Automated method and system for acquisition and preparation of microbial samples for identification and antibiotic susceptibility testing

Abstract

The present application is entitled Automated methods and systems for obtaining and preparing microbial samples for identification and antibiotic susceptibility testing. A method and automated apparatus for locating and selecting a microbial colony on a culture plate and subjecting the obtained sample to a variety of downstream tests, including tests to identify the microorganism and tests to identify the susceptibility of the microorganism to an antibiotic. The method includes the following automated steps: locating and selecting a colony of microorganisms on a culture plate; obtaining a sample of the selected colony of microorganisms; automatically preparing a suspension of the sample of microorganisms by dipping a picking tool with the sample into a suspension, after which the picking tool is oscillated in at least a vertical direction to release the sample from the picking tool into the suspension. The turbidity of the suspension is monitored to ensure that the concentration of microorganisms in the suspension is sufficient so that the suspension is used as a source of sample to conduct identification and antibiotic susceptibility tests of the microorganisms in the sample. The apparatus and system optionally provide downstream processing of the preparation of the sample for antibiotic susceptibility testing (AST). The apparatus includes further processing of the inoculation of an AST plate for AST testing. The further processing includes capping the inoculated plate and transferring it to an AST instrument.

Description

[0001] This application is a divisional application of the original application having a filing date of May 27, 2016, application number 201680030939.X, entitled "Automated method and system for obtaining and preparing microbial samples for identification and antibiotic susceptibility testing".

[0002] Cross Reference to Related Applications

[0003] This application claims the benefit of U.S. provisional application number 62 / 167,577, filed May 28, 2015, U.S. provisional application number 62 / 318,494, filed April 5, 2016, U.S. provisional application number 62 / 167,593, filed May 28, 2015, and U.S. provisional application number 62 / 269,545, filed December 18, 2015, the disclosures of which are incorporated herein by reference. BACKGROUND

[0004] Methods and systems for positioning and selecting microbial colonies and identifying microorganisms with mass spectrometry, in particular MALDI-TOF-MS (Matrix Assisted Laser Desorption and Ionization Time-of-Flight Mass Spectrometry), and systems for performing the same are known. Such systems and methods are described in WO2013 / 147610 to Botma et al., the disclosure of which is incorporated herein by reference.

[0005] MALDI analysis is a useful tool for solving structural problems in biochemistry, immunology, genetics and biology. The sample is ionized in the gas phase and the ion mass is measured using a time-of-flight (TOF) analyzer. The TOF analysis starts when the ions are formed and as they are accelerated to a certain constant kinetic energy as they enter the drift region. The ions reach a detector after a flight time that is proportional to the square root of their mass. A mass spectrum is generated because ions of different mass reach the detector at different times.

[0006] Mass spectrometry is a powerful tool in the fields of drug discovery and development, genotyping and proteomic research. MALDI, a special type of mass spectrometry, has found application in the field of characterization and identification of bacteria and microorganisms. The current trend in research is to analyze an increasing number of samples with a large number of individual samples ranging from micro-molar to atomic-mole levels. As a result, the samples also become smaller, which requires efficient and reliable acquisition of a suitable number of microorganisms and the ability to store the acquired number of samples on a target plate used in the MALDI instrument.

[0007] In a typical MALDI TOF MS operation, the sample to be analyzed is spotted or deposited on a MALDI target plate, which can be a metal or other material that allows the sample to ionize. The generally accepted method for preparing a MALDI target plate is to spot or swab the sample suspected of containing microorganisms directly from a plated culture medium onto the target plate. After the addition of the sample, a matrix reagent is often added to support ionization of the sample. In some cases an extractant is also added. In other cases, an off-line extraction step can be required before the sample is added to the target plate.

[0008] Once the target plate is prepared, it is placed in a fixed position in the MALDI instrument. The target plate has multiple deposition points (e.g., 24 to 384 deposition points on a single target plate) and the deposition points are fixed with respect to the orientation of the edge of the target plate. The target plate is placed on an X-Y stage so that the sample obtained from a microorganism colony can be deposited on a selected deposition point. A high voltage potential is maintained between the target plate and the metal grid. This voltage can be constant or pulsed, depending on the results desired and the degree of vacuum created within the chamber. A laser is fired into the sample / matrix, creating a cloud of ions. The voltage differential is used to accelerate the ions into a flight tube for analysis of the ions. The analysis correlates the time of flight with the mass of the ionized components.

[0009] There are multiple parameters that can affect the quality of the results, including the flatness of the target, the amount and type of matrix, the concentration of the sample, the conductivity of the sample target, the accuracy of the deposition point placement, and several other variable factors.

[0010] Because the process requires picking up a colony and depositing it directly on the plate, the picked sample cannot be used as a source of sample for other analyses. Therefore, if it is desired to perform other tests on the sample, another portion of the sample must be taken to perform the test. Because multiple colony picks are required for multiple tests, increased processing time and the potential for inconsistent results due to differences between the two picks of the sample are required. Therefore, there is still a need for an automated, efficient method and system for obtaining a sample of microorganisms from a colony and providing the obtained sample for multiple tests. SUMMARY

[0011] To solve at least one of the above mentioned problems, the present invention provides an automated method and system for positioning and selecting a microbial colony on a petri dish and identifying a microorganism in the selected colony using MALDI and at least one other test. The method comprises the following automated steps: positioning and selecting a microbial colony on a petri dish; obtaining a sample from the selected microbial colony; preparing a suspension of the obtained sample; dispensing a portion of the obtained sample on a target plate and placing the target plate in an instrument performing MALDI to identify the sample of the selected microbial colony; 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 use an existing automated AST method (BD Phoenix or Vitek) or can be Kirby-Bauer / disk diffusion, disk dilution, broth and agar dilution or other methods.

[0012] In one embodiment, the suspension is prepared in a cuvette. The suspension in the cuvette is checked using a nephelometer to determine if the value of the turbidity of the sample falls within a predetermined range of values that is determined to be suitable for the MALDI test. 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 transferred from the cuvette for MALDI, the suspension is checked again by the nephelometer and the turbidity of the suspension is determined again. This time the turbidity of the suspension is evaluated to determine if the turbidity falls within a range of turbidity values that is suitable for use in the sample in the second test (e.g. AST test). If not, the amount of diluent in the suspension is adjusted to provide a suspension with a suitable turbidity value.

[0013] All steps are performed automatically, which largely avoids the above mentioned problems, since automation avoids undesirable variations and errors, which lead to incorrect results from the MALDI instrument, additional costs and waste of time. By automating each step, these problems are at least largely overcome. It has been a prejudice in the art that at least a part of these steps can only be done manually, however contrary to this, the present invention provides for the first time the possibility that all necessary steps can be automated when positioning and selecting a microbial colony and performing microorganism identification in the selected colony using MALDI.

[0014] 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 a 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 prior to overlaying with an aliquot of a MALDI matrix solution. Another embodiment can include spotting of an extractant, such as formic acid, prior to the matrix reagent to enhance results.

[0015] The alternative method of using the suspension is further useful in cases where another test or analysis is performed on the sample of microbial colonies. In one embodiment of the method according to the present invention, the other analysis described above can be performed in a particularly reproducible and efficient manner, wherein the method further includes the automated steps of: taking a second aliquot of the sample suspension; depositing the second aliquot of the sample suspension into a broth of an AST test; and transferring the inoculated AST broth test tube to an instrument that performs a susceptibility test or other additional analysis. Thus the method of the present invention can be used to automatically take or pick up a sample and add it to an existing ID / AST instrument, including but not limited to BACTEC TM , Phoenix, MGIT, VITEK, and BacT / Alert.

[0016] An embodiment of fully integrated automated method comprises the aforementioned steps combined into a single process flow. In particular, a stage is provided for a culture dish carrying microorganisms. The culture dish is placed on the stage. An automated picking tool is provided with an automated positioning device equipped with a picking tool holder for supporting a picking tool (e.g. a pipette). The positioning device is installed to position the picking tool in a starting position above the culture dish and to automatically lower the picking tool towards the culture dish or to automatically raise the picking tool away from the culture dish, respectively, and to position the picking tool in a transfer position. The picking tool is placed on the picking tool holder of the positioning device. The picking tool is positioned in the starting position above the culture dish and is automatically lowered towards the culture dish into contact with the microorganisms to pick a sample of the microorganisms. The picking tool is automatically raised carrying the sample of the microorganisms away from the culture dish to the transfer position. A suspension medium automatic dispenser is provided for automatically dispensing a suspension medium within a suspension tube supported in a suspension tube holder. The automatic dispenser automatically dispenses an initial amount of the suspension medium into the suspension. The positioning device automatically moves the picking tool from a position above the culture dish to a position above the suspension. The positioning device lowers the picking tool into the suspension medium contained within the suspension tube or raises the picking tool away from the suspension medium contained within the suspension tube, respectively, and optionally positions the picking tool in a waiting position above the suspension tube. With the picking tool having the sample of the microorganisms immersed in the suspension medium, the positioning device oscillates the picking tool in linear vertical movements for a period of time. The picking tool is raised away from the suspension medium contained within the suspension tube to the waiting position after the period of time has ended. A turbidimeter (also referred to herein as a nephelometer) is provided to perform a measurement of the turbidity of the suspension medium contained within the suspension tube supported in the suspension tube holder. At least after the end of the oscillation time of the picking tool, the turbidity of the suspension medium contained within the suspension tube supported in the suspension tube holder is measured with the turbidimeter and a final measurement value is provided indicative of the measured turbidity.

[0017] A controller is provided communicatively connected to the positioning device, the transfer device, the suspension medium automatic dispenser and the turbidimeter to automatically control the movements of the positioning device, the movements of the transfer device, the operation of the suspension medium automatic dispenser and the operation of the turbidimeter, respectively. The controller controls and monitors the suspension and operates to provide a suspension having a turbidity within the specification as previously described.

[0018] The present invention further relates to an apparatus for automatically preparing a suspension of a sample of microorganisms for performing the aforementioned method for automatically selecting a colony of microorganisms on a culture dish and preparing a suspension of a sample of microorganisms and using the suspension for at least two tests of microorganism identification and antibiotic susceptibility. The apparatus has:

[0019] A stage for holding a petri dish;

[0020] A pick-up tool and a positioning device equipped with a pick-up tool holder supporting the pick-up tool. The positioning device is configured to position the pick-up tool in a starting position above a petri dish and to automatically lower and raise the pick-up tool towards and away from the petri dish, respectively, and to position the pick-up tool in a transfer position;

[0021] A suspension tube station for supporting a suspension tube;

[0022] A suspension medium automatic dispenser for automatically dispensing a suspension medium into a suspension tube supported in the suspension tube station;

[0023] A transfer device for automatically transferring the pick-up tool from the transfer position of the positioning device to a position above a suspension tube supported in the suspension tube station and lowering or raising the pick-up tool into or out of a suspension medium contained in the suspension tube and positioning the pick-up tool in a waiting position above the suspension tube supported in the suspension tube station, the transfer device being further configured to oscillate the pick-up tool in linear vertical movements for a period of time;

[0024] A turbidimeter for measuring the turbidity of a suspension medium contained in a suspension tube supported in the suspension tube station and providing a final detection value indicative of the measured turbidity;

[0025] A controller in communication with the positioning device, the transfer device, the suspension medium automatic dispenser and the turbidimeter for automatically controlling the movements of the positioning device, the movements of the transfer device, the operation of the suspension medium automatic dispenser and the operation of the turbidimeter, respectively.

[0026] The controller can:

[0027] a) determine whether the final turbidity measurement is higher than a first threshold value (maximum value) previously saved in the memory of the controller, if so, the controller is configured to implement step b) (dilution); or whether the final turbidity measurement is lower than or equal to the first threshold value and greater than or equal to a second threshold value (minimum value) previously saved in the memory of the controller, the first threshold value being greater than the second threshold value, if so, the controller is configured to implement step c) (acceptable turbidity); or whether the final measurement is lower than the second threshold value, if so, the controller is configured to implement step d) (concentration);

[0028] b) control the suspension medium dispenser to supply an additional amount of suspension medium into the suspension tube;

[0029] c) providing a signal that the suspension tube with the suspension can be removed from the suspension tube holder for further processing; or

[0030] d) placing said further pick-up tool in said pick-up tool holder of the positioning device in the manner of said first pick-up tool.

[0031] In another embodiment of the apparatus according to the application, the controller is configured to control the turbidimeter so as to start measuring the turbidity of the suspension medium contained in the suspension tube supported in the suspension tube holder by the turbidimeter before the pick-up tool is immersed in the suspension medium contained in the suspension tube.

[0032] In an advantageous embodiment of the apparatus according to the application, said first pick-up tool is provided as a further pick-up tool in step d); and the controller is configured to control the transfer device to position said further pick-up tool in said pick-up tool holder of the positioning device.

[0033] Preferably, the controller is configured to determine the amount of suspension medium to be added based on the initial amount of suspension medium, the final measurement value and the values of the first and / or second threshold value. In particular, the controller is configured to control the automatic dispenser of suspension medium in the aforementioned manner.

[0034] In case the apparatus according to the application comprises an automatic culture dish positioning and removal device for automatically positioning and removing the culture dish comprising the microorganism on and from the stage, respectively, the controller is configured to communicate with the automatic culture dish positioning and removal device to control the operation of the automatic culture dish positioning and removal device and to automatically position the culture dish comprising the microorganism on the stage, and in case the apparatus comprises an automatic suspension container positioning and removal device for automatically positioning and removing the suspension container on and from the suspension container platform, respectively, the controller is configured to communicate with the automatic suspension container positioning and removal device to control the operation of the automatic suspension container positioning and removal device and to automatically position the suspension container on the suspension container platform. In this case, the controller is then preferably configured to allow the automatic removal of the culture dish from the stage by the automatic culture dish positioning and removal device only after a signal has been provided that the suspension container with the suspension can be removed from the suspension tube container holder for further processing. Further, the controller is then preferably configured to automatically remove the suspension container from the suspension container platform by the automatic suspension container positioning and removal device only after a signal has been provided that the suspension container with the prepared suspension can be removed from the suspension container platform.

[0035] The present invention is further directed to a method of automatically depositing a droplet of a suspension comprising a sample of microbial colonies 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 of sample for another test (e.g., AST).

[0036] The apparatus has a pipetting tool and a positioning device having a pipetting tool holder for supporting the pipetting tool. The positioning device is configured to position the pipetting tool in a starting position above a suspension test tube containing a suspension of a sample of microbial colonies. The pipetting tool is automatically lowered into the suspension and raised from the suspension, respectively, and positioned in a transfer position.

[0037] The pipetting tool picks up an amount of the suspension and the pipetting tool with the amount of suspension is raised to the transfer position. The pipetting tool has a pressurizable chamber closed by a control valve to contain the amount of suspension medium.

[0038] A target plate holder is provided for supporting a target plate having at least one deposition spot.

[0039] The apparatus positions the target plate in the target plate holder.

[0040] The apparatus comprises a transfer device to automatically transfer the pipetting tool from the transfer position of the positioning device to a position above one of the deposition spots on the target plate and to lower the pipetting tool (e.g., pipetting tip) to a predetermined distance above the target plate and to pressurize the chamber (e.g., to a pressure in the range of about 0.5 bar to 1.1 bar, although this is by way of example and not limitation) and then to open the valve for a period of time to deposit a droplet of the suspension having a volume of about 0.5 μl to 3.0 μl on one of the deposition spots. Preferably, the pipetting tool is shaped such that the droplet of suspension on the target plate is deposited in a manner that does not splash.

[0041] The suspension test tube is then moved to a second position. In the second position, the turbidity of the suspension is adjusted for a second test (e.g., AST). The second position has a turbidimeter for determining whether the turbidity of the suspension is suitable for the second test. The pipetting tool is then used to take additional suspension and use the suspension to inoculate a container for another test (e.g., AST).

[0042] In one embodiment, an automated system is described for preparing individual sample suspensions, aliquots removed from the suspensions are used for identification (ID) and a second test of microorganisms in the sample. In another embodiment, the automated system prepares individual sample suspensions, aliquots removed from the suspensions are used for identification (ID) and antibiotic susceptibility testing (AST) of microorganisms in the sample. The system includes at least a first portion for performing the ID analysis. The first portion has a mechanism that either automatically transports the culture plates or manually receives the culture plates. The system includes an imaging device or is connected to an imaging device that optically probes the culture plates and, from the image, colonies of interest are identified. In an alternative embodiment, the image acquisition and colony selection are performed prior to the culture plates being received by the system. The system includes a mechanism that identifies the location of the colonies of interest on the plate and designates the colonies of interest for picking. The first portion includes an automated robotic picking tool. The system also includes a controller that is in communication with the robotic picking tool that instructs the robotic picking tool to acquire a pipette and then transport the pipette to a position above the colony of interest. The lid of the plate has been removed to facilitate picking of the colony. The robotic picking tool then lowers the pipette so that the tip contacts the colony of interest.

[0043] After the colony has been picked, the controller instructs the robotic picking tool to transport the picked sample to a first sample suspension preparation station. Optionally, the system captures a new image of the plate after the colony is picked to verify that the pick was from the correct location. The first sample suspension station has a suspension dispenser that dispenses sample suspension liquid into a suspension test tube or cuvette or other suitable container. The first sample suspension station has a nephelometer or other suitable device to measure the turbidity of the liquid in the suspension test tube or cuvette. The robotic picking tool releases the sample carried over from the culture plate into the suspension liquid. In some embodiments, the robotic picking tool oscillates the picking tool to facilitate release of the sample into the suspension. The nephelometer measures the turbidity of the suspension, where in response to a turbidity measurement outside of a predetermined turbidity value, the automated system adjusts the suspension so that it has an acceptable specific gravity (i.e., turbidity) for the ID analysis.

[0044] The first portion further includes a first robotic pipettor. The first robotic pipettor obtains a first aliquot of the suspension at the first station and inoculates a container for the ID analysis. The container (e.g., a MALDI plate) is then removed from the system and transported to a device that performs MALDI. The container can be transported mechanically or manually. The suspension test tube or cuvette is then transported to a location in the first portion where the remaining portion of the suspension is prepared for a second analysis (e.g., an AST analysis). The transport is performed in an automated manner using a conveyor belt.

[0045] The first portion has a second nephelometer at the second sample suspension platform to detect turbidity of the suspension. The first robotic pipettor is further configured to adjust the sample concentration in the suspension test tube or cuvette to a predetermined concentration for the second analysis, and to obtain a second aliquot of the sample suspension with the adjusted concentration, and to inoculate a sample tube with the second aliquot suspension for AST analysis. Such sample tubes are commonly referred to as AST broth tubes.

[0046] The system optionally has a second portion for preparing a plate for AST analysis. The automated system has automated machinery for transferring the inoculated sample tube from the first portion to the second portion. In one embodiment, the inoculated sample tube is lowered through a faceplate of the second sample suspension platform, and is transferred under the faceplate, emerging from under the faceplate of the second portion. The second portion has a second robotic pipettor that takes an aliquot from the inoculated sample, and inoculates an AST plate with the taken aliquot. The second portion also has a tool by which caps 99 (see Figure 26 ) are stored, dispensed, manipulated, and advanced into cap holes of the inoculated plate. The second portion also has automated means for placing the inoculated plate into an AST device configured to have at least two doors, a first door that receives the plate from the plate placing automated means. A second door is used for manually placing the inoculated plate by a user. The AST device need not be located at the second portion of the system, and can be contiguous therewith. The second portion also has a controller in communication with the AST instrument to request, schedule access to, and open the first door of the AST instrument. BRIEF DESCRIPTION OF DRAWINGS

[0047] The present invention will be further explained with reference to the following drawings.

[0048] Figure 1 is a front view of a system according to an embodiment of the disclosure including a system housing.

[0049] Figure 2 is a schematic view of a component layout within the system housing of Figure 1 according to an embodiment of the disclosure.

[0050] Figure 3 is a structural block diagram of the system of Figure 1 according to an embodiment of the disclosure, including exemplary components suitable for performing the methods described herein.

[0051] Figure 4A is a perspective view of an embodiment of a low volume single cuvette nephelometer.

[0052] Figure 4B is a perspective view of a low volume single cuvette nephelometer according to an embodiment of the disclosure. Figure 4ACross-sectional top view of a low volume single cuvette nephelometer.

[0053] Figure 5A Perspective view of a single cuvette according to another embodiment of the disclosure for use with a single cuvette nephelometer as shown in Figure 4A

[0054] Figure 5B Perspective view of a single cuvette according to another embodiment of the disclosure for use with a single cuvette nephelometer as shown in Figure 4A

[0055] Figure 6 Process flow diagram showing one process embodiment for preparing a sample using the nephelometer as shown in Figure 4A

[0056] Figure 7A Perspective view of a continuous cuvette nephelometer according to an embodiment of the disclosure.

[0057] Figure 7B Cross-sectional top view of a continuous cuvette nephelometer as shown in Figure 7A

[0058] Figure 8 Perspective view of a linear low volume multi-cuvette array / strip according to an embodiment of the disclosure for use with a continuous cuvette nephelometer as shown in Figure 7A

[0059] Figure 9 Partial transparent, perspective view of a stacked cuvette.

[0060] Figure 10 Perspective view of a nephelometer according to another embodiment of the disclosure.

[0061] Figure 11 Cross-sectional view of the nephelometer as shown in Figure 10

[0062] Further cross-sectional view of the nephelometer as shown in Figure 12 Figure 10 Figure 10 Another cross-sectional view of the nephelometer as shown in

[0063] Figure 13 Another cross-sectional view of the nephelometer as shown in Figure 10

[0064] Figure 14 Sample preparation decision chart, where sample preparation is based on measured sample turbidity.

[0065] ​​​​​​​​Figure 15 The pipette is shown removing the mucous sample from the target plate, where a string begins to form.

[0066] Figure 16 The pipette is shown removing the mucous sample from the target plate, where a string begins to form. Figure 15 The pipette is shown being further pulled away from the agar surface of the plate to further extend the string.

[0067] Figure 17A is a time graph showing the change in capacitance over time when the pipette picks up the sample without forming a string.

[0068] Figure 17B is a time graph showing the change in capacitance over time when the pipette picks up the sample and a string forms.

[0069] Figure 18 is a flow chart showing the automated process according to one embodiment of the present application.

[0070] Figure 19 is a flow chart comparing the timeline of the automated process of Figure 18 to the timeline of a manual implementation of the process.

[0071] Figure 20 is a schematic side view of the system of Figure 1 in use with a cartridge transfer instrument and a plurality of detection instruments.

[0072] Figure 21 is a diagram of an exemplary system for automated preparation, transfer, and detection of samples, including the system of Figure 20 , a cartridge transfer instrument, and a detection instrument, and further including an exemplary microbiological test cartridge and Figure 3 a controller 30.

[0073] Figure 22 is a rear perspective view of a cartridge gripper of a cartridge transfer instrument according to an embodiment of the present disclosure as it approaches a cartridge in a cartridge support structure.

[0074] Figure 23 is a side perspective view of the cartridge gripper of Figure 21 , detailing the rotatable coupling between the gripper plate and the arm of the automated cartridge transfer instrument.

[0075] Figure 24 is a front perspective view of the cartridge gripper of Figure 21 .

[0076] Figure 25 shows an exemplary microbiological test cartridge of Figure 21 .

[0077] Figure 26 shows a tray for temporary storage of cartridges.

[0078] Figure 27A is a front perspective view of one of the test instruments including a manual door Figure 20

[0079] Figure 27B-27D is a front perspective view of one of the test instruments including a manual door Figure 27A

[0080] Figure 28A is a front perspective view of one of the test instruments including a manual door Figure 3

[0081] Figure 28B is a front perspective view of one of the test instruments including a manual door Figure 3

[0082] Figure 28C is a front perspective view of one of the test instruments including a manual door Figure 3

[0083] Figure 29 is a front perspective view of one of the test instruments including a manual door

[0084] Figure 30 is a front perspective view of one of the test instruments including a manual door Figure 1 DETAILED DESCRIPTION

[0085] As used herein, a "cuvette" and / or "microcuvette" and / or "low volume cuvette" and / or "LVC" and / or "sample container" or "container" 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 particular space and orientation for testing or processing.

[0086] As used herein, an "algorithm" is one or more mathematical instructions that are used to process values of data to make decisions based on the mathematical values and then produce corrected or more accurate data values that represent the desired output.

[0087] As used herein, an "amplifier" is an electronic circuit used to take a small original electronic signal and increase its amplitude to produce a new signal that is proportionally larger that represents the original signal. Suitable amplifiers are well known to those skilled in the art and are not described in detail herein.

[0088] As used herein, an "analog-to-digital converter" or "A / D converter" is an electronic device that is capable of taking a varying electrical signal and converting it to a number that represents the original signal amplitude.

[0089] ​​​​​​As used herein, "dilution" means a solution or suspension resulting from the addition of a diluent to a concentrated solution or suspension, resulting in a new suspension or solution having a lower uniform concentration of sample in solution or suspension than the original solution or suspension.

[0090] As used herein, "laser" or "laser diode" is an electronic device that produces a concentrated or focused beam of light when an electrical current is applied.

[0091] As used herein, "optical attenuation filter" is a device placed in the optical path that absorbs and attenuates the amount of light as it passes through the filter, such that the light passing through the filter has a proportionally lower intensity than the original light source.

[0092] As used herein, "light emitting diode" or "LED" is an electronic device that emits a specific type and orientation of light when an electrical current is applied.

[0093] As used herein, "McFarland" is a unit of measure for the amount of solid particles dispensed within a fluid or liquid suspension.

[0094] As used herein, "nephelometer" is an instrument capable of detecting the amount of solid particles within a suspension. As used herein, "nephelometric assay" refers to a method that can detect the amount of suspended solids within a suspension.

[0095] As used herein, "photodiode" and / or "detector" is an electronic device used to detect the intensity of light in a given environment.

[0096] As used herein, "saturated" and / or "state of saturation" is the point at which a detector has reached the maximum amount of output signal that it is capable of producing. For example, adding more light to a photodetector reaches a point of oversaturation that cannot cause the detector output signal to produce any more change, the output signal has reached its maximum operating capacity.

[0097] As used herein, "suspension" is a solution in which solids are uniformly distributed in a liquid.

[0098] As used herein, "turbidity" is a measure of the amount of suspended solids in a solution (i.e., the cloudiness of a liquid sample).

[0099] Described herein are methods and systems for preparing individual suspensions from colonies of microorganisms, which are the source of samples for determining the ID and antibiotic susceptibility of selected colonies of microorganisms. Because samples for characterization and identification of microorganisms are often obtained from petri dishes with multiple colonies growing on the culture, it is important that the sample be obtained from the colony of interest. If the sample is obtained from a colony that is not of interest, the time and effective use of the MALDI instrument is compromised. The present invention contemplates an automated process for identifying and selecting a colony of interest from among multiple colonies present on a petri dish. This process of discriminating colonies can be performed at least partially automatically by providing a petri dish comprising multiple colonies of microorganisms, acquiring an initial image of the petri dish comprising all of the colonies of microorganisms, displaying the initial image of the petri dish comprising all of the colonies of microorganisms on a display, and selecting at least one colony of microorganisms from the initial image.

[0100] In this manner, the researcher or technician can select the colony of interest based on education and knowledge. In a particular embodiment, the petri dish is provided with a unique identifier to identify the petri dish, such as a barcode, and the method further comprises the step of storing the initial image of the petri dish comprising all of the colonies, storing information relating to the at least one selected colony of microorganisms, and storing the identifier of the petri dish in the memory of the central control computer. In another embodiment, the researcher or technician can manually enter processing instructions relating to the processing of the selected colony of microorganisms of the petri dish, which are stored in the memory of the central control computer for subsequent use.

[0101] In one embodiment, the colonies on the plate are imaged according to the methods described in provisional patent application number 62 / 151,681, filed April 23, 2015, entitled "Colony Contrast Gathering," also as described in PCT / US2016 / 028913 and PCT / EP2015 / 052017, entitled "A System and Method for Image Acquisition Using Supervised High Quality Imaging," which are incorporated herein by reference. The contrast of the different colonies relative to the culture medium provides the ability to discriminate the colonies, which facilitates automated colony picking. As noted elsewhere, the image of the culture plate can be obtained from a separate device prior to its receipt by the system described herein, or the system herein can be integrated with a module in which the image is acquired.

[0102] After the initial image of the petri dish is acquired, the petri dish is incubated for a period of time to allow the microorganisms on the plate, if present, to grow. In another embodiment of the present invention the method comprises the automated steps of positioning the petri dish on a stage for the petri dish, acquiring an image of the petri dish positioned on the stage, acquiring an identification of the petri dish, comparing the image acquired by the imaging device of the picking tool device with the stored initial image of the petri dish to obtain information related to the location of the selected microorganism colony, and optionally acquiring processing instructions related to the treatment to be performed on the selected microorganism colony. By comparing the images of the petri dish, when it is placed inside the picking tool device equipped with the initial image, the location of the selected colony can be automatically acquired, for example by computerized image comparison.

[0103] In another embodiment, alignment marks on the agar surface or the petri dish can be used to reposition the colonies. These alignment marks can be embedded on the plate during manufacturing, or applied by the user or the organic growth or incorporated on the petri dish or agar surface in any suitable manner. Using machine vision equipment, another reference point, for example the center of the petri dish, is detected from which the petri dish coordinates can be determined. A barcode is one example of an alignment. The location of the colonies on the petri dish can be determined with respect to their relative distance from the center and angular offset from the barcode zero offset. Once the relative position of the colonies is determined, the petri dish can be moved to another system where the next two steps are performed. The center of the petri dish is determined, for example by mechanical means. The barcode zero offset is detected, for example by rotating the petri dish while having a fixed sensor to detect the presence of the barcode label and scanning the barcode with a barcode scanner. At this point, the center of the petri dish is known and the barcode zero offset is known, so the location of the colonies mentioned previously can be easily calculated as they are stored as distance from the center of the petri dish and angular offset from the barcode label. The method as described here does not require a camera or computer vision system in the second system (the colony picking system in this example), or any other system that needs the colony location information. The zero offset used in this example is to a barcode label, but it can be to any unique alignment feature of or applied to the petri dish, as noted above.

[0104] An automated method and apparatus for picking microorganisms from a culture medium surface is described in U.S. Patent Publication No. 2014 / 0242570 (U.S. Serial No. 14 / 347,841), entitled "Method For Picking UP Cell Material And Assembly For Performing Said Method", to Botma et al., which is commonly owned and the contents of which are incorporated herein by reference.

[0105] As described by Botma et al., in a beneficial embodiment the method further comprises the steps of moving the picking tool away from the contact position towards a check position by a predetermined distance, and holding the picking tool in the check position, and measuring the capacitance of the system consisting of the picking tool and the support at the check position. In some cases the sample material to be picked is very sticky or slimy. When the picking tool after contacting such a sample material is moved away from the sample, a thread-like object can remain in contact between the picking tool and the residual sample material in the petri dish. Such a thread-like object can damage and possibly contaminate the picking tool device. By measuring the capacitance of the picking tool and the picking tool support at a check position, for example a few millimeters above the petri dish, the presence of such a thread-like object can be detected, so that appropriate measures can be taken. In those embodiments of the method in which the picking tool holder is arranged for removably holding a picking tool, when the picking tool holder is suitable for clamping and releasing a picking tool, an automated response to the detection of a residual thread-like object can be performed. For example, in case the measured capacitance at the check position is different from the initial capacitance at the initial position, the picking tool is released from the picking tool holder, so that the picking tool falls into the petri dish, after which the petri dish can be discarded. These steps can easily be performed in an automated manner, so that no time-consuming human intervention is necessary for discarding the picking tool and the petri dish.

[0106] In one embodiment, a pipette tip is used to pick a colony from the surface of a culture medium, such as agar, on which the colony is arranged. In one embodiment the pipette can suck the colony into the tip by suction. In other embodiments, suction is not used to suck the colony into the pipette tip, and only the contact force between the colony and the pipette tip forces the colony into the pipette tip.

[0107] In a further embodiment, the method according to the invention comprises the step of automatically preparing a suspension of the microbial sample. In this method the following steps are performed.

[0108] A first picking tool is provided together with a positioning device equipped with a picking tool holder for supporting the picking tool, such as the aforementioned pipette tip picking tool. The positioning device is configured to position the picking tool above the starting position of the selected microbial colony on the petri dish. The positioning device automatically lowers the picking tool towards the petri dish and raises the picking tool away from the petri dish, respectively, and positions the picking tool in the transfer position.

[0109] The first picking tool is positioned within the picking tool holder of the positioning device. The picking tool is then positioned at a starting position above the location of the selected microorganism colony on the petri dish. The picking tool is then automatically lowered to contact the microorganism colony to pick the microorganism sample. The picking tool is then automatically raised with the collected microorganism sample away from the petri dish to a transfer position.

[0110] A suspension tube holder is provided to support at least one suspension tube. The suspension tube is placed within the suspension tube holder. While referred to herein as a suspension tube, the container of the suspension can be a test tube, a vial, a cuvette, or other container used to hold a suspension solution.

[0111] A suspension medium automatic dispenser is provided to automatically dispense a suspension medium within the suspension tube supported by the suspension tube holder. The automatic dispenser automatically supplies an initial amount of the suspension medium into the suspension tube supported by the suspension tube holder. A transfer device, which can be separate from the positioning device or part of the positioning device, is also provided to automatically transfer the picking tool (having collected a sample) to a position above the suspension tube supported by the suspension tube holder. The transfer device lowers or raises the picking tool (and the sample carried by the picking tool) to enter or exit the suspension medium contained within the suspension tube. The transfer device also positions the picking tool at a waiting position above the suspension tube supported by the suspension tube holder, respectively.

[0112] The transfer device oscillates the first picking tool in linear vertical movement for a period of time while the first picking tool with the microorganism sample is immersed within the suspension medium such that the sample is released into the suspension medium and the suspension is mixed. After the period of time, the first picking tool is raised to exit the suspension medium contained within the suspension tube and to the waiting position. Alternatively, instead of oscillation to release the microorganism sample, repeated pipetting of the picking tool while partially immersed within the suspension medium can accomplish the release of the microorganism and the mixing of the suspension.

[0113] In the automated method, a turbidimeter is provided to detect the turbidity of the suspension medium contained within the suspension tube supported by the suspension tube holder. In one embodiment, the turbidimeter is as described in U.S. Provisional Application No. 62 / 056,911 and PCT / IB2015 / 00272 (published as WO2016 / 051267), both of which are commonly assigned and incorporated by reference in their entirety.

[0114] After the time period of the pick-up tool oscillation, the turbidity of the suspension medium contained within the suspension test tube supported by the suspension test tube holder is detected by the turbidimeter and a final detection value is provided indicative of the measured turbidity.

[0115] In further embodiments, a controller is provided in communication with the positioning device, the transfer device, the suspension medium automatic dispenser, and the turbidimeter. The controller automatically controls the movement of the positioning device, the movement of the transfer device, the operation of the suspension medium automatic dispenser, and the operation of the turbidimeter, respectively.

[0116] Reference is made to Figure 6 In one embodiment, the controller determines whether the final turbidity measurement is above a first threshold value (maximum value) previously saved in the controller memory. If so, step b) (dilution described below) is implemented. If the final turbidity measurement is less than or equal to the first threshold value and greater than or equal to a second threshold value (minimum value) previously saved in the controller memory, wherein the first threshold value is equal to or greater than the second threshold value, step c) (acceptable turbidity described below) is implemented. If the final measurement is less than the second threshold value, step d) (increased turbidity described below) is implemented.

[0117] In step b), the suspension medium automatic dispenser is automatically controlled to supply an additional amount of suspension medium in the suspension test tube. In step c), a signal is provided that the suspension test tube with the suspension can be removed from the suspension test tube holder for further processing.

[0118] According to step d), another pick-up tool is obtained and placed in the pick-up tool holder of the positioning device as described above. The positioning device positions the other pick-up tool in the starting position above the petri dish, automatically lowers the other pick-up tool toward the petri dish to contact the microorganisms to pick-up another sample of microorganisms, automatically raises the other pick-up tool with the sample of microorganisms away from the petri dish and to the transfer position, all as described for the first pick-up. Although the pick-up tool described herein is a pipette, other suitable pick-up 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 PCT / US2016 / 026625, filed April 8, 2016, which are incorporated herein by reference.

[0119] The transfer device automatically transfers a further pick-up tool with a further microorganism sample from the transfer position of the positioning device to a position above the suspension test tube supported by the suspension test tube holder and lowers the further pick-up tool into the suspension medium contained in the suspension test tube and oscillates the further pick-up tool in linear vertical movements for a time period while the further pick-up tool with the further microorganism sample is immersed in the suspension medium. After the time period the pick-up tool is raised out of the suspension medium contained in the suspension test tube and reaches the waiting position. After the time period of oscillation of the further pick-up tool the turbidity of the suspension medium contained in the suspension test tube supported by the suspension test tube holder is detected by the turbidimeter and a further final detection value is provided which represents the detected turbidity.

[0120] After the sample has been obtained, in another embodiment the pipetting system is able to perform a series of rapid pipetting and dispensing of the pipette tip in the suspension. For example, the pipetting system is able to repeat the series of pipetting up to about 20 times and dispensing about 250 μΙ_ of the 300 μΙ_ sample within 20 seconds. The repeated action creates a high shear force at the tip of the pipette. The high shear force causes the clumps or mucilaginous strands containing the microorganism sample to disperse to create a more uniform suspension.

[0121] In this way it is possible to prepare a suspension of a microorganism sample in an improved automated way, while by means of the controller and the turbidimeter it is possible to provide a suspension test tube containing a suspension medium which contains an amount of microorganisms which is always sufficient (and reproducibly) to perform a correct analysis of the microorganisms.

[0122] In another embodiment of the method for the automated preparation of a suspension of a microorganism sample according to the present application, the controller is configured such that the step of detecting the turbidity of the suspension medium contained in the suspension test tube supported by the suspension test tube holder by means of the turbidimeter is additionally performed for a time period of oscillation of the pick-up tool, wherein the turbidimeter is configured to provide online detection values representing the detected turbidity to the controller for the time period of oscillation of the pick-up tool. In this way it is possible to obtain an extremely fast automated detection of the amount of microorganisms in the suspension. In particular, if during the oscillation the online detection values of the turbidity are equal to or smaller than a first threshold value and equal to or larger than a second threshold value, the controller controls the movement of the transfer device such that the pick-up tool is raised to the waiting position and the controller further provides a signal that the suspension test tube with the suspension can be removed from the suspension holder for further processing. In this way, when the suspension medium contains a sufficient amount of microorganisms, the oscillation of the pick-up tool is stopped such that the method can be performed in a very time-efficient way.

[0123] The pick-up tool and the sensor of the turbidimeter are arranged relative to each other such that during oscillation of the pick-up tool, the pick-up tool does not obstruct the path of the turbidimeter.

[0124] In a further embodiment of the method of automatically preparing a suspension of a microbiological sample according to one embodiment herein, the controller is configured to control the turbidimeter such that the step of detecting the turbidity of the suspension medium contained in the suspension test tube supported by the suspension test tube holder by the turbidimeter starts before the pick-up tool is immersed in the suspension medium contained in the suspension test tube. In this way, it is for example possible to check whether the initial suspension medium used is contaminated. In addition, this provides an indication of the initial value of the turbidity, which is useful for determining the final detection value.

[0125] In another embodiment of the method of automatically preparing a suspension of a microbiological sample, the method further comprises the step of providing a suspension test tube holder for supporting a suspension test tube. The suspension test tube holder can be adapted to rotate a suspension test tube supported within the rotatable suspension test tube holder. In other embodiments the controller is configured such that it is communicatively connected with the rotatable suspension test tube holder to control the rotation of the suspension test tube holder. The controller is further configured such that the suspension test tube is rotated during the measurement of the turbidity of the suspension medium contained in the suspension test tube. The rotation of the suspension test tube allows turbidity measurements at a plurality of locations within the rotating test tube that are rotationally spaced apart from each other, which results in a more accurate final detection value of the turbidity of the suspension. Such rotation is not necessary for the release of the sample from the pick-up tool. The oscillation of the pick-up tool as described above is more than sufficient for the release of the sample.

[0126] Although another pick-up tool can be used that is different from the first pick-up tool, the method can be performed in an economic way: when in step d) the first pick-up tool is provided for use as the other pick-up tool; and the positioning of the other pick-up tool on the pick-up tool holder of the positioning device is performed by the transfer device under control of the controller.

[0127] In another embodiment of the method of automatically preparing a suspension of a microbiological sample according to the invention, a further amount of suspension medium is determined by the controller based on the initial amount of suspension medium, the final detection value and the first and / or second threshold value. This makes it possible to carefully control the amount of suspension medium used. As such, suspension medium is saved.

[0128] Because, in some embodiments, the picking tool oscillates in vertical linear movement relative to the suspension test tube, the horizontal cross-section of the suspension test tube can be relatively small. This makes it possible to use less suspension volume. In one embodiment the controller causes an initial amount of about 0.1 ml to 5 ml of suspension liquid to be dispensed, and preferably less than about 1 ml (in one example about 300 μL). In other embodiments the volume of suspension liquid is about 0.5 ml to about 2 ml. In one embodiment the volume dispensed is 300 μL. Such a relatively small amount of suspension medium is sufficient for preparing a correct suspension of a microbiological sample.

[0129] In this method of automatically preparing a suspension of a microbiological sample, it is possible to use test tubes, vials or cuvettes with a maximum cross-sectional diameter of about 2 to 12 mm (preferably about 3 mm) as suspension containers, which are relatively small compared to conventional suspension test tubes with a diameter of about 16 mm. The test tubes can have a square, rectangular or circular cross-section, and the actual cuvette shape is largely a matter of design choice. In one embodiment the test tubes are circular with a diameter of about 6 to about 12 mm. In one advantageous embodiment the diameter is about 10 mm. Due to such a relatively small suspension test tube, correct release of the sample from the picking tool is obtained: when the controller is configured to control the oscillation of the transfer device such that the picking tool oscillates at a frequency of about 5 Hz to about 250 Hz. The choice of frequency in this range is largely a matter of design choice, and will depend on the composition of the suspension to be prepared. For suspensions made from samples and solutions that easily disperse from each other, a frequency of 5-12 Hz is sufficient. For compositions that do not easily form a suspension, a frequency of about 100 Hz or higher is required. Preferably, the controller is configured to control the oscillation of the transfer device such that the picking tool oscillates with an amplitude of about 0.5 mm to about 4 mm, preferably about 2 mm to about 3 mm, most preferably about 1 mm, which results in the most optimal release of the sample from the picking tool. In embodiments in which the controller is configured to control the oscillation of the transfer device such that the time period during which the picking tool oscillates is about 3 seconds to about 120 seconds (preferably about 30-60 seconds), in virtually all cases the entire sample can be released from the picking tool. If the oscillation only takes about 3 to 10 seconds (6 seconds is about the average minimum oscillation time), this is beneficial for efficiency and throughput.

[0130] The values of frequency, amplitude and duration depend on the properties of the specific microorganism and, for example, its adhesion to the picking tool. In one embodiment, the imaging inspection can be used to infer whether at least a substantial part of the sample has been released from the picking tool by first using the preferred values mentioned above. If still some material remains on the picking tool, the process of vertical oscillation is repeated with different values within the given range.

[0131] The automated method of preparing a suspension of a microorganism sample additionally comprises providing a petri dish automatic positioning and removal device to automatically position and remove the petri dish from the stage. The controller is communicatively connected to the petri dish automatic positioning and removal device to control operation of the petri dish automatic positioning and removal device. In this way positioning of the petri dish (carrying the target microorganism) on the stage can be automatically performed under control of the controller. In other embodiments, a suspension test tube automatic positioning and removal device is provided to automatically position and remove the suspension test tube from the suspension test tube holder, respectively. The controller is communicatively connected to the suspension test tube automatic positioning and removal device to control operation of the suspension test tube automatic positioning and removal device, such that positioning of the suspension test tube in the suspension test tube holder can be automatically performed under control of the controller. Advantageously, the controller is then configured such that the petri dish can be allowed to be automatically removed from the stage by the petri dish automatic positioning and removal device only after a signal is provided that the suspension test tube with the suspension can be removed from the suspension test tube holder for further processing. In another embodiment, the controller is configured such that the suspension test tube holder can be automatically removed from the suspension test tube holder by the suspension test tube automatic positioning and removal device only after a signal is provided that the suspension test tube with the suspension can be removed from the suspension test tube holder for further processing.

[0132] In another embodiment of the method according to the application, an identification mark is provided on the suspension test tube. According to the method, the identification mark of the suspension test tube is saved in the memory of the central control computer together with the properties of the suspension associated with the characteristics of the petri dish from which the selected microorganism colony comes. This ensures that the method can not only be run automatically in a very efficient manner, but also that the obtained analysis results can be processed correctly and accelerated.

[0133] In a further embodiment of the method described herein, a pipetting tool is provided (separately or the pick-up tool is adapted to receive and use the pipette) to deposit an aliquot (or multiple aliquots) of the suspension onto the MALDI plate and also to deposit an aliquot of the suspension for other downstream analysis (e.g. AST). The positioning device is provided with a pipette holder for supporting the pipetting tool. The positioning device is configured to position the pipetting tool in a starting position above the suspension tube. The positioning device automatically lowers the pipetting tool to enter the suspension or raises the pipetting tool to exit the suspension, respectively, and positions the pipetting tool in a transfer position. The pipetting tool is received by the pipette holder of the positioning device. The positioning device positions the pipetting tool in a starting position above the suspension tube, lowers the pipetting tool to enter the suspension in the suspension tube, operates the pipetting tool to pick up an amount of the suspension, and raises the pipetting tool with the amount of the suspension to the transfer position. The pipetting tool has a pressurizable chamber, which is closed by a control valve, in order to contain the amount of the suspension medium.

[0134] The method provides a target plate holder for supporting a target plate, the target plate having at least one deposition point. The target plate is positioned on the target plate holder. A transfer device is provided for automatically transferring the pipetting tool from the transfer position of the positioning device to a position above one deposition point of the target plate and lowering the pipetting tool to a predetermined distance above the target plate. A pressure in the range of about 0.5 bar to 1.1 bar is applied to the chamber and then the valve is opened for a period of time so that a drop of the suspension having a volume of about 0.5 μl to 3.0 μl is deposited on the deposition point, in particular covering at most about half of one of the deposition points on the target plate. The pipetting tool is then raised from the target plate. Depending on the properties of the particular microorganism, e.g. viscosity, the pressure and the opening time are adjusted to obtain a drop of the suspension which can be reproducibly prepared and which can be accurately deposited on the target plate using an automated process.

[0135] In the aforementioned manner, the pipetting tool is used to obtain more suspension. The pipetting tool is then used to dispense the suspension into a container for another analysis (e.g. a container adapted for performing an antibiotic susceptibility test (AST)).

[0136] In order to avoid cross-contamination in a preferred embodiment of the method according to the application, the shape of the pipetting tool, in particular the shape of its dispensing tip, can be such that the drop of the suspension can be deposited on the target plate or other container in a splash-free manner. At present, in addition to selecting a suitable pressure in the range mentioned above and a suitable opening time of the valve in the range mentioned above, a suitable shape of the pipetting tool also ensures that the deposition of the drop of the suspension can be achieved in a splash-free manner, depending on the kind of microorganism used, in particular its viscosity.

[0137] In another embodiment of the method, identification marks are provided on the target plate and other containers of the sample test (e.g. AST), selectively on the deposition points of the target plate. According to the method, the identification marks of the target plate and the deposition points are stored in the memory of the central control computer together with the properties of the suspension associated with the characteristics of the petri dish from which the selected microorganism colony comes. The method not only enables an extremely efficient automatic operation, but also enables an accurate and fast processing of the analysis results obtained.

[0138] In another embodiment of the method, a prepared container, e.g. a container used for assisting in the execution of another test (e.g. AST), can be moved from a position where the microorganism suspension and other appropriate reagents are pipetted into the container to a second position, where a further pipette removes the mixture from the container and inoculates a cartridge for the test. The cartridge can be further positioned by a robot after inoculation into a support structure, which supports the cartridge until it is retrieved by a cartridge transfer instrument. If the conditions are met, the cartridge transfer instrument picks up or grips the cartridge from the support structure and transfers the cartridge to another support structure located within the test instrument (e.g. AST test instrument).

[0139] Mass spectrometry, in particular MALDI or MALDI-TOF-MS, is used for identifying microorganisms. In a MALDI-TOF-MS run, samples of microorganism colonies are spotted or deposited on a target plate, which is fixed to a fixed position within the MALDI instrument. The target plate usually has a plurality of deposition points (e.g. 24 to 384 deposition points on a single target plate). The deposition points have a fixed orientation with respect to the edge of the target plate. The target plate is positioned on an X-Y stage, so that the samples obtained from the microorganism colonies can be deposited on selected deposition points. The position of a particular sample deposition can be displayed by X-Y coordinates / parameters and stored in the memory of the central control computer.

[0140] Although details are not shown in Figure 2 , the target plate 42 is shown positioned below the transfer track 18, the position of which is indicated by B. The samples can be transferred from the petri dishes 3 and / or the suspension test tubes 11 along the transfer track 18 to the position B above the target plate, where the samples are lowered for deposition onto the deposition points on the target plate. This is different from Figure 1 other transfer mechanisms can also be considered. For example, a panel-mounted transfer mechanism can be used.

[0141] The present application will be described in detail below with reference to preparing a suspension containing a sample and depositing the suspension onto a deposition point on a target plate. Typically, microorganism colonies are automatically positioned and detected on a petri dish. A sample of a selected microorganism colony is obtained in an automated manner, e.g. by a picking tool in contact with the colony.

[0142] When performing microbial characterization and identification, typically a plurality of colonies is grown on a petri dish. In addition a plurality of different petri dishes is processed by the apparatus. Thus, the present application provides the ability to identify each petri dish individually, e.g. by a barcode, and in addition each colony of interest on a single petri dish is selected and assigned an identification tag. Prior to the automated steps of locating and selecting a microbial colony on a petri dish, a petri dish containing a plurality of microbial colonies is provided. An initial image of the petri dish is acquired. The image comprises all colonies of microorganisms. The apparatus or a device in communication with the apparatus displays the initial image of the petri dish comprising all colonies of microorganisms on a display and selects at least one microbial colony in the initial image. In this way, the researcher or the laboratory technician can select the colony of interest based on detailed education and knowledge. In one embodiment, the image information is processed and the colony for picking is identified based on specifications. Since a specific tag is provided for each petri dish to identify the petri dish, e.g. a barcode or the like, the initial image of the petri dish comprising the colony is saved and information related to the at least one selected target microbial colony is saved (preferably with a link to the (electronic) initial image). All the information and tags of the petri dish are saved in the memory of the central control computer in order to allow high accuracy and process integrity.

[0143] In this way, the only possible manual operation in the method and apparatus described herein is the operation of selecting the colony of interest. The processing of all sample related data is performed in an automated manner. Optionally, the researcher and the laboratory technician can manually input processing instructions regarding the selected microbial colony of the petri dish to be performed. The processing instructions are also saved in the memory of the central control computer for later use. After this step of manual operation is performed, all subsequent performed steps are performed in a reliable and efficient manner automatically.

[0144] For further processing of this automation, the petri dish is automatically positioned on a stage of a pick tool device for the petri dish comprising an imaging device. An image of the petri dish positioned in the pick tool device is obtained, which image, together with the identification of the petri dish, can be compared with the stored initial image of the petri dish, whereby information is obtained about the position of the selected microbial colony, or alternatively information is obtained about the processing instructions for the processing to be performed on the selected microbial colony. By comparing the image of the petri dish when placed on the pick tool device with the initial image, the position of the selected colony can be obtained automatically, for example by computerized image comparison. In addition, each target plate is provided with an identification mark, and optionally each deposition spot of the target plate has a separate identification mark or position identification. The containers for the AST are also provided with an identification mark, to enable the results to be associated with the correct sample. After saving the identification marks of the target plate and the deposition spots in the memory of the central control computer, together with the characteristics of the suspension associated with the identification of the petri dish from which the selected microbial colony was obtained, the obtained MALDI / AST results can be correctly associated with the particular microbial colony under test in an accurate and automated manner.

[0145] It has been found that the accuracy of the analysis results obtained when using a MALDI instrument to analyze the part of the deposition spot that was initially not covered by the sample is surprisingly much higher than the accuracy of the analysis results obtained when using a MALDI instrument to analyze the part of the deposition spot that was initially covered by the sample, when the sample covers at most about half of one of the deposition spots on the target plate. It is believed that the crystallization process that takes place after a drop of matrix material is applied to the sample covering part of the deposition spot ensures that the part of the deposition spot that is not covered also contains an amount of sample material, and that this amount is very suitable to provide excellent analysis results. The physical or chemical process that leads to this effect is not known at present, but can become more clear after understanding the basic processes behind the MALDI technique.

[0146] Sample preparation system and method

[0147] An embodiment of the method of the present application for preparing a suspension from a sample of a microbial colony picked from a petri dish will now be described together with an embodiment of a sample preparation system 1000 for carrying out the method.

[0148] Figure 1A system 1000 that performs the methods described herein is shown. The system 1000 includes a housing 1005 that provides an environment for the components that implement and perform the methods. In this regard, the components are distributed among multiple stages of the housing 1005. From left to right, the housing provides a receiving stage 1010, a picking stage 1020, a preparation stage 1030, and a transfer stage 1040. The receiving stage 1010 receives one or more petri dishes that can carry colonies of microorganisms of interest and automatically passes the petri dishes to the picking stage 1020. The picking stage 1020 automatically detects colonies of interest and picks samples from there. The preparation stage 1030 automatically prepares samples for testing, such as identification (ID) and antibiotic susceptibility testing (AST). The transfer stage 1040 automatically transfers prepared AST samples to AST cassettes (also referred to herein as plates), which are automatically transferred to an AST system.

[0149] In a typical method, an automated picking tool device 8 is provided to obtain a picking tool 6 and transfer the tool to a stage 2 that supports a petri dish 3 that has been placed on the stage. Prior to picking, a colony of interest 4 is identified on the petri dish 3 and its location thereon is determined. The picking tool 6 is informed of the location by the controller 30, moves over the colony of interest 4 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 tube 11, which is preferably performed prior to the picked colony sample 19 entering the tube 11. The picking tool 6' is then positioned so that a portion of the picking tool 6' that carries the picked sample 19 is immersed in the suspension 14. The picking tool 6' is agitated to release the microorganisms. A turbidimeter 20 monitors the turbidity of the suspension and provides such information to the controller 30, which cross-references the measured turbidity with the concentration specification for the suspension aliquot for the test to be performed, such as ID and AST. The target concentrations for ID and AST are described herein.

[0150] Once the suspension reaches the desired turbidity, an aliquot of the suspension is removed from the tube 11 and a plate 42 for performing an ID test is inoculated with the suspension. A pipetting tool 46 then takes another aliquot of the suspension for an AST. In some embodiments, the suspension can need to be further diluted with suspension liquid prior to removing the suspension for the AST. Once obtained, the pipetting tool 46 then dispenses the suspension into a container 82 for the AST test or other analysis, such as a molecular diagnostic analysis. The container can include reagents for the further test and can be bar code scanned by the tube gripper robot 50 prior to dispensing.

[0151] After that, the container 82 is transferred by the moving device 80 to a second position. At this position another pipetting tool 66 removes the suspension from the container 82 and inoculates a test cartridge 90. The cartridge 90 can be moved by the cartridge transfer robot 70 to a support structure of the cartridge filling unit 78 before inoculation. The cartridge filling unit 78 can initiate rotation of the cartridge 90 to reach an optimal angle for inoculation. A lid opening robot (not shown) can remove the lid from the cartridge 90 when needed for inoculation via the pipette 60. After the cartridge 90 is inoculated, it can be transferred by the transfer instrument 2000 to the test instrument 2050 (see Figure 20 ). Once the test is performed, the system can then output a final sample report indicating the quantification of sample growth and the results of the ID and AST.

[0152] The method is now described more particularly with reference to the system 1000 and its components. Figure 2 The picking platform 1020, 1030, and 1040 are schematically shown arranged within the housing 1005 of the system 1000.

[0153] The picking platform 1020 comprises a stage 2 for a petri dish 3 comprising microorganisms 4 on a nutrient layer 5, for example an agar gel layer. The petri dish 3 can be positioned onto the stage 2 via a moving arm (not shown) that transfers the petri dish 3 from the receiving platform 1010. The receiving platform 1010 can automatically receive a plurality of petri dishes from other upstream laboratory equipment and arrange them in a stacked arrangement before feeding the petri dishes 3 to the picking platform 1020.

[0154] Once the petri dish 3 is received at the picking platform 1020, colony identification and colony picking is performed. The platform 1020 comprises a positioning device 8 comprising a picking tool holder 9 for releasably supporting a picking tool, for example a disposable pipette tip. As shown, the picking tool holder 9 supports a first picking tool 6. The positioning device 8 is configured to position the first picking tool 6 in a starting position (indicated in solid line in Figure 2 ) above the petri dish 3 and is configured to automatically lower or raise the first picking tool 6 towards the petri dish 3 to exit the petri dish 3 so that the first picking tool 6 can be positioned in a position (indicated in dashed line) in which it contacts the microorganisms 4 and picks a sample 19 of the microorganisms 4. After the first picking tool 6 has picked the sample 19 (the first picking tool with the retained sample 19 is indicated in Figure 2After the first pick tool 6' is positioned as shown in Fig. 6, the positioning device 8 raises and positions the first pick tool 6' above the suspension tube 11 in a transfer position "A". The positioning device 8 preferably raises the pick tool 6' vertically to a start position before moving horizontally along the transfer track 18 to the transfer position A. This can help prevent contamination by a slime string that can form during sample picking. However, in other embodiments, the positioning device 8 can move both vertically and horizontally toward the transfer position A (as indicated by the arrow in Fig. 6) at the same time. Figure 2

[0155] In picking colonies for further testing, some colonies have a property of being slimy or slippery as mentioned above. This is referred to as slime consistency, which makes it difficult to remove the colony from the agar surface. As mentioned above, after a colony is contacted by a picking device, a slime string often forms between the pick tool and the colony on the agar surface (Figs. 7 and 8). This string can be difficult to break in a controlled manner and causes the problems mentioned above related to possible contamination of other samples and surfaces within the instrument. Figure 15 16

[0156] When picking colonies manually, the user will see the formation of the string and can make various hand motions to eliminate the string. This includes rotating the pick tool and / or rubbing the device on a clean portion of the plate. Because the string can be observed visually, the user will see when the string breaks and can continue with the testing. All of these checks can be performed with little risk of cross contamination.

[0157] In one embodiment of an automated process that addresses the presence of a slime string, the string is detected optically (e.g., a camera can be used to monitor and detect such a string), or by monitoring changes in an electric field. The string is electrically conductive relative to the surrounding air. Once the string is detected, one skilled in the art will recognize that any number of mechanical devices can be used to sever the string. Referring to Fig. 9, a culture plate 710 is shown disposed within an automated system 700. The culture plate 710 has agar 720 disposed thereon, on which a number of different colonies 730 are formed. A pipette tip 740 is lowered to contact one of the colonies, and as it is aspirated, a string 750 is formed. Referring to Fig. 10, as the pipette tip 740 continues to be raised away from the surface of the agar 720, the string 750 is elongated. Moving the pipette tip at this point will cause the string to be moved to another location within the system 700. This can cause cross contamination by the string at the other location within the system 700. Figure 15 Figure 16

[0158] ​​​​​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 aspirates from the surface of the plate to transfer the picked sample into suspension. The string will cause a difference in the charging of the capacitance as the picking tool is retracted from the sample.

[0159] The capacitance level sensor can sense a variety of solid, aqueous and organic liquids. Capacitance detection relies on a radio frequency signal applied to the capacitance circuit. By monitoring the capacitance (pF = picofarad), the formation of a slime string can be detected. Figure 17A is a plot of capacitance as the picking tool is lowered and contacts the agar surface. The capacitance rapidly decreases as the picking tool is raised and leaves the agar surface. Figure 17B The change in the capacitance signal as a slime string forms when the picking tool is removed from the surface is shown. The capacitance slowly decays as the string becomes thinner and thinner and finally breaks.

[0160] The conductance level sensor uses a low voltage level between two sensors. Because the slime string is electrically conductive, as long as the agar surface is connected to the picking tool through the slime string, a high electrical conductivity will be maintained.

[0161] The string can also be detected optically. A light signal passing through the plate is diffracted by the string formed between the plate and the pipette. The interruption of the signal can be detected by software and thereby indicate the presence of the string.

[0162] Any number of mechanical devices can be used to remove the string. The preferred solution is cost effective and does not generate aerosols or contaminate other plates in the system. The slime coating on some bacteria makes picking difficult for both manual and automated systems. The slime biofilm protects the organisms but makes their manipulation difficult. For this example, an additional automated step or feature is provided after the picking of the slime sample to eliminate the string and prevent contamination of the system.

[0163] In one embodiment, a resistively heated hot wire or blade is provided to cut the slime string. The wire or blade is heated to a temperature sufficient to sterilize the cutting device so that it can be reused continuously. The skilled artisan can select a temperature that will decontaminate the wire or blade by killing microorganisms but not so hot as to cause rapid evaporation of the picked sample which would cause aerosol release of the organisms.

[0164] Very cold temperatures can also be used to break the string. A small spray of liquid nitrogen to the pipette tip once the string is sensed will harden the string so that it breaks. In an alternative embodiment, a chilled cutting probe can be used to cut the slime string and obtain a clean break.

[0165] In another embodiment, a rotating disposable rod is used to break the string. When the string is detected, a cutting rod is used to contact it. In an alternative embodiment, the cutting rod can be rotated so that the mucus string wraps around the rod to ensure that the string is broken.

[0166] In another embodiment, an ultrasonic device is provided to break the string when the pipette tip forms a string as it is withdrawn from the agar plate. For example, an ultrasonic horn is attached to the pipette tip adapter. As the picking device is pulled away from the agar surface, a high frequency short pulse causes the mucus string to be easily sheared.

[0167] In another embodiment, the string is allowed to dry, thus becoming brittle and breaking. A small nozzle located next to the plate sprays air at the string, reducing the drying time. The drying time is controlled so that it does not significantly increase the time of any one pick.

[0168] In another embodiment, a strong current is passed through the string. The natural resistance of a thin mucus string will cause the greatest resistance in the thinnest (smallest electrical conductivity) part of the string. This increased resistance will cause the string to break. The current is chosen so that it is strong enough to break the string, but not so strong that it causes rapid vaporization, which would cause an aerosol release of the organic matter.

[0169] After the string is detected, the tip is advanced over the agar surface (3-6 mm above the surface). As the string falls on the agar and the tip continues to move, the string will elongate to the breaking point. However, because the string will break above the agar, there is no risk of cross contamination. In an alternative embodiment, a rapid zig-zag movement path is used as the tip changes direction over the agar so that the string breaks.

[0170] In an alternative embodiment, the tip punches into the agar of a plate with no growth. This will wipe the tip clean and remove the string. In another embodiment, the pipette tip is moved over the agar surface to the edge of the plate. The string can be effectively wiped off the edge of the plate and the string is removed.

[0171] In another embodiment, a small vacuum device near the tip is used when the mucus string is detected. The vacuum will create a vacuum within the string using a HEPA filtration system and eliminate environmental contamination.

[0172] In an alternative embodiment, the pipette is treated or coated with a mucus dissolving agent that can break down the high molecular weight glycoproteins found in the mucus string. One example of such an agent is n-acetyl-l-cysteine.

[0173] In another embodiment, the low energy laser is positioned away from the plate side. As the pipette leaves the plate area, the pipette tip is moved right over the laser beam. If the string is present, the slime string will then move through the light beam. The slime string will be heated to the point where the string will break.

[0174] In another embodiment, the tip can be rotated 360 degrees when the string is detected. The rotation will cut the slime string. In another embodiment, the pipette tip is moved up and down to contact the agar surface at the same location where the pick-up occurs, breaking the string. With each downward contact, the pipette tip aspirates a volume. This cuts the slime string and in effect draws most or some of the string into the pipette tip.

[0175] The pick-up station 1020 further includes a suspension tube holder 10 for supporting a suspension tube 11 that can hold a suspension medium 14. In the present embodiment, the suspension tube holder 10 is a rotatable suspension tube holder 11 to rotate the suspension tube about a vertical axis D. However, in some embodiments, the tube holder 10 can be stationary. The suspension medium 14, as shown, is dispensed from a suspension medium automatic dispenser 12 having a dispensing nozzle 13 for automatically dispensing the suspension medium 14 into the suspension tube 11 supported by the suspension tube holder 10. However, in some embodiments, an automatic pipettor, such as pipettor 40, can separately dispense the suspension medium 14 into the tube 11.

[0176] The positioning device 8 also includes incorporated therein a transfer device 15 for assisting in the automatic transfer of the sample 19 into the suspension medium 14. The transfer device 15 is connected to the pick-up tool holder 9 and is configured to linearly vertically move the oscillating pick-up tool 6' for a time period sufficient to allow the sample 19 to be released from the pick-up tool 6'. In the method, once the suspension tube 11 is inoculated with the suspension medium 14 and the pick-up tool 6' is positioned at the start position of the transfer position A above the tube 11, the positioning device 8 lowers the pick-up tool 6' into the suspension medium 14. As the sample 19 is immersed, the transfer device 15 is activated to oscillate the pick-up tool 6' so that the sample 19 is released into the suspension medium 14, as shown in Figure 2 the method. Thereafter, the positioning device 8 positions the pick-up tool 6 above the suspension tube 11 at a waiting position, which can be the same as the start position. In other embodiments, the waiting position and the start position can be different from each other.

[0177] The system 1000 further comprises a turbidimeter 20 for performing a turbidity measurement of the suspension medium 14 contained within the suspension cuvette 11 supported by the suspension cuvette holder 10. As is generally known in the art, the measurement provided by the turbidimeter can be used to gauge the concentration of a material, in this example the concentration of microorganisms suspended in the suspension medium. As shown in Figure 2 the turbidimeter 20 comprises a laser 21 that emits a laser beam towards and through the suspension medium 14, and a sensor 22 that detects the amount of laser light that is transmitted through the suspension medium 14. Preferably, there is also a sensor (not shown in the figures) that is arranged perpendicular to the path of the laser beam so that the amount of laser light that is scattered by the suspension can be detected.

[0178] The operation of the system 1000 is controlled by a controller 30. The controller 30 comprises a processor 32 and a memory 34 as schematically shown in Figure 3 Fig. 4. The controller 30 is communicatively connected to the positioning device 8, the transfer device 15, the suspension medium automatic dispenser 12 and the turbidimeter 20 to automatically control the movement of the positioning device 8, the movement of the transfer device 15, the operation of the suspension medium automatic dispenser 12 and the operation of the turbidimeter 20, respectively. In addition, the controller 30 can also be communicatively connected to other components of the apparatus, such as the pick-up tool holder 9, the laser 21 and the sensor 22.

[0179] In the embodiment shown in Figure 2 and 3 Fig. 4, the controller 30 is configured to control the turbidimeter 20 such that the turbidity measurement of the suspension medium 14 is started before the pick-up tool 6' is immersed in the suspension medium 14. In addition, the controller 30 also controls the rotatable suspension cuvette holder 10 to start rotating the suspension cuvette 11 supported in the holder 10 before the pick-up tool 6' is immersed in the suspension medium 14, and to keep the suspension cuvette 11 rotating during the turbidity measurement of the suspension medium 14. The controller 30 further controls the turbidimeter 20 such that the turbidity measurement is performed during the entire time period in which the pick-up tool 6' is oscillating. In this way, the turbidimeter 20 provides the controller 30 with an online measurement that represents the turbidity, and thus the concentration of microorganisms, measured during the time period in which the pick-up tool 6' is oscillating.

[0180] As mentioned above, the controller 30 comprises a memory 34 in which a first and a second threshold value are stored. The first threshold value is greater than or equal to the second threshold value. If the turbidity measurement provided by the turbidimeter is equal to or between the first and the second threshold value, the concentration / amount of micro-organisms in the suspension medium is sufficient for the suspension cuvette 11 containing the suspension 14 to be further processed. In this case, the controller 30 provides a signal that the suspension in the suspension cuvette 11 can be further processed when the turbidity measurement is between the first and the second threshold value. Furthermore, in this case the pick-up tool 6 is discarded, for example the positioning device can be moved over a waste bin and the pick-up tool holder is activated to release the pick-up tool 6 into the waste bin.

[0181] If the final measurement of the turbidimeter 20 is higher than the first threshold value previously stored in the memory 34 of the controller 30, it is determined that the concentration of micro-organisms is too high for the suspension in the suspension cuvette 11 to be further processed. In this case, the controller 30 controls the suspension medium brake dispenser 12 or some other medium dispenser to supply an additional amount of suspension medium 14 to the suspension cuvette 11. The additional amount of suspension medium 14 is based on the initial amount of suspension medium, the final measurement and the first and / or second threshold value, such that adding the additional amount of suspension medium to the suspension medium 14 already contained in the suspension cuvette 11 will result in a concentration of micro-organisms in the suspension medium 14 in the cuvette 11 that meets the requirements for further processing, which concentration can be confirmed by an additional or further measurement of the turbidity by the turbidimeter 20.

[0182] If the final measurement of the turbidimeter 20 is below the second threshold, i.e. the concentration of microorganisms 4 in the suspension medium 14 is too low, the controller 30 controls the positioning device 8 so that the first picking tool 6 picks an additional sample 19 of microorganisms 4 to further concentrate the suspension medium 14. Alternatively, the first picking tool 6 can be discarded and a second picking tool can be used to pick the additional sample. In this case, when the final measurement is determined to be less than the threshold, the controller controls the first picking tool 6 in the picking tool holder 9 of the positioning device 8 to lower from a starting position above the petri dish 3 towards the petri dish and contact the microorganisms 4 to pick an additional sample 19 of microorganisms 4. Thereafter, the first picking tool 6' with the additional sample 19 of microorganisms 4 is automatically raised away from the petri dish to the starting position at the transfer position A above the suspension test tube 11. Next, the picking tool 6' with the additional sample of microorganisms is lowered into the suspension medium 14 and oscillated by the transfer device 15 in linear vertical movements for a period of time to release the additional sample 19 of microorganisms 4 into the suspension medium 14. A turbidity measurement is again taken during the oscillation and the measured value is compared to the first and second thresholds stored in the memory 34 of the controller 30. In this case, the controller 30 is configured to control the movement of the positioning device 8 so that once the additional sample is at least partially removed from the picking tool 6, the picking tool 6 is raised to the waiting position if the on-line measurement of turbidity taken by the turbidimeter 20 during the oscillation is less than or equal to the first threshold and greater than or equal to the second threshold.

[0183] While, as described, the concentration of microorganisms in the suspension medium 14 can be increased by multiple subsequent colony pick-ups when the measured turbidity value is less than the threshold level, other procedures can also be performed without regard to the measured concentration being determined to be too low. In this regard, as described in more detail below, multiple dispersions of a low concentration suspension can be deposited on the same point on a MALDI plate. This has the effect of concentrating the microorganisms 4 on the MALDI plate rather than in the suspension medium 14.

[0184] The suspension test tube 11, or alternatively a vial or cuvette that can be used in particular in the apparatus of the present application, has a cross-section with a maximum dimension of about 2 to 12 mm, preferably about 3 mm. In these relatively small suspension test tubes, the controller 30 can control the suspension medium automatic dispenser 12, or other medium dispenser, so that the initial amount of suspension medium supplied is about 0.1-5 ml, preferably less than about 1 ml.

[0185] The oscillation of the transfer device 15 is controlled by the controller 30 such that the pick tool 6' oscillates at a frequency of between about 5 Hz and about 250 Hz, preferably about 100 Hz, with an amplitude of between about 0.5 mm and about 4 mm, preferably about 2 mm to about 3 mm. The controller 30 can be further configured to control the oscillation of the transfer device 15 such that the pick tool 6' oscillates for a time of between about 3 seconds and about 120 seconds, preferably about 30 seconds to about 60 seconds.

[0186] Nephelometer of an automated system and method

[0187] Various embodiments of a nephelometer will now be described. It should be appreciated that any one of these now described nephelometers can constitute the previously described nephelometer 20. In one embodiment, the nephelometer for use in the automated system 1000 can be the nephelometer described in U.S. Provisional Application 62 / 056,911, which is commonly assigned and incorporated by reference herein. In this embodiment, the suspension does not undergo oscillation when measuring turbidity.

[0188] Another embodiment of a nephelometer 100 is shown in Figure 4A and 4B The nephelometer 100 is a low volume nephelometer designed to accommodate individual suspension test tubes shown as cuvettes 110 placed inside a nephelometer base 101, which has a suspension 120 as shown in Figure 4A The 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 attenuation filter 160 (see best Figure 4B ). The cuvettes 110 with samples 120 are positioned in the center of the nephelometer 100 and inside the nephelometer base 101. The light source 130, the scatter detector 140, and the transmitted light detector 150 are positioned at 90 degree angles relative to each other around the cuvettes 110. The scatter detector 140 is positioned close to the cuvette 110 containing the sample suspension 120 and parallel to the incident light source 130. This minimizes the diffraction, refraction, and reflection effects of the scattered light. The transmitted light detector 150 is positioned at 180 degrees or opposite the light source 130. The transmitted light detector 150 can also be oriented perpendicular to the incident light beam or at different angles to reduce reflection effects from its surface. The light attenuation filter 160 is positioned between the cuvette 110 and the transmitted light detector 150. In this configuration, the sample suspension 120 is individually processed inside the cuvette 110, and the nephelometer 100 detects scattered and / or transmitted light at certain angles through the test sample 120.

[0189] Low volume cuvettes / cuvettes (or microcuvettes) are contemplated for use, which are designed to be used in conjunction with a low volume nephelometer, such as the nephelometer 100, to process relatively small amounts of biological and fluid suspensions. Figure 5Aand 5B Alternative embodiments of the low-volume cuvette are described. The cuvettes 110, 110' are molded from optically transparent plastic with minimal tapered sides 430, 440 that are optically smooth polished to facilitate orientation within the nephelometer 100. The cuvettes 110, 110' can be configured as individual units for single-use applications. However, in some embodiments, as further described below, where a series of cuvettes are used to prepare a suspension, the cuvettes 110, 110' can be configured for use with a linear array strip for such applications. Alternatively, the cuvettes 110, 110' can be configured for use with a matrix array designed to simultaneously process multiple samples. In the matrix embodiment, multiple series of suspensions are prepared in parallel.

[0190] As shown, the cuvettes 110, 110' have a lower portion 410 that has a relatively small volume compared to an upper portion 400. The suspension is initially prepared within the small volume portion 410. The suspension is thus first disposed within the interior of the lower portion 410 of the cuvette 110, 110'. A biological sample suspected of including a target microorganism is added and mixed with the fluid suspension to provide a test sample suspension 120. The turbidity of the suspension within the lower portion 410 is detected. In this regard, when the cuvette 110 or 110' is coupled with the nephelometer 100, light generated by the light source 130 passes through the sample suspension 120 disposed within the interior of the lower portion 410. The nephelometer 100 detects the light scattered by the lower portion 410 via the detectors 140 and 150, and detects the turbidity of the sample within the lower portion 410 of the cuvette based on the detected light.

[0191] Below the lower portion 410 of each cuvette 110, 110' is a "macro-particle" collection region 420 designed to receive macro-particles that settle from the sample suspension, which otherwise adversely affect the accuracy of the turbidity detection made by the nephelometer 100. Low volume samples additionally have insufficient volume to cause the micro-particle contaminants to settle from the portion of the suspension that is probed by the nephelometer. For example, light passing through a low volume suspension containing micro-particle contaminants cannot distinguish between the sample and the contaminants within the suspension and can produce 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 can inform a false dilution. An inaccurate McFarland value can also cause a sample to be processed downstream (e.g., by AST or MALDI) when the true McFarland value is known, the sample would not be further processed. That is, the true McFarland value would inform the operator that the sample is not suitable for MALDI or AST. Additionally, the presence of contaminants in the sample can interfere with the accurate concentration detection of the sample to be tested. As such, the cuvettes 110, 110' according to the illustrated embodiments provide this separate, micro-particle collection region 420 that is positioned outside the direct light path through the lower portion 410. Micro-particle contaminants settle within the collection region 420 and do not remain in the test region of the sample suspension, which occurs in the lower portion 410. The chamber length of the lower portion is in the range of about 5.5 mm and is designed to provide a chamber length sufficient for low volume samples to obtain proper turbidity detection. Once the test sample suspension is prepared, the lower portion is designed to provide sufficient chamber length 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 that is near optically transparent or other light-transmissive material known to those skilled in the art. Such materials allow light to pass through the walls 440 of the lower portion of the cuvette without interference.

[0192] Those skilled in the art will appreciate that there are three dimensions of design freedom in configuring the small volume portion 410 of the cuvette 110, 110'. The dimensions of the small volume portion 410 are largely 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-up tool) that introduces the sample into the lower portion of the cuvette. For example and without limitation, the dimensions of the lower portion of the cuvette are set to provide sufficient space for a 3 mm diameter pick-up tool to be dipped into the lower portion and rotated within the lower portion such that it does not touch the side walls of the cuvette 110, 110', creating scratches and surface aberrations that can degrade its optical transparency.

[0193] Of course, the dimensions of the lower portion 410 must be adapted to the optical detection of the sample. In particular, the dimensions of the lower portion 410 of the cuvette 110, 110' are set to work with the light source 130 and the detectors 140, 150 of the nephelometer 100. The dimensional constraints on the cuvette design are therefore functional to the architecture of the nephelometer 100.

[0194] Above the lower portion 410 is the upper portion 400, which is used to dilute the sample suspension placed in the container for further processing for downstream applications, such as AST. The upper portion 400 has greater width and length than the lower portion 410. Preferably, the internal dimensions of the container are designed to be suitable for the automatic mixing of biological samples and suspensions for further direct dilution of the test sample suspension inside the container (when needed). In operation, the stacked container design of the cuvette 110, 110' makes it possible to detect the turbidity of the sample suspension therein and, if the target turbidity is not reached, to further dilute the sample and repeat the turbidity measurement. This configuration makes it possible to perform sample dilution in real time (i.e. while the sample is being optically probed). In addition, the stacked container design makes it possible to detect the turbidity of low volume sample suspensions (e.g. of about 200 microliters to about 500 microliters volume of suspension) with the added advantage of having a greater volume suitable for sample dilution.

[0195] As shown, the top or upper portion 400 of each cuvette 110, 110' has an approximately square or rectangular boundary. The geometry of the upper portion 400 is largely a matter of design choice. The bottom or lower portion 410 also has an approximately square boundary. In this regard, the cuvettes 110, 110' are "nested" from top to bottom, as the upper portion 400 has a larger cross-sectional dimension relative to the lower portion 410. Alternative shapes of the cuvettes 110, 110' are contemplated, so long as the walls 440 of the bottom portion 410 are at an angle to one another (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 to one another (as compared to a circular tube) results in less aberration of the light signal and better mixing of the test sample. This is demonstrated in the described embodiments 110, 110', in which the lower portion 410 has four sides 440 that are perpendicular to one another, thereby defining a square. Additionally, the upper portion 400 also 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 a 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 cuvette 110, 110'. Additionally, the square configuration of the cuvette / container 110, 110' allows the light path to pass through and into the sample suspension at a right angle to the planar surfaces of the container 110, 110'. This configuration also minimizes the potential for diffraction and refraction of the light source 130 as it enters and exits the cuvette 110, 110'.

[0196] Different configurations of the cuvette 110, 110' are contemplated. In the described embodiments, the top portion 400 of the cuvette 110 tapers to the lower portion 410. The corners of the top portion 400 where the sidewalls 430 intersect are aligned with the corners of the lower portion 410 (as can be seen by the straight edge 401). The tapered edge 401 distinguishes the transition between the wider upper portion 400 and the narrower lower portion 410. Figure 5A In the described embodiments, the top portion 400 of the cuvette 110 tapers to the lower portion 410. The corners of the top portion 400 where the sidewalls 430 intersect are aligned with the corners of the lower portion 410 (as can be seen by the straight edge 401). The tapered edge 401 distinguishes the transition between the wider upper portion 400 and the narrower lower portion 410.

[0197] In the described embodiments, the top portion 400 of the cuvette 110 tapers to the lower portion 410. The corners of the top portion 400 where the sidewalls 430 intersect are aligned with the corners of the lower portion 410 (as can be seen by the straight edge 401). The tapered edge 401 distinguishes the transition between the wider upper portion 400 and the narrower lower portion 410. Figure 5B In another embodiment 110' shown, the corners of the upper portion 400 where the sidewalls 430 intersect are offset from the corners of the lower portion 410 where the sidewalls 440 intersect. The offset occurs at the tapered edge 401. Figure 5BThe offset edge 402 is shown. In one particular embodiment, the corner of the lower portion 410 is offset by 45 degrees from the corner of the top portion 400. Advantageously, this configuration allows the light source 130 and detectors 140, 150 to be arranged on either side of the cuvette 110' when the cuvette 110' is placed inside the turbidimeter base 101.

[0198] Now described as follows Figure 6 The flowchart illustrates a method for detecting turbidity using a turbidimeter 100 and cuvette 110. Cuvette 110 is placed manually or automatically inside the turbidimeter base 100. An initial suspension liquid (free of microorganisms) is placed inside cuvette 100. The fluid volume is approximately 200 μL to approximately 500 μL. Preferably, the initial suspension fluid volume is approximately 300 μL. If dilution is required to obtain a specific McFarland value, additional fluid can be added to cuvette 110. Next, a biological sample suspected of containing microorganisms is added to cuvette 110 and mixed with the suspension fluid to obtain a test sample suspension 120. The turbidimeter 100 detects the initial turbidity of the test sample 120, and the McFarland value is recorded in memory 34. If the initial turbidity reading is too high, the sample suspension is further diluted by adding additional suspension fluid. In one embodiment, dilution is performed automatically. The higher portion 400 of cuvette 110 allows the volume of the suspension fluid to exceed the volume of the lower portion 410. The turbidimeter 100 measures the turbidity of the diluted suspension. Once the predetermined McFarland value is obtained, the suspension is processed for downstream testing, storage, or disposal. The suspension can be diluted many times as needed to obtain the desired McFarland value.

[0199] The light from light source 130 is used to detect the suspension 120 (e.g., a test sample) arranged inside cuvette 110. Light that impacts a surface (e.g., the planar sidewall 440 of cuvette / container 110) is referred to herein as incident light. Light scattered from the particles in 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 planar sidewall 440 of cuvette / container 110).

[0200] During operation, the transmitted light is received by the transmitted light detector 150. In an exemplary embodiment, the transmitted light detector 150 is positioned in the incident light path to maximize the detection of light transmitted through the suspension. In cases where the surface of the detector 150 is highly reflective, the detector 150 can be positioned such that the detector surface is placed at a small angle (not 90 degrees) relative to the optical path axis. This angle optimizes the detection of transmitted light, ensuring that no light is reflected back into the suspension 120 or guided to other parts of the turbidimeter 100. The intensity of the light collected by the detector 150 is proportional to the turbidity of the suspension.

[0201] A light attenuating filter 160 is positioned directly in front of the transmitted light detector 150. The filter reduces the intensity of light incident on the detector 150 by an amount proportional to the intensity of the incident light beam. In an exemplary embodiment, the filter 160 allows the detector 150 to operate without saturation and provides a sufficient bandwidth of detector operating intensity to detect slight changes in transmitted light intensity.

[0202] The nephelometer 100 also detects the amount of scattered light. The scatter detector 140 is positioned so that its detection surface is parallel to the incident light path and along one side of the cuvette 110. Some of the light passing through the suspended sample 120 is scattered by the particles in the suspension. The side scatter detector 140 collects some 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 test suspension 120. One way to detect the turbidity of the suspension 120 is to process the amount of scattered light collected by the scatter detector 140 through a variety of algorithms known in the art. The data collected from the scatter detector 140 can be combined with the data collected from the transmitted light detector 150 in a variety of ways. For example, the signals can be physically combined, or the detector values can be mathematically combined in a manner that further improves the accuracy and reliability of the initial signal. The signals or data values can be combined additively, subtractively, differentially, etc. to provide a resulting signal indicative of the combined signal. This combination can be performed by the processor 32. When the signals of the detector values are combined in this manner, they are able to improve the resolution and accuracy of the collected data used to detect turbidity. Advantageously, the data collected from two separate detectors (scatter and transmitted light data) can provide more accurate results for small volume samples. Dual detection is advantageous in those embodiments where scatter detection is not sufficient. Detection of both transmitted and scattered light yields can be more accurate because the length of the light path through the small volume sample 120 is limited.

[0203] In an exemplary embodiment, the scatter detector 140 and the transmitted light detector 150 are standard high efficiency photodiode detectors. However, other detectors with similar characteristics can also be used. Suitable detectors include those that operate from the ultraviolet (UV) through the visible spectrum to the infrared (IR). Suitable detectors can be based on their linear response curve, size, reproducibility of results, and ability to operate / detect light paths in low light conditions and detect small changes 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.

[0204] In exemplary embodiments, the light source is a high intensity light emitting diode (LED) or diode laser. Preferably, the LED light has a frequency of about 650 nm. Preferably, the detector light has a wavelength in the red color band (i.e., about 620-750 nm). However, one skilled in the art can use probe light of different visible frequencies. Optionally, a focusing lens 170 Figure 4B is used to focus the light into a narrow beam (i.e., a beam having a diameter of about 3 mm). The focusing lens 170 is positioned in front of the light source 130. The use of the focusing lens 170 concentrates the light from the light source 130 into the sample area 410 of the cuvette and minimizes the amount of light that can be scattered from the test area. It is clear to one skilled in the art that light scattered outside the test area (i.e., the lower portion 410 of the cuvette 110) renders that scattering unusable for the purpose of detecting sample turbidity because of the high background signal. The focused light then enters the lower portion 410 of the cuvette 110 at an angle perpendicular to the surface of the cuvette 110 from the focusing lens 170 (not shown). The perpendicular angle mitigates unwanted diffraction and refraction that occurs when a beam of light passes from one medium (e.g., air) to another medium (e.g., the planar side of the cuvette). The light path of the focused beam is maintained as the light transmits through the suspension toward the detectors 140 and 150. In embodiments where the light source 130 is a diode laser, an additional lens 170 to focus the beam can not be needed. This is due in part to the nature of the laser, which provides a collimated and focused light for probing the suspension. The focusing lens 170 is used in embodiments where the light source 130 is an LED and collimated or focused light is needed or desired.

[0205] Figure 7A and 7B Another nephelometer embodiment 200 is shown, in which the cuvettes are advanced through the nephelometer in series. The system is designed to use a series of cuvettes (as described below) that are advanced through the nephelometer in a serial manner. Each cuvette 110 can be placed directly inside the nephelometer base 201 by placing the lower portion of the cuvette into the channel 220, as shown in Figure 7A Alternatively, each cuvette 110 can be first placed in a linear array of containers 300, and the linear array 300 Figure 8 containing a plurality of containers can be placed inside the nephelometer by passing through the channel 220. After the containers (either individually or in a linear array) are placed inside the nephelometer base, the suspensions are prepared in the cuvettes and the turbidity is detected as described above.

[0206] The nephelometer 200 also includes a light source 230, a focusing lens 270, a scatter detector 240, a transmittance light detector 250, and a light attenuation filter 260, as described above for Figure 4BThe cuvette 110 with sample 120 is positioned in the center of the device and inside the nephelometer base 201. The light source 230, the side scatter detector 240 and the transmission detector 250 are positioned at 90 degree angles relative to each other as shown above around the cuvette 110. The side scatter detector surface 240 is positioned parallel to the incident light beam of the light source 230. The side scatter detector 240 is positioned in close proximity to the test sample 120 and parallel to the incident light source which minimizes the diffraction, refraction and reflection effects of the scattered light. The transmission light detector 250 is positioned opposite the light source 230 and the incident light from the light source is transmitted toward the transmission light detector. The detector 250 can also be positioned perpendicular to the incident light path or at an angle that is off perpendicular by a few degrees to reduce the reflection effects from its surface. A light attenuation filter 260 is positioned between the cuvette 110 and the transmission light detector 250.

[0207] Figure 8 A series of cuvette arrays / vessels for an embodiment of the device of the present application (such as the nephelometer 200) is shown. This embodiment differs from the above described embodiment in that the suspension test tubes are placed in a rotating fashion for turbidity detection. The series of cuvette arrays 300 is a series of cuvette strips that move along a guide channel 220. The LED light source 230 is placed on one side of the guide channel 220 of the guide strip 300. The strip 300 is slidably engaged with the channel 220. The strip 300 can also include transport line holders or other structures 530 for facilitating stacking, packaging and shipping. Figure 9 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. When processing is complete the linear strip 300 can be indexed and advanced to the next cuvette and processing of the next sample continues using the same nephelometer. The cuvette strip 300 can be stored or discarded based on the needs of the individual user. In this embodiment, the individual nephelometer is designed to efficiently process multiple samples without the need to remove and replace individual cuvettes. The linear cuvette strip 300 can be designed in a variety of cuvette shapes, sizes and configurations. For example, the wells 320 of the strip 300 can be designed to be deeper or shallower, wider, narrower, longer, shorter, etc. depending on the design of the cuvette. Additionally, the wells can be connected to each other across the individual wells or individually inserted into wells positioned adjacent to each other. The placement of the plurality of cuvettes 110' with edges 402 within the linear array 300 allows for more efficient cuvette 110' transport through the nephelometer 200 as they can be processed and received by the nephelometer consecutively and do not require additional manipulation for detection.

[0208] In Figure 9Another series of cuvette embodiments described in the middle, the cuvette strips are stackable and can be separated into individual cuvettes or linear cuvette strips, depending on the structure of the nephelometer. In the described embodiments, cuvette 500 is carried by a holder 510. Holder 510 has a planar surface from which the cuvette is suspended. The planar surface has notches (not shown) to allow the cuvette to be separated into individual cuvettes or cuvette strips. The stacked cuvettes can also have a transmission line holder 530 (as described above). Note that to facilitate stacking, the lower portion 540 of cuvette 500 is received by a wider upper portion 550.

[0209] Figure 10 is a perspective view of a nephelometer 590 showing an aperture 575 for a light source 570, an aperture 635 for a scatter light detector, and an aperture 605 for a transmission light detector.

[0210] Figure 11 is a cross-sectional view of a nephelometer 590 showing a transmission light path through the lower portion 540 of cuvette 500. A light source (570, Figure 12 ) is received by an aperture 575 located on one side of cuvette receptacle 580 of nephelometer 590. Aperture 575 receives the light source. A sensor 600 Figure 12 ) is positioned within aperture 605 directly facing aperture 575, with the lower portion of cuvette 540 positioned between the two. The nephelometer has a cover 620.

[0211] Figure 12 is a cross-sectional view of a nephelometer 590 showing a scatter light path through the lower portion 540 of cuvette 500. A light source (570, Figure 12 ) is located on one side of cuvette receptacle 580 of nephelometer 590. A sensor 630 Figure 10 ) is positioned within aperture 635 perpendicular to light source 570, with the lower portion of cuvette 540 positioned between the two.

[0212] Figure 13 is a cross-sectional view of a nephelometer 590 showing a transmission light path through the lower portion 540 of cuvette 500. Light source 570 is received by an aperture 575 located on one side of cuvette receptacle 580 of nephelometer 590. Between sensor 600 and cuvette 500 is a light attenuation filter 640 placed in front of the transmission light detector to reduce the light intensity to a usable level so that the sensor does not saturate. Aperture 575 receives light source 570 and a lens 650 for focusing the light signal. Sensor 600 is positioned within aperture 605 directly facing aperture 575, with the lower portion of cuvette 540 positioned between the two.

[0213] In one embodiment, the samples are disposed within cuvettes and individually processed when placed into the nephelometer. After the samples are processed and the McFarland values are obtained, the cuvettes are removed from the nephelometer and replaced with new cuvettes. In this embodiment, one or more nephelometers can operate independently. In an alternative embodiment, the nephelometer is configured to pass a continuous series of cuvettes through the nephelometer for detection. Linear cuvette channels 220 receive the strips 300 of cuvette wells 320 Figure 4B ). The strips are passed through the nephelometer, stopping so that each cuvette is optically probed for detection, as described in detail elsewhere herein.

[0214] The method of detecting turbidity according to the present application is automated. The data collected from the detection can be further processed to yield meaningful results. In these embodiments, the signal from the detector is sent to a signal amplifier. The amplifier output is in communication with an analog to digital converter circuit, which outputs a digital representation of the signal, which is then processed using a variety of algorithms to determine if the measured value is at the target value. If the measured value is greater than the target value, then the sample is diluted as described above, and the turbidity is re-measured. This re-measurement can be performed manually by an operator or in an automated fashion, where the cuvette is removed from the nephelometer for dilution, and passed back to the nephelometer for additional detection. The method of processing the signal into a usable output is developed using different dilutions of different biological or non-biological samples and correlating the McFarland value to the concentration of the suspension. These data are then used to generate a data set, which is further analyzed by an algorithm that corrects for the linearity and offset of the data curve to yield a representative output value for the turbidity value and compared to the target value. This process is repeated until the target turbidity value is obtained, as described elsewhere herein.

[0215] The system 1000 can also comprise a conveyor, the end positions of which can form the stage 2 of the Petri dish; or a conveyor and a stage 2, which can be positioned relative to each other such that the Petri dish can be transported onto the stage and away from the stage by suitable operation of the conveyor. The conveyor is controlled by the controller 30 to automatically position and remove the Petri dish comprising the microorganism on and from the stage, respectively. Note that in other (not shown) embodiments, different means for automatically positioning and removing the Petri dish on and from the stage, respectively, can also be used. In particular, the controller 30 is configured to allow the Petri dish to be automatically removed from the stage by the Petri dish automatic positioning and removal device only after a signal has been provided that the suspension test tube with the suspension can be removed from the suspension test tube holder for further processing. This ensures that it is always possible to pick up a further sample if required.

[0216] As Figure 2As shown, the apparatus 1000 of the present application can also include a suspension test tube automatic positioning and removal device (not shown) for automatically positioning or removing a suspension test tube from the suspension test tube holder, respectively. The suspension test tube automatic positioning and removal device can include a gripping tool to releasably grasp the suspension test tube 11. Again, the controller 30 can be configured in communication with the suspension test tube automatic positioning and removal device to control the operation of the suspension test tube automatic positioning and removal device and to automatically position the suspension test tube 11 on the suspension test tube holder 10. The controller 30 is particularly configured to automatically remove the suspension test tube from the suspension test tube holder by the suspension test tube automatic positioning and removal device only after a signal is provided that the suspension test tube with the suspension can be removed from the suspension test tube holder for further processing. The suspension test tube automatic positioning and removal device can be moved along the track 18 independent of the movement of the positioning device 8. The suspension test tube 11 can be taken and the suspension test tube with the suspension medium including a sufficient concentration of microorganisms can be handed over to an apparatus for further processing, such as an incubator. It is noted that the multi-track system can guide the suspension test tube automatic positioning and removal device and the positioning device 8 to different locations where different components are present or different processes can be carried out.

[0217] The sample suspension thus prepared is used for the characterization or identification of the microorganism using MALDI and can optionally be used for other analyses, such as AST. For the identification of the microorganism using MALDI, an aliquot of the sample suspension is obtained using a pipetting or picking tool and the aliquot is transferred to the target plate 42. A drop can be taken by using the tool 46 which is held by the gripping tool 49 of the pipettor 40 and is then automatically lowered into the suspension at position A. When the tool 46 is raised out of the suspension, a drop of the suspension will adhere to the tip of the tool 46 which can be transferred along the track to position B where the tool 46 with the drop of the suspension is lowered until the drop of the suspension contacts the deposition spot 44 on the target plate 42. After the tool 46 has been raised away from the target plate 42, at least a portion of the drop of the suspension will remain at the deposition spot 44. Alternatively, the picking tool 46 can be used to pick up an amount of the suspension 14 from the suspension test tube 11, to transfer the amount to position B and to deposit a drop of the suspension on the target plate 42. After the drop of the suspension has been deposited on the target plate 42 and in particular after the drop has dried, a MALDI matrix solution is automatically overlaid on the sample amount or sample portion deposited on the target plate 42. For performing other tests or another analysis, a second drop of the sample suspension can be obtained in a similar manner and the drop can be automatically transferred and deposited, for example, on a test culture dish which is further transferred in an automated manner to carry out a susceptibility test or another analysis.

[0218] In one embodiment, the matrix solution is dispensed to multiple spots on the target plate 42. This increases throughput and reduces consumable costs, e.g., pipettes, etc. In this embodiment, a sufficient volume of matrix solution (i.e., matrix solution for many target spots) is drawn into a pipette, and the pipette is used to dispense multiple spots in sequence. Typically, dispensing low volumes of fluid in the range of 1 to 20 microliters requires that the fluid droplet be touched to the surface to be dispensed upon, such that the surface tension of the fluid touching the target plate will pull the droplet off the pipette tip 46. In the process of "triggering" the droplet onto the target plate surface, the pipette tip 46 can inadvertently touch the surface of the target plate 42. If the pipette tip 46 touches the target plate 42, there is a risk of transporting excess sample material from one target plate deposition spot to the next, causing cross contamination. In an attempt to prevent cross contamination, capacitive liquid detection is integrated into the pipettor 40 to detect when a droplet touches the target plate 42. Capacitive liquid detection is used to dispense multiple times from a single volume of matrix solution in a single pipette according to the following steps.

[0219] First, a new pipette tip is picked up. The dry tip is then moved to the target plate 42 and the plate is brought into contact with the pipette tip at a non-target spot location to determine and record the precise vertical (Z) position between the target plate 42 location and the tip interface.

[0220] Next, a sufficient volume of matrix solution is drawn from the matrix reagent container. The container has a septum that prevents evaporation of the matrix solution. After the matrix solution is drawn and the tip is removed from the matrix container, the septum wipes away any residual matrix liquid that can be covering the tip 46. This ensures that when the matrix solution is dispensed from the tip, a droplet will form at the end of the tip and will not move to the sides of the pipette tip 46.

[0221] The pipette tip 46 is then moved to the target plate 42. A droplet forms at the end of the tip 46. The tip 46 is moved downward in the vertical (Z) direction until the droplet contacts the target plate spot 44. The capacitive sensing circuit generates a signal when the droplet contacts the plate 42, indicating that the droplet is contacting the plate 42. The vertical (Z) position of the tip 46 is checked to confirm that the tip 46 is not contacting the target plate 42. If the tip 46 is not contacting the plate 42, the multiple dispensing process continues. If the tip 46 is contacting the plate 42, the tip 46 is ejected as waste, a new tip is picked up, and the dry tip is moved into position to contact the target plate to establish a new tip-to-plate vertical (Z) position, and the process continues until all of the target spots 44 of the plate 42 have been inoculated with matrix solution.

[0222] Taking samples for ID and AST from the same suspension

[0223] Preparation of suspensions for MALDI and AST testing from a colony pick is described in U.S. Patent No. 9,180,448, which is commonly assigned to the present application and is incorporated herein by reference in its entirety. The disclosure herein can refer to a sample preparation device (hereinafter sample preparation or preparation platform) such as the "Phoenix AP", or an AST system such as the BD Phoenix TM or to a mass spectrometry system such as MALDI, but it should be understood that the meaning of these terms is not limited to devices having these trademark names, but can also include devices having substantially similar functionality. Devices having substantially similar functionality can include the Vitek (bioMerieux) and MicroScan (Siemens Healthcare) ID / AST systems.

[0224] In one embodiment, the device described herein integrates the microorganism identification capabilities of a MALDI instrument and the data processing capabilities of an AST and laboratory analysis or processing system, such as the Phoenix, Phoenix AP, BACTEC, or EpiCenter system.

[0225] As described above, suspensions are prepared from microorganisms obtained from prepared plates 3 or from blood culture bottles. In one embodiment, suspension tubes 11 are over-inoculated with microorganisms 4. Tubes 11 are advantageously used as a source for both ID and AST. This ensures that not only are samples from the same patient, but also the same isolate, subjected to ID and AST testing.

[0226] Suspensions are prepared at a concentration suitable for MALDI. Suspensions suitable for MALDI typically have a McFarland value of about 2. An automated system is used to inoculate suspension tubes 11 by pick tool 6, monitor turbidity, and process the suspension to provide a suspension having a target turbidity. The automated process of providing a suspension having a target turbidity has been described in detail herein. The suspension is then used to inoculate MALDI plates 42 as described above. As noted above, system 1000 correlates culture dishes 3 with suspension tubes 11 and MALDI plates 42 by using machine-readable labels and codes. In one embodiment, the device scans the bar code on MALDI plates 42 and writes the plate ID to the RFID tag on the suspension tube holder. System 1000, using automated pipettor 40, automatically adds MALDI reagents (e.g., formic acid, matrix, etc.) to prepare the suspension dispensed onto MALDI plates 42 for analysis as described herein.

[0227] The system 1000 uses the automated pipettor 40, or dispensing nozzle 30, to then dispense additional solution (e.g., deionized water) into the test tube 11, and the nephelometer 20 monitors the turbidity to provide a suspension having a turbidity suitable for an AST or other diagnostic test (e.g., a molecular test). Automated systems and methods for providing a suspension having a target turbidity are described in detail herein and are not repeated. For an AST, the target turbidity is about 0.5 McFarland and is typically no less than about 0.25 McFarland. The pipettor 40 then transfers an aliquot into an AST test tube 82. The RFID tag on the test tube holder is updated with the adjustment result.

[0228] The AST test tube 82, as shown in Figure 2 , is supported by an AST test tube mover 80. The AST test tube mover 80 is a robot that is typically disposed below the system panel 7 and is configured to move in at least two dimensions, as shown by the vertical and horizontal arrows in Figure 2 . In particular, the AST test tube mover 80 is configured to support (e.g., by a container or gripper) the AST test tube 82 and to move the AST test tube 82 below the panel 7 and between the pre-designated locations at the preparation platform 1030 and the transfer platform 1040, respectively. In this regard, the panel 7 can have openings through which the mover 80 can raise and lower the AST test tube 82 at the pre-designated locations. Of course, it is also contemplated that the suspended test tube gripper robot 50 can move the AST test tube 82 from a suspended position above the panel 7, rather than from below the panel 7, between the preparation platform 1030 and the transfer platform 1040.

[0229] The AST test tube 82 can be stored in the platform 1040. Prior to aliquot transfer into the AST test tube 82, the test tube gripper robot 50 grabs the AST test tube 82 by the gripper tool 59 and moves from the storage location of the AST test tube 82 to the barcode scanner to register the test tube 82 by the controller 30. Thereafter, the gripper robot 50 drops the test tube 82 to the mover robot 80. The robot 80 then carries the test tube 82 below the panel 7 to location C at the preparation platform 1030, where the test tube 82 is raised at least partially above the panel. The pipettor 40 then retrieves an aliquot of the diluted suspension from the test tube 11, moves to location C, and then inoculates the AST test tube 82 at location C with the aliquot. The test tube mover 80 then carries the test tube 82 containing the suspension to the transfer platform 1040, as described in Figure 2 .

[0230] While at the platform 1040, another pipettor 60 takes an aliquot of the suspension from the AST test tube 82'. Prior to taking this aliquot, the cartridge transfer robot 70 grabs the empty AST cartridge 90 by the gripper tool 79 and moves the cartridge 90 from the storage location to the cartridge filling unit 78, which includes a cartridge support structure for supporting the cartridge 90. This unit 78 can be movable so that the cartridge 90 is rotated from a vertical configuration to an inclined configuration, as shown, to facilitate inoculation. A decapper (not shown) can also move away the lid of the sealed cartridge 90 prior to inoculation. The pipettor 60 then automatically inoculates the AST cartridge 90 with the diluted suspension. Both the AST suspension test tube 82 and the AST cartridge 90 are coded, which associates the suspension that will undergo AST analysis with the pick up of the suspension that was prepared. The facility has a data management system that associates the cartridge with the suspension used to inoculate the cartridge 90. The system 1000 reads the MALDI plate ID and the plate position of each suspension, and forms the necessary associations of the colonies picked from the identified petri dishes 3 with the suspensions prepared therefrom and the MALDI plate 42 and the position on the plate 42 where the relevant suspension was inoculated and the AST suspension test tube 82 and the AST cartridge 90 inoculated with the AST suspension. Automation actions are provided to inoculate the AST cartridge and to transfer the inoculated cartridge to the test instrument that performs AST on the inoculated cartridge. One exemplary cartridge transfer instrument for automatically moving the inoculated cartridge 90 into the AST test instrument and to move the tested cartridge out of the AST test instrument will be described below.

[0231] Preparation of MALDI plates using layering technique

[0232] In one embodiment of the present application, the suspensions are automatically deposited on the MALDI plate 42 using a dispensing / layering method. This method is described in U.S. Provisional Application No. 62 / 038,509, entitled "Method Of Sample Preparation For Maldi", filed on April 18, 2014, which is commonly assigned to the present application, which was filed as PCT / US21015 / 45506, published as WO2016028684. U.S. Provisional Application No. 62 / 038,509 and PCT / US21015 / 45506 are incorporated by reference in their entirety.

[0233] 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.

[0234] Bacterial suspensions are prepared as described elsewhere herein. The solution dispensing / layering method entails, as its name implies, forming two or more layers of solution for MALDI identification. A selected volume of sample is dispensed onto a MALDI plate 42 and dried. Next, at least a second aliquot of the suspension is dispensed (preferably of the same volume) onto the dried suspension. Dispensing is accomplished using the automated method described above. The second dispensed aliquot is dried. Optionally, more layers of the suspension can be deposited and dried. After the final layer of two or more layers of suspension 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 dispensing / layering method has been determined to provide acceptable MALDI results for liquid samples having a McFarland turbidity value significantly less than 2.0, for both gram positive and gram negative bacteria.

[0235] For example, in one embodiment, if a liquid bacterial suspension (prepared from a bacterial colony picked from an agar plate and suspended in water (mass spectrometry grade), as described above with reference) has a 0.5 McFarland value, which is significantly less than a 2.0 McFarland value, it is determined that the solution dispensing / layering sample preparation can be used to prepare the sample for MALDI.

[0236] After it is determined that the solution dispensing / layering preparation is to be used for MALDI, an amount of suspension is selected for each layer. In the example above of a sample having a 0.5 McFarland value, the volume of each layer selected is at least about 3 microliters and no more than about 4 microliters. The number of layers is controlled by the turbidity value and the sample volume. Once the volume of the layers is selected and deposited onto the MALDI plate, the sample is dried. The exact drying conditions are a matter of design choice and are selected to provide rapid drying while maintaining sample integrity for MALDI testing. Suitable drying conditions are readily determined by one skilled in the art. For example, the drying step can be accomplished at ambient temperature or with the aid of a hot plate (exemplary of about 40 degrees Celsius to about 45 degrees Celsius). After drying, a second layer of suspension is deposited on the first layer. The second layer has the same volume as the first layer. If desired, additional layers are added and dried. Because the layering method requires additional time and resources, the number of layers is limited to the number of layers needed to obtain accurate results from MALDI.

[0237] After the solution dispensing / layering sample is deposited, the sample target well is processed using typical MALDI procedures (addition of 70% formic acid and matrix).

[0238] It has been determined that the preparation process of the MALDI sample depends on a number of factors, but most importantly: i) the concentration of microorganisms in the suspension; ii) the volume of the suspension; and iii) the number of dispenses, if applicable. The concentration of microorganisms is reflected by the turbidity of the sample. Generally, the higher the turbidity, the higher the concentration of microorganisms.

[0239] As described elsewhere herein, the turbidity is detected by a nephelometer. Once the turbidity of the suspension is assessed as described above, a decision is made as to how to proceed with the sample preparation for MALDI. Such a decision is made by evaluating the turbidity information and the sample volume. In these embodiments, the sample information is input into a data set. This data set, preprogrammed with information regarding the sample preparation best suited for a particular sample, outputs a recommended MALDI sample preparation method.

[0240] In an automated system, the processor controls the MALDI preparation protocol, depending on the information the processor receives 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 the predetermined range of turbidity values, then the processor provides instructions to transfer a predetermined volume of the diluted sample to the MALDI plate 42. The automated system prepares the sample for MALDI based on the processor's instructions (i.e. formic acid is added prior to MALDI to fix the sample, then a MALDI matrix solution is applied over the sample, as described elsewhere herein). If the processor determines that the turbidity is outside the predetermined range, the processor provides instructions to prepare the MALDI sample using less than the normal volume (i.e. if 0.5 microliters is normally deposited on the MALDI plate 42, then for a high turbidity sample, only 0.25 microliters is deposited on the MALDI plate 42). If the processor determines that the turbidity is less than the predetermined range, the sample is deposited in layers on the MALDI plate 42, with sample drying between multiple depositions. As noted above, because the methods and apparatus described herein are fully automated embodiments, the system dispenses the suspension onto the MALDI plate 42 using the automated pipette described herein based on the processor's instructions.

[0241] Figure 14 A flow chart showing the automated process of multiple dispenses of the suspension onto the MALDI plate 42 is shown. The suspension is automatically prepared and its turbidity is evaluated as described elsewhere herein. If the measured turbidity is within the predetermined range, a predetermined volume aliquot is deposited onto the MALDI plate. If the measured turbidity is above the predetermined range, then a smaller volume of the sample is deposited on the MALDI plate 42. If the measured turbidity is less than the predetermined range, then the sample preparation scheme of multiple dispenses with drying between dispenses described above is employed.

[0242] 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 microliters is deposited on the MALDI plate 42. If the sample turbidity is higher than about 6, the amount of sample deposited on the MALDI plate 42 is reduced to about 1 microliter. If the sample turbidity is less than about 2, but in the range of about 1 to about 2, about 3 microliters of sample is deposited on the MALDI plate 42, dried, and a second 3 microliters of sample is deposited and dried. If the sample turbidity is about 0.5 to about 1, three "layers" of suspension, each of about 3 microliters, are deposited and dried. If the sample turbidity is about 0.25 to about 0.5, four "layers" of suspension (3 microliters each) are deposited and dried.

[0243] After the sample is deposited and dried, the sample is processed for MALDI, as described elsewhere herein.

[0244] Each suspension test tube includes a unique identification mark, which is saved in the memory of the central control computer, in association with the nature of the suspension (related to the identification of the Petri dish from which the selected microbial colony was obtained), for the purpose of accurately and in a rapid manner linking to the analysis results of the Petri dish and colony from which the results are derived, etc.

[0245] In another embodiment, the system has a predetermined range of turbidity, where dilution is not required for MALDI or AST. If the turbidity is within this predetermined range (e.g., approximately 0.5 to approximately 2 McFarland values), the suspension can be used to inoculate MALDI plate 42 using the stratification method described above (if the concentration of the suspension is not high enough, a single dispensing will be performed). In this embodiment, the volume of suspension inoculated into the suspension tube also depends on the measured change in turbidity. For example, if the McFarland value of the suspension is 0.5, then 25 μL is inoculated into the suspension tube. For the same specifications, if the turbidimeter detects 1 McFarland suspension, then only 12.5 μL of that suspension is used. The two dispensings deliver approximately the same amount of microorganisms into the suspension tube 11, but the volume of 0.5 McFarland suspension is twice the volume of 1 McFarland suspension. Therefore, 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, the amount of microorganisms inoculated into test tube 11, rather than the volume, determines whether the amount dispensed is sufficient. If the turbidimeter 20 determines that the suspension is within a predetermined range, the information is communicated to the controller 30, which then determines whether dispensing onto the MALDI plate 42 must be done via a stratification method, or whether a single dispensing is sufficient. The controller 30 will also refer to a lookup table to determine the volume of suspension to be dispensed into the AST test tube 82, which specifies the dispensing amount as a function of the measured turbidity.

[0246] Inoculation of the AST cartridge is described in U.S. Patent No. 6,096,272 to Clark et al., which is incorporated herein by reference. In practice, the suspension is inoculated into the AST inoculum liquid, which is then transferred into the test cartridge 90 via an automated mechanical transfer for fluid transfer as described above. The AST cartridge 90 is tilted, with the inoculation port at the top for filling (see [link to documentation]). Figure 25 Each well within the AST box 90 is inoculated with an AST inoculum. The inoculum flows down the AST box in a meandering manner, filling the well as the liquid front advances towards the absorbent pad. Each well is open, allowing liquid to fill the well. Each well has a sharp, rounded edge to separate a constant amount of liquid from excess liquid and to isolate each well from the liquid in adjacent wells 31. The pad absorbs excess liquid.

[0247] like Figure 2As shown, an amount of the suspension is taken from the suspension in the suspension test tube by a pipetting tool 46, which can be automatically gripped and positioned by a gripping tool (functionally a pipetting tool holder) 49. The pipettor 40 is configured to position the pipetting tool 46 to a start position above the suspension test tube 11 and to automatically lower and raise the pipetting tool 46 into and out of the suspension and to position the pipetting tool 46 above a transfer position B of the MALDI plate 42, respectively. When the pipetting tool 46 is lowered into the suspension in the suspension test tube 11, the pipetting tool 46 is operated in a manner known per se (e.g. using negative pressure) to pick up an amount of the suspension. The pipetting tool with the amount of the suspension is then raised to the transfer position. For holding the amount, the pipetting tool comprises a pressurized chamber which is closed by a control valve. The pipetting tool 46 is automatically transferred to a position B above one of the deposition points 44 of the target plate 42. In this position, the pipetting tool 46 is lowered to a predetermined 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 the suspension with a volume in the range of about 0.5 to 3.0 microliters is deposited on the deposition point 44, in particular covering at most about half of one of the deposition points on the target plate 42. After the droplet is deposited, the pipetting tool 46 is raised from the target plate 42 and can be transferred to a position where it can be discarded or cleaned for reuse.

[0248] Figure 19 A flow chart is shown comparing the timeline of the automated process of Figure 18 to the timeline of a comparable manual implementation process. The manual process is shown to take up to 48 hours and requires an incubation time of 18-24 hours only after which the plate is evaluated for growth. By comparison, because the automated process can detect even relatively small contrasts between colonies (compared to background and to each other), incubation requires only 12-18 hours before samples can be identified and prepared for further testing (e.g. AST, MALDI).

[0249] Further aspects of the previously described embodiments are described below. The aforementioned user interface provides the user with an image of the culture plate. The user can interact with the interface to pick colonies of interest from the plate.

[0250] When the user selects a colony, the device provides the user with a menu selection to select one or both of MALDI or AST for sample processing. Based on the size of the picking tool (i.e., pipette as described elsewhere herein), the device provides a pick tolerance to ensure that the colony is picked within the designated area. The pick is then locked in place and the colony is picked. In one embodiment, the pick tolerance is 5 mm in diameter. Using a pick tool with a 3 mm diameter, the distance pick ensures that a 1 mm diameter pick area within the pick tolerance will be picked. The processing selection is sent to the controller to enable tracking of the processing of the sample.

[0251] Figures 1-3 A system overview diagram showing the identification (MALDI-TOF) and AST (antibiotic susceptibility) preparation locations and user interface touch screen 1006. Figure 2 A petri dish 3 is shown with a colony 4 designated for picking and moved to the picking platform 1020 of the system. The petri dish 3 is arranged for colony picking in the manner described previously herein. The petri dish 3 is then scanned for tracking.

[0252] The lid of the petri dish 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 location to the inoculation location A where the colony is placed into the suspension test tube 11 as described above. The test tube or cuvette is as described previously herein. The pipette 6 brings the sample into the test tube 11 where the suspension liquid is already present. The relative amount of suspension is controlled so that the suspension meets a predetermined McFarland standard as described previously herein.

[0253] As the samples, suspensions, etc. are trackable throughout the processing of the methods and devices described herein, these are trackable by bar code as consumables of the described devices and methods. The devices will provide full automated inventory control of the consumables within the device.

[0254] As the suspension is deposited on the MALDI target plate 42, it can be spotted in layers as described elsewhere herein. The drying of the sample and extraction of the formic acid are also as described previously. The deposition of the MALDI spots on the target plate 42 followed by the deposition of the matrix solution is automated as described previously.

[0255] After aliquots for MALDI are obtained from the suspension, the suspension is further used to inoculate tube 82 for the AST assay. In one embodiment, a larger pipette 46 is used for AST tube inoculation compared to 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 AST suspension. The target turbidity value for AST in one embodiment is 0.5 McFarlane (McF). The AST tubes containing the AST broth are removed from the support. In one embodiment, the broth tubes also include Alamar Blue (Alamar Biosciences, Sacramento, Calif.). Alamar Blue is a redox colorimetric indicator that changes color from deep blue to bright pink in the presence of metabolically active, growing organic matter. The use of Alamar Blue in sensitivity analysis is well known to those skilled in the art and is not described in detail herein. As an example of the traceability of consumables, reagents, and samples throughout the system, the AST tubes 82 are scanned. The cap is removed from the AST broth tube using a cap opener, and the cap is then disposed of. The AST broth tube 82 is then transferred to the inoculation site on the AST plate 90. The inoculation site is as follows: Figure 2 As shown. Figure 2 An automated pipette 60 is shown, which inoculates an AST plate 90 with AST broth from the bottle on the left. A dye is provided to change the color of the broth. Figure 2 Pipette 46 is shown, which is used to inoculate AST test tubes with 0.5 McF suspension. Figure 2 The image shows pipette 46 mixing the suspension in AST tube 82 by repeatedly aspirating and dispensing the solution.

[0256] Figure 2 The automation of providing the AST plate 90 (another consumable) for inoculation is shown. Figure 2 The inoculation of AST plate 90 was also shown. The recapping of AST plate 90 is also automated. Figure 2 This demonstrates how the device provides a seamless process and workflow for sample preparation of both ID and AST. System 1000 can be modularly configured to perform identification (MALDI-TOF), antibiotic susceptibility testing (AST), or both. Consider integrating the device described herein with larger systems, such as TotalLab Automation's Work Cell Automation.

[0257] Box transfer

[0258] Thus, System 1000 can be used in conjunction with other laboratory systems / instruments to facilitate fully automated sample preparation and testing. For example... Figure 20As shown, system 1000 can be used in conjunction with automated cassette transfer instrument 2000 and one or more cassette testing instruments 2050a-d. As described above, system 1000 can automatically prepare AST cassettes 90 for testing. In the described embodiment, cassette transfer instrument 2000 is specifically configured to transport the thus prepared AST cassettes 90 from system 1000 to one of the plurality of AST cassette testing instruments 2050a-d.

[0259] AST box 90, such as Figure 21 , 25 As shown in Figure 26, any kit that can be used to test analytes / inoculants, such as BD Phoenix, can be used. TM ID / AST plates (Becton, Dickinson, and Co., Franklin Lakes, NJ). Regardless of the cartridge used, such a cartridge 90 typically includes an inlet 95 for inoculating the interior space of the cartridge 90 with an analyte, such as a microbial suspension or blood culture. This inlet 95 can be sealed with a removable cap or diaphragm 99. For example, system 1000 may include a capping / uncapping machine (not shown) located within a transfer platform 1040, which can open or recap the removable cap 99.

[0260] like Figure 21 As shown, controller 30 and system 1000 (as referenced above) Figure 3 The system 30 connects to the box transfer instrument 2000 and the box testing instrument 2050. The controller 30 coordinates and controls each of these systems / instruments 1000, 2000, 2050a-d to perform box preparation, box transfer, and sample testing. In this respect, the controller 30 is configured to perform specific tasks depending on the type of loading and unloading of the box testing instrument 2050. For example, the controller 30 may be configured to allow manual and / or automatic distribution / transfer of boxes 90 from the preparation system 1000 to the box testing instrument 2050, manual and / or automatic loading of the testing instrument 2050, and manual and / or automatic removal of boxes 90 from the testing instrument 2050, which may then be manually or automatically transferred to the storage bin 2006 or waste bin 2004. The controller 30 may take the form of the depicted desktop computer combined with a touch-screen tablet, for example... Figure 1 The plate 1006 shown, or some other forms known in the art, may be used. Alternatively, multiple controllers may be used. For example, controller 30 may be connected to instrument 1000 and cartridge transfer instrument 2000, while another controller (not shown) may be connected separately to test instrument 2050. Such controllers can communicate with each other to coordinate cartridge transfer.

[0261] Box transfer instrument

[0262] The cartridge transfer instrument 2000, best shown in Figure 20 FIG. 1, can be a multi-axis robot that includes a z-axis arm 2010, an x-axis arm 2012, a rotational component 2014, a cartridge gripper assembly 2015, and a vacuum pump 2002. The cartridge gripper assembly 2015 is connected to the rotational component 2014, which can rotate the cartridge gripper assembly 2015 so that it faces the system 1000 in one orientation and the test instruments 2050a-d in another orientation. In this regard, the rotational component 2014 can rotate the cartridge gripper assembly at least 180 degrees about the z-axis. The rotational component 2014 and the cartridge gripper assembly 2015 can be connected to the x-axis arm 2012, which is itself connected to the z-axis arm 2010. The z-axis arm 2010 can move the cartridge gripper assembly 2015 in a vertical direction so that any of the test instruments 2050a-d, which are depicted as stacked in a vertical arrangement, are accessed. 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-d.

[0263] Figures 22-24 The cartridge gripper assembly 2015 is described as generally including a movable arm 2030, a support arm 2038, and a gripper plate / component 2020. The movable arm 2030 is suspended from the support arm 2038 and is movable relative to the support arm 2038 along its axis, for example, by a bracket and pinion gear arrangement. The movable arm 2030 includes a curved top surface 2034, best shown in Figure 23 FIG. 2. The gripper plate 2020 is pivotally coupled to the movable arm 2030 proximate the curved top surface 2034 and pivots about the axis of the coupling 2036 that connects the two components. The curved top surface 2034 helps guide and support the gripper plate 2020 as it pivots between the first and second positions.

[0264] The pivoting function of the gripper plate 2020 about the pivot axis is further accomplished by the use of a torsion spring 2035, which is described in Figure 23 FIG. 3. The torsion spring 2035 is coiled around the coupling 2036 that couples the gripper plate 2020 and the movable arm 2030. In this manner, as force is applied to the gripper plate 2020 and the gripper plate 2020 is pivoted from the first position to the second position, the tension of the torsion spring 2035 increases. As the tension increases, the potential energy in the torsion spring 2035 increases to bias toward the first position. In this regard, the potential energy of the torsion spring 2035 in this illustrated configuration is lowest when the gripper plate 2020 is tilted back into the first position or rest position so that its cartridge contact surface 2026 is tilted relative to the vertical axis, best shown in Figure 23The 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 clamp plate 2020 when in the rest position can be greater or less than 30 degrees.

[0265] A force is applied to the bottom end of the plate 2020, which can cause the clamp plate 2020 to move from the rest position toward a second position or transfer position (not shown). When in the second position, the spring 2035 is taut such that when the force is released from the bottom of the plate 2020, the plate returns to the rest position. In the second position, the cartridge contact surface 2026 of the clamp plate is at a different angle than when it is in the first position. For example, the clamp 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 nearly any angle within the range of pivoting from the first position, and generally is any angle that brings the cartridge 2020 into flush contact with the opposing surface 2072. The opposing surface 2072, as Figure 22 shown, is a surface of the cartridge support structure 2070 that receives the cartridge 2070 from or hands off the cartridge 90 to the clamp assembly 2020. This cartridge support structure 2070 can be located in the testing instrument 2050 or also in the system 1000. Alternatively, the cartridge support structure can be a tray, such as the tray 2040, as Figure 26 shown. Thus, as described, the clamp plate 2020 can pivot between the rest position and the transfer position. The ability of the cartridge clamp plate 2020 to pivot enables the clamp plate 2020 to grip, retrieve, reposition, and release a target, such as the AST cartridge 90.

[0266] The cartridge contact surface 2026 is adapted to grip the cartridge 90 when it contacts it. In one example, the gripping is achieved by a negative air pressure applied on the cartridge contact surface 2026 of the clamp plate 2020. To obtain the negative air pressure, a suction cup 2028 is embedded on the cartridge contact surface 2026 of the plate (see Figure 24 ) and is connected to a pneumatic conduit 2037 that is fed through an opening in the plate 2026, which provides a vacuum from the vacuum pump 2002 (see Figure 20 ).

[0267] In a method of use, when the clamp plate 2020 is in the first position, the clamp plate 2020 is advanced by the moving arm 2030 toward the first cartridge support structure 2070, which can be located within the system 1000 (see Figure 22). At this point, the bottom edge of the clamp plate 2020 reaches the cartridge 90 and first contacts the cartridge 90 before the rest of the clamp plate 2020. The clamp plate 2020 advancing in this manner toward the cartridge 90 helps the clamp plate 2020 matingly engage the cartridge 90 and dislodge the cartridge 90 from the cartridge support 2070. As the clamp plate 2020 contacts the cartridge 90, the moving arm 2030 continues to advance, which exerts a force on the torsion spring and causes the clamp plate 2020 to pivot from the first position to the second position. The second position is reached when the cartridge contact surface 2026 of the clamp plate 2020 is nearly flush with the fixed opposing face 2072 holding the cartridge 90. In the second position, the cartridge contact surface 2026 is also generally flush with the cartridge surface 92 (see Figure 25 ) so that the vacuum pressure pushes the cartridge 90 against the clamp plate 2020 and holds the clamp plate 2020 thereon.

[0268] After that, when the cartridge 90 is secured to the clamp plate 2020, the clamp assembly 2015 is moved away from the cartridge support structure 2070, which removes the force that supported the plate 2020 in the second position, which causes the clamp plate 2020 and cartridge 90 to return to the first position under the bias of the spring 2036. This helps dislodge the cartridge 90 from the cartridge support structure 2070. In addition, the moving arm 2030 moves along the support arm 2038 in a direction away from the cartridge support structure 2070 so that the bumper surface 2039 (see Figure 24 ) on the support arm 2038 pushes against the upper surface of the cartridge 90 held by the clamp plate 2020. This causes the clamp plate 2020 to pivot back to the second position so that the cartridge 90 is oriented in a substantially vertical orientation. This provides clearance from the cartridge support structure 2070 for the clamp assembly 2015 to be rotated by the rotating member 2014 so that the cartridge 90 can be transferred to another cartridge support structure 2070 that receives it.

[0269] In this regard, the rotating component 2014 rotates the clamp assembly toward the second cassette support structure 2070, which can be located within the testing instrument 2050. Rotating in this manner causes the clamp plate 2020 to appear at the second cassette support structure 2070. When aligned with the second cassette support structure 2070, the moving arm 2030 advances toward the support structure 2070, which separates the shock absorber surface 2039 from the cassette, thereby releasing the force that holds the cassette 90 and clamp plate 2020 in the second position. This causes the cassette 90 and clamp plate 2020 to move to the first position as it advances toward the second support structure 2070. In this regard, the bottom end of the cassette 90 is first received by the second cassette support structure 2070. As the moving arm 2030 advances further, the resistance applied by the second cassette support structure 2070 helps to pivot the cassette 90 toward the second position so that it is approximately flush with the receiving surface 2072 of the support structure 2070. At this point, the cassette 90 is received by the support structure 2070 and the vacuum is turned off so that the clamp plate 90 moves away and back to the first position.

[0270] Alternative features of the cassette clamp assembly 2015 are contemplated. For example, in another embodiment, the pivoting function of the clamp plate 2020 is provided by using a compression spring (not shown). The compression spring is located between the clamp plate 2020 and the bottom surface 2032 of the moving arm 2030. In the first position, the compression spring has a relatively low potential energy. As the clamp plate 2020 pivots from the first position to the second position, the spring is compressed and the potential energy of the spring increases. In a variation, the compression spring is preloaded with enough compression force to ensure that the clamp plate 2020 does not tend to tilt in either direction before contacting the cassette support structure 2070. The compression within the spring is used to hold the clamp plate 2020 in place as the arm 2030 moves the plate 2020 from one position to the other.

[0271] In another embodiment, the pivoting function of the clamp plate 2020 is accomplished by using a tension spring (not shown). Like the compression spring described above, the tension spring is placed between the clamp plate 2020 and the arm 2030, however, in this case, the spring is placed above the top surface 2034 of the arm 2030. This ensures that as the clamp plate 2020 moves from the first position to the second position, the tension within the spring increases, storing more energy toward the latter. In a variation similar to the compression spring described above, the tension spring can be preloaded with tension in the first position.

[0272] In another embodiment, a resilient member (not shown) is used to provide the pivoting function. The resilient member is a structure that deforms from an equilibrium state when the clamp plate 2020 is pivoted from the first position to the second position, and then returns to its original dimensions when the clamp plate returns to the first position. The resilient member is preferably made of a material that has a sufficiently low Young's modulus to allow elastic deformation in response to the resistance generated by contact with the stationary instrument when the clamp plate contacts and is closer to the cartridge support structure 2070.

[0273] In another embodiment, a wave spring (not shown) provides the pivoting function. The wave spring is placed and operates with respect to the assembly 2015 similarly to a compression spring, and can additionally be positioned and connected in ways known to those skilled in the art.

[0274] In other embodiments, passive means different from those described above can be used to provide the pivoting function. Passive means of control are known to those skilled in the art and are not described in detail herein.

[0275] In further embodiments, active means can be used to provide the pivoting function. Examples of active control include linear actuators, such as electronic or pneumatic actuators, pistons, such as electric or pneumatic pistons, rotating ball screws and nuts, and rack and pinions. Any active means of control known to those skilled in the art can also be considered.

[0276] In any of the above embodiments, the clamp plate 2020 can be sized to accommodate the dimensions of a particular cartridge. In this way, the clamp plate is not limited to a particular width or length. Additionally, the thickness of the clamp plate is largely a matter of design choice, provided that it is sufficient to support the desired target load based on the material used.

[0277] Also, in any of the above embodiments, the cartridge contact surface 2026 of the clamp plate 2020 can be adapted to be flush with different cartridge types, shapes, and sizes. For example, the contact surface 2026 can feature a concave or convex shape across the length of the clamp plate 2020.

[0278] In any of the above embodiments, the clamp plate 2020 can be adapted to include a plurality of surface features for grasping a particular target. For example, Figure 25 The illustrated cartridge 90 includes a plurality of features that can be used for grasping by the clamp plate 2020. These include a slit 93 near the center region of the cartridge running in a generally longitudinal direction, a concave region 94 within the center portion of the top surface of the cartridge 90, and a protuberance 96 at the uppermost and lowermost ends of the cartridge 90.

[0279] To accommodate the features of these cassettes, the contact surface 2026 can include a convex shape and positioning to accommodate the associated features of the cassette 90. The clamp plate 2020 can also be configured such that it expands between the protuberances 26 to exert opposing forces on the longitudinal axis of the cassette 90. Alternatively, the clamp plate 2020 can be adapted to grip the protuberances 96. Another clamp plate configuration can also be considered, such as one with opposing fingers, that can be adapted to grip the sides 98 of the cassette 90.

[0280] In any of the above embodiments, the surface 2026 of the clamp plate 2020 can include a structure that maintains the alignment of the target that has been grasped and retrieved. In one example, the improved alignment of the retrieved cassette 90 relative to the clamp plate 2020 is provided by rails 2027 that run parallel to the sides of the plate 2020 and can extend from the top to the bottom of the plate, as shown in Figure 24

[0281] Automated loading of sample cassettes for AST

[0282] Figures 27A-27D An exemplary cassette testing instrument 2050 is described. In particular, the cassette testing instrument 2050 described is an AST instrument. However, it should be understood that the principles described herein can be applied to any laboratory instrument where automated input and movement of sample cassettes is desired.

[0283] The cassette testing instrument 2050 generally includes a housing 2052 defining a chamber therein and a first or manual door 2060 and a second or automated door 2066 for accessing the chamber. The housing 2052 can include a cassette holder 2054 disposed within the chamber that includes a plurality of containers or cassette support structures 2073 for receiving individual cassettes 90. The cassette holder 2054 and containers can be moved within the chamber by activating a container drive 2078 (e.g., a motor and a conveyor belt) such that each container is accessible from the door opening for receiving or removing a cassette 90. In one example, the cassette holder 2054 can be a drum with a plurality of containers 2073 that rotates about a shaft.

[0284] As shown in Figure 27A the first door 2060 is generally located on a first side (in this embodiment, the front) of the instrument 2050 and is manually operable. The first door 2060 is hinged to the housing 2052 and includes a mechanical or magnetic door latch 2064 or locking pin that can be automatically locked by a lock mechanism 2074 during operation of the testing instrument to prevent the first door 2060 from opening. In alternative embodiments, rather than being hingedly connected to the housing 2052, the first door 2060 can be slidably connected to a rail that allows the door to slide open or closed.

[0285] As shown in Figures 27B-27D ​As shown, the second door 2066 is normally located on the second side of the instrument 2050 (in this embodiment, considered the back side) and can be automatically operated. The second door 2066 is slidably disposed within a rail 2056, allowing the door 2066 to slide from one side to the other, and is coupled to a linear or door drive 2076, such as a lead screw, rack and pinion, pneumatic cylinder / piston, motorized linear drive, or some other mechanical or electromechanical device. The door drive 2076 opens and closes the door 2064. The rail 2056 at least partially defines the extent to which the second door opens. The second door 2066 can expose one or more of the cartridge holders 2054. Two independently operated rear automated doors are contemplated to allow the flexibility of exposing only the top cartridge holders or the bottom cartridge holders. This can require the use of additional rails 2056 and door drives 2076.

[0286] The testing instrument 2050 can include additional doors, such as third and fourth doors, which can be disposed on the sides of the instrument 2050 and can be manually or automatically operated. Additionally, the second door 2066 can be alternatively disposed on the side of the instrument 2050 adjacent to the front side on which the first door 2060 is located. In another embodiment, the automated door 2066 can be integrated into the manual door 2060, such that during manual operation, the automated door 2066 moves with the manual door 2060, and during automated operation, only the automated door 2066 opens and closes while the manual door 2060 remains closed. Of course, it is also contemplated that the testing instrument 2050 can have only one door, which can be automatically operated.

[0287] As mentioned above, the testing instrument 2050 can be incorporated into a broader system as a component subsystem, the broader system including the transfer instrument 2000, and the preparation system 1000 and being controlled by the controller 30. As such, the testing instrument 2050 can include components 2070 that are communicatively connected to and / or controlled by the controller 30. The components are shown in Figure 28A and generally include, but are not limited to, a user input interface 2071, a display interface 2072, and locking mechanisms 2074, door drives 2076, and container drives 2078.

[0288] As mentioned above, the testing instrument 2050 can be incorporated into a broader system as a component subsystem, the broader system including the transfer instrument 2000, and the preparation system 1000 and being controlled by the controller 30. As such, the testing instrument 2050 can include components 2070 that are communicatively connected to and / or controlled by the controller 30. The components are shown in Figure 27AAs shown, the housing 2052 can also include a user input interface 2071 and a display interface 2072. The user interface 2071 can be one or more 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 testing instrument 2050 is in an automatic mode, the controller 30 operates the door drive 2076 of the second door 2066 while the controller 30 operates the locking mechanism 2074 to keep the first door 2060 locked. The input interface 2071 can be configured so that the user can override the automatic mode so that when the testing cycle is complete, the controller 30 deactivates the door drive 2076 and operates the locking mechanism 2074 to allow the user to open the first door 2060. Additionally, the user input 2071 can be configured to allow the user to further specify whether the cartridge 90 is to be loaded or unloaded. The controller 30 can then determine whether the appropriate cartridge 90 or receptacle is properly present at the manual door opening.

[0289] The display interface 2072 can be a screen or LED light. When the user requests a manual mode through the user input interface 2071, the display interface 2072 can appear a warning that a test is still in progress within the instrument 2050 and that the first door 2060 cannot be opened until the test is complete. The display interface 2072 can also display information or indicate when the first door 2060 is not locked to begin a manual loading or unloading. The display interface 2072 can also display the current mode of the instrument 2050, whether it is manual or automatic.

[0290] Additionally, the cartridge transfer instrument 2000 can include components 2041 that are communicatively connected to and / or operated by the controller 30. The components are shown in Figure 28B and generally include, but are not limited to, a cartridge gripper 2020, cartridge displacement drive(s) 2030, and cartridge position sensor(s) 2048.

[0291] As mentioned above, the cartridge gripper plate 2020 can include a vacuum port / suction cup 2028 that provides suction to secure and release the cartridge 90. The opening / closing operation of the vacuum pump 2002 that provides the suction can be controlled by the controller 30.

[0292] The cartridge displacement drive 2030 controls the movement of the transfer instrument 2000. Thus, in the case where the transfer instrument 2000 is a robot as described above, the displacement drive 2030 provides the robotic degrees of freedom movement to move the gripper plate 2020 and any cartridge 90 attached thereto. The controller 30 controls the drive 2030 to direct the movement of the cartridge and also to deactivate the drive 2030 when the manual mode of the testing instrument 2050 is activated.

[0293] To determine the position and orientation of the cartridge 90 attached to the gripper plate 2020, the transfer instrument 2000 can include a cartridge position sensor 2048 in communication with the controller 30 on a feedback circuit to assist in guiding the transfer of the cartridge.

[0294] As mentioned above, the controller 30 can be a desktop computer or some other computing device and can include a display interface 2072 and a user interface 2071 such as a keyboard and mouse. Also, as shown, the computing architecture of the controller typically includes a processor 32 and a memory 34. As shown, Figure 28C the controller can also include a subsystem interface 36. Figure 28C

[0295] The subsystem interface 36, which can 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, instructions and data are communicated between the controller 30 and the components 2070 via the subsystem interface 36.

[0296] The memory / data store 34 can include RAM, ROM, flash memory, etc. The memory 34 includes processor control instructions 37 and stored data 38. The processor control instructions 37 include instructions related to the operation of, for example, the locking mechanism 2074, the door drive 2076, the container drive 2078, and the input interface 2071. The stored data 38 can include cartridge identification (e.g., bar code information or serial number), corresponding container identification, and timing information, test start time, and test length.

[0297] In one embodiment of the method including automated cartridge transfer, the cartridge 90 is automatically loaded and automatically unloaded from the testing instrument 2050. In this embodiment, the preparation system 1000 prepares the AST cartridge 90 by inoculating the cartridge 90 with the sample as described in detail above. More particularly, the cartridge 90 is automatically inoculated by one or more robots that can remove the access cap 99 (e.g., cap) of the cartridge 90. The pipettor 60 dispenses the analyte into the cartridge 90. Alternatively, the access can be covered by a septum, in which case the robot can use a needle to pierce the septum to inoculate the interior of the cartridge 90. Thereafter, the cartridge 90 can be placed in a pick-up position, for example, in the transfer platform 1040, and the preparation system 1000 notifies the controller 30 that the cartridge 90 is ready for testing and the time is ready to be completed.

[0298] ​Afterwards, the controller 30 operates the cartridge transfer instrument 2000, which picks up the inoculated cartridge 90 and transfers it by the gripper assembly 2015 and displacement drive 2030 to a predetermined receptacle for such cartridges within the testing instrument 2050. The controller 30 also activates the door drive 2076 and receptacle drive 2078, which opens the second door 2066 of the testing instrument 2050 and moves the receptacle into alignment with the second door opening using feedback from the cartridge position sensor 2048.

[0299] The transfer instrument 2000 then places the cartridge 90 into the receptacle and communicates the specific cartridge / receptacle position (which is related to other definitions of the cartridge) to the controller 30. The transfer instrument 2000 then retrieves another cartridge 90 as instructed by the controller 30. Typically, the receptacles will be filled with cartridges 90 before testing. When the cartridge holder 2054 is filled with cartridges 90 as instructed by the controller 30, this is sensed by the transfer instrument 2000 or the testing instrument 2050 and communicated to the controller 30. The controller 30 then operates the receptacle drive 2078, which provides more empty receptacles to the second door opening. Once all of the receptacles are filled with cartridges as instructed by the controller 30, the controller 30 activates the door drive 2076, closes the second door 2066 and instructs the testing instrument 2050 to begin testing, in this embodiment the test is an AST.

[0300] Once testing is complete, the controller 30 activates the door drive 2076 and operates the cartridge transfer instrument 2000, or another cartridge transfer instrument, to remove the tested cartridges from their respective receptacles within the testing instrument 2050 by the cartridge displacement drive 2030. Such cartridges can be moved to a storage bin 2006. Alternatively, the transfer instrument 2000 can dump the tested cartridges into the waste bin 2004. This process can be performed continuously 24 hours / day, 7 days a week.

[0301] In another method embodiment, the test instrument 2050 can be manually loaded or unloaded. Initially, the instrument 2050 can be set to an automatic mode in which the transfer instrument 2000 implements automatic loading and unloading as described above with respect to the first method embodiment of automatic transfer. However, in the event that the user elects to manually load or unload the test instrument 2050, the user can engage the user interface 2071 to set the instrument 2050 and the overall system into a manual mode. Once the user interface 2071 is engaged, the test instrument 2050 notifies the controller 30 which disables the door drive 2076 and determines whether a test is currently in progress. Other subsystems can be disabled by the controller 30, such as the cartridge transfer instrument 2000. If no test is in progress, the controller 30 activates the lock mechanism 2074 which unlocks the first door 2060. If a test is in progress, the controller 30 keeps the door 2060 locked and notifies or indicates to the user through 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 the test instrument 2050 can be manually loaded or unloaded with inoculated cartridges 90. The user can then open the first door 2060 and begin manually loading or unloading the test instrument 2050 with inoculated cartridges 90.

[0302] In some embodiments, the user interface 2071 provides additional functionality, such as specifying whether manual loading or unloading is required, rather than simply activating a manual mode. It is also contemplated that specific cartridges 90 can be identified for removal. In the event that the user commands manual loading, the controller 30, along with activating the lock mechanism 2074 to unlock the first door 2060, activates the container drive 2078 to move one or more empty containers into alignment with the first door opening. Conversely, in the event that manual unloading is selected, the controller 30 operates the container drive 2078 to bring cartridges that have been tested into alignment with the first door opening so that they can be manually unloaded.

[0303] Alternative system

[0304] Various modifications, additions and permutations of the features discussed above can be employed without departing from the present application. For example, Figure 29 An alternative preparation system 1000' is described. The system 1000' is similar to the system 1000 in that it includes a housing which houses several platforms, such as the receiving platform 1010, the preparation platform 1030, and the transfer platform 1040. However, the system 1000' is different in that it has a picking platform 1020'. As described previously with respect to the system 1000, the platform 1020 includes a positioning device 8 which carries a picking tool 6 and uses the picking tool 6 to pick a sample from a colony 4 on a plate 3 and transfer the picked colony to a suspension tube 11. The positioning device 8 includes a transfer device 15 which is used to oscillate the picking tool when it is immersed in the suspension medium.

[0305] Platform 1020', on the other hand, separates the positioning device 8 and the transfer device 15. In this respect, the positioning device 8 and the transfer device 15 are independently connected to the transfer track 18. The transfer device 15 includes a transfer bracket 16 equipped with a gripping tool 17 to releasably grip the pickup tool 6. In this way, the transfer tool 15 can be moved to the positioning device 8, allowing the gripping tool 17 to take over the pickup tool 6 from the positioning device 8. The pickup tool bracket 9 releases the pickup tool 6 after the gripping tool 17 has gripped the pickup tool. Figure 29 In the illustrated embodiment, the pickup tool 6', which has previously picked up a sample of microorganisms 4, is positioned by the transfer device 15 at the starting position indicated by the solid line above the suspension tube 11. The transfer device 15 is configured to lower the pickup tool 6' into the suspension medium 14 contained within the suspension tube 11, at which position, as... Figure 29 As shown by the dashed line, the pickup tool 6' carrying sample 19 is immersed in the suspension medium 14. In this position, the transfer device 15 is activated to linearly and vertically move the oscillating pickup tool 6' for a period of time, sufficient to release the sample from the second pickup tool 6'. Afterwards, the transfer device 15 positions the pickup tool 6', now freed of its contents, in a waiting position above the suspension tube 11. Figure 29 The illustrated embodiment corresponds to the starting position of the transfer device 15. Subsequently, the transfer device can release the pickup tool 6 above the waste bin, during which time the positioning device 8 can retrieve the second pickup tool and the second sample. Therefore, in this embodiment, the positioning device pushes the pickup tool onto the transfer device 15, rather than retaining the pickup tool by transferring the sample to the suspension medium. This embodiment can be used in situations where the pickup tool does not require active suction or vacuum to hold the sample.

[0306] Another embodiment of the preparation system 3000 is shown in Figure 30The system 3000 is similar to the system 1000 in that it comprises a housing which houses several platforms, e.g. a receiving platform (not shown), a picking platform 4020, a preparation platform 4030, and a transfer platform 4040. In addition, the picking platform 4020 comprises a positioning device 3008 carrying a picking tool 3006, and the preparation platform comprises a pipettor 2040 carrying a pipette tip 3046. However, unlike the system 1000, in which the suspension tube 11 is held in the same general location for both inoculation with the picked sample 19 by the positioning device 8 and retrieval of the suspended sample by the pipettor 40, the positioning device 3008 and the pipettor 3040 are incorporated into their respective platforms 3020, 3030. In other words, the pipettor 3040 does not move from the preparation platform 4030 to the picking platform 4020 to retrieve the suspended sample from the suspension tube 3011, but rather the suspension tube 3011 is moved from the picking platform 4020 to the preparation platform 4030 after being inoculated by the picking tool 3006.

[0307] This is achieved by a suspension tube mover 3070. The suspension tube mover 3070 is a robot generally arranged below the faceplate 3007, and is configured to move in at least two dimensions, as indicated by the double-headed arrows. Figure 30 In particular, the suspension tube mover 3007 is configured to support (e.g. by a container or a clamp) the suspension tube 3011, and to move the suspension tube 3011 below the faceplate 3007, and between pre-designated locations A and A' located at the picking and preparation platforms, respectively. In this regard, the faceplate can have an opening therethrough, where the mover can raise or lower the suspension tube at these pre-designated locations.

[0308] In addition, each of these locations A and A' has a turbidimeter 3020, 3060, e.g. one of the aforementioned turbidimeters, which comprises a laser or light emitter 3021, 3061 and a detector 3022, 3062. Thus, the first location A located at the picking platform 4020 comprises a first turbidimeter 3020, and the second location A' located at the preparation platform 4030 comprises a second turbidimeter 3060. In one embodiment, the turbidimeter is an 8-channel device which can simultaneously detect turbidity in 8 cuvettes, e.g. by comprising multiple light sources and detectors.

[0309] In a method of using system 3000, a petri dish 3003 is moved from the receiving platform to the picking platform 4020 and can be positioned on the platform 3002. The petri dish 3003 includes a culture medium 3005 and one or more colonies of microorganisms. A target colony 3004 is selected and a picking tool 3006 is positioned above the target colony 3004. The picking tool 3006 is lowered to retrieve a sample 3019 from the target colony 3004 by the positioning device 3008. Thereafter, the positioning device 3008 moves the picked colony 3019 to a position A above the suspension test tube 2011 within the picking platform 4020. The positioning device 3008 lowers the picked microorganism 3019 and immerses it in the suspension medium within the suspension test tube 3011. The transfer device 3015 agitates the picking tool 3006 to release the microorganism into the suspension medium. The nephelometer 3020 detects the turbidity of the suspension. Additional picking of microorganisms 3004 can be performed until a desired turbidity is obtained.

[0310] Once the desired turbidity is obtained by successive colony picking, the test tube mover 3070 lowers the suspension test tube 3011' with the microorganism suspension therein until it is positioned below the faceplate 3007. The mover then moves the test tube 3011' to a second test tube position A' at the preparation platform 4030. Thereafter, the mover 3070 raises the test tube 3011' through the opening of the faceplate 3007 to prepare the MALDI plate 3042. The MALDI plate is prepared by the pipette 3040 which moves to a position A' above the test tube 3011' and retrieves an aliquot of the suspension therefrom. The pipette 3040 then moves the aliquot to a position B above the MALDI plate 3042 where the pipette 3040 deposits the aliquot to the MALDI plate 3042 at a predetermined position 3044 as described in detail above. Once the MALDI plate 3042 is prepared, the pipette 3040 can then aspirate deionized water or some other suspension medium into the suspension test tube 2011' at the second test tube position A'. The nephelometer 3060 at the second test tube position A' detects the turbidity. Once the desired McFarland number for the AST is obtained, the pipette 3040 aspirates an aliquot from the suspension test tube 2011' at position A' and then transfers the aliquot to position C where the pipette 3040 uses the suspension to inoculate the AST broth test tube 3082. The AST broth test tube 3082 is moved to the transfer platform 4040 by the AST test tube mover 3080 in a manner similar to the mover 3070 by lowering the test tube below the faceplate 3007 and passing it under the faceplate to a position within the transfer platform 4040. From here, the sample within the AST test tube 3082 is inoculated into the AST cartridge 90 as previously described.

[0311] In other embodiments of the system 3000, the preparation platform 4030 can include two cuvette positions, such that the system includes a total of three suspension cuvette positions, one within the pick-up platform 4020 and two within the preparation platform 4030. In this embodiment, one of the cuvette positions within the preparation platform 4030 can be used for MALDI plate preparation, while the other position within the preparation platform 4030 can be used for preparation of an AST cuvette 3082. In this regard, the cuvette position for MALDI preparation can not have a nephelometer, as the suspension turbidity for MALDI preparation has already been determined by the nephelometer 3020 of the pick-up platform 4020. However, the suspension cuvette position for AST cuvette preparation will have a nephelometer 3060, such that the pipette 2040 is assisted in diluting the suspension to the appropriate McFarland number for the AST.

Claims

1. An automated system for preparing single-sample suspensions, wherein aliquots are transferred from the suspension for identification (ID) and antibiotic susceptibility testing (AST) of microorganisms in the sample, the system comprising: A first portion for preparing a sample for ID analysis, the first portion comprising: The sample pickup platform, the sample suspension preparation platform, the first inoculation platform for inoculating a first aliquot of a sample plate with a prepared suspension for ID analysis, and the second inoculation platform for inoculating a second aliquot of a sample tube with a prepared suspension, the sample pickup platform including a robotic pickup tool, a stage for receiving culture plate media, and a controller communicating with a device for acquiring an image of the culture plate media, capable of identifying the location of colonies of interest on the culture plate media from the image, the robotic pickup tool being configured to, in response to instructions from the controller, acquire a pipette tip, lower the pipette tip until it contacts the colony of interest, raise the pipette tip now carrying the colony of interest, and move the pipette tip carrying the colony to the suspension preparation platform; The suspension preparation platform includes a position for receiving suspension cuvettes, a diluent dispenser configured to dispense suspension diluent into the suspension cuvettes, and a turbidimeter for measuring the concentration of the sample in the suspension cuvettes after the robotic tool has released the picked-up sample into the diluent. A suspension tube mover configured to move the suspension cuvette between a pre-specified position on the sample pickup platform and the suspension preparation platform; A first robotic pipette is configured to obtain a first aliquot from a prepared suspension and dispense the first aliquot into a container for ID analysis. The first robotic pipette is further configured to dilute the prepared suspension to one of the turbidity values ​​within a predetermined range for AST testing. A photodetector is used to detect whether a line of sample is formed when the robotic pickup tool is raised from the culture plate medium, wherein the controller defines a pickup tolerance area for positioning on the culture plate medium, wherein the colony to be picked up is specified based on the size of the robotic pickup tool. The first robotic pipette is further configured to obtain a second aliquot of the prepared and diluted suspension and to inoculate the prepared and diluted suspension into sample tubes for the AST test; and A transfer mechanism for transferring inoculated AST sample tubes to a second part, the second part comprising: A second robotic pipette obtains an aliquot from an inoculated AST sample tube and distributes the aliquot onto an AST plate; and A robotic mechanism that grasps the plate and places it in a device for AST testing.

2. The system according to claim 1, wherein the robotic mechanism within the second portion for placing inoculated AST plates into equipment for AST testing is a plate gripper comprising a gripper plate coupled to an arm of a three-axis stepper motor controlled fixture such that the gripper plate is adapted to pivot about an axis of the coupling from a first position to a second position; and wherein the gripper plate comprises a gripping surface adapted to grip a target.

3. The system according to claim 1, further comprising a mechanism to sever the string, the mechanism selected from the group consisting of a cutting tool, an ultrasonic device, a drying device, and a freezing device.

4. The system according to claim 3, wherein the cutting tool is selected from the group consisting of a laser, a bar, a wire, and a blade.

5. The system according to claim 4, wherein the cutting tool is heated.

6. An automated system for preparing a single sample suspension from which aliquots are removed for identification (ID) analysis of microorganisms in the sample and antibiotic susceptibility testing (AST) analysis of microorganisms in the sample, the system comprising: a control tool comprising a processor and a memory; a first portion for preparing a sample for ID analysis, the first portion comprising: a tool for receiving a culture plate in a petri dish; the system configured to communicate with an imaging tool for obtaining an image of the culture plate and a tool for identifying colonies on the culture plate and designating colonies for automated sample picking from the obtained image; a first automated device for automatically handling picking tools; a tool for communicating the location of the colonies from the control tool to the first automated device; wherein the control tool controls picking of the colonies and communicates the picked sample to a first sample suspension preparation platform through the first automated device, wherein the first automated device further comprises a suspension test tube mover configured to communicate a suspension test tube to a pre-designated location in the first sample suspension preparation platform; a tool for providing a suspension liquid into the suspension test tube at the first sample preparation platform, wherein the automated picking tool is configured to deposit the picked sample within the suspension liquid in the suspension test tube to form a suspension; a nephelometer for detecting turbidity of the suspension liquid in the suspension test tube at the first platform, wherein the automated system, in response to a turbidity detection value outside of a predetermined turbidity value, adjusts one of the amount of sample or the amount of suspension in the suspension test tube to provide a suspension having a sample concentration within a predetermined range; a second automated device, wherein the second automated device is a pipettor that acquires a first aliquot suspension in the suspension test tube at the first platform and inoculates within a container for ID analysis; a first tool to communicate the suspension test tube with the remaining portion of the suspension from the first sample suspension preparation platform to a second sample suspension preparation platform; a detector for detecting whether a string of sample is formed when the first automated device transfers the pick-up tool carrying the sample from the culture dish; wherein the second automated device is further configured to adjust the concentration of the remaining portion of the suspension within the tube to a predetermined concentration for a second analysis, and to obtain a second aliquot of the sample suspension having the adjusted concentration, and to inoculate a tube for the AST analysis with the second aliquot of the sample suspension; and a second portion for inoculating a plate for the AST analysis; wherein the automated system has a second tool for transferring the inoculated sample tube from the first portion to the second portion; the second portion has a third automated device that is a pipette that takes an aliquot from the inoculated tube for the AST analysis and inoculates an AST plate with the taken aliquot; and a loader robot that loads the inoculated AST plate into a device that performs the AST analysis.

7. The system of claim 6, wherein the AST device comprises at least two gates, a first gate that receives the plate from the loader robot for transferring the inoculated AST plate, wherein the loader robot is a plate gripper that comprises a gripper plate coupled to an arm of a three-axis stepper motor controlled fixture such that the gripper plate is adapted to pivot about an axis of the coupling from a first position to a second position; and wherein the gripper plate comprises a clamping surface that is adapted to clamp a target.

8. The system of claim 7, wherein the string is detected by optically or electronically monitoring the pick-up tool as the pick-up tool is raised away from the culture dish.

9. The system of claim 8, further comprising a machine for severing the string, wherein the machine cuts the string by one of cutting the string, sonicating the string, drying the string, or freezing the string.

10. The system of claim 9, wherein the machine for cutting the string is selected from the group consisting of a laser, a rod, a wire, and a blade.

11. The system of claim 9, wherein the machine for cutting the string is heated.

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