processor, dispenses the suspension onto the MALDI plate 42 using the automated pipettor described herein and above.

[0189] 14 shows a process flow for an automated process for multiple dispensing of a suspension onto a MALDI plate 42. The suspension is automatically prepared and its turbidity assessed as described elsewhere herein. If the measured turbidity is within a predetermined range, an aliquot having a predetermined volume is deposited onto the MALDI plate. If the measured turbidity is greater than the predetermined range, a smaller volume of sample is deposited onto the MALDI plate 42. If the measured turbidity is less than the predetermined range, the sample preparation protocol described above using multiple dispenses with drying between dispenses is used.

[0190] In one exemplary embodiment, a sample is obtained and a suspension is prepared. The turbidity is measured. If the turbidity (in McFarland units) is between about 2 and about 6, about 3 μl is deposited onto the MALDI plate 42. If the sample turbidity is greater than about 6, the amount of sample deposited onto the MALDI plate 42 is reduced to about 1 μl. If the sample turbidity is less than about 2 but within the range of about 1 to about 2, about 3 μl of sample is deposited onto the MALDI plate 42 and dried, and a second 3 μl sample is deposited and dried. If the sample turbidity is about 0.5 to about 1, three "layers" of suspension, each about 3 μl, are deposited and dried. If the sample turbidity is about 0.25 to about 0.5, four "layers" of suspension, each 3 μl, are deposited and dried.

[0191] After the sample is deposited and dried, the sample is processed for MALDI as described elsewhere herein.

[0192] Each suspension tube is provided with a unique identification mark which is stored in the memory of the central control computer together with the characteristics of the suspension with a link to the identity of the culture dish from which the selected colony of the microorganism was obtained, thereby inter alia correctly and quickly linking the obtained analytical results to the culture dish and colony associated with that result.

[0193] In yet a further embodiment, the system has a predetermined range of turbidity within which no dilution is required for either MALDI or AST. If the turbidity is within this predetermined range (e.g., about 0.5 McFarland to about 2 McFarland), the suspension can be used to inoculate the MALDI plate 42 using the layering method as described above (unless the suspension is concentrated enough for a single aliquot). In this embodiment, the volume of suspension inoculated into the suspension tube similarly varies depending on the measured turbidity. For example, if the McFarland value for the suspension is 0.5, a volume of 25 μl is inoculated into the suspension tube. For that same specification, if the nephelometer measures a 1 McFarland suspension, only 12.5 μl of that suspension would be used. Both aliquots deliver approximately the same amount of microorganisms into the suspension tube 11, but the volume of the 0.5 McFarland suspension is twice the volume of the 1 McFarland suspension. Thus, there is an inverse relationship between the McFarland value of the suspension and the volume of suspension inoculated into the AST tube 82. The higher the McFarland value, the smaller the volume of suspension inoculated into the suspension tube 11. This is because, for AST, it is the amount of microorganisms inoculated into the tube 11, not the volume, that determines whether the dispensed amount is appropriate. If the nephelometer 20 determines that the suspension is within the predetermined range, that information is communicated to the controller 30, which then determines whether dispensing onto the MALDI plate 42 should be performed by layering or whether a single dispense is sufficient. The controller 30 will determine the volume of suspension to dispense into the AST tube 82 by reference to a lookup table that will similarly specify the dispensed amount as a function of the measured turbidity.

[0194] Inoculation of cartridges for ASTs is described in U.S. Patent No. 6,096,272 to Clark et al., which is incorporated herein by reference. In practice, a suspension is inoculated with an AST inoculum fluid, which is then transferred into the test cartridge 90 using the automated fluid transfer mechanism described above. The AST cartridge 90, with its top inoculation port for loading, is tilted (see FIG. 25). Each well in the AST cartridge 90 is inoculated with the AST inoculum fluid. The inoculum fluid flows down the AST cartridge in a serpentine fashion, filling the wells as the liquid front advances toward the absorbent pad. Each well is drained, allowing the liquid to fill the well. Each well has a sharp, circular rim that separates a consistent amount of liquid from excess and isolates each well from the liquid in adjacent wells 31. The pad absorbs the excess liquid.

[0195] As shown in FIG. 2 , a certain amount of suspension can be taken from the suspension in the suspension tube by a pipetting tool 46, which can be automatically held and positioned by a gripping means (functioning as a pipetting tool holder) 49. The pipettor 40 is arranged to position the pipetting tool 46 at a start position above the suspension tube 11, to automatically lower and lift the pipetting tool 46 into and out of the suspension, and to position the pipetting tool 46 at a transfer position B above the MALDI plate 42. When the pipetting tool 46 is lowered into the suspension in the suspension tube 11, it operates in a manner known per se (e.g., using a vacuum) to pick up a certain amount of suspension. The pipetting tool with that amount of suspension is then raised to the transfer position. To hold that amount, the pipetting tool comprises a pressurizable chamber that is closed by a controlled valve. The pipetting tool 46 is automatically transferred to position B above one of the deposition spots 44 on the target plate 42. In this position, the pipetting tool 46 is lowered a predefined distance above the target plate 42, after which the chamber is pressurized to a pressure in the range of about 0.5 bar to 1.1 bar. The valve is then opened for a time such that a droplet of suspension having a volume in the range of about 0.5 μl to 3.0 μl is deposited on the deposition spot 44, specifically covering at most about half of one of the deposition spots on the target plate 42. After the droplet is deposited, the pipetting tool 46 can be lifted from the target plate 42 and transported to a location where the pipetting tool 46 can be discarded or cleaned for reuse.

[0196] Figure 19 shows a flowchart comparing the timeline of the automated process of Figure 18 with that of an equivalent process performed manually. The manual process has been shown to take up to 48 hours, requiring an 18-24 hour incubation period, only after which the plates are evaluated for growth. In contrast, the automated process can detect even relatively low contrast between colonies (compared to the background and each other), requiring only 12-18 hours of incubation before specimens can be identified and prepared for further testing (e.g., AST, MALDI).

[0197] Further aspects of the above-described embodiments are described below: The above-described user interface provides a user with an image of a culture plate, and the user can interact with the interface to pick a colony of interest from the plate.

[0198] Once the user selects a colony, the instrument provides a menu selection for the user to select one or both of MALDI, AST, or both for sample processing. Based on the size of the pick tool (i.e., a pipette, as described elsewhere herein), the instrument provides a pick tolerance to ensure the colony is picked within a designated area. The pick tool is then locked onto the target, and the colony is picked. In one embodiment, the pick tolerance diameter is 5 mm. With a pick tool having a 3 mm diameter, this distance ensures that a 1 mm diameter area within the pick tolerance area will be picked. The processing selection is sent to the controller, allowing the processing of the sample to be traced.

[0199] The system overviews in Figures 1-3 show the identification (MALDI-TOF) and AST (antibiotic susceptibility testing) preparation stations and the user interface touch screen 1006. Figure 2 shows that a dish 3 with colonies 4 thereon is designated for picking, which is moved to the system's pick station 1020. The dish 3 is aligned for colony picking in the manner described herein above. The dish 3 is then scanned for traceability.

[0200] The dish cover is then removed and the pipette pick tool 6 is moved over the selected colony 4. The positioning device 8 moves the pipette pick tool 6 from the pick position to the inoculation position A where the colony is placed in the suspension tube 11 as described above. The tube or cuvette is described herein above. The pipette 6 brings the sample into the tube 11 with the suspension already present therein. The relative amounts of the suspension are controlled so that the suspension meets the predetermined McFarland standard as described herein above.

[0201] Just as samples, suspensions, etc. can be traced throughout the processing of the methods and devices described herein, consumables in the devices and methods can be tracked by barcodes. The device will allow for fully automated inventory management of consumables within the device.

[0202] When the suspension is deposited onto the MALDI target plate 42, it can be spotted in multiple layers as described elsewhere herein. Drying of the sample and formic acid extractant is also described above. The deposition of the MALDI spots onto the target plate 42, followed by the deposition of the matrix solution, is automated as described above.

[0203] After an aliquot for MALDI is obtained from the suspension, the suspension is further used to inoculate tubes 82 for AST testing. In one embodiment, a larger pipette 46 is used for inoculating the AST tubes than for colony picking. In one embodiment, a 50 μl pipette is used for colony picking, and a 1 ml pipette tip is used to prepare the suspension for AST. The target turbidity for AST, in one embodiment, is 0.5 McFarland (McF). The AST tube with AST broth therein is removed from the rack. In one embodiment, the broth tube also contains Alamar Blue (Alamar Biosciences, Sacramento, CA). Alamar Blue is a redox colorimetric indicator that changes color from deep blue to bright pink in the presence of metabolically active growth organisms. The use of Alamar Blue in susceptibility assays is well known to those skilled in the art and will not be described in detail herein. The AST tube 82 is scanned as an example of system-wide traceability of consumables, reagents, and samples. The cap is removed from the AST broth tube using a decapper, the cap is then discarded, and the AST broth tube 82 is then transported to a location where the AST panel 90 is inoculated. The inoculation location is shown in FIG. 2. FIG. 2 shows the automated pipettor 60 used to inoculate the AST panel 90 with the AST broth in the left bottle. A dye is used to change the color of the broth. FIG. 2 shows the pipette 46 used to inoculate the AST tube 82 with a 0.5 McF suspension. FIG. 2 shows that the pipette 46 is used to mix the suspension in the AST tube 82 by repeatedly aspirating and dispensing the solution.

[0204] FIG. 2 illustrates automation of providing an AST panel 90 (another consumable) for inoculation. FIG. 2 similarly illustrates inoculation of the AST panel 90. Recapping of the AST panel 90 is similarly automated. FIG. 2 thus illustrates how the device provides a seamless process and workflow for sample preparation for both ID and AST. System 1000 can be configured as a modular assembly to perform only one of identification (MALDI-TOF), antibiotic susceptibility testing (AST), or both. It is contemplated that the devices described herein can be integrated into larger systems, such as workcell automation for full laboratory automation.

[0205] Cartridge Transfer Thus, system 1000 can be used in conjunction with other laboratory systems / instruments to help fully automate sample preparation and testing. As shown in FIG. 20, system 1000 may be utilized with an automated cartridge transfer instrument 2000 and one or more cartridge testing instruments 2050a-2050d. As noted above, system 1000 can automatically prepare AST cartridges 90 for testing. In the illustrated embodiment, cartridge transfer instrument 2000 is specifically configured to transport such prepared AST cartridges 90 from system 1000 to one of the multiple AST cartridge testing instruments 2050a-2050d.

[0206] The AST cartridge 90 can be any cartridge available for testing an analyte / inoculum, as shown in FIGS. 21, 25, and 26. For example, the cartridge 90 can be any cartridge for performing antibiotic susceptibility testing, such as the BD Phoenix™ ID / AST Panel (Becton, Dickinson, and Co., Franklin Lakes, New Jersey). Regardless of which cartridge is used, such cartridge 90 generally includes an inlet 95 for inoculating the interior space of the cartridge 90 with an analyte, which may include, for example, a microbial suspension or a blood culture. Such an inlet 95 may be sealed by a removable cap or septum 99. For example, the system 1000 may include a capper / decapper (not shown) in the transfer station 1040 for decapping and recapping the removable cap 99.

[0207] As shown in FIG. 21 , controller 30 is coupled to system 1000 (described above with respect to FIG. 3 ), cartridge transfer instrument 2000, and cartridge testing instrument 2050. Controller 30 regulates and controls each of these systems / instruments 1000, 2000, 2050a-2050d to perform cartridge preparation, cartridge transfer, and sample testing. In this regard, controller 30 is configured to perform certain tasks depending on the type of loading and unloading of cartridge testing instrument 2050. For example, controller 30 may be configured to enable manual and / or automated dispensing / transfer of cartridge 90 from preparation system 1000 to cartridge testing instrument 2050, manual and / or automated loading of testing instrument 2050, and manual and / or automated removal of cartridge 90 from testing instrument 2050, which may then be manually or automatically transferred to storage 2006 or waste 2004. Controller 30 may be in the form of a desktop computer as shown, incorporated into a touchscreen panel such as panel 1006 shown in FIG. 1, or some other form known in the art. Alternatively, multiple controllers may be utilized. For example, controller 30 may be connected to instrument 1000 and cartridge transfer instrument 2000, while another controller (not shown) may be separately connected to test instrument 2050. Such controllers may communicate with each other to regulate cartridge transfer.

[0208] Cartridge Transfer Equipment The cartridge transfer device 2000 may be a multi-axis robot including a z-axis arm 2010, an x-axis arm 2012, a rotating member 2014, a cartridge gripper assembly 2015, and a vacuum pump 2002, as best shown in FIG. 20 . The cartridge gripper assembly 2015 is connected to the rotating member 2014, which can rotate the cartridge gripper assembly 2015 to orient the system 1000 in one orientation and the test instruments 2050a-2050d in another orientation. In this regard, the rotating member 2014 can rotate the cartridge gripper assembly at least 180 degrees about the z-axis. The rotating member 2014 and cartridge gripper assembly 2015 may themselves be connected to an x-axis arm 2012 that is connected to the z-axis arm 2010. The z-axis arm 2010 can move the cartridge gripper assembly 2015 vertically to access any one of the test instruments 2050a-2050d, which are shown as stacked in a vertical arrangement. Additionally, the x-axis arm 2012 can move the cartridge gripper assembly 2015 in the x-axis between the system 1000 and the instruments 2050a-2050d.

[0209] 22-24 show the cartridge gripper assembly 2015, which generally includes a movable arm 2030, a support arm 2038, and a gripper plate / member 2020. The movable arm 2030 is suspended from the support arm 2038 and is movable along its axis relative to the support arm 2038, such as by a rack and pinion mechanism. The movable arm 2030 includes a curved upper surface 2034, as best shown in FIG. 23. The gripper plate 2020 is pivotally coupled to the movable arm 2030 adjacent the curved upper surface 2034 and pivots about the axis of a coupling 2036 connecting the two. The curved upper surface 2034 helps to guide and support the gripper plate 2020 as it pivots between a first position and a second position.

[0210] The ability of the gripper plate 2020 to pivot about the pivot axis is further achieved through the use of a torsion spring 2035, shown in FIG. 23. The torsion spring 2035 is wound around a coupling 2036 that couples the gripper plate 2020 to the movable arm 2030. Thus, a force is applied to the gripper plate 2020, increasing the tension in the torsion spring 2035 as the gripper plate 2020 pivots from the first position to the second position. As the tension increases, the potential energy of the torsion spring 2035 increases, urging it toward the first position. In this regard, because the potential energy of the spring 2035 in the illustrated configuration is lowest when the gripper plate 2020 is tilted back to its first or rest position, the cartridge contacting surface 2026 of the gripper plate 2020 is at an angle to the vertical axis, as best shown in FIG. 23. In the first position, the cartridge contact surface 2026 is preferably at about 30 degrees relative to the vertical axis, however, it is contemplated that the angle of the gripper plate 2020 may be greater or less than 30 degrees when in the rest position.

[0211] A force applied to the bottom end of the plate 2020 may move the gripper plate 2020 from the rest position toward a second or transfer position (not shown). When in the second position, the spring 2035 is tensioned such that when the force is released from the bottom end of the plate 2020, the plate returns to the rest position. In the second position, the cartridge contact surface 2026 of the gripper plate is positioned at an angle different from that of the first position. For example, the gripper plate 2020 is oriented such that the cartridge contact surface 2026 is preferably substantially vertical in the second position. However, the second position can be at nearly any angle within a pivot range from the first position, and generally any angle that brings the gripper plate 2020 into flush contact with the opposing surface 2072. The opposing surface 2072 is the surface of a cartridge retaining structure 2070 that receives a cartridge 2070 from the gripper assembly 2015 or hands off a cartridge 90 to the gripper assembly 2020, as shown in FIG. 22 . Such a cartridge retaining structure 2070 may be located in the testing instrument 2050 as well as in the instrument 1000. Alternatively, the cartridge retaining structure may be a tray, such as the tray 2040 shown in FIG. 26 . As such, as will be described, the gripper plate 2020 may pivot between a rest position and a transfer position. It is the ability of the cartridge gripper plate 2020 to pivot that allows the gripper plate 2020 to grip, remove, reposition, and release an object, such as the AST cartridge 90.

[0212] The cartridge contact surface 2026 is adapted to grip the cartridge 90 upon contact with the cartridge contact surface 2026. In one example, gripping is achieved by applying negative air pressure to the cartridge contact surface 2026 of the gripper plate 2020. To obtain the negative air pressure, suction cups 2028 are recessed into the cartridge contact surface 2026 of the plate (see FIG. 24) and connected to air pressure conduits 2037 that feed through openings in the cartridge contact surface 2026, and which supply vacuum from the vacuum pump 2002 (see FIG. 20).

[0213] In a method of use, the gripper plate 2020 is advanced by the movable arm 2030 toward a first cartridge retaining structure 2070, which may be located within the system 1000, while the gripper plate 2020 is in the first position (see FIG. 22 ). In this regard, the bottom edge of the gripper plate 2020 reaches and contacts the cartridge 90 prior to any other portion of the gripper plate 2020. This advancing of the gripper plate 2020 toward the cartridge 90 causes the gripper plate 2020 to matingly engage the cartridge 90 and assist in removing the cartridge 90 from the cartridge holder 2070. Once the gripper plate 2020 contacts the cartridge 90, the movable arm 2030 continues to advance, applying a force to the torsion spring and pivoting the gripper plate 2020 from the first position to the second position. The second position is reached when the cartridge contacting surface 2026 of the gripper plate 2020 is substantially flush with the stationary opposing surface 2072 that holds the cartridge 90. In the second position, the cartridge contacting surface 2026 is also substantially flush with the cartridge surface 92 (see FIG. 25 ), so that the vacuum urges the cartridge 90 against the gripper plate 2020 and holds the gripper plate 2020 on the cartridge 90.

[0214] Thereafter, once the cartridge 90 is fastened to the gripper plate 2020, the gripper assembly 2015 moves away from the cartridge retaining structure 2070, which removes the force holding the plate 2020 in the second position, which returns the gripper plate 2020 and cartridge 90 to the first position under the bias of the spring 2036. This aids in removing the cartridge 90 from the cartridge retaining structure 2070. Additionally, as the movable arm 2030 moves along the support arm 2038 in a direction away from the cartridge retaining structure 2070, the bumper surface 2039 (see FIG. 24 ) on the support arm 2038 presses against the upper surface of the cartridge 90 held by the gripper plate 2020. This pivots the gripper plate 2020 back to the second position, so that the cartridge 90 is oriented in a substantially vertical orientation. This provides clearance from the cartridge holding structure 2070 for the gripper assembly 2015 to rotate by the rotating member 2014 so that the cartridge 90 can be transported to another cartridge holding structure 2070 for receiving the cartridge 90.

[0215] In this regard, the rotating member 2014 rotates the gripper assembly toward a second cartridge retaining structure 2070, which may be located within the testing instrument 2050. This rotation allows the gripper plate 2020 to be presented to the second cartridge retaining structure 2070. Upon alignment with the second cartridge retaining structure 2070, the movable arm 2030 is advanced toward the retaining structure 2070, which disengages the bumper surface 2039 from the cartridge, thereby releasing the force holding the cartridge 90 and gripper plate 2020 in the second position. This causes the cartridge 90 and plate 2020 to move toward the first position as they advance toward the second retaining structure 2070. In this regard, the bottom end of the cartridge 90 is received first by the second cartridge retaining structure 2070. As the movable arm 2030 advances further, the resistance applied by the second cartridge retaining structure 2070 helps to pivot the cartridge 90 toward the second position so that it is approximately flush with the receiving surface 2072 of the retaining structure 2070. At this point, the cartridge 90 is received by the retaining structure 2070, the vacuum is turned off, and the gripper plate 90 is allowed to move away and return to the first position.

[0216] Alternative features of the cartridge gripper assembly 2015 are contemplated. For example, in another embodiment, the pivoting capability of the gripper plate 2020 is provided through the use of a compression spring (not shown). The compression spring is located between the gripper plate 2020 and the lower surface 2032 of the movable arm 2030. In a first position, the compression spring has a relatively low potential energy. As the gripper plate 2020 pivots from the first position to the second position, the spring is compressed, increasing the potential energy of the spring. In one variation, the compression spring is preloaded with sufficient compression to ensure that the gripper plate 2020 does not experience tilt in either direction before contacting the cartridge retaining structure 2070. The compression of the spring helps to hold the gripper plate 2020 in place as the arm 2030 moves the plate 2020 from one location to another.

[0217] In yet another embodiment, the pivoting ability of the gripper plate 2020 is achieved through the use of a tension spring (not shown). Similar to the compression spring described above, the tension spring is installed between the gripper plate 2020 and the arm 2030. However, in this case, the spring is installed on the upper surface 2034 of the arm. This ensures that as the gripper plate 2020 moves from the first position to the second position, tension increases in the spring, storing more energy for the gripper plate 2020. In a variation similar to that for the compression spring described above, the tension spring may be preloaded in tension in the first position.

[0218] In another embodiment, an elastomeric member (not shown) is used to provide the pivoting function. The elastomeric member is a structure that deforms from an equilibrium state as the gripper plate 2020 pivots from a first position to a second position, and then returns to its original size as the gripper plate returns to the first position. The elastomeric member is preferably made of a material with a sufficiently low Young's modulus to allow elastic deformation in response to resistance forces generated by contact with the stationary equipment as the gripper plate contacts and moves closer to the cartridge retaining structure 2070.

[0219] In another embodiment, a wave spring (not shown) provides the pivoting function. The installation and operation of the wave spring relative to assembly 2015 is similar to that of a compression spring and is generally positioned and attached in a manner known to those skilled in the art.

[0220] In other embodiments, passive means other than those described above may be used to provide the turning function. Passive control forms are well known to those skilled in the art and will not be described in detail herein.

[0221] In further embodiments, active means may be used to provide the pivoting function. Examples of active control include linear actuators, such as electric or pneumatic actuators, pistons, such as electric or pneumatic pistons, rotary ball screws and nuts, and racks and pinions. Similarly, any other form of active control known to one of ordinary skill in the art is contemplated.

[0222] In any of the above embodiments, the gripper plate 2020 can be sized to fit a particular cartridge size. Thus, the dimensions of the gripper plate are not limited to a particular width or length. Furthermore, the thickness of the gripper plate is primarily a matter of design choice, provided that the thickness is sufficient given the material used to support the anticipated object load.

[0223] Similarly, in any of the above embodiments, the cartridge contact surface 2026 of the gripper plate 2020 can be adapted to be flush with different cartridge types, shapes, and sizes. For example, the contact surface 2026 can be characterized by a concave or convex shape over the length of the gripper plate 2020.

[0224] In any of the above embodiments, the gripper plate 2020 may be adapted to include various surface features for gripping particular objects. For example, the cartridge 90 shown in FIG. 25 includes various features that can be used for gripping by the gripper plate 2020. These include, among other features, a gap 93 near a generally longitudinally extending central region of the cartridge, a recessed region 94 in a central portion of the top surface of the cartridge 90, and bumps 96 on the upper and lower ends of the cartridge 90.

[0225] To accommodate these cartridge features, the contact surface 2026 may include protrusions shaped and positioned to fit corresponding features on the cartridge 90. The gripper plate 2020 may likewise be constructed to expand between the bumps 26 to apply forces in opposite directions on the longitudinal axis of the cartridge 90. In other words, the gripper plate 2020 may be adapted to grip the bumps 96. It is also contemplated that another gripper plate structure, such as a structure with opposable fingers, may be adapted to clamp the sides 98 of the cartridge 90.

[0226] In any of the above embodiments, the surface 2026 of the gripper plate 2020 may include structure to maintain alignment of the gripped and ejected object. In one example, improved alignment of the ejected cartridge 90 relative to the gripper plate 2020 is provided by rails 2027, which may run parallel to the sides of the plate 2020 and extend from the top to the bottom of the plate, as shown in FIG.

[0227] Automated loading of sample cartridges for AST 27A-27D illustrate an exemplary cartridge testing instrument 2050. In particular, the cartridge testing instrument 2050 shown is an AST instrument. However, it should be understood that the principles described herein may be applied to any laboratory instrument in which automated input and output of sample cartridges is desired.

[0228] The cartridge testing instrument 2050 generally includes a housing 2052 defining a cavity therein and a first or manual door 2060 and a second or automatic door 2066 for accessing the cavity. The housing 2052 may include a cartridge holder 2054 disposed within the cavity including a plurality of receptacles or cartridge retention structures 2073 for receiving individual cartridges 90. The cartridge holder 2054 and receptacles may be movable within the cavity by activation of a receptacle actuator 2078 (e.g., a motor and belt) such that each receptacle is presentable to the door, which opens to receive or remove a cartridge 90. In one example, the cartridge holder 2054 may be a drum having a plurality of receptacles 2073 that are rotatable about an axis.

[0229] 27A , the first door 2060 is generally located on a first side (considered the front in this embodiment) of the instrument 2050 and is manually operable. The first door 2060 is hingedly mounted to the housing 2052 and includes a mechanical or magnetic latch 2064 or deadbolt that can be locked by an automatic locking mechanism 2074 during operation of the test instrument to prevent the first door 2060 from being opened. In an alternative embodiment, rather than being hingedly connected to the housing 2052, the first door 2060 can be slidably mounted on a track that allows the door to slide open and close.

[0230] As shown in FIGS. 27B-27D , the second door 2066 is generally located on a second side (considered the rear in this embodiment) of the device 2050 and is automatically operable. The second door 2066 is slidably positioned within a track 2056, allowing the door 2066 to slide from side to side, and is coupled to a linear or door actuator 2076, such as a lead screw, rack and pinion, pneumatic cylinder / piston, electric linear actuator, or some other mechanical or electromechanical device. Such a door actuator 2076 opens and closes the door 2064. The track 2056 at least partially defines the extent of the second door opening. The second door 2066 may expose one or more cartridge holders 2054. Two independently operating rear automatic doors are envisioned, allowing the flexibility to expose only the upper or lower cartridge holders. This may require the use of an additional track 2056 and door actuator 2076.

[0231] Testing equipment 2050 may include additional doors, such as a third door and a fourth door, which may be disposed on a side of equipment 2050 and may be operated manually or automatically. Furthermore, second door 2066 may alternatively be disposed on a side of equipment 2050 adjacent the front side where first door 2060 is located. In another embodiment, automatic door 2066 may be integrated with manual door 2060, such that during manual operation, automatic door 2066 moves with manual door 2060, and during automatic operation, only automatic door 2066 opens and closes while manual door 2060 remains closed. Of course, it is also contemplated that testing equipment 2050 may have only one door that can be operated automatically.

[0232] As noted above, the test equipment 2050 may be incorporated as a component subsystem into a broader system that includes the transfer equipment 2000 and the preparation system 1000 and is controlled by the controller 30. Accordingly, the test equipment 2050 may include mechanisms 2070 that communicate with and / or are operated by the controller 30. Such mechanisms are shown in FIG. 28A and generally include, but are not limited to, a user input interface 2071, a display interface 2072, as well as a locking mechanism 2074, a door actuator 2076, and a receptacle actuator 2078.

[0233] As shown in FIG. 27A , the housing 2052 may also include a user input interface 2071 and a display interface 2072. The user interface 2071 may be one or more push buttons or a touch screen that allows a user / operator to input commands or requests, such as a manual override request or instruction. For example, when the test instrument 2050 is in automatic mode, the controller 30 operates the door actuator 2076 for the second door 2066, while the controller 30 operates the locking mechanism 2074 to keep the first door 2060 locked. The input interface 2071 may be configured to allow a user to override the automatic mode, such that, once a test cycle is completed, the controller 30 disables the door actuator 2076 and operates the locking mechanism 2074, allowing the user to open the first door 2060. Additionally, the user input interface 2071 may be configured to allow a user to further specify whether the cartridge 90 is loaded or unloaded. The controller 30 can then determine whether the appropriate cartridge 90 or receptacle is properly presented for manual door opening.

[0234] Display interface 2072 may be a screen or LED light. When a user requests manual mode via user input interface 2071, display interface 2072 may present a warning that a test is still running within instrument 2050 and that first door 2060 cannot be opened until the test is completed. Display interface 2072 may also display a message or indicate when first door 2060 is unlocked to allow for manual loading or unloading. Display interface 2072 may also display the current mode of instrument 2050, whether manual or automatic.

[0235] Additionally, cartridge transfer device 2000 may include mechanisms 2041 in communication with and / or operated by controller 30. Such mechanisms are shown in FIG. 28B and generally include, but are not limited to, cartridge gripper 2020, cartridge translation actuator(s) 2030, and cartridge position sensor(s) 2048.

[0236] As mentioned above, the cartridge gripper plate 2020 may include vacuum ports / suction cups 2028 that provide suction to unlatch the cartridge 90. The on / off operation of the vacuum pump 2002 that provides such suction may be controlled by the controller 30.

[0237] The cartridge translation actuator 2030 controls the movement of the transfer device 2000. Thus, if the transfer device 2000 is a robot as described above, the translation actuator 2030 provides the robot with an automatic degree of freedom of movement to move the gripper plate 2020 and any cartridges 90 attached to the gripper plate 2020. The controller 30 controls the actuator 2030 to direct cartridge movement, and also deactivates the actuator 2030 when the manual mode of the test device 2050 is engaged.

[0238] To determine the position and orientation of the cartridge 90 attached to the gripper plate 2020, the transfer device 2000 may include a cartridge position sensor 2048, which communicates with the controller 30 on a feedback loop to help direct cartridge transfer.

[0239] As noted above, the controller 30 may be a desktop computer or some other computing device and may include a display interface 2072 and a user interface 2071, such as a keyboard and mouse. Similarly, as shown in Figure 28C, the computing architecture of the controller generally includes a processor 32 and memory 34. The controller may also include a subsystem interface 36, as shown in Figure 28C.

[0240] A subsystem interface 36, which may include an external bus, couples the controller 30 to the preparation system 1000, the cartridge transfer instrument 2000, and the cartridge testing instrument 2050. In particular, commands and data are communicated between the controller 30 and components 2070 via the subsystem interface 36.

[0241] Memory / data storage 34 may include RAM, ROM, flash memory, etc. Memory 34 includes processor control instructions 37 and stored data 38. Processor control instructions 37 include, for example, instructions related to the operation of locking mechanism 2074, door actuator 2076, receptacle actuator 2078, and input interface 2071. Stored data 38 may include cartridge identification information (such as barcode information or serial number), corresponding receptacle identification information, and timing information, test start time, and test length.

[0242] In one embodiment of the method including automated cartridge transfer, the cartridge 90 is automatically loaded into and unloaded from the testing device 2050. In such an embodiment, the preparation system 1000 prepares the AST cartridge 90 by inoculating the cartridge 90 with a sample, as described in detail above. More specifically, the cartridge 90 is automatically inoculated by one or more robots, which may remove the inlet cover 99 (e.g., cap) of the cartridge 90. A pipettor 60 dispenses the analyte into the cartridge 90. Alternatively, the inlet may be covered by a septum, in which case the robot may use a needle to inoculate the interior of the cartridge 90 through the septum. The cartridge 90 may then be placed at a pick-up location, such as in the transfer station 1040, and the preparation system 1000 notifies the controller 30 that the cartridge 90 is ready to be tested and when preparation is complete.

[0243] The controller 30 then operates the cartridge transfer device 2000, which picks up the inoculated cartridge 90 and transfers it, via the gripper assembly 2015 and translation actuator 2030, to a designated receptacle for such cartridge within the testing instrument 2050. The controller 30 similarly activates the door actuator 2076 and the receptacle actuator 2078, with the door actuator 2076 opening the second door 2066 of the testing instrument 2050 and the receptacle actuator 2078 moving the receptacle into alignment with the opening second door using feedback from the cartridge position sensor 2048.

[0244] The transport device 2000 then places the cartridge 90 into the receptacle and communicates the specific cartridge / receptacle location (relative to other details of the carriage) to the controller 30. The transport device 2000 then retrieves additional cartridges 90 as commanded by the controller 30. Typically, the receptacle is completely filled with cartridges 90 before testing. Once the cartridge holder 2054 is filled with cartridges as commanded by the controller 30, this is detected by the transport device 2000 or the test device 2050 and communicated to the controller 30. The controller 30 then operates the receptacle actuator 2078, which presents more empty receptacles to the second door opening. Once all of the receptacles are filled with cartridges as commanded by the controller 30, the controller 30 activates the door actuator 2076, which closes the second door 2066 and commands the test equipment 2050 to begin the test, which in this embodiment is an AST.

[0245] Once testing is complete, the controller 30 activates the door actuator 2076 to operate the cartridge transport device 2000 or another cartridge transport device to remove the tested cartridges from their respective receptacles within the testing device 2050 via the cartridge translation actuator 2030. These cartridges may be moved to storage 2006. Alternatively, the transport device 2000 may dump the tested cartridges into a waste container 2004. This process may be carried out continuously 24 hours a day, 7 days a week.

[0246] In another method embodiment, the test device 2050 may be loaded or unloaded manually. Initially, the instrument 2050 may be set to an automatic mode, in which the transport instrument 2000 performs automatic loading and unloading as described with respect to the first method embodiment of automatic transport. However, if the user chooses to perform manual loading and unloading of the test instrument 2050, the user may engage the user interface 2071 to set the instrument 2050 and the entire system to manual mode. When the user interface 2071 is engaged, the test instrument 2050 notifies the controller 30, which disables the door actuator 2076 and determines whether a test is currently being performed. Other subsystems, such as the cartridge transport instrument 2000, may be deactivated by the controller 30. If a test is not being performed, the controller 30 activates the locking mechanism 2074, which unlocks the first door 2060. If a test is being performed, the controller 30 keeps the door 2060 locked and notifies or indicates to the user via the display interface 2072 that the door 2060 cannot be opened. Once the test is complete, the controller 30 unlocks the first door 2060 and notifies the user that it is permissible to proceed. The user can then open the first door 2060 and begin manually loading or unloading the inoculated cartridge 90 into the testing instrument 2050.

[0247] In some embodiments, the user interface 2071 provides additional functionality beyond simply activating the manual mode, such as specifying whether manual loading or manual unloading is desired. It is also contemplated that a particular carriage 90 may be identified for removal. If the user commands manual loading, the controller 30 activates the locking mechanism 2074, unlocks the first door 2060, and activates the receptacle actuator 2078 to move one or more empty receptacles into alignment with the first door opening. Conversely, if manual unloading is selected, the controller 30 operates the receptacle actuator 2078 to present the tested cartridge to the first door opening for manual unloading.

[0248] System Alternatives Numerous variations, additions, and combinations of the features discussed above may be utilized without departing from the present invention. For example, FIG. 29 illustrates an alternative preparation system 1000′. System 1000′ is similar to system 1000 in that it includes a housing that houses several stations, such as receiving station 1010, preparation station 1030, and transfer station 1040. However, system 1000′ differs with respect to its pick station 1020′. As described above with respect to system 1000, station 1020 includes a positioning device 8 that carries a pick tool 6 and uses such pick tool 6 to pick samples from colonies 4 on plate 3 and transfer such picked colonies to suspension tube 11. Such positioning device 8 includes a transfer device 15 that is used to vibrate the pick tool when immersed in the suspension medium.

[0249] Station 1020', on the other hand, separates the positioning device 8 and the transfer device 15. In this regard, the positioning device 8 and the transfer device 15 are independently connected to a transfer track 18. The transfer device 15 comprises a transfer holder 16 with a gripping tool 17 for releasably holding the pick tool 6. Thus, the transfer device 15 may be moved to the positioning device 8, whereby the gripping tool 17 can take over the pick tool 6 from the positioning device 8. The pick tool holder 9 releases the pick tool 6 after the gripping means 17 has gripped the pick tool. In the embodiment shown in FIG. 29, the pick tool 6', which has previously picked a sample of microorganisms 4, is positioned by the transfer device 15 above the suspension tube 11 in a start position shown by solid lines. The transfer device 15 is arranged to lower the pick tool 6' into the suspension medium 14 contained in the suspension tube 11, at which point the pick tool 6' with the sample 19 is immersed in the suspension medium 14, as shown by dashed lines in FIG. 29. In this position, the transfer device 15 is activated to vibrate the pick tool 6' in a linear vertical motion for a period sufficient to release the sample from the pick tool 6'. The transfer device 15 then positions the released pick tool 6 in a waiting position above the suspension tube 11. That waiting position is identical to the start position of the transfer device 15 in the embodiment shown in FIG. 29. The transfer device may then release the pick tool 6 above the waste receptacle, during which time the positioning device 8 may have already removed a second pick tool and a second sample. Thus, in this embodiment, the positioning device hands the pick tool to the transfer device 15 rather than holding it through the transfer of the sample to the suspension medium. Such an embodiment may be utilized when the pick tool does not require active suction or vacuum to retain the sample.

[0250] Another embodiment of a preparation system 3000 is shown in FIG. 30. System 3000 is similar to system 1000 in that it includes a housing that houses several stations, such as a receiving station (not shown), a pick station 4020, a preparation station 4030, and a transfer station 4040. Additionally, pick station 4020 includes a positioning device 3008 that carries a pick tool 3006, and the preparation station includes a pipettor 2040 that carries a pipette tip 3046. However, unlike system 1000, in which suspension tubes 11 remain in the same general location for inoculation of picked samples 19 by positioning device 8 and removal of suspended samples by pipettor 40, positioning device 3008 and pipettor 3040 are retracted to their respective stations 3020, 3030. In other words, the pipettor 3040 does not move from the preparation station 4030 to the pick station 4020 to receive the suspended sample from the suspension tube 3011; rather, the suspension tube 3011 moves from the pick station 4020 to the preparation station 4030 after inoculation by the pick tool 3006.

[0251] This is accomplished by suspension tube mover 3070, a robot disposed entirely below deck 3007 that is configured to move in at least two dimensions, as indicated by the double-headed arrow in FIG. 30. In particular, suspension tube mover 3007 is configured to hold suspension tube 3011, such as by a receptacle or gripper, and move suspension tube 3011 below deck 3007 between predesignated positions A and A', located at the pick station and preparation station, respectively. In this regard, the deck may have openings through which the mover can lift and lower suspension tubes to these predesignated positions.

[0252] Additionally, each of these positions A and A' includes a nephelometer 3020, 3060, such as one of the nephelometers mentioned above, including a laser or light emitter 3021, 3061 and a detector 3022, 3062. Thus, a first position A located at the pick station 4020 includes a first nephelometer 3020, and a second position A' located at the preparation station 4030 includes a second nephelometer 3060. In one embodiment, the nephelometer is an eight-channel device that can measure turbidity in eight cuvettes simultaneously, such as by including multiple light sources and detectors.

[0253] In a method of using the system 3000, a culture dish 3003 may be moved from the receiving station to the pick station 4020 and placed on the platform 3002. Such dish 3003 contains a medium 3005 and one or more microbial colonies. A target colony 3004 is selected, and a pick tool 3006 is positioned above the target colony 3004. The pick tool 3006 is lowered to remove a sample 3019 of the target colony 3004 by a positioning device 3008. The positioning device 3008 then moves the picked colony 3019 to position A above the suspension tube 2011 in the pick station 4020. The positioning device 3008 lowers the picked microorganism 3019 and immerses it in the suspension medium in the suspension tube 3011. The transfer device 3015 vibrates the pick tool 3006 to release the microorganism into the suspension medium. A nephelometer 3020 measures the turbidity of the suspension. Further picking of microorganisms 3004 may be performed until the desired turbidity is achieved.

[0254] Once the desired turbidity is achieved through successive colony picks, tube mover 3070 lowers suspension tube 3011' with the microbial suspension therein until it is below deck 3007. The mover then moves tube 3011' to a second tube position A' located in preparation station 4030. Mover 3070 then lifts tube 3011' through an opening in deck 3007 for preparation of MALDI plate 3042. The MALDI plate is prepared by pipettor 3040, which moves to position A' above tube 3011' and removes an aliquot of the suspension from tube 3011'. Pipettor 3040 then moves the aliquot to position B above MALDI plate 3042, where it deposits the aliquot at a predetermined location 3044 on MALDI plate 3040, as described in detail above. Once the MALDI plate 3042 is prepared, pipettor 3040 can then aspirate deionized water or some other suspension medium into suspension tube 2011′ at second tube position A′. Nephelometer 3060 at second tube position A′ measures turbidity. Once the desired McFarland number is achieved for the AST, pipettor 3040 aspirates an aliquot from suspension tube 2011′ at position A′ and then transfers the aliquot to position C, where pipettor 3040 inoculates the suspension into AST broth tube 3082. AST broth tube 3082 is moved to transfer station 4040 by AST tube mover 3080 in a manner similar to that of mover 3070 by lowering the tube below deck 3007 and transporting the tube below deck to a position within transfer station 4040. From there, the sample in AST tube 3082 is inoculated into AST cartridge 90, as described above.

[0255] In other embodiments of system 3000, preparation station 4030 may include two tube positions such that the system includes a total of three suspension tube positions (one in pick station 4020 and two in preparation station 4030). In such an embodiment, one of the tube positions in preparation station 4030 may be utilized for MALDI plate preparation, while the other position in preparation station 4030 may be utilized for preparing AST tubes 3082. In this regard, the tube position for MALDI preparation may not have a nephelometer, as the suspension turbidity for MALDI preparation would likely be determined by nephelometer 3020 in pick station 4020. However, the suspension tube position for AST tube preparation would have nephelometer 3060 to assist pipettor 2040 in diluting the suspension to the appropriate McFarland number for AST.

Claims

1. 1. A laboratory device comprising: a housing defining an interior space for receiving a plurality of cartridges containing analytes for testing; a first door coupled to a first side of the housing and configured to receive the plurality of cartridges for testing into the interior of the housing, the plurality of cartridges being received by manual operation; a second door coupled to a second side of the housing, the second door configured for automatic operation; a door actuator coupled to the second door and configured to operate the second door to receive a plurality of cartridges for testing into the interior of the housing, the cartridges being received by automated operation; Equipped with When the first door and the second door are closed, the first door and the second door define a portion of the first side of the housing and a portion of the second side of the housing, respectively.

2. 10. The laboratory equipment of claim 1, wherein the first door includes a hinge coupled to the housing, the first door being rotatable about the hinge upon application of a manually applied force at a location offset from the hinge.

3. 3. The laboratory equipment of claim 2, wherein the housing includes a track at least partially defining a second door opening, the second door coupled to the track and slidable along the track upon activation of the door actuator.

4. 4. The laboratory equipment of claim 3, wherein the door actuator is controlled by an automatic controller.

5. 4. The laboratory equipment of claim 3, wherein the door actuator is a linear actuator.

6. 6. The laboratory equipment of claim 5, wherein the linear actuator is one of a lead screw, a rack and pinion, and a pneumatic piston.

7. 10. The laboratory instrument of claim 1, wherein the housing is a housing in which antibiotic susceptibility testing is performed.

8. 1. An automated system for testing an analyte, comprising: a plurality of cartridges having an interior space configured to receive an analyte; a testing instrument having a housing, a first door, a second door, and a door actuator, the housing defining an interior space for receiving the plurality of cartridges, the first door coupled to a first side of the housing and configured for manual operation, and the second door coupled to a second side of the housing and coupled to the door actuator, the second door configured for automatic operation; a controller coupled to the door actuator; Equipped with When the first door and the second door are closed, the first door and the second door define a portion of the first side of the housing and a portion of the second side of the housing, respectively.

9. 10. The automated system of claim 8, further comprising a cartridge transporter in communication with the controller, the cartridge transporter adapted to transport the cartridge for automated preparation and testing.

10. The automated system of claim 9 further comprising a cartridge preparation station coupled to the controller.

11. 9. The automated system of claim 8, wherein the first door includes a hinge coupled to the housing, the first door being rotatable about the hinge upon application of a manually applied force at a location offset from the hinge.

12. 12. The automated system of claim 11, wherein the housing includes a track at least partially defining a second door opening, the second door being slidably engaged with the track and slidable along the track upon activation of the door actuator by the controller.

13. 13. The automated system of claim 12, wherein the door actuator is a linear actuator.

14. 14. The automated system of claim 13, wherein the linear actuator is one of a lead screw, a rack and pinion, and a pneumatic piston.

15. 9. The automated system of claim 8, wherein the testing instrument is an identification and antibiotic susceptibility testing instrument.

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