Apparatus, system, and method for preparing output samples using aeration.

Abstract

Methods, devices, and systems for preparing an output sample of bacteria are disclosed. In one aspect, a method is disclosed that includes introducing an aliquot of a sample containing bacteria into a sample container, where the aliquot of sample in the sample container is a contained sample in fluid communication with a reference sensor and an active sensor. The method also includes incubating the contained sample and aerating the contained sample at a flow rate of between 7.0 μL / sec / mL of the contained sample and 10.0 μL / sec / mL of the contained sample. The method further includes monitoring a change in the ORP of the contained sample using a reader electrically coupled to the reference sensor and the active sensor, and cooling the contained sample when it is determined that the concentration of bacteria in the contained sample has reached a desired or target concentration or within an acceptable margin of error.

Description

Technical Field

[0001] Cross - reference to related applications

[0001] This application claims priority to U.S. Patent Application No. 63 / 141,057, filed on January 25, 2021, and U.S. Patent Application No. 63 / 212,600, filed on June 18, 2021, the contents of which are hereby incorporated by reference in their entirety. This application also incorporates by reference U.S. Patent Application Publication No. 2019 / 0293529A1, published on September 26, 2019, and U.S. Patent Application Publication No. 2021 / 0131993A1, published on May 6, 2021.

[0002] Technical field

[0002] The present disclosure generally relates to the preparation of diagnostic samples, and more specifically, to devices, systems, and methods for preparing an output sample of bacteria at a target or desired concentration (or within an acceptable error margin) using ORP monitoring and aeration.

Background Art

[0003] Background

[0003] Infections caused by antibiotic - resistant bacteria are a major problem for healthcare workers in hospitals, nursing homes, and other healthcare settings. The rapid detection of the susceptibility of such bacteria to antibiotics is extremely important to prevent the spread of resistance profiles. New technologies (e.g., matrix - assisted laser desorption ionization time - of - flight mass spectrometry (MALDI - TOF MS), real - time polymerase chain reaction (real - time PCR), etc.) have been developed to identify bacteria in samples such as positive blood cultures, but the first step in most antibiotic susceptibility testing (AST) protocols still involves the preparation of an output sample or inoculum having a concentration that matches the McFarland standard.

[0004]

[0004] Existing methods and equipment used to prepare such output samples include costly, time-intensive (e.g., up to 24 hours) and labor-intensive microbial culture techniques. However, these methods often require manual interpretation by skilled personnel and are susceptible to technical or clinician errors. In addition, certain samples, including animal or human blood, are often difficult to assess using popular optical techniques due to the opacity of the samples. Furthermore, such optical techniques often require expensive equipment. Moreover, while some methods consider preparing output samples using universal look-up tables (LUTs), one drawback of methods that rely solely on universal LUTs is that the time to reach the target concentration is greatly influenced by the growth rate of bacteria in the source sample. This means that the time required to prepare output samples can vary greatly depending on the bacteria in the source sample. This makes reliance on such methods impractical in busy laboratory settings. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005]

[0005] As a result of the above limitations and constraints, improved apparatus, systems, and methods are needed to quickly and efficiently prepare bacterial output samples of desired or target concentrations for downstream testing. [Means for solving the problem]

[0006] overview

[0006] Disclosed are various methods, devices, and systems for preparing output samples of bacteria at a desired or target concentration. In one embodiment, the desired or target concentration or an acceptable error margin (±0.5log) of the desired or target concentration. 10A method for preparing a bacterial sample within a sample may include introducing an aliquot of the bacterial sample into a sample container, the aliquot of the sample in the sample container being a containing sample in fluid communication with a reference sensor and an active sensor; incubating and aerating the containing sample, the containing sample being aerated at a flow rate of 7.0 microliters (μL) / sec / millimeter (mL) to 10.0 μL / sec / mL; monitoring changes in the oxidation-reduction potential (ORP) of the containing sample using readers electrically coupled to the reference sensor and the active sensor; and cooling the containing sample if it is determined that the bacterial concentration in the containing sample has reached a desired or target concentration or within an acceptable margin of error.

[0007]

[0007] The method may include searching a species-independent lookup table (LUT) from a database, the species-independent LUT containing species-independent ORP changes associated with species-independent bacterial concentrations, and the species-independent LUT is generated from multiple component LUTs containing ORP changes and bacterial concentrations measured using multiple reference bacterial samples incubated and aerated at flow rates of 7.0 μL / sec / mL to 10.0 μL / sec / mL for each of the reference bacterial samples.

[0008]

[0008] The method may further include, if one of the species-independent ORP change amounts is associated with one of the species-independent bacterial concentrations equal to a desired or target concentration, selecting one of the species-independent ORP change amounts associated with the one of the species-independent bacterial concentrations equal to the desired or target concentration as a threshold ORP change amount, and determining that the concentration of bacteria in the containing sample has reached the desired or target concentration or within an acceptable error margin when the change in ORP of the containing sample monitored by the reader reaches the threshold ORP change amount.

[0009]

[0009] A species-independent LUT can be generated from at least three component LUTs, including a first LUT, a second LUT, and a third LUT, each of which is either a species-specific LUT or a lineage-specific LUT. The first LUT, the second LUT, and the third LUT can be generated using ORP and bacterial concentration measurements performed on a first reference bacterial sample, a second reference bacterial sample, and a third reference bacterial sample. The first reference bacterial sample may contain bacteria of a first species, the second reference bacterial sample may contain bacteria of a second species different from the first species, and the third reference bacterial sample may contain bacteria of a second species and a third species different from the first species.

[0010]

[0010] Each of the lineage-specific LUTs can be generated by monitoring the change in ORP of at least one reference bacterial sample over a period of time, periodically performing optical density (OD) measurements of at least one reference bacterial sample over the same period, converting the results of the OD measurements to the reference bacterial concentration using a conversion factor, and relating the reference bacterial concentration to the change in ORP of at least one reference bacterial sample.

[0011]

[0011] This method is expressed by the following formula:

number

[0012]

[0012] The method uses the following formula:

Number

[0013]

[0013] The sample can include at least one of a body fluid and a bacterial culture derived from the body fluid. The output sample can be prepared without any prior knowledge of the species of bacteria in the inclusion sample or without having previously determined the species of bacteria in the inclusion sample. The bacteria in the inclusion sample can be facultative anaerobes or obligate aerobes. Further, the bacteria in the inclusion sample can be Gram-negative bacteria. The desired or target concentration can be 1.4×10 8 CFU / mL to 1.6×10 8 CFU / mL.

[0014]

[0014] This method may further include diluting a source sample containing bacteria at a dilution ratio of 1:10 to 1:100 to produce a diluted sample. The aliquot of the sample introduced into the sample container may be an aliquot of the diluted sample.

[0015]

[0015] The reference sensor may include a reference electrode material and a wick, which are in fluid communication with the contained sample such that at least some of the contained sample in the chamber cavity of the sample container is drawn toward the reference electrode material by the wick, and the contained sample is in fluid contact with the reference electrode material. The active sensor may be coupled to at least a portion of the chamber sidewall of the sample container. The active electrode material of the active sensor may face the chamber cavity so that the contained sample is in fluid contact with the active electrode material when the contained sample fills the chamber cavity. The ORP of the contained sample may be determined by the reader based on the potential difference measured between the active electrode material and the reference electrode material when the reference sensor and the active sensor are electrically coupled to the reader.

[0016]

[0016] The inclusion sample can be incubated at an incubation temperature of approximately 33°C to 37°C. The inclusion sample can be aerated according to an aeration cycle. The aeration cycle may include an aeration period followed by a non-aeration period. The aeration period may be longer than the non-aeration period. For example, the aeration period may be about 7 minutes to about 10 minutes, and the non-aeration period may be about 3 seconds to about 10 seconds.

[0017]

[0017] The contained sample may be aerated using an electric piston pump. The electric piston pump may be housed in a leader. Aerating the contained sample may further include introducing ambient air into the sample container through an opening defined along the base of the sample container.

[0018]

[0018] Desired or target concentration or an acceptable error margin (±0.5log) of the desired or target concentration 10A system for preparing output samples of bacteria within a sensor is also disclosed. The system may comprise a sensor device including a container chamber configured to hold aliquots of a bacterial sample, wherein the aliquots of the sample in the container chamber are containing samples in fluid communication with a reference sensor and an active sensor; and a reader configured to receive the sensor device, the reader also configured to incubate and aerate the containing sample when the sensor device is located within the reader, the containing sample being aerated at a flow rate of 7.0 microliters (μL) / sec / millimeter (mL) to 10.0 μL / sec / mL of the containing sample. One or more processors of the reader are configured to monitor changes in the oxidation-reduction potential (ORP) of the containing sample when the reader is electrically coupled to the reference sensor and the active sensor of the sensor device, and to cool the containing sample when it is determined that the concentration of bacteria in the containing sample has reached a desired or target concentration or within an acceptable error margin. [Brief explanation of the drawing]

[0019] Brief explanation of the drawing [Figure 1A]

[0019] A front view of one embodiment of a sensor device that uses ORP monitoring and aeration to prepare an output sample of bacteria at a desired or target concentration (or within an acceptable margin of error). [Figure 1B]

[0020] A cross-sectional side view of a portion of the sensor device is shown. [Figure 1C]

[0021] This shows a magnified perspective view of the active sensor attached to the side wall of the sensor device's chamber. [Figure 1D]

[0022] This shows a cross-sectional view of the sensor device filled with the sample. [Figure 2A]

[0023] This document illustrates one embodiment of a reader that can be used to monitor the ORP of a sample contained within a sensor device. [Figure 2B]

[0024] This shows specific functional components of the reader with the reader housing removed for viewing purposes. [Figure 2C]

[0025] This shows a partial side cross-sectional view of the sensor device located within the reader. [Figure 3]

[0026] One embodiment of a method for preparing a bacterial output sample at a desired or target concentration (or within an acceptable error margin of the desired or target concentration) is shown. [Figure 4]

[0027] This shows one embodiment of a species-independent lookup table (LUT) generated from multiple species-independent lookup tables (LUTs). [Figure 5]

[0028] This shows one embodiment of a species-independent LUT generated from six lineage-specific LUTs. [Figure 6A]

[0029] The results from 41 trial runs performed to evaluate the effectiveness of the methods and systems disclosed herein for preparing bacterial output samples at desired or target concentrations (or within an acceptable margin of error of the desired or target concentration) are shown below. [Figure 6B]

[0029] The results from 41 trial runs performed to evaluate the effectiveness of the methods and systems disclosed herein for preparing bacterial output samples at a desired or target concentration (or within an acceptable error margin of the desired or target concentration) are shown below. [Figure 7A]

[0030] This graph shows the effect of aeration on the bacterial growth rate of facultative anaerobic bacteria. [Figure 7B]

[0030] This graph shows the effect of aeration on the bacterial growth rate of obligate aerobic bacteria. [Figure 8A]

[0031] This table shows that aeration can reduce the variation in growth rates of different bacterial species. [Figure 8B]

[0032] This table shows that the average bacterial doubling time can be calculated from the doubling times of bacteria specific to multiple species. [Figure 9A]

[0033] This is an ORP growth curve showing the ORP change of an inclusion sample measured by a reader over a certain period. [Figure 9B]

[0034] This is a bacterial growth curve showing the change in bacterial concentration in a sample over a certain period of time. [Modes for carrying out the invention]

[0020] Detailed explanation

[0035] Variations of the apparatus, devices, systems, and methods disclosed herein will be best understood from the detailed description when read in conjunction with the accompanying drawings. It should be emphasized that, in general practice, various features in the drawings may not be to a constant scale. Conversely, for clarity, the dimensions of various features may be arbitrarily enlarged or reduced, and not all features are visible or depicted in every drawing. The drawings are construed for illustrative purposes only and are not intended to limit the claims to those defined or shown.

[0021]

[0036] Figures 1A to 1D show one embodiment of a sensor device 100 that uses oxidation-reduction potential (ORP) monitoring and aeration to prepare an output sample of bacteria at a desired or target concentration (or within an acceptable error margin).

[0022]

[0037] In some embodiments, the source sample may be obtained from a patient or subject. For example, the source sample may be obtained from a human patient or subject. In other embodiments, the source sample may be obtained from a non-human animal patient or test subject.

[0023]

[0038] In certain embodiments, the source sample may include body fluids collected, extracted, or otherwise obtained from a patient or subject, or bacterial cultures derived therefrom. More specifically, the body fluids may be at least one of blood, urine, serum, plasma, saliva, sputum, semen, breast milk, synovial fluid, cerebrospinal fluid, wound material, mucus, feces with fluid, vaginal secretions, synovial fluid, pleural fluid, ascites, pericardial fluid, and amniotic fluid.

[0024]

[0039] In additional embodiments, the source sample may be a swab obtained from a patient or subject, in which case the swab or a portion thereof is resuspended in a liquid bacterial culture medium or nutrient medium. More specifically, the swab may be a wound swab, a rectal swab, or a vaginal swab.

[0025]

[0040] In other embodiments, the source sample may be an environmental sample or a food / beverage sample. For example, the source sample may include an environmental sample taken from a stream, river, lake, sea, contamination site, quarantine zone, emergency area, or a combination thereof. In other embodiments, the source sample may include a food sample taken from a food preparation facility, cafeteria, or waste disposal facility.

[0026]

[0041] In all such embodiments, the source sample may contain or include bacteria. In certain embodiments, the source sample may be a bacterial culture derived from at least one of a patient sample, a biological sample, an environmental sample, and a food sample. For example, the source sample may be a bacterial culture derived from or resuspended from a bodily fluid (or swab) obtained from a patient or subject.

[0027]

[0042] As a more specific example, the source sample may be a bacterial culture or resuspended bacterial culture derived from the blood of a patient or subject who has tested positive for bacterial growth. Such a source sample may also be called a positive blood culture. In this disclosure, a positive blood culture (or PBC) is a bacterial culture derived from blood taken from a patient or subject who has tested positive for bacterial growth. For example, a patient may exhibit symptoms of sepsis (e.g., high fever, chills, etc.), and blood (e.g., 5 to 10 mL) may be taken from the patient and transferred to a commercial blood culture vessel or container containing bacterial growth medium (e.g., 30 to 40 mL of growth medium). The blood culture vessel or container can then be incubated at 35°C ± 2°C to allow bacteria to grow. If the patient's blood is contaminated with bacteria, the bacteria will grow in the vessel or container. Next, bacterial growth can be monitored using a blood culture system or apparatus (for example, by monitoring bacterial CO2 production in the container or vessel), and the system and apparatus can determine that the sample has been tested "positive" for bacterial growth if a critical CO2 threshold is met. Depending on the type of bacteria and the rate of bacterial growth, the blood culture can become positive in 7 hours to 3 days. Such a "positive blood culture" can then serve as a source sample.

[0028]

[0043] As will be disclosed in more detail in the following sections, variations of the apparatus, devices, systems, and methods disclosed herein can be used with ORP monitoring and aeration to prepare bacterial output samples or standardized inoculum from a source sample at a desired or target concentration (or within an acceptable margin of error).

[0029]

[0044] Figure 1A shows a front view of one embodiment of the sensor device 100. The sensor device 100 can be designed or configured as a sample container comprising a container chamber 102 and a container cap 104 that is detachably attached to or fixed (e.g., screwed or press-fitted) to the container chamber 102.

[0030]

[0045] The sensor device 100 may further include an active sensor 106 fixed, bonded, or otherwise coupled to at least a portion of the container chamber 102, and a reference sensor 108 integrated with the container cap 104 or manufactured as part of the container cap 104.

[0031]

[0046] The container chamber 102 can be made in part from an inert or non-conductive material. In some embodiments, the container chamber 102 may include, or be made in part from, a polymer material, a ceramic material, or glass, or a combination thereof. More specifically, the container chamber 102 may include, or be made in part from, polyvinyl chloride (PVC), poly(methyl methacrylate) (PMMA), polydimethylsiloxane (PDMS), or a combination thereof.

[0032]

[0047] Figure 1B shows a cross-sectional side view of a portion of the sensor device 100. For clarity, the reference electrode material 132 and wick components 134 of the reference sensor 108 (see, for example, Figure 1D) are not shown in Figure 1B.

[0033]

[0048] Figure 1B may include a chamber side wall 110 surrounding a chamber cavity 112 configured to receive and hold a contained sample 113 (see, for example, Figure 1D). The contained sample 113 may refer to an aliquot of a source sample that has been filtered and / or diluted and introduced into the chamber cavity 112 of a container chamber 102 (see, for example, Figure 3).

[0034]

[0049] As shown in Figure 1B, the active sensor 106 can be fixed, bonded, or otherwise attached to the chamber side wall 110 of the container chamber 102. In other embodiments not shown, the active sensor 106 can be attached to the bottom of the container chamber 102 or otherwise positioned along the bottom.

[0035]

[0050] The active sensor 106 can be coupled to at least a portion of the chamber side wall 110 in a window opening 114 defined along the chamber side wall 110. The chamber side wall 110 may have a groove 116 surrounding the window opening 114. The groove 116 can be defined along the outside of the chamber side wall 110.

[0036]

[0051] Regarding the arrangement of the active sensor 106, the active sensor 106 can be configured such that no portion of the active sensor 106 extends into the chamber cavity 112, as shown in Figures 1B and 1C. The active sensor 106 can be made of an active electrode layer 118 or a conductive substrate partially covered with an active electrode material. The active electrode layer 118 of the active sensor 106 faces the chamber cavity 112, and the sample contained in the chamber cavity 112 can be brought into fluid contact with the active electrode layer 118 through at least a portion of the chamber side wall 110 surrounding the window opening 114.

[0037]

[0052] Figure 1C shows a magnified perspective view of an active sensor 106 bonded to the chamber sidewall 110. In the embodiment shown in Figure 1C, the active sensor 106 is bonded to a groove 116 in the chamber sidewall 110. At least a portion of the active electrode layer 118 of the active sensor 106 can cover a window opening 114 defined along the chamber sidewall 110 such that this portion of the active electrode layer 118 covering the window opening 114 is positioned to be in fluid communication with the chamber cavity 112 of the container chamber 102. When the container chamber 102 is filled with the contained sample 113 (see, for example, Figure 1D), the contained sample 113 can be in fluid contact with the portion of the active electrode layer 118 covering the window opening 114.

[0038]

[0053] In some embodiments, the cavity volume of the chamber cavity 112 can be approximately 0.8 mL to approximately 1.2 mL. In a more specific example, the chamber cavity 112 can be approximately 1.0 mL. In other embodiments, the cavity volume of the chamber cavity 112 can be greater than 1.2 mL.

[0039]

[0054] Figure 1C also shows that the active sensor 106 may have sides covered with adhesive 120. Since the active sensor 106 may contain multiple layers, the adhesive 120 can protect specific layers of the active sensor 106 from undesirable contact with the contained sample 113. The adhesive 120 can act as a barrier to prevent the contained sample 113 from contacting the side 122 of the active sensor 106. In other embodiments contemplated by this disclosure, though not shown, the groove 116 of the chamber sidewall 110 may be sized such that the active sensor 106 fits tightly into the groove 116 and the wall of the groove 116 is adjacent to or borders the side 122 of the active sensor 106. This can ensure that only the exposed portion of the active electrode layer 118 contacts the contained sample 113, leading to more accurate measurement of the solution properties (e.g., ORP and pH) of the contained sample 113.

[0040]

[0055] To bond the active sensor 106 to the container chamber 102, a bead of adhesive 120 can be applied to the inner ledge 124 and / or side border 126 of the groove 116, and then the active sensor 106 can be press-fitted into the groove 116 using the end effector of a pick-and-place machine. The active sensor 106 can be press-fitted or otherwise facilitated into the groove 116 until the outward-facing surface of the active sensor 106 is flush with the outer surface of the chamber side wall 110.

[0041]

[0056] The adhesive 120 then hardens, fixing the active sensor 106 in place. In some embodiments, the adhesive 120 can be a medical-grade UV-curing adhesive. For example, the adhesive 120 can be Dymax® 1405M-T-UR-SC adhesive (curable using LED light with a wavelength of approximately 405 nm). In other embodiments, the adhesive 120 can be any low-outgassing medical-grade adhesive.

[0042]

[0057] As described above, the active sensor 106 can be made of a conductive substrate partially covered with an active electrode layer 118 or an active electrode material. The active sensor 106 can be positioned such that the active electrode layer 118 faces the chamber cavity 112 and fluidly contacts the sample in the chamber cavity 112 with the active electrode layer 118 through at least a portion of the chamber sidewall 110 surrounding the window opening 114. In this embodiment, the active sensor 106 (including the active electrode layer 118) is located radially outward from the side facing the interior of the chamber sidewall 110 or the cavity, and the side portion 122 of the active sensor 106 is not exposed to the contained sample 113.

[0043]

[0058] Once the container chamber 102 is filled with the sample 113, a reader 200 (see, for example, Figures 2A-2C) communicatively coupled to the sensor device 100 can measure or monitor the oxidation-reduction potential (ORP) of the sample 113. In these embodiments, the active electrode layer 118 can be a redox-sensitive material. For example, the redox-sensitive material may be platinum (Pt), gold (Au), a redox-sensitive metal oxide, or a combination thereof, or may include them. More specifically, the redox-sensitive material may be silicon dioxide (SiO2), aluminum oxide (Al2O3), titanium dioxide (TiO2), tantalum pentoxide (Ta2O5), hafnium dioxide (HfO2), iridium dioxide (IrO2), ruthenium dioxide (RuO2), zirconium dioxide (ZrO2), or a combination thereof, or may include them.

[0044]

[0059] In other embodiments, the pH of the contained sample 113 can also be measured or monitored by the reader 200. When the solution property of the contained sample 113 to be measured or monitored is pH, the active electrode layer 118 can be a pH-sensitive material. For example, the pH-sensitive material can be silicon dioxide (SiO2), aluminum oxide (Al2O3), titanium dioxide (TiO2), tantalum oxide / pentoxide (Ta2O5), hafnium dioxide (HfO2), iridium dioxide (IrO2), ruthenium dioxide (RuO2), zirconium dioxide (ZrO2), or a combination thereof, or may include them.

[0045]

[0060] Although not shown, it is intended by this disclosure that the sensor device 100 can be designed to measure both the pH and ORP of the contained sample 113 simultaneously. For example, the container chamber 102 of the sensor device 100 may have a plurality of window openings 114 defined along the chamber side wall 110 of the container chamber 102. Each of these window openings 114 can then be covered with a different active sensor 106 (for example, one window opening 114 can be covered with an active sensor 106 having an active electrode layer 118 made of redox-sensitive material, and another window opening 114 can be covered with an active sensor 106 having an active electrode layer 118 made of pH-sensitive material).

[0046]

[0061] The sensor device 100 may have a device height. In some embodiments, the device height can be about 20.0 mm to about 50.0 mm. In other embodiments, the device height can be about 25.0 mm to about 35.0 mm. For example, the device height can be about 31.3 mm.

[0047]

[0062] Figure 1D shows that the reference sensor 108 may be fabricated as a container cap 104 or may be integrated into a part of the container cap 104. The reference sensor 108 may include a reference conduit 128 including a reference conduit cavity 130 (see Figure 1B). The reference conduit cavity 130 may have first and second openings at both ends. The reference conduit 128 may be an elongated channel or passage configured to extend into the chamber cavity 112 of the container chamber 102.

[0048]

[0063] The reference sensor 108 may also include a reference electrode material 132 and a wick or wick component 134 that are in fluid communication with the chamber cavity 112. The reference conduit cavity 130 can accommodate the wick component 134. At least some of the contained sample 113 can be pulled towards the reference electrode material 132 by the wick component 134.

[0049]

[0064] The reference conduit 128 can be tapered such that the volume of the reference conduit cavity 130 tapers or narrows from the reference conduit base 136 to the reference conduit tip 138 (see Figure 1B). The shape of the wick component 134 can match or conform to the shape of the reference conduit cavity 130. The shape of the wick component 134 can be configured such that it tapers or narrows from the wick base 140 to the wick tip 142.

[0050]

[0065] The wick component 134 can extend through the length of the reference conduit cavity 130. In some embodiments, the wick component 134 can fill or occupy all of the space within the reference conduit cavity 130. In other embodiments, the wick component 134 can fill or occupy only a portion of the space within the reference conduit cavity 130.

[0051]

[0066] At least a portion of the wick component 134 is in fluid communication with the chamber cavity 112 of the container chamber 102, so that when the container chamber 102 is filled with the contained sample 113, at least some of the contained sample 113 in the container chamber 102 is drawn up toward the wick base portion 140 by at least a portion of the wick tip portion 142, absorbed, or otherwise drawn out. The wick component 134 can be made of a polymer material that draws the contained sample 113 toward the reference electrode material 132 by capillary action.

[0052]

[0067] In some embodiments, at least a portion of the wick tip 142 may extend beyond the reference conduit tip 138 so that the wick tip 142 protrudes or extends into the chamber cavity 112 of the container chamber 102. In these embodiments, the wick tip 142 may extend or protrude into the containing sample 113 when the container chamber 102 is filled with the containing sample 113.

[0053]

[0068] In other embodiments, the wick tip 142 is positioned near or above the reference conduit tip 138 so that the wick tip 142 does not protrude or extend into the chamber cavity 112 of the container chamber 102. In these embodiments, the wick tip 142 can still be in fluid communication with the container chamber 102, and the contained sample 113 can still reach or come into contact with the wick tip 142 by being drawn into the reference conduit 128 by capillary action or by perturbing or shaking the container chamber 102.

[0054]

[0069] As described above, the wick component 134 can be made from a partially porous material. The wick component 134 can be made from a material having pores ranging in size from 15 μm to about 150 μm (e.g., about 50 μm). In some embodiments, the wick component 134 can be made from a partially polymer material. As a more specific example, the wick component 134 can be made from a partially porous polymer material having pores ranging in size from 15 μm to about 150 μm. In one embodiment, the wick component 134 can be made from partially high-density polyethylene (HDPE). For example, the wick component 134 can be made from HDPE having pores of about 50 μm. In other embodiments, the wick component 134 can be made from partially natural fibers. For example, the wick component 134 can be made from cellulose fibers, pulp, paper, cotton, or a combination thereof.

[0055]

[0070] The wick component 134 may also be treated with a surfactant such that at least the surface of the wick component 134 is covered with the surfactant. In some embodiments, the wick component 134 may be immersed in a solution saturated with or containing a surfactant before being introduced into the reference conduit cavity 130. The surfactant may be configured to increase the hydrophilicity of the wick component 134 (i.e., to make the substantially hydrophobic surface of the wick component 134 more hydrophilic). In some embodiments, the surfactant may be a fluorinated surfactant. In other embodiments, the surfactant may be one or more nonionic surfactants such as poloxamers. As a more specific example, the surfactant may include Pluronic® F-68.

[0056]

[0071] In one embodiment, the reference conduit 128 may have a substantially conical or frustoconical shape with a reference conduit cavity 130, and the reference conduit cavity 130 may also have a substantially conical or frustoconical shape. In other embodiments, the reference conduit 128 may have a substantially elongated pyramidal shape with a polygonal base. For example, the reference conduit 128 may have a substantially elongated triangular pyramidal shape, a square pyramidal shape, or a pentagonal pyramidal shape. In additional embodiments, the reference conduit 128 may have a substantially cylindrical shape with a substantially cylindrical reference conduit cavity 130. In these embodiments, the reference conduit 128 may have a tapered reference conduit tip 138 (see, for example, Figure 1B).

[0057]

[0072] As shown in Figure 1D, at least a portion of the wick component 134 can be in fluid contact with the contained sample 113 in the container chamber 102. At least some of the contained sample 113 can be drawn up by the wick component 134 toward the wick base end 140. The reference electrode material 132 can be positioned toward the wick base end 140.

[0058]

[0073] Figure 1D also shows that at least a portion of the active electrode layer 118 can be in fluid contact with the contained sample 113 in the container chamber 102. When the wick component 134 pulls up or sucks up the contained sample 113, the contained sample 113 can reach the reference electrode material 132, and the charge carriers in the contained sample 113 can establish an electrical connection between the reference electrode material 132 of the reference sensor 108 and the active electrode layer 118 of the active sensor 106. When both the reference sensor 108 and the active sensor 106 are electrically coupled to the reader 200 (see, for example, Figures 2A-2C), the reader 200 can be used to measure the solution properties (e.g., ORP or pH) of the contained sample 113.

[0059]

[0074] The solution properties (e.g., ORP or pH) of the contained sample 113 can be determined based on the potential difference measured between the active sensor 106 and the reference sensor 108 when the reference sensor 108 and the active sensor 106 are electrically connected to the reader 200. For example, the reference sensor 108 can provide a more stable half-cell potential than the active sensor 106 when both the reference electrode material 132 and the active electrode layer 118 are in fluid contact with the contained sample 113 in the container chamber 102.

[0060]

[0075] In some embodiments, the reference electrode material 132 may be a conductive ink applied to or dispensed onto the wick base end 140. The conductive ink applied to or dispensed onto the wick base end 140 can be solidified by curing. More specifically, the conductive ink may be silver-silver chloride (Ag-AgCl) ink.

[0061]

[0076] At least a portion of the reference electrode material 132 can be bonded to the wick component 134. For example, the reference electrode material 132 may be a hardened and solidified mass located at the wick base end 140. In certain embodiments, the reference electrode material 132 may be located in the center of the container cap 104. In some embodiments, at least a portion of the reference electrode material 132 may protrude or extend beyond the container cap 104.

[0062]

[0077] One advantage of the wick component 134 disclosed herein is that the wick component 134 can draw up the sample, allowing the contained sample 113 to move through the pores of the wick component 134 toward the reference electrode material 132 by capillary action. For example, the contained sample 113 can be drawn up to the wick base end 140, where it fluidly contacts the reference electrode material 132. If the reference electrode material 132 is made of a material such as silver-silver chloride (Ag-AgCl), the wick component 134 prevents silver ions (Ag) from freely diffusing into the contained sample 113 in the container chamber 102 if the wick component 134 is not present. +) can act as a barrier or obstruction to such silver ions. Such silver ions may be harmful to bacteria in the containing sample 113 or may affect bacterial growth in other ways. The wick component 134 can act as a barrier or obstruction to such ions by slowing or stalling the diffusion of harmful silver ions into the containing sample 113. A wick component 134 having the dimensions and shape disclosed herein may be effective in slowing or stalling the diffusion of such harmful ions.

[0063]

[0078] When the reference sensor 108 is implemented as a container cap 104, the container cap 104 may have dimensions defined by a cap width (or diameter) and a cap height. In some embodiments, the gap width may be about 10.0 mm to about 20.0 mm. For example, the cap width may be about 15.7 mm. In some embodiments, the cap height may be about 5.0 mm to about 20.0 mm. For example, the cap height may be about 10.5 mm. When the container cap 104 is tightened, fixed, or otherwise coupled to the container chamber 102, the sensor device 100 may have a device height measured from the bottom of the container chamber 102 to the top of the cap 144 of the container cap 104.

[0064]

[0079] The wick component 134 may have a wick height measured from the wick base 140 to the wick tip. In some embodiments, the wick height can be approximately 10.0 mm to approximately 20.0 mm. More specifically, the wick height can be approximately 14.0 mm to approximately 15.0 mm. For example, the wick height can be approximately 14.8 mm.

[0065]

[0080] As shown in Figure 1D, the reference electrode material 132 can be positioned or disposed at least partially within a central recess, descent, or concave area of ​​the container cap 104 above the wick component 134. If the reference sensor 108 is a cured or solidified conductive ink or solution (e.g., Ag-AgCl ink), the recess, descent, or concave area can act as a space to receive the liquid ink or solution to cure.

[0066]

[0081] In some embodiments, the reference electrode material 132 may have a reference electrode height and a reference electrode width. The reference electrode height may be about 0.2 mm to 1.0 mm. For example, the reference electrode height may be about 0.4 mm. The reference electrode width may be about 2.0 mm to about 5.0 mm. For example, the reference electrode width may be about 3.0 mm. One advantage of the reference sensor 108 disclosed herein is that the reference sensor 108 can act as a stable reference electrode or provide a stable reference potential for up to 10 hours of testing or operation.

[0067]

[0082] Figure 1D also shows that the sensor device 100 may also have an aeration port 146 or opening defined along the underside of the container chamber 102. In other embodiments not shown, the aeration port 146 may be defined along the chamber side wall 110 of the container chamber 102.

[0068]

[0083] The aeration port 146 can be covered by a first gas permeable membrane 148. The aeration port 146 and the first gas permeable membrane can be configured to allow gas 150 to enter the container chamber 102.

[0069]

[0084] In some embodiments, gas 150 can be ambient air (e.g., air in a laboratory, clinical setting, or testing facility). In other embodiments, gas 150 can include a combination of pressurized oxygen, carbon dioxide, nitrogen, and argon. Aeration of the sample can accelerate the growth of bacterial populations in the contained sample 113 by providing an oxygen-rich environment within the container chamber 102.

[0070]

[0085] In alternative embodiments not shown, the aeration port 146 may be defined along the top 144 of the container cap 104, and the gas 150 may be introduced into the container chamber 102 from the top of the container chamber 102.

[0071]

[0086] Gas 150 (e.g., ambient air) can be introduced into the container chamber 102 by an electric piston pump, a syringe pump, or another type of pump / micropump device integrated within the leader 200. Gas 150 (e.g., ambient air) can be introduced into the container chamber 102 through the aeration port 146 and the first gas permeable membrane 148 or directed in any other way at a flow rate of 7.0 microliters (μL) / second / milliliter (mL) to 10.0 μL / second / mL of the contained sample 113. As a more specific example, gas 150 (e.g., ambient air) can be introduced into the container chamber 102 through the aeration port 146 and the first gas permeable membrane 148 or directed in any other way at a flow rate of 8.8 μL / second / mL of the contained sample 113. In some embodiments, the gas 150 (e.g., ambient air) can be introduced into the container chamber 102 through the aeration port 146 and the first gas-permeable membrane 148 at specific duty cycles or intervals, or directed in other ways.

[0072]

[0087] In certain embodiments, the second gas-permeable membrane 152 can cover at least a portion of the underside of the container cap 104. The second gas-permeable membrane 152 can also prevent any liquid in the container chamber 102 from spilling out of the container chamber 102, while allowing any gas 150 that has been introduced into or otherwise introduced into the container chamber 102 to escape.

[0073]

[0088] In some embodiments, the first gas permeable membrane 148 and the second gas permeable membrane 152 can be made of the same material. The first gas permeable membrane 148 and the second gas permeable membrane 152 can be made of hydrophobic gas permeable membranes or thin sheets. For example, both the first gas permeable membrane 148 and the second gas permeable membrane 152 can be made of polytetrafluoroethylene (PTFE), or may contain PTFE.

[0074]

[0089] As shown in Figure 1D, the container cap 104 can be detachably or removablely coupled or fastened to the container chamber 102 by screwing it to a portion of the container chamber 102 via a threaded connection 154. The container cap 104 (which functions as part of the reference sensor 108) is fastened or coupled to the container chamber 102 via the threaded connection 154, and can create an airflow path 156 as gas 150 (e.g., ambient air) enters the container chamber 102 through the first gas-permeable membrane 148. The air then exits the container chamber 102 through the second gas-permeable membrane 152 and an air gap 158 defined between the threads of the container cap 104 and the container chamber 102.

[0075]

[0090] The container cap 104 can be made in part from a transparent or clear material or a transparent or clear non-conductive material. In other embodiments, the container cap 104 can be made in part from a translucent or see-through material. For example, at least a portion of the wick component 134 can be visible through the side of the container cap 104. This allows a user or operator of the sensor device 100 to observe the suction of the contained sample 113 from the wick tip 142 to the wick base 140 when the container cap 104 is tightened onto the container chamber 102, ensuring that at least some of the contained sample 113 is reachable by the reference electrode material 132 located at the wick base 140. In some embodiments, the container cap 104 can be made in part from a clear or transparent polymer material, glass, or a combination thereof.

[0076]

[0091] In some embodiments, the container chamber 102, the container cap 104, or a combination thereof can be made in part from an inert polymer material. For example, the container chamber 102, the container cap 104, or a combination thereof can be made in part from at least one of polyoxymethylene, polyamide, polyethylene, acrylonitrile butadiene styrene, polycarbonate, or copolymers or composites thereof. In other embodiments, the container chamber 102, the container cap 104, or a combination thereof can be made in part from a glass material such as borosilicate glass or a ceramic material.

[0077]

[0092] In some embodiments, the active sensor 106 can also be insert-molded into a portion of the chamber sidewall 110 when the container chamber 102 is made of a polymer material. For example, the active sensor 106 can be insert-molded into the chamber sidewall 110 while the container chamber 102 is formed by injection molding.

[0078]

[0093] If the active sensor 106 is insert-molded into a portion of the chamber side wall 110 of the container chamber 102, the active sensor 106 may have a side portion 122 enclosed by the polymer material used to make the chamber side wall 110.

[0079]

[0094] For example, the active sensor 106 can be insert-molded such that the active electrode layer 118 faces the chamber cavity 112 and the sample 113 contained in the chamber cavity 112 is brought into fluid contact with the active electrode layer 118 through at least a portion of the chamber side wall 110 surrounding the window opening 114.

[0080]

[0095] Figure 1D also shows that the side of the active sensor 106 opposite to the active electrode layer 118 can be used to contact the conductive contacts or conductive connections of the reader 200 (see, for example, Figures 2A to 2C). As will be discussed in more detail in the following sections, this side of the active sensor 106 can be called the conductive layer 160.

[0081]

[0096] In some embodiments, the conductive layer 160 may be a layer of gold. In other embodiments, the conductive layer 160 may be made of another type of conductive metal, such as platinum, nickel, copper, an alloy, or a combination thereof.

[0082]

[0097] Although not shown in the figures, this disclosure envisions that a portion of the chamber sidewall 110 surrounding the window opening 114 (for example, see Figures 1B to 1D for the location of the window opening 114) can be focally melted (e.g., by ultrasonic welding), and the active sensor 106 can be fixed or otherwise bonded to the chamber sidewall 110 by pressing the active sensor 106 into the molten portion of the chamber sidewall 110. Once the molten portion of the chamber sidewall 110 cools, the active sensor 106 is fixed or bonded to the chamber sidewall 110.

[0083]

[0098] In some embodiments, the active sensor 106 may be substantially shaped as a flattened or truncated rectangular prism. In other embodiments, the active sensor 106 may be substantially shaped as a disc or a flattened or truncated polygonal prism (e.g., a flattened or truncated pentagonal or hexagonal prism).

[0084]

[0099] If the active sensor 106 is substantially shaped like a rectangular prism, it may have a sensor length dimension, a sensor width dimension, and a sensor height dimension. In some embodiments, the sensor length dimension may be about 100 μm to 6.0 mm, the sensor width dimension may be about 100 μm to 6.0 mm, and the sensor height dimension may be about 10 μm to 0.70 mm. For example, if the active sensor 106 is substantially shaped like a rectangular prism, it may have a sensor length dimension of about 6.0 mm, a sensor width dimension of about 6.0 mm, and a sensor height dimension of about 0.61 mm.

[0085]

[0100] In some embodiments, the active sensor 106 may have an active electrode layer 118 made of a precious metal. For example, the active electrode layer 118 may be made of platinum, gold, or a combination or composite thereof.

[0086]

[0101] The active electrode layer 118 can be bonded to one surface of the conductive substrate via an adhesive layer. The conductive substrate can be made of a conductive material such as stainless steel (SS). For example, the conductive substrate can be SS316. In other embodiments, the conductive substrate can be made of aluminum, copper, or any combination or composite of aluminum, copper, or stainless steel.

[0087]

[0102] In some embodiments, the adhesive layer may be a thin layer of chromium (Cr). Alternatively, the adhesive layer may be a thin layer of gold, nickel, titanium, or tantalum. The adhesive layer may be disposed between the conductive substrate and the active electrode layer 118.

[0088]

[0103] In an alternative embodiment, the active electrode layer 118 can be directly disposed on one surface of the conductive substrate without an adhesive layer.

[0089]

[0104] The active electrode layer 118 can have an active electrode layer thickness of approximately 50 nm to approximately 500 nm (for example, approximately 400 nm). The adhesive layer can have an adhesive layer thickness of approximately 5 nm to approximately 50 nm (for example, approximately 20 nm). The ratio of the adhesive layer thickness to the active electrode layer thickness can be approximately 1:10 to approximately 1:20.

[0090]

[0105] The conductive substrate can have a substrate layer thickness. The substrate layer thickness can be approximately 10 μm to approximately 0.70 mm (for example, approximately 0.61 mm).

[0091]

[0106] In other embodiments, the active electrode layer 118 can be made of a metal oxide. For example, the active electrode layer 118 can be made of tantalum pentoxide (Ta2O5). In other embodiments, the active electrode layer 118 can be made of silicon dioxide (SiO2), silicon nitride (Si3N4), aluminum oxide (Al2O3), titanium dioxide (TiO2), hafnium dioxide (HfO2), iridium dioxide (IrO2), ruthenium dioxide (RuO2), zirconium dioxide (ZrO2), or a combination or composite thereof. In these embodiments, the conductive substrate can be made of a conductive material such as stainless steel (SS). For example, the conductive material can be SS316. The conductive material can also be made of aluminum, copper, or any combination or composite of aluminum, copper, or stainless steel.

[0092]

[0107] The deposited layers can be selected to achieve specific desired sensitivity or specificity for a particular sample. Other surface modification techniques, such as bio-functionalization using self-assembled monolayers (SAMs), antibodies, bound antibody fragments, bound aptamers, bound DNA, and plasma treatment, can also be employed to modify the surface properties of the deposited layers, and consequently their specificity and sensitivity.

[0093]

[0108] In certain embodiments, the active sensor 106 can take advantage of the miniaturization and efficiency of printed circuit board (PCB) manufacturing techniques. For example, the active sensor 106 can be made from a non-conductive PCB substrate partially covered with an active electrode layer 118. In some embodiments, the non-conductive PCB substrate can be made from polyimide. In other embodiments, the non-conductive PCB substrate can be made from a glass fiber reinforced epoxy laminate material such as FR4 composite material. In certain embodiments, the PCB substrate can be a flexible PCB material.

[0094]

[0109] In some embodiments, the active electrode layer 118 can be made of a precious metal. For example, the active electrode layer 118 can be made of platinum, gold, or a combination or composite thereof. Platinum or gold can be electrodeposited or sputter-deposited onto the PCB substrate.

[0095]

[0110] The active electrode layer 118 may have an active electrode layer thickness of at least 50 nm. In certain embodiments, the active electrode layer 118 may have an active electrode layer thickness of at least 400 nm. When the active electrode layer 118 is made of platinum, the active sensor 106 can be used to measure or monitor the ORP of a sample.

[0096]

[0111] In an alternative embodiment, a platinum layer deposited on a non-conductive PCB substrate can be modified using surface modification techniques to transform the platinum layer into a pH-sensitive layer. For example, the platinum layer can be oxidized using oxygen plasma treatment to create a platinum oxide (PtO2) layer. The resulting platinum oxide layer can respond to hydrogen ions and can be used as a pH-sensitive layer. In this embodiment, the active sensor 106 can be used to measure or monitor the pH of a sample.

[0097]

[0112] The PCB substrate can have conductive contacts or a conductive layer 160 patterned on the substrate surface opposite to the active electrode layer 118. In some embodiments, the conductive layer 160 may be a layer of gold. In other embodiments, the conductive layer 160 may be made of another type of conductive metal such as platinum, nickel, copper, or an alloy or composite thereof.

[0098]

[0113] In some embodiments, the active electrode layer 118 can be electrically coupled to the conductive layer 160 by one or more conductive vias. In one embodiment, the conductive vias can be made partially of copper or a copper alloy. In other embodiments, the conductive vias can be made of another type of conductive metal, such as gold.

[0099]

[0114] In some embodiments, each active sensor 106 may have at least one conductive via located in the center of the sensor package. In other embodiments, the conductive via may be located near the periphery or edge of the sensor package.

[0100]

[0115] Conductive vias can be formed by electroplating, deposition, or a combination thereof. Furthermore, additional features or patterns may be formed on the PCB substrate using standard PCB etching processes.

[0101]

[0116] Figure 2A shows one embodiment of a reader 200 configured to monitor or measure the solution properties (e.g., ORP or pH) of a sample 113 contained in a container chamber 102 of a sensor device 100. The reader 200 and sensor device 100 may be part of a system 301 (see, for example, Figure 3) that prepares an output sample of bacteria at a desired or target concentration (or within an acceptable error margin).

[0102]

[0117] The reader 200 may include a reader housing 202 configured to house certain functional components of the reader 200, including a main controller 208 (see, e.g., Figure 2B), a signal readout control unit 210 (see, e.g., Figure 2B), a thermal control module 212 (see, e.g., Figures 2B and 2C), and an aeration control module 214 (see, e.g., Figures 2B and 2C). The reader housing 202 may also expose a touchscreen display 204 configured to display certain information to the user, allowing the user to input commands and input a desired or target concentration 308 (see, e.g., Figure 3) to the reader 200. For example, the display 204 of the reader 200 may display a message or text indication to the user indicating that an output sample of the desired or target concentration (or within an acceptable margin of error) has been successfully prepared (i.e., the bacteria in the contained sample 113 have reached the desired or target concentration level, or have reached the desired or target concentration level within an acceptable margin of error). For example, the display 204 can also display a countdown timer that shows the user the remaining time until the output sample is ready (i.e., the remaining time until the bacteria in the contained sample 113 reach the desired or target concentration 308, or reach a concentration level within an acceptable error margin).

[0103]

[0118] The lid 206 or cover of the reader 200 can be opened or lifted to expose a container receiving space configured to accommodate or receive the sensor device 100 (the container receiving space is the space occupied by the sensor device 100 in Figure 2C).

[0104]

[0119] Figure 2B shows specific functional components of the reader 200 with the reader housing 202 removed for clarity. As shown in Figure 2B, the reader 200 may include a thermal control module 212 and an aeration control module 214. The thermal control module 212 may be configured to incubate the sensor device 100 filled with a sample. The thermal control module 212 can incubate the sensor device 100 by heating at least a portion of the sensor device 100 via a heating block 220 (see, for example, Figure 2C). In some embodiments, the heating block 220 may heat the side of the container chamber 102 opposite to the active sensor 106. In certain embodiments, the heating block 220 may partially surround or embrace the container chamber 102 to heat the sensor device 100.

[0105]

[0120] In some embodiments, the heating block 220 can be made partially of aluminum. In other embodiments, the heating block 220 can be made partially of another type of thermally conductive metal material.

[0106]

[0121] The sensor device 100 can be heated to an incubation temperature of approximately 30°C to approximately 40°C (for example, approximately 35°C ± 2°C). The sensor device 100 can be incubated for an incubation period. The incubation period can range from 15 minutes to more than 2 hours. The incubation period can be adjusted based on the type of bacteria suspended in the source sample.

[0107]

[0122] The thermal control module 212 can also be used to cool the contained sample 113 to a cooling temperature when the solution properties of the contained sample 113 (e.g., ORP or pH) change by a threshold amount indicating that the bacteria in the contained sample 113 have reached a desired or target concentration, or have reached a desired or target concentration level within an acceptable error margin. In other embodiments, the thermal control module 212 can be used to cool the contained sample 113 to a cooling temperature when the elapsed time reaches a specific time limit or time threshold. In some embodiments, the thermal control module 212 can cool the contained sample 113 in the sensor device 100 to a cooling temperature of about 4°C to about 25°C.

[0108]

[0123] When the bacteria in the contained sample 113 reach a desired or target concentration (or within an acceptable margin of error), the contained sample 113 in the sensor device 100 can be considered an output sample ready for further downstream testing (e.g., antibiotic susceptibility testing). In certain embodiments, the reader 200 may be equipped with an auditory component (e.g., a speaker) that generates an auditory signal (i.e., sounds an alarm) to notify the user or laboratory technician that the output sample is ready and ready for further downstream testing.

[0109]

[0124] In some embodiments, the thermal control module 212 can be controlled by the main controller 208 of the reader 200. In other embodiments, the thermal control module 212 can be controlled by another controller or module within the reader 200 or by the signal readout control unit 210.

[0110]

[0125] In some embodiments, a nutrient solution or stimulant can be introduced into the container chamber 102 before the sensor device 100 is incubated. For example, the nutrient solution may be a solution containing bacto-tryptone, yeast extract, beef extract, cation-modified Müller-Hinton broth (CAMHB), starch, caseic acid hydrolysate, calcium chloride, magnesium chloride, sodium chloride, blood or hemolysis containing horse hemolysis (LHB), a CAMHB-LHB mixture, glucose, or a combination thereof. The nutrient solution can be used to counteract the buffering effect of ions or substances present in the sample if the sample consists of body fluids.

[0111]

[0126] The aeration control module 214 can be configured to aerate the sample 113 contained in the container chamber 102 by supplying gas 150 (e.g., ambient air, see Figure 1D) into the chamber cavity 112. The gas 150 can be supplied into the container chamber 102 through aeration ports 146 defined along the bottom or base of the container chamber 102 (see also Figures 1B and 1D).

[0112]

[0127] As described above, the container cap 104 of the sensor device 100 (which functions as part of the reference sensor 108) can be fixed to or coupled to the container chamber 102 by a threaded connector 154 that allows a portion of the airflow path 156 to be created between the container cap 104 and the threads of the container chamber 102. The gas 150 (e.g., ambient air) enters the container chamber 102 through the first gas-permeable membrane 148 to the aeration port 146, and then first aerates the contained sample 113, and then exits the container chamber 102 through the second gas-permeable membrane 152 and the air gap 158 defined between the threads of the container cap 104 and the container chamber 102.

[0113]

[0128] Figure 2C shows that the leader 200 may be equipped with a gas nozzle 222 connected to the lower part of the sensor device 100, which can aerate the contained sample 113 in the container chamber 102. The gas nozzle 222 may be located at the end or tip of the gas transport conduit 224. The gas transport conduit 224 can connect the gas nozzle 222 to the aeration control module 214. In some embodiments, at least a portion of the gas transport conduit 224 may be located along the perimeter of the base or lower part of the leader 200, or may be wound around it.

[0114]

[0129] In some embodiments, the aeration control module 214 may include one or more filters (e.g., inline filters, conduit filters, pipe filters, and / or hose filters) for filtering the ambient air drawn into the aeration control module 214. In additional embodiments, the gas transport conduit 224 may include an inline filter configured to filter the ambient air and remove particulate matter from the ambient air before it reaches the sensor device 100 and / or the gas nozzle 222.

[0115]

[0130] As shown in Figure 2C, the gas nozzle 222 can be connected to the aeration port 146 at the bottom of the container chamber 102 via the nozzle interface 226. In some embodiments, the nozzle interface 226 can be an O-ring. In other embodiments, the nozzle interface 226 can be another type of gasket or fluid sealing interface.

[0116]

[0131] In some embodiments, gas 150 can be ambient air (e.g., air in a laboratory, clinical setting, or testing facility). In other embodiments, gas 150 can include a combination of pressurized oxygen, carbon dioxide, nitrogen, and argon. Aeration of the sample can accelerate the growth of bacterial populations in the sample by providing an oxygen-rich environment within the container chamber 102.

[0117]

[0132] Aeration of the contained sample 113 can increase the oxygen supply to the bacteria, thereby accelerating the growth rate of such bacteria in the contained sample 113 within the sensor device 100. Furthermore, aeration of the contained sample 113 can also separate the bacteria from the inner wall of the container chamber 102, thereby preventing the formation of a biofilm.

[0118]

[0133] Aeration is important for accelerating the growth rate of bacteria in the sensor device 100, but it has also been found that excessive aeration or aeration of the contained sample 113 at high flow rates can have certain detrimental effects on the ORP signal monitored by the reader 200. For example, while aeration of the contained sample 113 can in most cases accelerate the growth rate of bacteria (especially aerobic bacteria) in the contained sample 113, excessive aeration can suppress the ORP signal and introduce errors into ORP measurement. Furthermore, excessive aeration can cause arbitrary changes in the ORP value (Δ ORP These particles may be too small to be of any value in distinguishing between different bacterial concentration levels.

[0119]

[0134] Furthermore, insufficient aeration or low flow rate aeration of the contained sample 113 may cause the contained sample 113 to become stagnant, and may also cause the sample preparation time to fall below the optimal level, i.e., to become slower.

[0120]

[0135] Therefore, the sample 113 contained in the sensor device 100 should be aerated at a flow rate within an optimal range to avoid the above-mentioned drawbacks. One such range, which may be found depending on the application, is a flow rate of 7.0 microliters (μL) / second / milliliter (mL) to 10.0 μL / second / mL for the sample 113. More specifically, the sample 113 can be aerated at a flow rate of approximately 8.8 (±0.9) μL / second / mL.

[0121]

[0136] In some embodiments, the contained sample 113 can be aerated using an electric piston pump. The electric piston pump can be housed or included within the leader 200. In certain embodiments, the electric piston pump can be completely housed or included within the leader 200. For example, the electric piston pump can be housed or included within the aeration control module 214.

[0122]

[0137] The electric piston pump can be operated by one or more stepper motors and lead screw drive units. The electric piston pump can be controlled by a dedicated controller, a main controller 208, or a combination thereof. In a more specific example, the electric piston pump may be a modification of the Cavro® Pulssar piston pump. In other embodiments, the contained sample 113 can be aerated using a syringe pump or an electric syringe pump.

[0123]

[0138] In some embodiments, the sample 113 contained within the sensor device 100 can be aerated according to an aeration cycle. The aeration cycle may include a non-aeration period after the aeration period during which no gas or ambient air is supplied to the container chamber 102. In certain embodiments, the aeration period may be longer than the non-aeration period. For example, the aeration period may be about 7 to 10 minutes, and the non-aeration period may be about 3 to 10 seconds.

[0124]

[0139] One technical problem that the present invention faces is that in electric piston pumps, when the pump piston reaches the tip of the pump chamber or barrel, the pump piston often needs to be retracted or returned to the home position. To address this technical problem, one technical solution discovered and developed by the present applicants is to use a non-aeration period to retract or return the pump piston to the home position.

[0125]

[0140] In some embodiments, the aeration control module 214 can be controlled by a main controller 208 (see, for example, Figure 2B). In other embodiments, the aeration control module 214 can be controlled by another controller or module in the reader 200 or by a signal readout control unit 210. For example, the amount of gas 150 (e.g., ambient air) supplied into or otherwise directed into the container chamber 102 is determined by changes in the solution properties (e.g., ORP or pH) of the contained sample 113 detected by the reader 200, or by the absence of any such changes.

[0126]

[0141] Figure 2C also shows that when the sensor device 100 is located within the container receiving space, the reference electrode contact 216 of the reader 200 can be positioned or moved to contact the reference electrode material 132 located on the container cap 104 of the sensor device 100 (see, for example, Figure 1D). Furthermore, when the sensor device 100 is located within the container receiving space, the active electrode contact 218 of the reader 200 can be positioned or moved to contact the conductive layer 160 (see, for example, Figures 1C and 1D) or conductive contact of the active sensor 106.

[0127]

[0142] In some embodiments, the reference electrode contact 216 and the active electrode contact 218 may include one or more conductive pogo or spring-biased pins, conductive leaf contacts, or combinations thereof. More specifically, the conductive pogo pins or leaf contacts may be made of copper, nickel, stainless steel, or an alloy thereof.

[0128]

[0143] The reference electrode contact 216 and the active electrode contact 218 can be electrically coupled to the signal readout control unit 210. The signal readout control unit 210 may include one or more processors, chipsets, or chip modules programmed to convert and read signals obtained from the active sensor 106 and the reference sensor 108 of the sensor device 100. For example, the signal readout control unit 210 can determine the ORP of the sample 113 contained in the sensor device 100 based on the potential difference measured between the active electrode layer 118 and the reference electrode material 132.

[0129]

[0144] The active electrode layer 118 is selected to readily interact with oxidized / reduced molecules in the containing sample 113 (i.e., without requiring an activation barrier or providing additional energy). The active electrode layer 118 is inert in that it does not participate in any redox reactions (e.g., a platinum or gold layer / material) and redox sensitive or redox active in that it functions as both an electron source and sink (in response to the redox state of the containing sample 113). The active electrode layer 118 is considered inert because the electron transfer process does not change the electrode material or its oxidation state. Electrons spontaneously move from the containing sample 113 to the active electrode layer 118 and from the active electrode layer 118 to the containing sample 113. A higher concentration of oxidizing molecules (positive ORP value) suggests that these molecules are more likely to accept electrons from the active electrode layer 118, while a higher concentration of reducing molecules (negative ORP value) suggests that these molecules are more likely to donate electrons to the active electrode layer 118. Therefore, ultimately, electrons are lost in the active electrode layer 118, leading to a positive ORP value, or electrons become excess in the active electrode layer 118, leading to a negative ORP value.

[0130]

[0145] During bacterial metabolism and growth, different redox-active species are generated. That is, bacterial growth and / or metabolic processes involve transformations that reduce oxidized molecules. As the amount of bacteria in the sample 113 increases, the concentration of reduced molecules / compounds increases. This lowers the ORP of the sample 113.

[0131]

[0146] As a more specific example, the amount of electron donors in the contained sample 113 (e.g., the amount of energy carriers such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2)) from Table 1 below may change due to bacterial growth in the contained sample 113.

[0132] [Table 1]

[0133]

[0147] The reference electrode material 132 is selected to maintain a constant potential throughout measurement / monitoring and to be unaffected by, or not participate in, any redox changes occurring in the contained sample 113.

[0134]

[0148] The potential difference measured between the active electrode layer 118 and the reference electrode material 132 is measured in an open-circuit configuration. That is, no current flows through the system, and the potential difference is measured using a very high impedance voltage measuring circuit or high impedance voltmeter integrated into the reader 200 (for example, integrated into the signal readout control unit 210).

[0135]

[0149] The ORP of sample 113 is measured without adding any reporter molecules or any redox mediators.

[0136]

[0150] Figure 3 shows one embodiment of a method 300 for preparing an output sample of bacteria 302 at a desired or target concentration 308 (or within an acceptable margin of error).

[0137]

[0151] Bacteria 302 can be a genus selected from the following group: Acinetobacter, Acetobacter, Actinomyces, Aerococcus, Aeromonas, Agrobacterium, Anaplasma, Azorhizobium, Azotobacter, Bacillus, Bacillus Bacteriodes, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Calymmatobacterium, Campylobacter, Chlamydia, Chlamydophila, Citrobacter, Clostridium stridium), Corynebacterium, Coxiella, Ehrlichia, Enterobacter, Enterococcus, Escherichia, Francisella, Fusobacterium, Gardnerella, Haemophilus, Helicobacter ter), Klebsiella, Lactobacillus, Legionella, Listeria, Methanobacterium, Microbacterium, Micrococcus, Morganella, Moraxella, Mycobacterium, Mycoplasma,Neisseria, Pandoraea, Pasteurella, Peptostreptococcus, Porphyromonas, Prevotella, Proteus, Providencia, Pseudomonas, Ralstonia, Raoultella, Rhizobium, Rickettsia, Rosalia Halimatophores (halimaea), Rothia, Salmonella, Serratia, Shewanella, Shigella, Spirillum, Staphylococcus, Strenotrophomonas, Streptococcus, Streptomyces, Treponema, Vibrio, Wolbachia, and Yersinia.

[0138]

[0152] More specifically, bacterium 302 can be a species consisting of the following groups: Acinetobacter baumannii, Actinobacillus spp., Actinomycetes, Actinomyces spp. (not limited to Actinomyces israelii and Actinomyces naeslundii), Aeromonas spp. (not limited to Aeromonas hydrophila, Aeromonas veronii biovar sobria, Aeromonas sobria), and Aeromonas caviar This includes species such as *Bacillus caviae*, *Anaplasma phagocytophilum*, *Alcaligenes xylosoxidans*, *Actinobacillus actinomycetemcomitans*, *Bacillus spp.* (not limited to *Bacillus anthracis*, *Bacillus cereus*, *Bacillus subtilis*, *Bacillus thuringiensis*, and *Bacillus stearothermophilus*), and *Bacteroides spp.* (not limited to *Bacteroides fragilis*). (including fragilis), Bartonella species (Bartonella spp.) (not limited to Bartonella bacilliformis and Bartonella henselae), Bifidobacterium species (Bifidobacterium spp.)), Bordetella spp. (not limited to Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), Borrelia spp. (not limited to Borrelia recurrentis and Borrelia burgdorferi), Brucella spp. (not limited to Brucella abortus, Brucella canis, Brucella melintensis, and Brucella suis), Burkholderia spp. (not limited to Brucella pseudomalley) This includes *Campylobacter pseudomallei* and *Burkholderia cepacia*, *Campylobacter* spp. (not limited to *Campylobacter jejuni*, *Campylobacter coli*, *Campylobacter lari*, and *Campylobacter fetus*), *Capnocytophaga* spp., *Cardiobacterium hominis*, *Chlamydia trachomatis*, *Chlamydophila pneumoniae*, and *Chlamydophila sitassi*. Citrobacter spp., Coxiella burnetii, Corynebacterium spp.)(not limited to Corynebacterium diphtheriae, Corynebacterium jeikeum, and Corynebacterium), Clostridium spp. (not limited to Clostridium perfringens, Clostridium difficile, Clostridium botulinum, and Clostridium tetani), Eikenella corrodens, Enterobacter spp. (not limited to Enterobacter aerogenes) This includes aerogenes, Enterobacter agglomerans, Enterobacter cloacae, and opportunistic Escherichia coli, and opportunistic Escherichia coli is not limited to enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enterogaggregative E. coli, and uropathogenic E. coli), Enterococcus spp.(Not limited to Enterococcus faecalis and Enterococcus faecium), Ehrlichia spp. (Not limited to Ehrlichia chafeensia and Ehrlichia canis), Erysipelothrix rhusiopathiae, Eubacterium spp., Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus Helicobacter species (spp.) (not limited to Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus, and Haemophilus parahaemolyticus), Helicobacter species (Helicobacter spp.) (not limited to Helicobacter pylori, Helicobacter cinaedi, and Helicobacter fennelliae), Kingella kingii, Klebsiella species (Klebsiella spp.)(Not limited to Klebsiella pneumoniae, Klebsiella granulomatis, and Klebsiella oxytoca), Lactobacillus spp., Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospira interrogans, Peptostreptococcus spp., Moraxella catarrhalis, Morganella spp., Mobiluncus Mycobacterium spp. (not limited to Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium bovis, and Mycobacterium marinum), Mycoplasma spp. (not limited to Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma genitalium) (including genitalium), Nocardia species (Nocardia spp.))(not limited to Nocardia asteroides, Nocardia cyriacigeorgica, and Nocardia brasiliensis), Neisseria spp. (not limited to Neisseria gonorrhoeae and Neisseria meningitidis), Pasteurella multocida, Plesiomonas shigelloides, Prevotella spp., Porphyromonas spp., Prevotella melaninogenica, Proteus spp. (not limited to Proteus vulgaris) (including Proteus vulgaris and Proteus mirabilis), Providencia spp. (not limited to Providencia alcalifaciens, Providencia rettgeri, and Providencia stuartii), Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcus equi, Rickettsia spp.)(not limited to Rickettsia rickettsii, Rickettsia akari, and Rickettsia prowazekii, Orientia tsutsugamushi (formerly Rickettsia tsutsugamushi), and Rickettsia typhi), Rhodococcus spp., Stenotrophomonas maltophilia, Salmonella spp. (not limited to Salmonella enterica, Salmonella typhi, Salmonella paratifida) (including paratyphi, Salmonella enteritidis, Salmonella cholerasuis, and Salmonella typhimurium), Serratia spp. (not limited to Serratia marcesans and Serratia liquifaciens), Shigella spp. (not limited to Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei), Staphylococcus spp.)(not limited to Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, Staphylococcus saprophyticus), Streptococcus species (Streptococcus spp.)(not limited to, Streptococcus pneumoniae) (e.g., chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin-resistant serotype 9V Streptococcus pneumoniae) Streptococcus pneumoniae), erythromycin-resistant serotype 14 Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, tetracycline-resistant serotype 19F Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus (Streptococcus pneumoniae) and trimethoprim-resistant serotype 23F pneumococcus p. Streptococcus pneumoniae, chloramphenicol-resistant serotype 4, spectinomycin-resistant serotype 6B, streptomycin-resistant serotype 9V, optochin-resistant serotype 14, rifampicin-resistant serotype 18C, penicillin-resistant serotype 19F Streptococcus pneumoniae) or trimethoprim-resistant serotype 23F Streptococcus pneumoniae), Streptococcus agalactiae, Streptococcus mutans, Group A streptococcus (Streptococcus pyogenes), Group B streptococcus (Streptococcus pyogenes), Group C streptococcus (Streptococcus agalactiae), Streptococcus anginosus, Group D streptococcus (Streptococcus equismilis,Group D Streptococci), Streptococcus bovis, Group F Streptococci, and Group G Streptococcus anginosusGroup G (including Streptococci), Spirillum minus, Streptobacillus moniliformi, Treponema spp. (not limited to Treponema carateum, Treponema petenue, Treponema pallidum, and Treponema endemicum), Tropheryma whippelii, Ureaplasma urealyticum, Veillonella spp., Vibrio spp. (not limited to Vibrio cholerae) This includes Vibrio cholerae, Vibrio parahemolyticus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis, Vibrio metchnikovii, Vibrio damsela, and Vibrio furnisii, Xanthomonas maltophilia, and Yersinia species. (spp.) (Not limited to Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis).

[0139]

[0153] Method 300 can be used to prepare an output sample of bacterium 302 at a desired or target concentration (or within an acceptable margin of error) if bacterium 302 is an obligate aerobic or obligate aerobic bacterium. Method 300 can also be used to prepare an output sample of bacterium 302 at a desired or target concentration (or within an acceptable margin of error) if bacterium 302 is a facultative anaerobic bacterium. Method 300 can further be used to prepare an output sample of bacterium 302 at a desired or target concentration (or within an acceptable margin of error) if bacterium 302 is a Gram-negative bacterium.

[0140]

[0154] One unexpected finding by the applicants is that the methods and systems disclosed herein work particularly well for preparing desired or targeted (or within an acceptable margin of error) output samples from source samples containing bacteria classified or considered to be obligate aerobic or obligate aerobic. For example, in certain species of obligate or obligate aerobic bacteria such as Acinetobacter baumannii (ABa) and Pseudomonas aeruginosa (PAe), the methods disclosed herein showed a significant reduction in sample preparation time for such obligate or obligate aerobic bacteria compared to methods without aeration.

[0141]

[0155] Another unexpected finding by the applicants is that the methods and systems disclosed herein work particularly well for preparing desired or targeted (or within an acceptable margin of error) output samples from source samples containing bacteria classified or considered to be facultative anaerobic bacteria, such as Escherichia coli (ECo), Serratia marcescens (SMa), and Proteus mirabilis (PMi). For example, with certain species of facultative anaerobic bacteria, the methods disclosed herein showed a significant reduction in sample preparation time compared to methods without aeration.

[0142]

[0156] Another unexpected discovery by the applicants is that the methods and systems disclosed herein work particularly well for preparing desired or targeted (or within an acceptable margin of error) output samples from source samples containing bacteria classified or considered to be facultative anaerobes or obligate / obligate aerobes. For example, the applicants have found that the method disclosed herein works particularly well for preparing desired or targeted output samples within an acceptable margin of error from source samples containing the following Gram-negative bacterial species: ECo, SMa, PMi, Proteous vulgaris (PVu), ABa, PAe, Klebsiella pneumoniae (KPn), Enterobacter cloacae (ECl), Klebsiella oxytoca (KOx), Klebsiella aerogenes (KAe), Citrobacter braakii (CBr), Citrobacter freundii (CFr), and Citrobacter coseri koseri)(CKo).

[0143]

[0157] In alternative embodiments, the method 300, device, and system 301 disclosed herein may also be used to prepare output samples of mold or fungi at desired or target concentrations (or within an acceptable margin of error). The fungi may be genera selected from the group consisting of: Candida and Cryptococcus.More specifically, fungi can be species selected from the following groups: Candida spp. (not limited to Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei), Aspergillus spp. (not limited to Aspergillus fumigatous, Aspergillus flavus, and Aspergillus clavatus), and Cryptococcous spp. (not limited to Cryptococcus neoformans) (including Neoformans, Cryptococcus gattii, Cryptococcus laurentii, and Cryptococcus albidus), Fusarium spp. (not limited to Fusarium oxysporum, Fusarium solani, Fusarium verticillioides, and Fusarium proliferatum), Rhizopus oryzae, Penicillium marneffei, Coccidiodes imithis immitis, as well as Blastomyces dermatitidis.

[0144]

[0158] Method 300 may include, in step 300A, diluting an aliquot of the source sample containing bacteria 302 by a dilution factor. As described above, the source sample may be obtained from a patient or subject. In some embodiments, the source sample may be obtained from a human patient or subject. In other embodiments, the source sample may be obtained from a non-human animal patient or test subject. In certain embodiments, the source sample may include body fluids or bacterial cultures derived therefrom collected, extracted, or otherwise obtained from a patient or subject. Body fluids may be at least one of blood, urine, serum, plasma, saliva, sputum, semen, breast milk, synovial fluid, cerebrospinal fluid such as joint fluid, wound material, mucus, feces with fluid, vaginal secretions, synovial fluid, pleural fluid, ascites, pericardial fluid, and amniotic fluid. For example, the source sample may be a bacterial culture derived from or resuspended from body fluids (or swabs) obtained from a patient or subject tested positive for bacterial growth. More specifically, the source sample can be a positive blood culture medium (PBC).

[0145]

[0159] In additional embodiments not shown in Figure 3, the source sample may be filtered before step 300A. This filtering step may include filtering the source sample using a laboratory filter, benchtop filter, medical filter, microfluidic filter, syringe filter, blood filter, urine filter, or a combination thereof, to filter out debris, inorganic materials, and larger cellular components, including blood cells or epithelial cells, from the source sample.

[0146]

[0160] Aliquots of the source sample can be diluted using diluent 306. Diluent 306 may contain a growth medium (e.g., bacterial growth medium) or a growth inducer. In some embodiments, diluent 306 may be a solution containing cation-modified Müller-Hinton broth (CAMHB), glucose-supplemented Müller-Hinton broth (MHG), CAMHB-LHB mixture, bacto-tryptone, trypsin-containing soybean digest, yeast extract, beef extract, starch, caseic acid hydrolysate, glucose or other carbohydrates, or a combination thereof. The growth inducer may contain carbon-based inducers, nitrogen-based inducers, minerals, trace elements, biological growth factors, or any combination thereof. For example, the growth inducer may contain, but is not limited to, carbohydrates such as glucose or starch, ammonia, magnesium, amino acids, casamino acids, vitamins, peptides, blood, or a combination thereof. In one exemplary embodiment, diluent 306 may contain tryptone, yeast extract, sodium chloride, starch, water, and glucose.

[0147]

[0161] The dilution ratio can be approximately 1:1 to approximately 1:100 or approximately 1:1 to approximately 1:1000. In some embodiments, the dilution ratio can be approximately 1:10 to approximately 1:100. More specifically, the dilution ratio can be approximately 1:10 to approximately 1:50. For example, if the source sample is a positive blood culture, the dilution ratio can be approximately 1:30. As a more specific example, 30 μL of the source sample can be diluted in 1 mL of diluent 306.

[0148]

[0162] Figure 3 shows that in step 300A, only one aliquot of the source sample is diluted. However, it is intended by this disclosure that additional aliquots of the source sample can be diluted at the same or different dilution ratios to produce additional diluted samples 304 (e.g., a second diluted sample, a third diluted sample, a fourth diluted sample, etc.). These additional diluted samples 304 can be used to produce an internal control or redundant sample.

[0149]

[0163] Method 300 may also include, in step 300B, introducing an aliquot of the diluted sample 304 containing bacteria 302 into the container chamber 102 of the sensor device 100. The amount of diluted sample 304 introduced may depend on the volume of the chamber cavity 112 of the container chamber 102 (see, for example, Figure 1). For example, a 1 mL aliquot of diluted sample 304 may be introduced into the container chamber 102 of the sensor device 100.

[0150]

[0164] Aliquots of the diluted sample 304 in the container chamber 102 can be in fluid communication with both the active sensor 106 and the reference sensor 108 of the sensor device 100. In this disclosure, aliquots of the diluted sample 304 in the container chamber 102 are referred to as the contained sample 113.

[0151]

[0165] As described above, the active sensor 106 can be coupled to at least a portion of the chamber side wall 110 of the container chamber 102. The active sensor 106 may also include an active electrode material or active electrode layer 118 facing the cavity 112 such that the contained sample 113 is in fluid contact with the active electrode material or active electrode layer 118 when the contained sample 113 fills the chamber cavity 112 of the container chamber 102.

[0152]

[0166] Furthermore, as described above, the reference sensor 108 may include a reference electrode material 132 and a wick or wick component 134 that are in fluid communication with the chamber cavity 112 of the container chamber 102. When the container chamber 102 is filled with the contained sample 113, at least some of the contained sample 113 in the container chamber 102 can be drawn up, absorbed, or otherwise drawn up by at least a portion of the wick component 134 in the direction of the wick base end 140. Since the reference electrode material 132 is positioned at the wick base end 140 (see, for example, Figure 1D), the contained sample 113 can come into fluid contact with the reference electrode material 132 via the wick component 134.

[0153]

[0167] Method 300 may further include, in step 300C, placing the assembled sensor device 100 (which is assembled when the container cap 104 is tightened onto the container chamber 102 filled with the sample) into the container receiving space of the reader 200. For example, the user can lift the lid 206 of the reader 200 and insert the assembled sensor device 100 into the container receiving space of the reader 200.

[0154]

[0168] As described above, when the sensor device 100 is located within the container receiving space, the reference electrode contact 216 of the reader 200 (see, for example, Figure 2C) can electrically contact, or move to electrically contact, the reference electrode material 132 located on the container cap 104 of the sensor device 100 (see, for example, Figure 1D). Furthermore, when the sensor device 100 is located within the container receiving space, the active electrode contact 218 of the reader 200 can electrically contact, or move to electrically contact, the conductive layer 160 (see, for example, Figures 1C, 1D, and 2C) or conductive contact of the active sensor 106. In this way, both the active sensor 106 and the reference sensor 108 can be electrically coupled to the reader 200.

[0155]

[0169] The reference electrode contact 216 and the active electrode contact 218 can be electrically coupled to a signal readout control unit 210 (see, for example, Figure 2B). The signal readout control unit 210 may include one or more processors, chipsets, or chip modules programmed to convert and read signals obtained from the active sensor 106 and reference sensor 108 of the sensor device 100. For example, the signal readout control unit 210 may determine the ORP of the sample 113 contained in the sensor device 100 based on the potential difference measured between the active electrode layer 118 and the reference electrode material 132.

[0156]

[0170] At this point, a user of the reader 200 (e.g., a laboratory technician or clinician) can input a desired or target concentration 308 into the reader 200. For example, the user can select a preset bacterial concentration level or input a desired or target concentration 308 by applying a specific touch input to the display 204 of the reader 200. Alternatively, the user can input the desired or target concentration 308 via a keyboard or other type of input device communicatively coupled to the reader 200. Alternatively, the user can input the desired or target concentration 308 into a computing device 310 (e.g., a tablet or laptop) communicatively coupled to the reader 200. The computing device 310 can transmit the desired or target concentration 308 to the reader 200 via a wireless communication protocol or a wired connection.

[0157]

[0171] The desired or target concentration 308 may be a bacterial concentration level required as part of a downstream test protocol, such as an antimicrobial agent or antibiotic susceptibility test (AST). In certain embodiments, the desired or target concentration 308 may be expressed or indicated as colony-forming units (CFU) per mL. In other embodiments, the desired or target concentration 308 may be expressed or indicated in relation to the MacFarland standard (e.g., 0.5 MacFarland, 1.0 MacFarland, 2.0 MacFarland, etc.).

[0158]

[0172] In some embodiments, the desired or target concentration 308 is approximately 1.4 × 10⁻⁶ 8 CFU / mL ~ 1.6 × 10 8 It can be CFU / mL. For example, the desired or target concentration of 308 is approximately 1.5 × 10⁻⁶. 8 The concentration can be CFU / mL (also known as the 0.5 MacFarland standard). In other embodiments, the desired or target concentration 308 is 1.6 × 10⁻⁶. 8 CFU / mL or more than 1.4 × 10 8 It can be less than CFU / mL.

[0159]

[0173] In some embodiments, the user may also input specific information regarding the classification (e.g., genus, family, or order) or characteristics of the bacterium 302. For example, the user may perform a Gram stain test on the bacterium 302 before introducing the diluted sample 304 into the sensor device 100. The user can then input to the reader 200 whether the bacterium 302 is Gram-positive or Gram-negative based on the Gram stain test. In some cases, the reader 200 may retrieve one or more lookup tables (LUTs) that match the classification or characteristics of the bacterium 302 provided by the user.

[0160]

[0174] Method 300 may also include incubating and aerating the contained sample 113 in step 300D. In some embodiments, the contained sample 113 in the sensor device 100 may undergo incubation and aeration simultaneously. In other embodiments, the contained sample 113 in the sensor device 100 may begin an incubation period without first aeration, or begin an aeration period without first incubation. In all such embodiments, there may be a period in which the contained sample 113 in the sensor device 100 undergoes both incubation and aeration.

[0161]

[0175] The sample 113 contained within the sensor device 100 can be incubated at an incubation temperature. In some embodiments, the incubation temperature can be about 30°C to about 40°C (e.g., about 35°C ± 2°C). In other embodiments, the incubation temperature can be about 25°C to about 30°C. As described above, the sensor device 100 containing the sample 113 can be incubated while housed in the reader 200. For example, the thermal control module 212 of the reader 200 can control the incubation of the sample-filled sensor device 100. The reader 200 can incubate the sensor device 100 by heating at least a portion of the sensor device 100 via a heating block 220 (see, for example, Figure 2C). In some embodiments, the heating block 220 can heat the side of the container chamber 102 opposite to the active sensor 106. In certain embodiments, the heating block 220 can heat a portion of the lower part or base of the container chamber 102, or partially surround or embrace the container chamber 102 to heat the sensor device 100.

[0162]

[0176] The sample 113 contained in the sensor device 100 can be aerated at an aeration flow rate or gas dispense rate. The aeration flow rate can range from 7.0 microliters (μL) / second / milliliter (mL) to 10.0 μL / second / mL of the sample 113. More specifically, the sample 113 contained in the sensor device 100 can be aerated at a flow rate of approximately 8.8 (±0.9) μL / second / mL of the sample 113. As described above, the sample 113 can be aerated using an electric piston pump. The electric piston pump can be housed or included in the leader 200. Aeration of the sample 113 can be controlled by the aeration control module 214 of the leader 200.

[0163]

[0177] In some embodiments, the sample 113 contained within the sensor device 100 can be aerated according to an aeration cycle. The aeration cycle may include a non-aeration period after the aeration period during which no gas or ambient air is supplied to the container chamber 102 (see, for example, Figure 2B).

[0164]

[0178] In certain embodiments, the aeration period can be longer than the non-aeration period. For example, the aeration period can be about 7 to 10 minutes, and the non-aeration period can be about 3 to 10 seconds. As a more specific example, the sample 113 contained in the container chamber 102 can be repeatedly aerated for about 8 minutes at a flow rate or dispensing rate of about 10.0 μL / sec / mL, followed by a non-aeration period of about 5 seconds.

[0165]

[0179] Method 300 may also include, in step 300E, monitoring the ORP change of the contained sample 113 in the sensor device 100. The ORP of the contained sample 113 can be monitored or measured as soon as the sensor device 100 is positioned in the reader 200 and the user inputs a desired or target concentration 308. The ORP of the contained sample 113 can be monitored or measured during the incubation period and / or aeration period.

[0166]

[0180] As described above, in some embodiments, the signal readout control unit 210 of the reader 200 can monitor the ORP of the contained sample 113 in the container chamber 102 of the sensor device 100. For example, as part of the ORP monitoring process, the ORP of the contained sample 113 can be sampled or identified multiple times per second, and such ORP values ​​can be recorded together with the elapsed time 313. The reader 200 can display the ORP change as a function of the elapsed time 313 as an ORP growth curve 311. In a more specific example, the ORP growth curve 311 can be rendered and displayed to the user via the display 204 of the reader 200 or via the display of a computing device 310 communicably coupled to the reader 200. As the amount of bacteria in the contained sample 113 increases (bacterial concentration increases), the amount of reducing molecules / compounds in the contained sample 113 also increases. This causes the ORP of the contained sample 113 to decrease or the ORP value to become more negative.

[0167]

[0181] Method 300 may further include, in step 300F, retrieving a species-independent lookup table (LUT) 312 from a database. The species-independent LUT 312 may be retrieved in response to the user entering a desired or target concentration 308. In other embodiments, the species-independent LUT 312 may be retrieved when the ORP of the contained sample 113 is started to be monitored by the reader 200. For example, one or more processors of the reader 200 may be programmed to retrieve the species-independent LUT 312 from the memory or storage unit of the reader 200. In other embodiments, one or more processors of the reader 200 may be programmed to retrieve the species-independent LUT 312 from a database stored in the computing device 310 or from a cloud database.

[0168]

[0182] The species-independent LUT 312 may include multiple species-independent ORP change amounts 314 and species-independent bacterial concentrations 316. Each species-independent bacterial concentration 316 may have a species-independent ORP change amount 314 associated with the species-independent bacterial concentration 316.

[0169]

[0183] A species-independent LUT 312 can be constructed or generated from multiple species-specific LUTs 406 and / or lineage-specific LUTs (see, for example, Figure 4). For example, the various independent ORP changes 314 and various independent bacterial concentrations 316 can each be the average of multiple ORP changes or bacterial concentrations across multiple LUTs. The construction or generation of a species-independent LUT 312 from species-specific LUTs 406 and / or lineage-specific LUTs 404 will be discussed in more detail in a later section.

[0170]

[0184] Method 300 may further include determining in step 300G whether the desired or target concentration 308 is included in the species-independent LUT 312. For example, one or more processors of the reader 200 may query the bacterial concentration field (i.e., species-independent bacterial concentration 316) in the species-independent LUT 312 using the desired or target concentration 308 entered by the user. If one or more processors of the reader 200 determine that the desired or target concentration 308 is included in the species-independent LUT 312, then in step 300H, one or more processors of the reader 200 may select one of the species-independent ORP change amounts 314 as the threshold ORP change amount 318 when one of the species-independent ORP change amounts 314 is associated with one of the species-independent bacterial concentrations 316 that is equal to or substantially equal to the desired or target concentration 308. In this way, the leader 200 relies primarily on the species-independent LUT 312 to set the threshold ORP change amount 318.

[0171]

[0185] However, if one or more processors of the reader 200 determine that the desired or target concentration 308 is not included in the species-independent LUT 312, then in step 300I, one or more processors of the reader 200 will reduce the time to the target concentration (t target )320 can be calculated. The calculation of the time to target concentration 320 will be discussed in more detail in a later section.

[0172]

[0186] In certain embodiments, one or more processors of the reader 200 may choose to calculate the time to target concentration 320 even if the desired or target concentration 308 is included in the species-independent LUT 312. For example, one or more processors of the reader 200 may choose this calculation based on a specific heuristic or pre-set rule that determines when a threshold ORP change 318 from the species-independent LUT 312 may be considered too high / too large or potentially error-prone. In this case, one or more processors of the reader 200 may decide to calculate the time to target concentration 320 rather than relying on a specific ORP value (i.e., species-independent ORP change 314) from the species-independent LUT 312. For example, one or more processors of the reader 200 may determine, based on real-time or near-real-time analysis of the behavior of the ORP growth curve 311 (e.g., whether the monitored ORP signal is beginning to flatten), that a specific smaller species-independent ORP change 314 is more accurate or less error-prone. In this case, one or more processors of the reader 200 may determine that a specific, smaller, species-independent ORP change 314 from the species-independent LUT 312 is more reasonable or less prone to error, and may use such an ORP change when calculating the time 320 to the target concentration.

[0173]

[0187] Method 300 may also include determining in step 300J that the bacteria in the contained sample 113 have reached a desired or target concentration 308 (or have reached a desired or target concentration 308 within an acceptable margin of error). For example, one or more processors of the reader 200 may determine that the bacteria in the contained sample 113 have reached a desired or target concentration 308 (or have reached a desired or target concentration 308 within an acceptable margin of error) if the ORP change of the contained sample 113, which is being monitored by the reader 200 in real time or near real time, has reached a threshold ORP change 318 (or has reached a threshold ORP change 318 within an acceptable margin of error) (see also step 300H) or if the elapsed time 313 has reached a calculated time to the target concentration 320 (see also step 300I).

[0174]

[0188] Method 300 may further include cooling the contained sample 113 in the sensor device 100 if, in step 300K, it is determined that the concentration of bacteria in the contained sample 113 has reached a desired or target concentration 308 (or has reached a desired or target concentration 308 within an acceptable margin of error). The contained sample 113 can be cooled to a cooling temperature of approximately 4°C to approximately 25°C. The sensor device 100 can be cooled within the reader 200. For example, a thermal control module 212 can also be used to cool the contained sample 113 to approximately 4°C to approximately 25°C. Cooling the contained sample 113 is necessary to prevent the bacteria in the contained sample 113 from continuing to grow or to prevent the bacterial concentration from rising further.

[0175]

[0189] Step 300K may also include the reader 200 alerting the user that the bacteria in the contained sample 113 have reached a desired or target concentration 308 (or within an acceptable margin of error) and that the output sample is now ready for downstream testing. For example, the reader 200 may be equipped with a speaker which can generate an audible alert or sound an alarm to notify the user that the bacteria in the contained sample 113 have reached a desired or target concentration 308 (or within an acceptable margin of error). In an additional embodiment, the reader 200 may render a visual or graphic alert via the display 204 to notify the user that the bacteria in the contained sample 113 have reached a desired or target concentration 308 (or within an acceptable margin of error) and that the output sample is now ready for downstream testing.

[0176]

[0190] As shown in Figure 3, a laboratory technician or clinician can use Method 300 to prepare an output sample of a desired or target concentration 308 (or within an acceptable margin of error) without any prior knowledge of the bacterial species in the containing sample 113 or without the need to identify the bacterial species in the containing sample 113. Since the laboratory technician or clinician no longer needs to subject the source sample or containing sample 113 to a separate species identification protocol, this can significantly reduce sample preparation time or the amount of human effort required to prepare the output sample.

[0177]

[0191] The method steps shown in Figure 3 do not require a specific order to achieve the desired result. Furthermore, certain steps or processes may be omitted or performed in parallel to achieve the desired result. In addition, other devices or apparatus may be used instead of the devices or apparatus shown in Figure 3.

[0178]

[0192] Figure 4 shows that a species-independent LUT 312 can be generated from multiple component LUTs 402. In some embodiments, a species-independent LUT 312 can be generated from at least three component LUTs 402. For example, a species-independent LUT 312 can be generated from five to eight component LUTs 402. In other embodiments, a species-independent LUT 312 can be generated from nine or more component LUTs 402.

[0179]

[0193] Each component LUT402 can be either a lineage-specific LUT404 or a species-specific LUT406. A species-specific LUT406 can be generated from multiple lineage-specific LUT404 containing bacteria of the same species. Each lineage-specific LUT404 can be compiled using ORP and bacterial concentration measurements measured simultaneously with a reference bacterial sample 408.

[0180]

[0194] In some embodiments, a species-independent LUT 312 can be generated from multiple (at least three) lineage-specific LUTs 404. In other embodiments, a species-independent LUT 312 can be generated from multiple (at least three) lineage-specific LUTs 406. In additional embodiments, a species-independent LUT 312 can be generated from a mixture of lineage-specific LUTs 404 and lineage-specific LUTs 406.

[0181]

[0195] For example, at least three component LUT402 may include a first LUT, a second LUT, and a third LUT. Each of the first, second, and third LUTs may be either a lineage-specific LUT404 or a species-specific LUT406. The first, second, and third LUTs may be generated using simultaneous ORP and bacterial concentration measurements performed or measured on a first reference bacterial sample, a second reference bacterial sample, and a third reference bacterial sample. The first reference bacterial sample may contain bacteria of a first species, the second reference bacterial sample may contain bacteria of a second species different from the first species, and the third reference bacterial sample may contain bacteria of a third species different from both the first and second species.

[0182]

[0196] Each component LUT 402 may include a component LUT ORP change 410 and a component LUT bacterial concentration 412. In some embodiments, the component ORP change 410 may be the same as the species-independent ORP change 314. In these embodiments, the component LUT bacterial concentration 412 associated with each component ORP change 410 can be averaged across multiple component LUTs 402, thereby obtaining a species-independent bacterial concentration 316.

[0183]

[0197] For example, Figure 5 shows a species-independent LUT 312 generated from six lineage-specific LUTs 404. More specifically, the six lineage-specific LUTs 404 could include LUTs representing strains ECo PSC-91, KPn PSC-38, ABa UCLA-126, PAe PSC-30, PVu UCLA-32, and SMa CDC-91. The average of the lineage bacterial concentrations (or various component LUT bacterial concentrations 412) across the six LUTs is calculated to obtain each of the species-independent bacterial concentrations 316 included as part of the species-independent LUT 312.

[0184]

[0198] Although not shown in Figure 5, this disclosure intends that species-independent LUT312 can also be produced from multiple species-specific LUT406 or mixtures of species-specific LUT406 and lineage-specific LUT404. For example, a species-specific LUT406 for SMa can be produced from multiple lineage-specific LUT404 for SMa, including LUTs representing SMa CDC-27, SMa CDC-91, SMa CDC-99, SMa CDC-121, SMa CDC-122, SMa CDC-130, or combinations thereof. As another example, a species-specific LUT406 for Staphylococcus aureus (SAu) can also be produced from multiple lineage-specific LUT404 for SAu, including LUTs representing SAu wild strain, SAu CDC-483, SAu CDC-475, SAu ATCC43300, or combinations thereof.

[0185]

[0199] Referring again to Figure 4, the method 400 for producing species-independent LUT312 can be started by preparing at least three reference bacterial samples 408. Then, at least three component LUT402 can be prepared using at least three reference bacterial samples 408.

[0186]

[0200] In some embodiments, six to eight reference bacterial samples 408 can be prepared. In other embodiments, nine or more reference bacterial samples 408 can be prepared. The accuracy of the LUT (including either species-specific LUT 406 or species-independent LUT 312) can be improved or enhanced when more reference bacterial samples 408 are used to generate such LUTs.

[0187]

[0201] Reference bacterial samples 408 can be prepared by resuspending plated colonies of known bacterial species and / or strains in a liquid growth medium such as diluent 306. Aliquots (e.g., 1 mL) of the resuspended bacterial sample can then be introduced into an instance of the sensor device 100. As shown in Figure 4, each reference bacterial sample 408 can be introduced into its own sensor device 100. Reference bacterial samples 408 can also be prepared so that each sample contains the same initial concentration of bacteria. For example, the initial concentration of bacteria in each reference bacterial sample 408 is approximately 1 × 10⁻⁶. 7 (1e7) CFU / mL or 5 × 10 7 It can be (5e7) CFU / mL.

[0188]

[0202] The ORP of each reference bacterial sample 408 can be monitored by the reader 200. For example, a sensor device 100 containing the reference bacterial sample 408 can be placed in the container receiving space of the reader 200, and the reader 200 can be programmed to monitor the ORP changes of the reference bacterial sample 408 over a certain period of time. Simultaneously with this monitoring, the optical density (OD) of the reference bacterial sample 408 can also be measured at specific time intervals 414. For example, the specific time interval 414 can be every few minutes, for example, every 15 minutes. In other embodiments, the specific time interval 414 can be every 5 minutes, every 10 minutes, every 20 minutes, or every 30 minutes. For example, the ORP of the reference bacterial sample 408 can be monitored over a period of 180 minutes. Simultaneously with the monitoring, the OD of this reference bacterial sample 408 can be measured periodically every 15 minutes during this 180-minute period.

[0189]

[0203] The reader 200 can incubate and aerate the reference bacterial sample 408 in the same manner as the contained sample 113. The reader 200 can incubate the reference bacterial sample 408 at an incubation temperature of approximately 30°C to approximately 40°C (e.g., approximately 35°C ± 2°C). The reader 200 can also aerate the reference bacterial sample 408 at a flow rate of 7.0 μL / sec / mL to 10.0 μL / sec / mL. More specifically, the reference bacterial sample 408 in the sensor device 100 can be aerated at a flow rate of approximately 8.8 (±0.9) μL / sec / mL.

[0190]

[0204] In some embodiments, the reference bacterial sample 408 in the sensor device 100 can be aerated according to an aeration cycle. The aeration cycle may include a non-aeration period after the aeration period during which no gas or ambient air is supplied to the container chamber 102. In certain embodiments, the aeration period may be longer than the non-aeration period. For example, the aeration period may be about 7 to 10 minutes, and the non-aeration period may be about 3 to 10 seconds. As a more specific example, the reference bacterial sample 408 in the container chamber 102 of the sensor device 100 may be repeatedly aerated for about 8 minutes at a flow rate or dispensing rate of about 10.0 μL / sec / mL of the reference bacterial sample 408, followed by a non-aeration period of about 5 seconds.

[0191]

[0205] In some embodiments, the OD measurement 417 can be performed at a wavelength of 600 nm using a spectroscopic measuring device 416 or system (e.g., a UV-Vis spectroscopic measuring device) (OD600 measurement). In certain embodiments, the sensor device 100 can be removed from the reader 200 at the end of each specific time interval 414, and the reference bacterial sample 408 can be transferred to another container or tube fitted with the spectroscopic measuring device 416 or system. In other embodiments, the sensor device 100 can be designed or otherwise configured to work directly with a particular type of spectroscopic measuring device 416 or system so that the OD of the reference bacterial sample 408 can be measured even when the reference bacterial sample 408 is in the container chamber 102 of the sensor device 100.

[0192]

[0206] In some embodiments, the spectroscopic measurement device 416 or system can be communicatively coupled to a computing device 310, which in turn is communicatively coupled to a reader 200. The computing device 310 records the results of OD measurement 417 and ORP monitoring and can store them in the memory of the computing device 310 or in a cloud-based database accessible by the computing device 310.

[0193]

[0207] In other embodiments, the spectroscopic measuring device 416 or system can be communicatively coupled to the reader 200, which can store the results of the OD measurement 417 along with the change in ORP.

[0194]

[0208] The OD measurement 417 can be converted to a reference sample bacterial concentration 418 (expressed in CFU / mL units) using a conversion factor. For example, one or more processors of the computing device 310 can be programmed to convert the results of the OD measurement 417 to the reference sample bacterial concentration 418 using a conversion factor. For example, the result of the OD measurement 417 is multiplied by the numerical conversion factor (e.g., OD × (1.76 × 10)). 9 This allows the sample bacterial concentration to be converted to a reference sample of 418. The conversion factor is usually instrument-dependent and varies from instrument to instrument.

[0195]

[0209] In certain embodiments, a plate count assay or flow cytometry assay can be performed instead of or in addition to the OD measurement 417 to determine the reference sample bacterial concentration 418.

[0196]

[0210] The computing device 310 can then generate a strain-specific LUT 404 by associating the measured ORP change of the reference bacterial sample 408 with each reference sample bacterial concentration 418 (converted from OD measurement 417). For example, each reference sample bacterial concentration 418 can be associated with the measured ORP change of the reference bacterial sample 408 identified by the reader 200. Furthermore, the reference sample bacterial concentration 418 can then be included as a component LUT bacterial concentration 412 of a particular strain-specific LUT 404, and the ORP change of the reference bacterial sample 408 can be included as the ORP change amount 410 of the component LUT of this particular strain-specific LUT 404.

[0197]

[0211] Next, this process can be repeated for each of the other reference bacterial samples 408 until at least three lineage-specific LUTs 404 are combined. In some embodiments, many lineage-specific LUTs 404 are created and then used to create multiple species-specific LUTs 406. Such species-specific LUTs 406, or combinations of species-specific LUTs 406 and lineage-specific LUTs 404, can be used to create a species-independent LUT 312.

[0198]

[0212] As described above, the LUTs (including any of species-independent LUT 312, lineage-specific LUT 404, and species-specific LUT 406) can be stored as part of a database software program in the memory of the reader 200, a computing device 310 communicably coupled to the reader 200, or a combination thereof. In other embodiments, the LUTs can be stored as part of a database software program in a computing cloud or a remote server and / or computing device 310 accessible by the reader 200 via a network.

[0199]

[0213] In some embodiments, multiple species-independent LUT312s can be prepared. In these embodiments, the species-independent LUT312s can be organized by genus, family, order, class, phylum, kingdom, or domain. Furthermore, specific species-independent LUT312s can be organized, selected, or searched based on microbial characteristics such as Gram type, or on functional capabilities such as the ability to hydrolyze specific proteins or molecules.

[0200]

[0214] Figures 6A and 6B show the results from 41 trial runs performed to evaluate the effectiveness of the method 300 and system 301 (sensor device 100 and reader 200) disclosed herein for preparing output samples at a desired or target concentration 308 (or within an acceptable error margin). All output samples were 1.5 × 10⁻⁶ as the desired or target concentration 308. 8Preparations were made using CFU / mL. As shown in Figure 6A, 41 trial runs included source samples containing 10 different species of Gram-negative bacteria. These species included PAe, ABa, ECo, KPn, ECl, KOx, PMi, KAe, SMa, and CFr. The bacterial species in such source samples were determined to ensure that Method 300 performs equally well against different types of bacteria. Those skilled in the art will understand that the bacterial species associated with the source sample do not need to be identified before preparing the output sample using Method 300.

[0201]

[0215] When preparing the output sample, the desired or target concentration is 308 (1.5 × 10⁻¹⁰). 8 Based on CFU / mL, the threshold ORP change is 318 (ΔORP Threshold A value of -60mV was selected from the species-independent LUT312 shown in Figure 5. The larger graph in Figure 6A shows these results along with the final output sample concentrations plotted for various bacterial species. The final output sample concentrations were determined using OD measurement and / or conventional bacterial culture plating methods. The smaller graph in Figure 6A is a combined box plot of these results.

[0202]

[0216] The average output sample concentration is 1.43 × 10⁻⁶. 8 (±0.15log 10 The result was CFU / mL. The goal of the trial run was that at least 95% of the output sample concentration should be 1.5 × 10⁻⁶. 8 Desired value: CFU / mL or ±0.5 log of the target concentration of 308. 10 It meant going inside.

[0203]

[0217] Figure 6B shows that 100% of the concentrations of the 41 output samples is 1.5 × 10⁻⁶. 8 Desired value: CFU / mL or ±0.5 log of the target concentration of 308. 10 Within that range, 95.1% of the 41 output sample concentrations were 1.5 × 10⁻⁶. 8 ±0.3 log of CFU / mL 10 Within that range, 78.0% of the 41 output sample concentrations were 1.5 × 10⁻⁶. 8±0.2 log of CFU / mL 10 This table shows that it was within the scope. Most downstream test protocols use 0.5 log 10 To consider the bacterial concentration error margin to be well within an acceptable range, all output samples generated from these 41 trial runs can be used for further downstream testing.

[0204]

[0218] These results demonstrate that the method 300 and system 301 (sensor device 100 and reader 200) disclosed herein are effective in preparing output samples within an acceptable error margin of the desired or target concentration 308. Furthermore, these results demonstrate that the method 300 and system 301 disclosed herein can also be effective in preparing output samples within an acceptable error margin of the desired or target concentration 308 from source samples containing bacterial species not included in the reference bacterial sample 408 used to prepare the species-independent LUT 312. That is, the species-independent LUT 312 on which the output sample is based is truly "species-independent" and has broad applicability beyond the species used to prepare the species-independent LUT 312.

[0205]

[0219] As will be discussed in the following sections, much of the effectiveness of Method 300 and System 301 disclosed herein may be attributable to the aeration protocol disclosed herein.

[0206]

[0220] Figures 7A and 7B are graphs showing the effect of aeration on the bacterial growth rates of Escherichia coli (ECo) and Acinetobacter baumannii (ABa), respectively. Aerated samples were aerated at a flow rate of approximately 8.8 μL / sec / mL, the same as that of sample 113, while stagnant samples were not aerated. UV-Visible optical density measurements were performed over time to track the growth behavior of such samples.

[0207]

[0221] Figure 7A shows that aeration has only a slight effect on the growth of ECo (facultative anaerobic bacteria), but as shown in Figure 7B, aeration has a much greater effect on the growth of obligate aerobic bacteria such as ABa. Therefore, aeration offers a dual advantage: it reduces the time required to prepare samples, i.e., preparation time, by accelerating the growth of certain types of bacteria (i.e., obligate or obligate aerobic bacteria), and it also makes the bacterial growth rate more uniform regardless of the type of bacteria in the sample.

[0208]

[0222] As mentioned above, it should be emphasized that excessive aeration of the contained sample 113 can adversely affect the ORP signal monitored by the reader 200. Therefore, the contained sample 113 in the sensor device 100 should be aerated at a flow rate within an optimal range. One such range discovered by the applicants is a flow rate of 7.0 μL / sec / mL to 10.0 μL / sec / mL of the contained sample 113. More specifically, the contained sample 113 can be aerated at a flow rate of approximately 8.8 (±0.9) μL / sec / mL.

[0209]

[0223] In some embodiments, the sample 113 contained within the sensor device 100 can be aerated according to an aeration cycle. The aeration cycle may include a non-aeration period after the aeration period during which no gas or ambient air is supplied to the container chamber 102. In certain embodiments, the aeration period may be longer than the non-aeration period. For example, the aeration period may be about 7 to 10 minutes, and the non-aeration period may be about 3 to 10 seconds.

[0210]

[0224] Figure 8A is a table showing that aeration reduces the variability in growth rates among different bacterial species. More specifically, the table in Figure 8A shows that aeration can reduce the overall coefficient of variation (CV) of bacterial doubling times across various species. All samples shown in Figure 8A were aerated at an aeration flow rate of approximately 8.8 μL / sec / mL for each sample.

[0211]

[0225] For three samples containing facultative anaerobic bacterial species (ECo, SMa, and PVu), the percentage change in bacterial doubling time ranged from 15% to 21%. However, for two samples containing obligate aerobic bacterial species, Aba and PAe, the percentage changes in bacterial doubling time were much higher, at 41% and 84%, respectively. Furthermore, when examining results for all bacterial species, the CV of bacterial doubling time was extremely high at 109% when such samples were not aerated (or remained stagnant). However, with aeration, the CV of bacterial doubling time decreased to 12%, and the bacterial growth behavior (which is evident throughout the bacterial doubling time) became very similar. This is important for the success of the species-independent method 300, as it ensures that all results are obtained within similar timeframes, regardless of whether the bacteria in the source sample are facultative anaerobic or obligate aerobic.

[0212]

[0226] Figure 8B shows that aeration can be used to reduce the overall CV of bacterial doubling time, thus reducing the average doubling time (t doubling_average ) multiple bacterial doubling times (t doubling This shows that it can be calculated from the average doubling time (t). For example, the average doubling time (t) doubling_average ) is at least three bacterial doubling times (t doubling It can be calculated by taking the average of (t). As a more specific example, the table in Figure 8B shows the bacterial doubling times (t) of five different bacterial species. doubling By taking the average of ), the average doubling time (t doubling_average This demonstrates that it is possible to calculate ( ).

[0213]

[0227] The doubling time for each bacterium (e.g., ECo, SMa, PVu, Aba, PAe, etc.) can be calculated using OD measurements performed on various reference bacterial samples 408 (see, e.g., Figure 4). As described above, the OD measurements can be converted to bacterial concentrations (in CFU / mL units) using conversion factors. The resulting change in bacterial concentration can then be plotted as a function of time, and the plot can be fitted to an exponential model such as the one provided in Equation 1 below:

number

[0214]

[0228] In equation 1 above, N is the converted bacterial concentration, t is the time in minutes, and A and k are fitting parameters. Bacterial doubling time (t doubling To determine the bacterial doubling time, A corresponds to the initial bacterial concentration (i.e., at t=0) and is not relevant to determining the bacterial doubling time, so it is not a problem.

[0215]

[0229] Bacterial doubling time (t doubling The relationship between ) and k is given by the following equation 2:

number

[0216]

[0230] As mentioned above, then, multiple bacterial doubling times (t doubling The mean doubling time (t) is taken by taking the average of ). doubling_average ) can be calculated. Average doubling time (t doubling_average ) If the desired or target concentration 308 is not included in the species-independent LUT312, the time to reach the target concentration (t target )320 (see, for example, step 300I in Figure 3) is necessary to calculate. For example, the user needs 3.0 × 10 8 A CFU / mL can be entered as the desired or target concentration 308, which exceeds any species-independent bacterial concentration 316 within the species-independent LUT312 on which the reader 200 relies (for example, the species-independent LUT312 shown in Figure 5 is 1.8 × 10⁶). 8 (It only goes up to CFU / mL).

[0217]

[0231] As described above, in certain embodiments, one or more processors of the reader 200 may choose to calculate the time to target concentration 320 even if the desired or target concentration 308 is included in the species-independent LUT 312. For example, one or more processors of the reader 200 may choose to perform this calculation based on a specific heuristic or pre-set rule that determines when a threshold ORP change 318 from the species-independent LUT 312 may be considered too high / too large or potentially error-prone. In this case, one or more processors of the reader may decide to calculate the time to target concentration 320 rather than relying on a specific species-independent ORP change 314 from the species-independent LUT 312. For example, one or more processors of the reader 200 may determine that a specific smaller species-independent ORP change 314 identified earlier in the ORP monitoring is more accurate or error-prone than a larger ORP change 314 acquired later as part of the ORP monitoring. One or more processors in the reader 200 can make this determination based on real-time or near-real-time analysis of the behavior of the ORP growth curve 311 (e.g., whether the monitored ORP signal is beginning to flatten out). In this case, one or more processors in the reader 200 can determine that a particular smaller species-independent ORP change 314 from the species-independent LUT 312 is more reasonable or less prone to error, and can choose to use such an ORP change when calculating the time to target concentration 320.

[0218]

[0232] One or more processors of the leader 200 calculate the time to reach the target concentration (t) using the following equation 3. target ) It can be programmed to calculate 320:

number

[0219]

[0233] In the above formula 3, t target (or time to target concentration 320) is when the contained sample 113 is desired or the target concentration 308 (Ntarget ) represents the amount of time required to reach ), where N1 is the species-independent bacterial concentration contained in the species-independent LUT, and t1 is the change in species-independent ORP (Δ) of the contained sample 113, associated with N1 from the species-independent LUT 312. ORP ) represents the amount of time required for a change of only t doubling_average t1 is the average bacterial doubling time. t1 can be determined from real-time ORP monitoring performed by the leader 200 on the sample 113.

[0220]

[0234] For example, Figure 9A is an ORP growth curve showing the ORP change of the contained sample 113 measured by the reader 200 over a period of approximately 60 minutes. In this particular contained sample 113, the user measured 3.0 × 10⁻¹⁶ 8 A CFU / mL is input as the desired or target concentration 308, which exceeds any species-independent bacterial concentration 316 within the species-independent LUT 312 on which the reader 200 relies (e.g., the species-independent LUT 312 shown in Figure 5). If the reader 200 determines that the desired or target concentration 308 is not included in the species-independent LUT 312, one or more processors of the reader 200 calculate the time (t) to reach the target concentration. target )320 can be chosen to calculate. One or more processors of the leader 200 take a single pair of entries from the species-independent LUT 312 (i.e., a single species-independent bacterial concentration 316 and its associated species-independent ORP change 314) and use formula 3 above to calculate the time to the target concentration (t target It can be programmed to calculate 320.

[0221]

[0235] For example, from the species-independent LUT312 shown in Figure 5, N1 is 1 × 10 8 CFU / mL (or 1.0E+8) can be selected. Then, one or more processors of the reader 200 monitor the ORP of the contained sample 113 in real time (see Figure 9A), and when the ORP of the contained sample 113 is -30mV (N1), the species-independent ORP change (Δ) is associated with the ORP of the contained sample 113. ORPt1 can be determined based on the amount of time required for the change to occur by the amount shown in Figure 5. As shown in Figure 9A, t1 can be determined to be 32 minutes based on real-time ORP monitoring. These values ​​can be used to determine the mean doubling time (t doubling_average Substituting this along with 28.4 minutes (see Figure 8B) into Equation 3, we get the time to reach the target concentration (t target )320 can be calculated as 76.4 minutes.

[0222]

[0236] In this example, the reader 200 can alert the user (e.g., a laboratory technician or clinician) when an output sample at the desired or target concentration 308, or an output sample within an acceptable error margin of the desired or target concentration 308, is ready.

[0223]

[0237] Figure 9B is a bacterial growth curve showing the change in bacterial concentration in the above-mentioned sample 113 as a function of time. The bacterial concentration can be obtained by converting the OD measurement values ​​over time for the above-mentioned sample 113.

[0224]

[0238] As shown in Figure 9B, at 76.4 minutes, the bacterial concentration in the above-mentioned sample 113 was approximately 2.4 × 10⁻⁶. 8 The concentration is CFU / mL. Bacterial concentration: 2.4 × 10⁻⁶ 8 CFU / mL (or 2.4E+8) is the desired or target concentration of 3 × 10⁻¹⁴. 8 0.1 log of CFU / mL (or 3.0E+8) 10 Since this is within the range, such final bacterial concentrations are sufficiently within an acceptable error margin (e.g., ±0.5 log) of the desired or target concentration 308. 10 It is considered to be within ).

[0225]

[0239] This example demonstrates the usefulness of Method 300 and System 301 when the desired or target concentration 308 exceeds any bacterial concentration included as part of the species-independent LUT 312. However, as mentioned above, N target Even if it is included as part of a species-independent LUT312, the time to reach the target concentration (t target)320 can also be calculated. For example, N target (N) is greater than N1 target >N1) Time to reach target concentration (t target )320 can be calculated.

[0226]

[0240] In fact, N target If N1 equal to is selected, the time to reach the target concentration (t target )320 is simply equal to t1. That is, the time to reach the target concentration (t target )320 simply means that the ORP of the contained sample 113 is associated with the species-independent ORP change (Δ) from the species-independent LUT312. ORP This is the time required for only that much change to occur.

[0227]

[0241] The time to reach the target concentration (t) target It is important to note that the calculation of the time to reach the target concentration (t) is only valid after the bacterial growth rates in all samples (including the containing sample 113 and all reference bacterial samples 408 used to prepare the species-independent LUT 312) have become more uniform through the specific aeration protocol disclosed herein. target The calculation of ) makes significant use of the benefits of aeration in arriving at an accurate final result.

[0228]

[0242] Several embodiments have been described. Nevertheless, it will be understood by those skilled in the art that various changes and modifications can be made to this disclosure without departing from the spirit and scope of the embodiments. Elements of systems, devices, apparatus, and methods shown with any embodiment are illustrative with respect to that particular embodiment and can be used in combination or otherwise in other embodiments within this disclosure. For example, the steps of any method illustrated or described herein do not require a specific order or sequence illustrated or described in order to achieve the desired result. In addition, other step operations may be provided, or steps or operations may be omitted or removed from the described method or process to achieve the desired result. Furthermore, any component or part of any apparatus or system described or illustrated herein may be removed, omitted or removed to achieve the desired result. In addition, certain components or parts of systems, devices, or apparatus illustrated or described herein have been omitted for the sake of brevity and clarity.

[0229]

[0243] Therefore, other embodiments are also within the scope of the following claims, and this specification and / or drawings can be considered illustrative rather than restrictive.

[0230]

[0244] Each of the individual variations or embodiments described and illustrated herein has discrete components and features that can be readily separated or combined with any of the features of other variations or embodiments. Modifications can be made to adapt specific situations, materials, substance compositions, processes, process operation, or steps to the object, spirit, or scope of the invention.

[0231]

[0245] The methods described herein may be performed in any logically possible order of the subsequent chapters and in the order of the events described. Furthermore, additional steps or actions may be provided, or steps or actions may be omitted, to achieve the desired results.

[0232]

[0246] Furthermore, where a range of values ​​is provided, any intermediate values ​​between the upper and lower limits of that range, as well as any other stated and intermediate values ​​within that stated range, are encompassed within the present invention. Also, any selective feature of a described variation of the present invention may be stated and claimed independently or in combination with any one or more of the features described herein. For example, a statement of the range 1 to 5 should be considered to disclose subranges such as 1 to 3, 1 to 4, 2 to 4, 2 to 5, 3 to 5, in addition to individual numbers within that range, such as 1.5, 2.5, and any whole or partial increments between them.

[0233]

[0247] All existing materials referenced herein (e.g., publications, patents, patent applications) are incorporated herein by reference in their entirety, unless otherwise inconsistent with the spirit of the present invention (in which case the provisions of this specification shall prevail). Referenced materials are provided simply because they were disclosed prior to the filing date of this application. Nothing in this specification should be construed as acknowledging that the present invention does not have prior rights to such materials by prior invention.

[0234]

[0248] A singular reference to an item includes the possibility that there may be multiple such items. More specifically, as used herein and in the appended claims, the singular forms “a,” “an,” and “the foregoing” also include references to the plural unless otherwise specified in the context. It should be further noted that claims may be written to exclude any optional elements. This statement is therefore intended to serve as a prerequisite for using exclusive terms such as “only,” “only,” etc., or for using “negative” limitations in relation to the description of elements of a claim. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0235]

[0249] The phrase “at least one” refers to any combination of one or more items or components (or a list of items or components) when such a phrase modifies multiple items or components. For example, “at least one of A, B, and C” means (i) A; (ii) B; (iii) C; (iv) A, B, and C; (v) A and B; (vi) B and C; or (vii) A and C.

[0236]

[0250] For understanding the scope of this disclosure, the term “equipped with” and its derivatives, as used herein, are intended to be open-ended terms that specify the presence of the described features, elements, components, groups, integers, and / or steps, but do not exclude other features, elements, components, groups, integers, and / or steps. The same applies to similar terms such as “contains,” “has,” and their derivatives. The terms “part,” “section,” “part,” “member,” “element,” or “component” can have a dual meaning when used in the singular, referring to one part or more parts. As used herein, the directional terms “forward, backward, upward, downward, vertical, horizontal, down, across, transverse, and longitudinal,” and any other similar directional terms, refer to the position of the device or equipment or the direction of the device or equipment as it is translating or moving.

[0237]

[0251] Finally, as used herein, terms of degree such as “substantially,” “about,” and “approximately” mean a reasonable deviation from the specified value (e.g., a deviation of up to ±0.1%, ±1%, ±5%, or ±10%, as appropriate) such that the specified value or the final result does not differ significantly or substantially from the specified value. For example, “about 1.0 cm” can be interpreted as “1.0 cm” or “between 0.9 cm and 1.1 cm.” When used to refer to a number or value that is part of a range, terms of degree such as “about” or “approximately” may be used to modify both the smallest and largest number or value.

[0238]

[0252] This disclosure is not intended to be limited to the specific forms described, This specification is intended to include substitutions, modifications, and equivalents of the variations or embodiments described herein. Furthermore, the scope of this disclosure fully encompasses other variations or embodiments that may become apparent to those skilled in the art in view of this disclosure.

Claims

1. A method for preparing a sample of bacteria at a desired or target concentration or within an acceptable error margin of the said desired or target concentration, The process involves introducing aliquots of the sample containing the bacteria into a sample container, wherein the aliquots of the sample in the sample container are contained samples that are in fluid communication with the reference sensor and the active sensor. The incubation and aeration of the contained sample, wherein the contained sample is aerated at a flow rate of 7.0 microliters (μL) / second / milliliter (mL) to 10.0 μL / second / mL of the contained sample. The change in the oxidation-reduction potential (ORP) of the contained sample is monitored using a reader electrically coupled to the reference sensor and the active sensor. When it is determined that the concentration of the bacteria in the contained sample has reached the desired or target concentration or within an acceptable margin of error, the contained sample is cooled. A method that includes this.

2. The method according to claim 1, further comprising searching a species-independent lookup table (LUT) from a database, wherein the species-independent LUT includes a species-independent ORP change associated with a species-independent bacterial concentration, and the species-independent LUT is generated from a plurality of component LUTs including ORP changes and bacterial concentrations measured using a plurality of the reference bacterial samples incubated and aerated at flow rates of 7.0 μL / sec / mL to 10.0 μL / sec / mL for each of the reference bacterial samples.

3. If one of the species-independent ORP change amounts is associated with one of the species-independent bacterial concentrations equal to the desired or target concentration, then one of the species-independent ORP change amounts associated with one of the species-independent bacterial concentrations equal to the desired or target concentration is selected as the threshold ORP change amount. When the change in ORP of the contained sample monitored by the reader reaches the threshold ORP change, it is determined that the concentration of the bacteria in the contained sample has reached the desired or target concentration or within an acceptable error margin. The method according to claim 2, further comprising:

4. The method according to claim 2, wherein the species-independent LUT is produced from at least three component LUTs, each of which is either a species-specific LUT or a lineage-specific LUT, and the first, second, and third LUTs are produced using ORP measurements and bacterial concentration measurements performed on a first reference bacterial sample, a second reference bacterial sample, and a third reference bacterial sample, respectively, the first reference bacterial sample comprising bacteria of a first species, the second reference bacterial sample comprising bacteria of a second species different from the first species, and the third reference bacterial sample comprising bacteria of a third species different from the second and first species.

5. Each of the aforementioned system-specific LUTs is: Monitoring the change in ORP of at least one reference bacterial sample over a certain period of time, The optical density (OD) of at least one reference bacterial sample is measured periodically over the same period as the aforementioned period. Converting the OD measurement results to the reference sample bacterial concentration using a conversion coefficient, The reference sample bacterial concentration is associated with the change in ORP of the at least one reference bacterial sample, The method according to claim 4, which is produced by performing the following.

6. The following formula: [Math 1] Using, the time (t ｔａｒｇｅｔ ) to the target concentration representing the amount of time required for the contained sample to reach bacteria at the desired or target concentration (N ｔａｒｇｅｔ ), where N ｔａｒｇｅｔ is not included in the species-independent LUT, and N １ is the species-independent bacterial concentration included in the species-independent LUT, and t １ is the time required for the ORP of the contained sample to change by the amount of species-independent ORP change (Δ １ ) associated with N ＯＲＰ from the species-independent LUT, and t １ is identified from the real-time ORP monitoring performed by the reader on the contained sample, and t ｄｏｕｂｌｉｎｇ＿ａｖｅｒａｇｅ is the average bacterial doubling time, to calculate If the elapsed time is equal to the time to reach the target concentration, it is determined that the concentration of the bacteria in the contained sample has reached the desired or target concentration or within an acceptable margin of error. The method according to claim 2, further comprising:

7. The following formula: [Math 2] Using this method, the contained sample reaches the desired or target concentration (N ｔａｒｇｅｔ The time to reach the target concentration (t) represents the amount of time required to reach the bacteria. ｔａｒｇｅｔ This involves calculating ), where N ｔａｒｇｅｔ and N １ Both are included in the aforementioned species-independent LUT, N ｔａｒｇｅｔ is N １ Larger than (N ｔａｒｇｅｔ >N １ ), t １ The ORP of the contained sample is N from the species-independent LUT. １ The species-independent ORP change associated with (Δ ＯＲＰ ) represents the time required for the change to occur, １ This is identified from real-time ORP monitoring performed by the reader on the contained sample, t ｄｏｕｂｌｉｎｇ＿ａｖｅｒａｇｅ This is the average bacterial doubling time, which can be calculated as follows: If the elapsed time is equal to the time to reach the target concentration, it is determined that the concentration of the bacteria in the contained sample has reached the desired or target concentration or within an acceptable margin of error. The method according to claim 2, further comprising:

8. The method according to claim 1, wherein the reference sensor comprises a reference electrode material and a wick, the reference electrode material and the wick are in fluid communication with the contained sample such that at least a portion of the contained sample in the chamber cavity of the sample container is drawn toward the reference electrode material by the wick, and the contained sample is in fluid contact with the reference electrode material, the active sensor is coupled to at least a portion of the chamber sidewall of the sample container, the active electrode material of the active sensor faces the chamber cavity such that the contained sample is in fluid contact with the active electrode material when the contained sample fills the chamber cavity, and the ORP of the contained sample is determined by the reader based on the potential difference measured between the active electrode material and the reference electrode material when the reference sensor and the active sensor are electrically coupled to the reader.

9. The method according to claim 1, wherein the bacteria are facultative anaerobic bacteria or obligate aerobic bacteria.

10. The method according to claim 1, wherein the bacteria are Gram-negative bacteria.

11. The method according to claim 1, wherein the sample of the bacteria at the desired or target concentration is prepared without any prior knowledge of the species of bacteria in the sample or without having previously identified the species of bacteria in the sample.

12. The method according to claim 1, wherein the sample comprises at least one of a body fluid and a bacterial culture solution derived from the body fluid.

13. The aforementioned desired or target concentration is 1.4 × 10 ８ CFU / mL~1.6×10 ８ The method according to claim 1, wherein the concentration is CFU / mL.

14. The method according to claim 1, wherein the contained sample is incubated at an incubation temperature of 33°C to 37°C.

15. The aforementioned acceptable error margin is ±0.5 log １０ The method according to claim 1.

16. The method according to claim 1, further comprising diluting the source sample containing the bacteria at a dilution ratio of 1:10 to 1:100 to produce a diluted sample, wherein the aliquot of the sample introduced into the sample container is an aliquot of the diluted sample.

17. The method according to claim 1, wherein the contained sample is aerated according to an aeration cycle, the aeration cycle includes a non-aeration period following an aeration period, and the aeration period is longer than the non-aeration period.

18. The method according to claim 17, wherein the aeration period is approximately 7 minutes to approximately 10 minutes, and the non-aeration period is approximately 3 seconds to approximately 10 seconds.

19. The method according to claim 1, wherein the contained sample is aerated using an electric piston pump, and the electric piston pump is housed in the leader.

20. The method according to claim 1, further comprising aerating the contained sample by introducing ambient air into the sample container through an opening defined along the base of the sample container.

21. A system for preparing bacterial samples at a desired or target concentration or within an acceptable error margin of the said desired or target concentration, A sensor device comprising a container chamber configured to hold aliquots of a sample containing the bacteria, wherein the aliquots of the sample in the container chamber are a sample containing fluids in communication with a reference sensor and an active sensor, A reader configured to receive the sensor device, the reader is also configured to incubate and aerate the contained sample when the sensor device is located within the reader, the contained sample is aerated at a flow rate of 7.0 microliters (μL) / second / milliliter (mL) to 10.0 μL / second / mL of the contained sample, and one or more processors of the reader, When the reader is electrically coupled to the reference sensor and the active sensor of the sensor device, the change in the oxidation-reduction potential (ORP) of the contained sample is monitored, When it is determined that the concentration of the bacteria in the contained sample has reached the desired or target concentration or within an acceptable margin of error, the contained sample is cooled. A leader configured to perform the following actions: A system equipped with these features.

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