Methods and systems for rapid testing for yeast

Abstract

Methods of preparing a sample for an assay for rapid quantification of a biologic contaminant. The method includes adding the sample to a vessel and inserting the vessel into a centrifuge. The method further includes centrifuging the sample to yield a first supernatant and a first pellet, discarding the first supernatant, and dissolving the first pellet in a first volume of a buffer to yield a first suspension volume. The method further includes centrifuging the first suspension volume to yield a second supernatant and a second pellet, discarding the second supernatant, and dissolving the second pellet in a second volume of the buffer to yield a final sample suspension. The method also includes adding the final sample suspension to an assay for rapid quantification for microbial contaminants.

Description

METHODS AND SYSTEMS FOR RAPID TESTING FOR YEASTCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is being filed as a PCT International patent application and claims priority to U.S. Provisional Patent Application Serial Number 63 / 589,831 filed October 12, 2023, the subject matter of which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] Fruit juice is transported long distances in tankers from supplier to processing plant, making it susceptible to microbial spoilage, especially during warmer months. High external temperatures cause the internal temperature of the tanker to rise, creating conditions for proliferation of microbes. Each year, this issue has implications for the juice industry, impacting inventory and leading to financial loss.

[0003] Traditional microbiology plating methods to enumerate yeast in juice generally take several days to return a result. This timeframe is too long considering that the juice needs to be accepted by the processing plant and processed quickly, or rejected outright if the product has deteriorated in transit. Other methods include evaluating the taste, odor and appearance of the juice, but this provides limited information about the microbiological state of the product.SUMMARY

[0004] Examples presented herein relate to a method of preparing a sample for an assay for rapid quantification of a biologic contaminant. The method includes adding the sample to a vessel and inserting the vessel into a centrifuge. The method further includes centrifuging the sample to yield a first supernatant and a first pellet, discarding the first supernatant, and dissolving the first pellet in a first volume of a buffer to yield a first suspension volume. The method further includes centrifuging the first suspension volume to yield a second supernatant and a second pellet, discarding the second supernatant, and dissolving the second pellet in a second volume of the buffer to yield a final sample suspension. The method also includes adding the final sample suspension to an assay for rapid quantification for microorganisms.

[0005] In other embodiments herein, the sample is a fruit juice. In still other embodiments herein, the sample is one or more of a pome fruit juice or a berry juice. In other embodiments herein, the sample includes at least one of apple juice, pear juice, nashi juice, or quince juice. In other embodiments herein, the sample includes at least one of blueberry juice, raspberry juice, goji berry juice, acai juice, aronia berry juice, and strawberry juice, or cranberry juice.

[0006] In other embodiments herein, the method further includes diluting the sample. In further embodiments herein, diluting the sample is performed at least one of prior to adding the sample to the receptacle, prior to inserting the receptacle into the centrifuging vessel, and / or prior to centrifuging the sample. In other further embodiments herein, the sample is diluted in the buffer at a ratio selected from a group consisting of 1 : 1000, 1 :500, 1 : 100, 1 :50, 1 :20, 1 : 10, 1 :5, 1 : 1, 5:1, 10: 1, 20: 1, 50: 1, 100: 1, or 1000: 1. In still further embodiments herein, the sample is diluted in the buffer a ratio selected from a group consisting of 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, and 15: 1. In other further embodiments herein, the sample includes a citrus juice. In still further embodiments herein, the sample includes at least one of orange juice, lemon juice, lime juice, grapefruit juice, and pineapple juice.

[0007] In other embodiments herein, the buffer is selected from a group consisting of calcium buffer, carbonic buffer, hydroxymethyl buffer, nitrate buffer, phosphate buffer and sulfonic buffer. In other embodiments herein, the assay is an adenosine triphosphate assay. In other further embodiments herein, the assay is a luciferase system.

[0008] In other embodiments herein, the method further includes incubating the final sample suspension in the assay. In further embodiments herein, incubating the final sample suspension includes an incubation period. In still further embodiments herein, the incubation period is selected from a group consisting of: 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 12 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, and 60 minutes.

[0009] In other further embodiments herein, the method further includes agitating the final sample suspension. In other embodiments herein, agitating the final sample suspension includes vortexing the final sample suspension. In still further embodiments herein, agitating the final sample suspension is performed at least one of prior to incubating the final sample suspension in the assay and concurrently with incubating the final sample suspension in the assay.

[0010] In other embodiments herein, the method further includes reading the final suspension using a luminometer to obtain a result, wherein the result is measured in relative light units (RLU). In still further embodiments herein, the method further includes interpreting the result by converting the relative light units (RLU) output by the luminometer to a colony forming unit (CFU) measurement.

[0011] In other embodiments herein, the biological contaminant is at least one of a bacteria, a mildew, a mold, a yeast, and a virus. In further embodiments herein, the biological contaminant is a microorganism.

[0012] In other embodiments herein, the vessel is one of a flask, a phial, a tube, and a vial.

[0013] Other examples presented herein relate to a method of rapidly quantifying a biologic contaminant in a liquid sample. The method includes adding the liquid sample to a vessel and inserting the vessel into a centrifuge. The method further includes centrifuging the liquid sample to yield a first pellet and resuspending the first pellet in a first volume of a buffer to yield a first suspension volume. The method also includes centrifuging the first suspension volume to yield a second pellet and resuspending the second pellet in a second volume of the buffer to yield a final sample suspension. The method also includes assaying the final sample suspension to generate a bioluminescence proportional to the presence of the biological contaminant, measuring the bioluminescence generated in the final sample suspension, and converting the bioluminescence measurement to a quantification of the biological contaminant.

[0014] In other embodiments herein, assaying the final sample suspension includes incubating the final sample suspension. In yet other embodiments herein, incubating the final sample suspension is performed for less than one hour. In other further embodiments herein, incubating the final sample suspension is performed for less than 30 minutes. In other embodiments herein, incubating the final sample suspension is performed for 10 minutes.

[0015] In other embodiments herein, the method further includes agitating the final sample suspension. In still other embodiments herein, agitating the final sample suspension includes vortexing the final sample suspension. In other embodiments herein, agitating the final sample suspension is performed at least one of prior to incubating the final sample suspension in the assay and concurrently with incubating the final sample suspension in the assay.

[0016] In other embodiments herein, the method further includes determining, based on the quantification of the biological contaminant, to reject a source of the liquid sample.

[0017] In other embodiments herein, the liquid sample is a fruit juice. In still other embodiments herein, the liquid sample is one or more of a pome fruit juice or a berry juice. In yet other embodiments herein, the liquid sample includes at least one of apple juice, pear juice, nashi juice, or quince juice. In other embodiments herein, the liquid sample includes at least one of blueberry juice, raspberry juice, goji berry juice, acai juice, aroni a berry juice, and strawberry juice, or cranberry juice.

[0018] In other embodiments herein, the method further includes diluting the liquid sample. In other embodiments herein, diluting the sample is performed at least one of prior to adding the sample to the receptacle, prior to inserting the receptacle into the centrifuging vessel, and prior to centrifuging the sample. In other embodiments herein, the sample is diluted in the buffer at a ratio selected from a group consisting of 1 : 1000, 1 :500, 1 : 100, 1 :50, 1 :20, 1 : 10, 1 :5, 1 : 1, 5: 1, 10: 1, 20: 1, 50: 1, 100: 1, or 1000: 1. In still other embodiments herein, the sample is diluted in the buffer a ratio selected from a group consisting of 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12:1, 13: 1, 14: 1, and 15: 1.

[0019] In other embodiments herein, the sample includes a citrus juice. In other embodiments herein, the sample includes at least one of orange juice, lemon juice, lime juice, and grapefruit juice. In some embodiments, the sample includes a composite fruit juice, such as pineapple juice or a berry juice such as strawberry, raspberry, or blackberry.

[0020] In other embodiments herein, the buffer is selected from a group consisting of calcium buffer, carbonic buffer, hydroxymethyl buffer, nitrate buffer, phosphate buffer and sulfonic buffer. In other embodiments herein, the assay is an adenosine triphosphate assay. In still other embodiments herein, the assay is a luciferase system. In other embodiments herein, the biological contaminant is at least one of a bacteria, a mildew, a mold, a yeast, and a virus. In still other embodiments herein, the biological contaminant is a yeast. In other embodiments herein, the vessel is one of a flask, a phial, a tube, and a vial.

[0021] Other examples presented herein relate to a method of determining whether a sample contains less than a threshold amount of a biological contaminant. The method includes adding the sample to a vessel and inserting the vessel into a centrifuge. The method further includes centrifuging the sample to yield a first pellet, dissolving the firstpellet in a first volume of a buffer to yield a first suspension volume, centrifuging the first suspension volume to yield a second pellet, and dissolving the second pellet in a second volume of the buffer to yield a final sample suspension. The method also includes assaying the final sample suspension to generate bioluminescence proportional to the biological contaminant’s presence, measuring the bioluminescence generated in the final sample suspension, converting the bioluminescence measurement to a quantification of the biological contaminant, and comparing the quantification of the biological contaminant to the threshold amount for the biological contaminant.

[0022] A variety of additional inventive aspects will be set forth in the description that follows. The inventive aspects can relate to individual features and to combinations of features. It is to be understood that both the forgoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the broad inventive concepts upon which the embodiments disclosed herein are based.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings, which are incorporated in and constitute a part of the description, illustrate several aspects of the present disclosure. A brief description of the drawings is as follows:

[0024] FIG. 1 illustrates a flowchart of an example method for preparing a sample for an assay for rapid quantification of a biologic contaminant.

[0025] FIG. 2 illustrates a flowchart of an example method for preparing a sample with dilution for an assay for rapid quantification of a biologic contaminant.

[0026] FIG. 3 illustrates a flowchart of an example method of performing a quantification assay on a prepared sample.Selected Definitions

[0027] As used herein, “weight percent,” “wt%, “percent by weight,” “% by weight,” and variations thereof refer to the concentration of a substance as the weight of that substance divided by the total weight of the composition and multiplied by 100.

[0028] As used herein, “volume percent,” “vol%, “percent by volume,” “% by volume,” and variations thereof refer to the concentration of a substance as the volume of that component divided by the total weight of the solution and multiplied by 100. Itshould be understood that, where “percent”, “%,” and the like are unspecified herein, they should generally be understood as a “volume percent,” “vol%,” etc.

[0029] As used herein, “g” represents gram; “L” represents liter; “mg” represents “milligram (10-3 gram);” “mL” represents milliliter (10-3 liter); “pl” represents microliter (10-6 liter).

[0030] The term “about” is used in conjunction with numeric values to include normal variations in measurements as expected by persons skilled in the art, and is understood to have the same meaning as “approximately” and to cover a typical margin of error, such as ± 10 % of the stated value. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial composition. Whether or not modified by the term “about,” the claims include equivalents to the quantities.

[0031] It should be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing “a compound” includes having two or more compounds that are either the same or different from each other. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0032] In the interest of brevity and conciseness, any ranges of values set forth in this specification contemplate all values within the range and are to be construed as support for claims reciting any sub-ranges having endpoints which are real number values within the specified range in question. By way of a hypothetical illustrative example, a disclosure in this specification of a range of from 1 to 5 shall be considered to support claims to any of the following ranges: 1-5; 1-4; 1-3; 1-2; 2-5; 2-4; 2-3; 3-5; 3- 4; and 4-5.

[0033] The term “substantially free” may refer to any component that the composition or method of the disclosure lacks or mostly lacks. When referring to “substantially free” it is intended that the component is not intentionally added to compositions or methods of the disclosure. Use of the term “substantially free” of a component allows for trace amounts of that component to be included in compositions or methods of the disclosure because they are present in another component. However, it is recognized that only trace or de minimus amounts of a component will be allowed when the composition is said to be “substantially free” of that component. Moreover,the term if a composition is said to be “substantially free” of a component, if the component is present in trace or de minimus amounts it is understood that it will not affect the effectiveness of the composition. It is understood that if an ingredient is not expressly included herein or its possible inclusion is not stated herein, the disclosure composition may be substantially free of that ingredient. Likewise, the express inclusion of an ingredient allows for its express exclusion thereby allowing a composition to be substantially free of that expressly stated ingredient.

[0034] The processes, methods, treatments, formulations, and compositions of the present disclosure may comprise, consist essentially of, or consist of the components and ingredients of the present invention as well as other ingredients described herein. As used herein, “consisting essentially of’ means that the methods and compositions may include additional steps, components or ingredients, but only if the additional steps, components or ingredients do not materially alter the basic and novel characteristics of the claimed processes and compositions.DETAILED DESCRIPTION

[0035] Examples presented herein relate to a rapid method to quantify the number of a biological contaminant in a sample in less than about three hours or less than about two hours, or less than about one hour, for example to identify the impending risk of spoilage and / or fermentation. Advantages of the disclosed method include enabling quick decisions around accepting or rejecting an incoming product.

[0036] A number of commercially available rapid tests to quantify viable microorganisms in beverages are presently available. However, these tests are unable to produce a suitable result using fruit juice as the matrix. The tests are generally characterized as unsuitable for high acid, unfilterable substrates, such as fruit juice. These substrates may pose challenges to the test systems, resulting in inhibition or enhancement due to low pH or the presence of bioactive metabolites. While examples discussed herein focus on fruit juice, it is noted that other substrates, which may exhibit similar characteristics, such as high background noise from the matrix, may also be advantageously analyzed using the methods disclosed herein.

[0037] Disclosed herein are systems and methods for rapid quantification of yeast and other microorganisms in substrates which may otherwise overwhelm the analysis. The disclosed methods significantly alter the execution of the rapid analysis as generallyunderstood by those of skill in the art and enables rapid quantification systems to be effectively used with historically problematic substrates.

[0038] Methods of quantifying microorganisms sometimes require a step of enrichment, where the concentration of microorganisms present in a sample is raised to a readily measurable level. For some microorganisms, such as bacteria, this enrichment period may be on the scale of several hours. For others, such as yeast, the enrichment step may require an incubation period may be hours to at least overnight.

[0039] To remove components of the solution interfering with readings and reduce background readings, a series of centrifugations of a sample are performed and the supernatant, containing the background generating components, is discarded. In addition, the microorganisms present in a larger sample are rapidly concentrated through the sequence of centrifugations as traditional enrichment methods may be ineffective for the evaluation of juice, due to the desired time frame for the analysis and the characteristics of the juice. Eliminating the need for an enrichment step significantly increases the speed of the analysis, enabling the quantification to be effectively performed at on-site locations, such as at a loading dock when a shipment of fruit juice is received. With the enrichment step eliminated, no incubator or other enrichment device is necessary, increasing the mobility and speed of the analysis.

[0040] To reduce background noise from the matrix, the methods disclosed herein introduces a dilution step. Following dilution, the sample is centrifuged to neutralize interference from matrix components. Finally, the sample is concentrated to negate the effect of the initial dilution. Once a sample is prepared according to the disclosed method, it can be effectively analyzed using microorganism quantification analysis. Even accounting for an incubation period that may be required after reagents are introduced to the sample, sample preparation and analysis according to the disclosed methods may still be performed in under an hour, as compared to the multiple hours required by methods requiring enrichment. In some embodiments, total time from drawing the initial sample to a result is less than about 120 minutes, less than about 100 minutes, less than about 80 minutes, less than about 60 minutes, less than about 50 minutes, less than about 40 minutes, less than about 30 minutes, less than about 20 minutes, or less than about 10 minutes.

[0041] Advantages of the methods disclosed herein over conventional methods are numerous. As noted above, the total time for analysis is significantly reduced, frommultiple hours, often 7-8 hours at a minimum, to less than an hour and in many cases no more than 40 minutes. The methods disclosed herein reduce the time needed for the analysis by eliminating the time-intensive enrichment of samples, which further may reduce costs and increase mobility of the analysis by eliminating the need for an enrichment media or device. The method disclosed herein enables analysis of highly acidic and / or unfilterable substrates, such as fruit juice, using known methods and reagents, or reagent combinations, to quantify microorganisms, which otherwise suffer from inhibition or enhancement of results due to the characteristics of the substrate. Further, the methods disclosed herein expand the scope of microorganisms that can be quantified beyond bacteria, including yeast. Finally, the disclosed methods are capable of detecting psychrophilic yeast which yeast plate count methods performed at conventional temperature is not.

[0042] FIG. 1 illustrates a flowchart of an example method 100 for preparing a sample for an assay for rapid quantification of a biologic contaminant. The biological contaminant may be any known or discovered cause of spoilage, but in many cases may be a microorganism. In examples, the biological contaminant is any of a bacteria, a mildew, a mold, or a yeast, . In some particular examples, the biological contaminant is a yeast.

[0043] At operation 102, a sample is added to a vessel. The sample may be drawn from a source to be tested, such as a tank or shipment of juice received as a storage or distribution facility. Samples may be drawn manually by a technician or automatically, such as periodic quality control samples taken by a production or packing device. In some embodiments, the sample is a liquid sample and the volume of the sample to be drawn is determined according to the final assay to be performed on the prepared sample. For example, the volume of a liquid sample drawn is between 1 pl and 1 L, between 10 pl and 500 ml, between 50 pl and 100 ml, between 100 pl and 5 ml. In other examples, the volume of liquid sample drawn is less than 1 L, less than 500 ml, less than 300 ml, less than 100 ml, less than 1 ml, less than 500 pl, less than 100 pl, less than 50 pl, or less than 20 pl.

[0044] In embodiments, the vessel may be a centrifuge vessel. The centrifuge vessel may be any appropriate vessel and may be selected to accommodate an available or desired centrifuge. In some examples, the vessel is a flask, a phial, a tube, a vial or an Eppendorf tube. The centrifuge vessel will be the appropriate size for the volume ofliquid sample drawn. For example, the centrifuge vessel may be IL, 500 ml, 100 ml, 2 ml 1ml, 500 pl, or 100 pl.

[0045] In some embodiments, the sample is a fruit juice. In some examples, the sample is one or more of a pome fruit juice or a berry juice. For example, the sample may include at least one of apple juice, pear juice, nashi juice, or quince juice. In other examples, the sample may include at least one of blueberry juice, raspberry juice, goji berry juice, acai juice, aronia berry juice, and strawberry juice, or cranberry juice. The sample may be a combination of juices. The sample may be single strength or concentrated.

[0046] At operation 104, the sample is centrifuged for a first time, yielding a first supernatant and a first pellet. Time and setting, such as speed and / or force, for the first centrifuge may be determined according to factors such as sample size, available equipment, etc. In some embodiments, the sample is centrifuged for 10 minutes at 3,000 x g (relative centrifugal force (RCF)). The centrifugation may occur for less than about 30 minutes, less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, less than about 5 minutes, or less than about 3 minutes. The centrifugation may occur at between about 1000 x g and about 10,000 x g, between about 2000 x g and about 5000 x g, or about 2500 x g and about 3500 x g. In some cases, the sample is removed immediately when centrifugation is completed, and the method proceeds to operation 106. At operation 106, the first supernatant is discarded.

[0047] At operation 108, the first pellet is dissolved in a first volume of a buffer to yield a first suspension volume. In some examples, the buffer includes one or more of calcium buffer, carbonic buffer, hydroxymethyl buffer, nitrate buffer, phosphate buffer and sulfonic buffer. In some embodiments, the first volume of buffer may be equal to the original sample volume. For example, a 1 ml sample may be drawn and centrifuged, and the first volume of buffer may be 1 ml. In some embodiments, the dissolving of the first pellet to yield a first suspension volume is enhanced by the use of agitation, e.g., using a vortex.

[0048] At operation 110, the first suspension volume is centrifuged to yield a second supernatant and a second pellet. The second centrifuge step may be determined according to the same factors as the first centrifuge step, operation 104. In some examples, the second centrifuge step may match the first centrifuge step. In some embodiments, the sample is centrifuged for 10 minutes at 3,000 x g RCF. In some cases, the sample isremoved immediately when centrifugation is complete and the method proceeds to operation 112. At operation 112, the second supernatant is discarded.

[0049] At operation 114, the second pellet is dissolved in a second volume of the buffer to yield a final sample suspension. The final sample suspension may use the same buffer as for the first sample suspension. In some cases, a different buffer may instead be used. In some embodiments, the second volume of buffer may be equal to the original sample volume and or the first volume of buffer. For example, a 1 mL sample may be drawn and centrifuged, and the second volume of buffer may be about 100 ul, or about 200 ul, or about 300 ul, or about 400 ul, or about 500 ul, or about 1 mL, or any volume in between.

[0050] At operation 116, the final sample suspension is added to an assay for rapid quantification for yeast. In embodiments, the assay may instead be added to the final sample suspension, or some combination such that some reagents are added the sample suspension and the sample solution is added to some reagents. The assay may be any appropriate assay for the quantification of microorganisms or other biological contaminants. Some non-limiting examples of suitable assays include manually counting colony forming units, absorbance-based assays, microscopy, flow cytometry, methylene blue dye reduction test (MBRT), and the start growth time (SGT) method. In embodiments, a particular assay may be selected based on available equipment and time. In examples, an absorbance-based adenosine triphosphate (ATP) assay, such as a luciferase system, may be used.

[0051] In some cases, the method 100 may be modified to include a dilution step to reduce background from the sample. FIG. 2 illustrates a flowchart of an example method 200 for preparing a sample with dilution for an assay for rapid quantification of a biologic contaminant.

[0052] At operation 201, the sample is diluted. In the example of FIG. 2, the step of diluting the sample is shown before the sample is added to the vessel at operation 202 and may advantageously ensure a sample of uniform dilution is centrifuged. In embodiments, diluting the sample may be performed prior to adding the sample to the vessel, prior to inserting the vessel into the centrifuge, or prior to centrifuging the sample.

[0053] In some embodiments, the sample is diluted at a ratio of 1 : 10. The dilution ratio may be selected according to characteristics of the juice, such as strength and acidity. Some non-limiting example of appropriate dilution ratios are 1 : 1000, 1 :500,1 : 100, 1 :50, 1 :20, 1 : 10, 1 :5, 1 : 1, 5: 1, 10: 1, 20: 1, 50: 1, 100: 1, or 1000: 1. Other nonlimiting examples of suitable dilution ratios include 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, and 15: 1.

[0054] At operation 202, a sample is added to a vessel. In embodiments, the vessel may be a centrifuge vessel. The sample may be drawn from a source to be tested, such as a tank or shipment of juice received as a storage or distribution facility. Samples may be drawn manually by a technician or automatically, such as periodic quality control samples taken by a production or packing device.

[0055] In embodiments, the vessel may be a centrifuge vessel. The centrifuge vessel may be any appropriate vessel and may be selected to accommodate an available or desired centrifuge. In some examples, the vessel is a flask, a phial, a tube, or a vial.

[0056] In some embodiments, the sample is a fruit juice. Embodiments incorporating the dilution step may be particularly advantageous when sampling a substrate with low pH, such as citrus juices. In some examples, the sample may include orange juice, lemon juice, lime juice, grapefruit juice, and pineapple juice.

[0057] At operation 204, the sample is centrifuged for a first time, yielding a first supernatant and a first pellet. Time and setting, speed and / or force, for the first centrifuge may be determined according to factors such as sample size, available equipment, etc. In some embodiments, the sample is centrifuged for 10 minutes at 3,000 x g RCF. In some cases, the sample is removed immediately when centrifugation is complete, and the method proceeds to operation 206. At operation 206, the first supernatant is discarded.

[0058] At operation 208, the first pellet is dissolved in a first volume of a buffer to yield a first suspension volume. In some examples, the buffer includes one or more of calcium buffer, carbonic buffer, hydroxymethyl buffer, nitrate buffer, phosphate buffer and sulfonic buffer. In some embodiments, the first volume of buffer may be equal to the original sample volume. For example, a 1 ml sample may be drawn and centrifuged, and the first volume of buffer may be 1 ml.

[0059] At operation 210, the first suspension volume is centrifuged to yield a second supernatant and a second pellet. The second centrifuge step may be determined according to the same factors as the first centrifuge step, operation 204. In some examples, the second centrifuge step may match the first centrifuge step. In some embodiments, the sample is centrifuged for 10 minutes at 3,000 x g RCF. In some cases, the sample isremoved immediately when centrifugation is complete, and the method proceeds to operation 212. At operation 212, the second supernatant is discarded.

[0060] At operation 214, the second pellet is dissolved in a second volume of the buffer to yield a final sample suspension. The final sample suspension may use the same buffer as for the first sample suspension. In some cases, a different buffer may instead be used. In some embodiments, the second volume of buffer may be equal to the original sample volume and / or the first buffer volume. The second buffer volume may be adjusted according to the dilution ratio or may be statically reduced in the case of diluted samples. For example, a 1 ml sample may be drawn and centrifuged, and the first volume of buffer may be 1 ml, while the second buffer volume may be reduced, e.g., 200 pl.

[0061] At operation 216, the final sample suspension is added to an assay for rapid quantification for yeast. In embodiments, the assay may instead be added to the final sample suspension, or some combination such that some reagents are added the sample suspension and the sample solution is added to some reagents. The assay may be any appropriate assay for the quantification of microorganisms or other biological contaminants may be used. Some non-limiting examples of suitable assays include manually counting colony forming units, absorbance-based assays, microscopy, flow cytometry, MBRT, and the SGT method. In embodiments, a particular assay may be selected based on available equipment and time. In examples, an absorbance-based ATP assay, such as a luciferase system, may be used.

[0062] FIG. 3 illustrates a flowchart of an example method 300 of performing a quantification assay on a prepared sample. The sample may be prepared according to either method 100 or method 200. As noted above, the assay may be any appropriate assay for the quantification of microorganisms or other biological contaminants. Example method 300 is directed to a particular example using an ATP assay measured based on relative light (e.g., using a luminometer), including assaying the final sample suspension to generate a bioluminescence proportional to the presence of the biological contaminant, measuring the bioluminescence generated in the final sample suspension, and converting the bioluminescence measurement to a quantification of the biological contaminant, according to the operations and considerations discussed below.

[0063] At operation 302, the final sample suspension is added to the assay. Operation 302 may be associated with operation 116 of FIG. 1 or operation 216 of FIG. 2. In some embodiments, the assay may include a single reagent or a pre-mixedcombination of reagents. Adding the sample to the assay may include mixing of reagents or combining a first pre-mixed combination of reagents to additional reagents or a second or more combination of reagents. Adding the sample to the assay may include mixing the sample with the assay reagents, e.g., by vortexing.

[0064] The volume of the final sample suspension added to the assay may be determined according to a number of factors, including the size of the original sample drawn, a concentration of the original sample drawn, the preparation of the sample (e.g., diluted or undiluted), the size of the container used for the assay, the concentration of the reagents used or available, etc.

[0065] At operation 304, the final sample suspension is incubated in the assay. The incubation may be performed for a predetermined incubation period at a predetermined temperature, e.g., 30 degrees C. The incubation period may be determined according to factors such as sample size and reagents used. In some embodiments, the incubation period is generally less than one hour, or even less than 30 minutes, in keeping with the rapid nature of the assay. Some non-limiting examples of appropriate incubation periods include 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 12 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, and 60 minutes.

[0066] In some embodiments, incubation at operation 304 incorporates additional elements to mix the assay during incubation. In some embodiments, the final sample suspension is agitated during incubation with the assay, such as by vortexing. Agitation may be performed prior to incubating the final sample suspension in the assay and / or concurrently with incubating the final sample suspension in the assay

[0067] At operation 306, the assay result is measured. In some embodiments, depending on the assay and / or the reagents used, measuring may be conducted immediately following the conclusion of the incubation period. In the present example, measuring the result of the assay includes measuring the bioluminescence generated in the final sample suspension following the incubation period. The measurement may be taken with any appropriate tool. For example, a luminometer is used to measure low light, such as bioluminescence, production in relative light units (RLU).

[0068] At operation 308, the result is converted. In the present example, the measurement is converted from the RLU output by the luminometer to a colony forming unit (CFU) measurement. RLU values are not in themselves the end result of a bio- luminogenic test. They must be correlated with a number of CFU to obtain the actualnumber of biological contaminant units or microorganisms in the sample. This conversion may generally be using a standard curve, calibrated to the biological contaminant to be quantified. For example, bacteria are generally smaller than the average yeast, and correlated CFU values associated with bacteria would not provide an accurate results to quantify yeast in a sample, or vice versa.

[0069] At operation 310, based on the final quantification of biological contaminants determined at operation 308, a final disposition for the sample is determined. In some embodiments, the final quantification may be compared with a predetermined threshold. The predetermined threshold may be associated with a standard, such as a food safety standard. If the quantification exceeds the threshold, the sample may be rejected and the source, e.g., tank, batch, etc. may be likewise rejected. In some embodiments, the threshold may be a predetermined number of RLUs. For example, thresholds of 500 RLU, 1000 RLU, or 2000 RLU may be implemented in various embodiments. If the quantification does not exceed the threshold, the sample may be accepted and the source likewise accepted.

[0070] In embodiments, standards and thresholds may be related to sensory standards in conjunction with measured microorganism content. For example, a sample may be further evaluated by taste and smell to determine an effect on the sample from the measured microorganism count.

[0071] Having described the preferred aspects and implementations of the present disclosure, modifications and equivalents of the disclosed concepts may readily occur to one skilled in the art. However, it is intended that such modifications and equivalents be included within the scope of the claims which are appended hereto.EXAMPLESExample 1

[0072] The quantification of yeast in orange, lemon, lime, grapefruit, and pineapple juices according to embodiments of the present disclosure using MicroSnap™ Total detection devices. MicroSnap™ Total is a rapid bioluminogenic test for detection and enumeration of bacteria that is available from Hygiena® LLC, Camarillo, California.

[0073] The MicroSnap™ detection devices were brought to roughly room temperature.

[0074] Each of the above-mentioned juices was diluted by adding 1 ml of juice to 9 ml of Butterfield’s Buffer resulting in 10 ml diluted mixtures for each of the juices. Butterfield’s Buffer is a common phosphate buffer (pH 7.2) commercially available from many companies, e.g., Thermo Fisher Scientific as Remel Butterfield’s Phosphate Buffer (Catalog number: R112520). The resulting 10 ml diluted mixtures for each juice were mixed thoroughly.

[0075] 1 ml from each 10 ml diluted mixture was added to a microcentrifuge tube.Each 1 ml centrifuge tube for each dilution was centrifuged at 3000 x g (RCF) for 10 minutes. Each microcentrifuge tube containing a diluted juice mixture was immediately removed from the centrifuge and the supernatant was quickly discarded by pouring off all liquid and keeping the pellet.

[0076] 1 ml of sterile Butterfield’s Buffer was added to each microcentrifuge tube and the pellet was resuspended by vortexing the microcentrifuge tube. Each 1 ml centrifuge tube for each dilution was centrifuged a second time at 3000 x g (RCF) for 10 minutes. Each microcentrifuge tube containing a diluted juice mixture was immediately removed from the centrifuge and the supernatant was quickly discarded by pouring off all liquid and keeping the pellet.

[0077] 200 pl of sterile Butterfield’s Buffer was added to each microcentrifuge tube and the pellet was resuspended by vortexing the microcentrifuge tube. Following vortexing (resuspension of pellet in 200 pl of buffer), 200 pl from each microcentrifuge tube was added to the MicroSnap™ Total detection device. In a preferred embodiment, only 4 samples in duplicate (8 tubes) are tested at one time due to the time-sensitivity of the procedure.

[0078] Break the seal on the ampoule of liquid at the top of the MicroSnap™ Total detection device and allow the liquid to mix with the sample below. Vortex the mixture and then squeeze the top of the device again to ensure that all liquid is at the bottom of the tube. Incubate each MicroSnap™ device for 10 minutes at 30°C.

[0079] Turn on the Ensure™ luminometer (available from Hygiena® LLC, Camarillo, California) just before the 10-minute incubation period ends. Allow the luminometer to warm up for 15 seconds.

[0080] Remove samples from the MicroSnap™ Total detection device immediately following the 10-minute incubation and place the MicroSnap™ Total detection device in the luminometer. Press “ok” on the luminometer to show results in Relative Light Units(RLU). In a preferred embodiment, results for duplicate samples of the same juice must be within 35% difference of each: % difference = (larger reading - lower reading) / lower reading. If the two readings are within the 35% range then take the average of the two readings in RLU.

[0081] The average of the RLU reading for each juice is then correlated with the colony forming units for yeast at different concentrations. In embodiments, correlation tables may be specific to a particular kind of device or model or model number.Example 2

[0082] Quantification of Yeast in Apple and Cranberry Juices according to embodiments of the present disclosure using MicroSnap™ Total detection devices.

[0083] The MicroSnap™ detection devices were brought to roughly room temperature.

[0084] 1 ml of each of the above-mentioned juices was added (without dilution) to a microcentrifuge tube. Each 1 ml centrifuge tube for each juice was centrifuged at 3000 x g (RCF) for 10 minutes. Each microcentrifuge tube containing a juice was immediately removed from the centrifuge and the supernatant was quickly discarded by pouring off all liquid and keeping the pellet.

[0085] 1 ml of sterile Butterfield’s Buffer was added to each microcentrifuge tube and the pellet was resuspended by vortexing the microcentrifuge tube. Each 1 ml centrifuge tube for each juice was centrifuged a second time at 3000 x g (RCF) for 10 minutes. Each microcentrifuge tube containing a juice was immediately removed from the centrifuge and the supernatant was quickly discarded by pouring off all liquid and keeping the pellet.

[0086] 1 ml of sterile Butterfield’s Buffer was added to each microcentrifuge tube and the pellet was resuspended by vortexing the microcentrifuge tube. Following vortexing (resuspension of pellet in 1 ml of buffer), 200 pl from each microcentrifuge tube was added to the MicroSnap™ device. In a preferred embodiment, only 4 samples in duplicate (8 tubes) are tested at one time due to the time-sensitivity of the procedure.

[0087] Break the seal on the ampoule of liquid at the top of the MicroSnap™ device and allow the liquid to mix with the sample below. Vortex the mixture and then squeeze the top of the device again to ensure that all liquid is at the bottom of the tube. Incubate each MicroSnap™ device for 10 minutes at 30°C.

[0088] Turn on the Ensure™ luminometer (available from Hygiena® LLC,Camarillo, California) just before just before the 10-minute incubation period ends. Allow the luminometer to warm up for 15 seconds.

[0089] Remove samples from the MicroSnap™ device immediately following the 10-minute incubation and place the MicroSnap™ device in the luminometer. Press “ok” on the luminometer to show results in Relative Light Units (RLU). In a preferred embodiment, results for duplicate samples of the same juice must be within 35% difference of each: % difference = (larger reading - lower reading) / lower reading. If the two readings are within the 35% range then take the average of the two readings in RLU.

[0090] The average of the RLU reading for each juice is then correlated with the colony forming units for yeast at different concentrations. In embodiments, correlation tables may be specific to a particular kind of device or model or model number. One such table is provided below, in Table 1, as an example. Correlation tables may be used to generate a graph from which a line of best fit may be determined and used to evaluate RLU readings of samples.Table 1CLAUSES

[0091] The following numbered clauses define further example aspects and features of the present disclosure:

[0092] Clause 1. A method of preparing a sample for an assay for rapid quantification of a biologic contaminant, the method including: adding the sample to a vessel; inserting the vessel into a centrifuge; centrifuging the sample to yield a first supernatant and a first pellet; discarding the first supernatant; dissolving the first pellet in a first volume of a buffer to yield a first suspension volume; centrifuging the first suspension volume to yield a second supernatant and a second pellet; discarding the second supernatant; dissolving the second pellet in a second volume of the buffer to yield a final sample suspension; and adding the final sample suspension to an assay for rapid quantification for yeast.

[0093] Clause 2. The method of clause 1, wherein the sample is a fruit juice.

[0094] Clause 3. The method of clause 1 or 2, wherein the sample is one or more of a pome fruit juice or a berry juice.

[0095] Clause 4. The method of clause 3, wherein the sample includes at least one of apple juice, pear juice, nashi juice, or quince juice.

[0096] Clause 5. The method of clause 3, wherein the sample includes at least one of blueberry juice, raspberry juice, goji berry juice, acai juice, aronia berry juice, and strawberry juice, or cranberry juice.

[0097] Clause 6. The method of any of clauses 1-5, further including diluting the sample.

[0098] Clause 7. The method of clause 6, wherein diluting the sample is performed at at least one of prior to adding the sample to the receptacle, prior to inserting the receptacle into the centrifuging vessel, and / or prior to centrifuging the sample.

[0099] Clause 8. The method of clause 6, wherein the sample is diluted in the buffer at a ratio selected from a group consisting of 1 : 1000, 1 :500, 1 : 100, 1 :50, 1 :20, 1 : 10, 1 :5, 1 : 1, 5: 1, 10: 1, 20: 1, 50: 1, 100: 1, or 1000: 1.

[0100] Clause 9. The method of clause 8, wherein the sample is diluted in the buffer a ratio selected from a group consisting of 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, and 15: 1.

[0101] Clause 10. The method of clause 6, wherein the sample includes a citrus juice.

[0102] Clause 11. The method of clause 10, wherein the sample includes at least one of orange juice, lemon juice, lime juice, grapefruit juice, and pineapple juice.

[0103] Clause 12. The method of any of clauses 1-11, wherein the buffer is a selected from a group consisting of calcium buffer, carbonic buffer, hydroxymethyl buffer, nitrate buffer, phosphate buffer and sulfonic buffer.

[0104] Clause 13. The method of any of clauses 1-12, wherein the assay is an adenosine triphosphate assay.

[0105] Clause 14. The method of clause 13, wherein the assay is a luciferase system.

[0106] Clause 15. The method of clause 13, further including incubating the final sample suspension in the assay.

[0107] Clause 16. The method of clause 15, wherein incubating the final sample suspension includes an incubation period.

[0108] Clause 17. The method of clause 16, wherein the incubation period is selected from a group consisting of: 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 12 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, and 60 minutes.

[0109] Clause 18. The method of clause 15, further including agitating the final sample suspension.

[0110] Clause 19. The method of clause 18, wherein agitating the final sample suspension includes vortexing the final sample suspension.[OHl] Clause 20. The method of clause 18, wherein agitating the final sample suspension is performed at at least one of prior to incubating the final sample suspension in the assay and / or concurrently with incubating the final sample suspension in the assay.

[0112] Clause 21. The method of clause 15, further including reading the final suspension using a luminometer to obtain a result, wherein the result is measured in relative light units (RLU).

[0113] Clause 22. The method of clause 21, further including interpreting the result by converting the relative light units (RLU) output by the luminometer to a colony forming unit (CFU) measurement.

[0114] Clause 23. The method of clause 15, wherein a total time to prepare, incubate, and analyze the sample comprises one of less than 120 minutes, less than 100minutes, less than 80 minutes, less than 60 minutes, less than 50 minutes, and less than 40 minutes.

[0115] Clause 24. The method of any of clauses 1-23, wherein the biological contaminant is at least one of a bacteria, a mildew, a mold, a yeast.

[0116] Clause 25. The method of clause 24, wherein the biological contaminant is a yeast.

[0117] Clause 26. The method of any of clauses 1-25, wherein the vessel is one of a flask, a phial, a tube, and a vial.

[0118] Clause 27. A method of rapidly quantifying a biologic contaminant in a liquid sample, the method including: adding the liquid sample to a vessel; inserting the vessel into a centrifuge; centrifuging the liquid sample to yield a first pellet; dissolving the first pellet in a first volume of a buffer to yield a first suspension volume; centrifuging the first suspension volume to yield a second pellet; dissolving the second pellet in a second volume of the buffer to yield a final sample suspension; assaying the final sample suspension to generate a bioluminescence proportional to a presence of the biological contaminant; measuring the bioluminescence generated in the final sample suspension; and converting the bioluminescence measurement to a quantification of the biological contaminant.

[0119] Clause 28. The method of clause 27, wherein assaying the final sample suspension includes incubating the final sample suspension.

[0120] Clause 29. The method of clause 28, wherein incubating the final sample suspension is performed for less than one hour.

[0121] Clause 30. The method of clause 29, wherein incubating the final sample suspension is performed for less than 30 minutes.

[0122] Clause 31. The method of clause 30, wherein incubating the final sample suspension is performed for 10 minutes.

[0123] Clause 32. The method of clause 27 or 28, further including agitating the final sample suspension.

[0124] Clause 33. The method of clause 32, wherein agitating the final sample suspension includes vortexing the final sample suspension.

[0125] Clause 34. The method of clause 32, wherein agitating the final sample suspension is performed at at least one of prior to incubating the final sample suspension in the assay and / or concurrently with incubating the final sample suspension in the assay.

[0126] Clause 35. The method of clause 27 or 28, wherein a total time to prepare, incubate, and assay the sample comprises one of less than 120 minutes, less than 100 minutes, less than 80 minutes, less than 60 minutes, less than 50 minutes, and less than 40 minutes.

[0127] Clause 36. The method of any of clauses 26-35, further including determining, based on the quantification of the biological contaminant, to reject a source of the liquid sample.

[0128] Clause 37. The method of any of clauses 26-36, wherein the liquid sample is a fruit juice.

[0129] Clause 38. The method of clause 37, wherein the liquid sample is one or more of a pome fruit juice or a berry juice.

[0130] Clause 39. The method of clause 38, wherein the liquid sample includes at least one of apple juice, pear juice, nashi juice, or quince juice.

[0131] Clause 40. The method of clause 38, wherein the liquid sample includes at least one of blueberry juice, raspberry juice, goji berry juice, acai juice, aronia berry juice, and strawberry juice, or cranberry juice.

[0132] Clause 41. The method of any of clauses 26-40, further including diluting the liquid sample.

[0133] Clause 42. The method of clause 41, wherein diluting the sample is performed at at least one of prior to adding the sample to the receptacle, prior to inserting the receptacle into the centrifuging vessel, and prior to centrifuging the sample.

[0134] Clause 43. The method of clause 41, wherein the sample is diluted in the buffer at a ratio selected from a group consisting of 1 : 1000, 1 :500, 1 : 100, 1 :50, 1 :20, 1 : 10, 1 :5, 1 : 1, 5: 1, 10: 1, 20: 1, 50: 1, 100: 1, or 1000: 1.

[0135] Clause 44. The method of clause 43, wherein the sample is diluted in the buffer a ratio selected from a group consisting of 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, and 15: 1.

[0136] Clause 45. The method of clause 41, wherein the sample includes a citrus juice.

[0137] Clause 46. The method of clause 45, wherein the sample includes at least one of orange juice, lemon juice, lime juice, grapefruit juice, and pineapple juice.

[0138] Clause 47. The method of any of clauses 27-46, wherein the buffer is a selected from a group consisting of calcium buffer, carbonic buffer, hydroxymethyl buffer, nitrate buffer, phosphate buffer and sulfonic buffer.

[0139] Clause 48. The method of any of clauses 27-47, wherein the assay is an adenosine triphosphate assay.

[0140] Clause 49. The method of clause 48, wherein the assay is a luciferase system.

[0141] Clause 50. The method of any of clauses 27-49, wherein the biological contaminant is at least one of a bacteria, a mildew, a mold, a yeast, and a virus.

[0142] Clause 51. The method of clause 50, wherein the biological contaminant is a yeast.

[0143] Clause 52. The method of any of clauses 27-51, wherein the vessel is one of a flask, a phial, a tube, and a vial.

[0144] Clause 53. A method of determining whether a sample contains less than a threshold amount of a biological contaminant, the method including: adding the sample to a vessel; inserting the vessel into a centrifuge; centrifuging the sample to yield a first pellet; dissolving the first pellet in a first volume of a buffer to yield a first suspension volume; centrifuging the first suspension volume to yield a second pellet; dissolving the second pellet in a second volume of the buffer to yield a final sample suspension; assaying the final sample suspension to generate bioluminescence proportional the biological contaminant’s presence; measuring the bioluminescence generated in the final sample suspension; converting the bioluminescence measurement to a quantification of the biological contaminant; and comparing the quantification of the biological contaminant the threshold amount for the biological contaminant.

[0145] Clause 54. The method of clause 53, wherein a total time from adding the sample to the vessel to determining whether the sample contains less than the threshold amount of the biological contaminant is one of less than 120 minutes, less than 100 minutes, less than 80 minutes, less than 60 minutes, less than 50 minutes, and less than 40 minutes.

[0146] Clause 55. A method of rapidly quantifying a biologic contaminant in a liquid sample, the method including: diluting the liquid sample with a first volume of a buffer; adding the diluted liquid sample to a vessel; inserting the vessel into a centrifuge; centrifuging the diluted liquid sample to yield a first pellet; dissolving the first pellet in a second volume of the buffer to yield a first suspension volume; centrifuging the firstsuspension volume to yield a second pellet; dissolving the second pellet in a third volume of the buffer to yield a final sample suspension; assaying the final sample suspension to generate a bioluminescence proportional a presence of the biological contaminant; measuring the bioluminescence generated in the final sample suspension; and converting the bioluminescence measurement to a quantification of the biological contaminant.

[0147] Clause 56. The method of clause 55, wherein the sample is diluted in the buffer at a ratio selected from a group consisting of 1 : 1000, 1 :500, 1 : 100, 1 :50, 1 :20, 1 : 10, 1 :5, 1 : 1, 5: 1, 10: 1, 20: 1, 50: 1, 100: 1, or 1000: 1.

[0148] Clause 57. The method of clause 56, wherein the sample is diluted in the buffer a ratio selected from a group consisting of 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, and 15: 1.

[0149] Clause 58. The method of clause 55, wherein the sample is a citrus juice.

[0150] Clause 59. The method of clause 58, wherein the sample includes at least one of orange juice, lemon juice, lime juice, and grapefruit juice.

[0151] Clause 60. The method of clause 55, wherein the sample comprises pineapple juice.

[0152] Clause 61. The method of any of clauses 55-60, wherein the buffer is a selected from a group consisting of calcium buffer, carbonic buffer, hydroxymethyl buffer, nitrate buffer, phosphate buffer and sulfonic buffer.

[0153] Clause 62. The method of any of clauses 55-61, wherein the assay is an adenosine triphosphate assay.

[0154] Clause 63. The method of clause 62, wherein the assay is a luciferase system.

[0155] Clause 64. The method of any of clauses 55-63, wherein the biological contaminant is at least one of a bacteria, a mildew, a mold, a yeast, and a virus.

[0156] Clause 65. The method of clause 64, wherein the biological contaminant is a yeast.

[0157] Clause 66. The method of any of clauses 55-65, wherein the vessel is one of a flask, a phial, a tube, and a vial.

[0158] Clause 67. The method of and of clauses 55-66, wherein assaying the final sample suspension includes incubating the final sample suspension.

[0159] Clause 68. The method of clause 67, wherein incubating the final sample suspension is performed for less than one hour.

[0160] Clause 69. The method of clause 68, wherein incubating the final sample suspension is performed for less than 30 minutes.

[0161] Clause 70. The method of clause 69, wherein incubating the final sample suspension is performed for 10 minutes.

[0162] Clause 71. The method of clause 67, further comprising agitating the final sample suspension.

[0163] Clause 72. The method of clause 71, wherein agitating the final sample suspension includes vortexing the final sample suspension.

[0164] Clause 73. The method of clause 71, wherein agitating the final sample suspension is performed at at least one of prior to incubating the final sample suspension in the assay and / or concurrently with incubating the final sample suspension in the assay.

[0165] Clause 74. The method of any of clauses 67-73, further including determining, based on the quantification of the biological contaminant, to reject a source of the liquid sample.

[0166] Clause 75. The method of any of clauses 67-74, wherein the liquid sample is a fruit juice.

[0167] Clause 76. The method of clause 55, wherein the liquid sample is one or more of a pome fruit juice or a berry juice.

[0168] Clause 77. The method of clause 76, wherein the liquid sample includes at least one of apple juice, pear juice, nashi juice, or quince juice.

[0169] Clause 78. The method of clause 76, wherein the liquid sample comprises at least one of blueberry juice, raspberry juice, goji berry juice, acai juice, aronia berry juice, and strawberry juice, or cranberry juice.

[0170] Clause 79. The method of clause 55, wherein a total time to quantify the biologic contaminant in the liquid sample includes one of less than 120 minutes, less than 100 minutes, less than 80 minutes, less than 60 minutes, less than 50 minutes, and less than 40 minutes.

Claims

CLAIMSWhat is claimed is:

1. A method of preparing a sample for an assay for rapid quantification of a biologic contaminant, the method comprising: adding the sample to a vessel; inserting the vessel into a centrifuge; centrifuging the sample to yield a first supernatant and a first pellet; discarding the first supernatant; dissolving the first pellet in a first volume of a buffer to yield a first suspension volume; centrifuging the first suspension volume to yield a second supernatant and a second pellet; discarding the second supernatant; dissolving the second pellet in a second volume of the buffer to yield a final sample suspension; and adding the final sample suspension to an assay for rapid quantification for yeast.

2. The method of claim 1, wherein the sample is a fruit juice.

3. The method of claim 1 or 2, wherein the sample is one or more of a pome fruit juice or a berry juice.

4. The method of claim 3, wherein the sample comprises at least one of apple juice, pear juice, nashi juice, or quince juice.

5. The method of claim 3, wherein the sample comprises at least one of blueberry juice, raspberry juice, goji berry juice, acai juice, aroni a berry juice, and strawberry juice, or cranberry juice.

6. The method of any of claims 1-5, further comprising diluting the sample.

7. The method of claim 6, wherein diluting the sample is performed at at least one of prior to adding the sample to the receptacle, prior to inserting the receptacle into the centrifuging vessel, and / or prior to centrifuging the sample.

8. The method of claim 6, wherein the sample is diluted in the buffer at a ratio selected from a group consisting of 1:1000, 1:500, 1:100, 1:50, 1:20, 1:10, 1:5, 1:1, 5:1, 10:1,20:1, 50:1, 100:1, or 1000:1.

9. The method of claim 8, wherein the sample is diluted in the buffer a ratio selected from a group consisting of 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, and 15:1.

10. The method of claim 6, wherein the sample comprises a citrus juice.

11. The method of claim 10, wherein the sample comprises at least one of orange juice, lemon juice, lime juice, grapefruit juice, and pineapple juice.

12. The method of any of claims 1-11, wherein the buffer is a selected from a group consisting of calcium buffer, carbonic buffer, hydroxymethyl buffer, nitrate buffer, phosphate buffer and sulfonic buffer.

13. The method of any of claims 1-12, wherein the assay is an adenosine triphosphate assay.

14. The method of claim 13, wherein the assay is a luciferase system.

15. The method of claim 13, further comprising incubating the final sample suspension in the assay.

16. The method of claim 15, wherein incubating the final sample suspension comprises an incubation period.

17. The method of claim 16, wherein the incubation period is selected from a group consisting of: 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 12 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, and 60 minutes.

18. The method of claim 15, further comprising agitating the final sample suspension.

19. The method of claim 18, wherein agitating the final sample suspension comprises vortexing the final sample suspension.

20. The method of claim 18, wherein agitating the final sample suspension is performed at at least one of prior to incubating the final sample suspension in the assay and / or concurrently with incubating the final sample suspension in the assay.

21. The method of claim 15, further comprising reading the final suspension using a luminometer to obtain a result, wherein the result is measured in relative light units (RLU).

22. The method of claim 21, further comprising interpreting the result by converting the relative light units (RLU) output by the luminometer to a colony forming unit (CFU) measurement.

23. The method of claim 15, wherein a total time to prepare, incubate, and analyze the sample comprises one of less than 120 minutes, less than 100 minutes, less than 80 minutes, less than 60 minutes, less than 50 minutes, and less than 40 minutes.

24. The method of any of claims 1-23, wherein the biological contaminant is at least one of a bacteria, a mildew, a mold, a yeast, and a virus.

25. The method of claim 24, wherein the biological contaminant is a yeast.

26. The method of any of claims 1-25, wherein the vessel is one of a flask, a phial, a tube, and a vial.

27. A method of rapidly quantifying a biologic contaminant in a liquid sample, the method comprising: adding the liquid sample to a vessel; inserting the vessel into a centrifuge; centrifuging the liquid sample to yield a first pellet; dissolving the first pellet in a first volume of a buffer to yield a first suspension volume; centrifuging the first suspension volume to yield a second pellet; dissolving the second pellet in a second volume of the buffer to yield a final sample suspension; assaying the final sample suspension to generate a bioluminescence proportional a presence of the biological contaminant; measuring the bioluminescence generated in the final sample suspension; and converting the bioluminescence measurement to a quantification of the biological contaminant.

28. The method of claim 27, wherein assaying the final sample suspension comprises incubating the final sample suspension.

29. The method of claim 28, wherein incubating the final sample suspension is performed for less than one hour.

30. The method of claim 29, wherein incubating the final sample suspension is performed for less than 30 minutes.

31. The method of claim 30, wherein incubating the final sample suspension is performed for 10 minutes.

32. The method of claim 27 or 28, further comprising agitating the final sample suspension.

33. The method of claim 32, wherein agitating the final sample suspension comprises vortexing the final sample suspension.

34. The method of claim 32, wherein agitating the final sample suspension is performed at at least one of prior to incubating the final sample suspension in the assay and / or concurrently with incubating the final sample suspension in the assay.

35. The method of claim 27 or 28, wherein a total time to prepare, incubate, and assay the sample comprises one of less than 120 minutes, less than 100 minutes, less than 80 minutes, less than 60 minutes, less than 50 minutes, and less than 40 minutes.

36. The method of any of claims 27-35, further comprising determining, based on the quantification of the biological contaminant, to reject a source of the liquid sample.

37. The method of any of claims 27-36, wherein the liquid sample is a fruit juice.

38. The method of claim 37, wherein the liquid sample is one or more of a pome fruit juice or a berry juice.

39. The method of claim 38, wherein the liquid sample comprises at least one of apple juice, pear juice, nashi juice, or quince juice.

40. The method of claim 38, wherein the liquid sample comprises at least one of blueberry juice, raspberry juice, goji berry juice, acai juice, aroni a berry juice, and strawberry juice, or cranberry juice.

41. The method of any of claims 26-40, further comprising diluting the liquid sample.

42. The method of claim 41, wherein diluting the sample is performed at at least one of prior to adding the sample to the receptacle, prior to inserting the receptacle into the centrifuging vessel, and prior to centrifuging the sample.

43. The method of claim 41, wherein the sample is diluted in the buffer at a ratio selected from a group consisting of 1 : 1000, 1 :500, 1 : 100, 1 :50, 1 :20, 1 : 10, 1 :5, 1 : 1, 5: 1, 10: 1, 20: 1, 50: 1, 100: 1, or 1000: 1.

44. The method of claim 43, wherein the sample is diluted in the buffer a ratio selected from a group consisting of 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, and 15: 1.

45. The method of claim 41, wherein the sample comprises a citrus juice.

46. The method of claim 45, wherein the sample comprises at least one of orange juice, lemon juice, lime juice, grapefruit juice, and pineapple juice.

47. The method of any of claims 27-46, wherein the buffer is a selected from a group consisting of calcium buffer, carbonic buffer, hydroxymethyl buffer, nitrate buffer, phosphate buffer and sulfonic buffer.

48. The method of any of claims 27-47, wherein the assay is an adenosine triphosphate assay.

49. The method of claim 48, wherein the assay is a luciferase system.

50. The method of any of claims 27-49, wherein the biological contaminant is at least one of a bacteria, a mildew, a mold, a yeast, and a virus.

51. The method of claim 50, wherein the biological contaminant is a yeast.

52. The method of any of claims 27-51, wherein the vessel is one of a flask, a phial, a tube, and a vial.

53. A method of determining whether a sample contains less than a threshold amount of a biological contaminant, the method comprising: adding the sample to a vessel; inserting the vessel into a centrifuge;centrifuging the sample to yield a first pellet; dissolving the first pellet in a first volume of a buffer to yield a first suspension volume; centrifuging the first suspension volume to yield a second pellet; dissolving the second pellet in a second volume of the buffer to yield a final sample suspension; assaying the final sample suspension to generate bioluminescence proportional the biological contaminant’s presence; measuring the bioluminescence generated in the final sample suspension; converting the bioluminescence measurement to a quantification of the biological contaminant; and comparing the quantification of the biological contaminant the threshold amount for the biological contaminant.

54. The method of claim 53, wherein a total time from adding the sample to the vessel to determining whether the sample contains less than the threshold amount of the biological contaminant comprises one of less than 120 minutes, less than 100 minutes, less than 80 minutes, less than 60 minutes, less than 50 minutes, and less than 40 minutes.

55. A method of rapidly quantifying a biologic contaminant in a liquid sample, the method comprising: diluting the liquid sample with a first volume of a buffer; adding the diluted liquid sample to a vessel; inserting the vessel into a centrifuge; centrifuging the diluted liquid sample to yield a first pellet; dissolving the first pellet in a second volume of the buffer to yield a first suspension volume; centrifuging the first suspension volume to yield a second pellet; dissolving the second pellet in a third volume of the buffer to yield a final sample suspension; assaying the final sample suspension to generate a bioluminescence proportional a presence of the biological contaminant; measuring the bioluminescence generated in the final sample suspension; andconverting the bioluminescence measurement to a quantification of the biological contaminant.

56. The method of claim 55, wherein the sample is diluted in the buffer at a ratio selected from a group consisting of 1:1000, 1:500, 1:100, 1:50, 1:20, 1:10, 1:5, 1:1, 5:1, 10:1,20:1, 50:1, 100:1, or 1000:1.

57. The method of claim 56, wherein the sample is diluted in the buffer a ratio selected from a group consisting of 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, and 15:1.

58. The method of claim 55, wherein the sample comprises a citrus juice.

59. The method of claim 58, wherein the sample comprises at least one of orange juice, lemon juice, lime juice, and grapefruit juice.

60. The method of claim 55, wherein the sample comprises pineapple juice.

61. The method of any of claims 55-60, wherein the buffer is a selected from a group consisting of calcium buffer, carbonic buffer, hydroxymethyl buffer, nitrate buffer, phosphate buffer and sulfonic buffer.

62. The method of any of claims 55-61, wherein the assay is an adenosine triphosphate assay.

63. The method of claim 62, wherein the assay is a luciferase system.

64. The method of any of claims 55-63, wherein the biological contaminant is at least one of a bacteria, a mildew, a mold, a yeast, and a virus.

65. The method of claim 64, wherein the biological contaminant is a yeast.

66. The method of any of claims 55-65, wherein the vessel is one of a flask, a phial, a tube, and a vial.

67. The method of and of claims 55-66, wherein assaying the final sample suspension comprises incubating the final sample suspension.

68. The method of claim 67, wherein incubating the final sample suspension is performed for less than one hour.

69. The method of claim 68, wherein incubating the final sample suspension is performed for less than 30 minutes.

70. The method of claim 69, wherein incubating the final sample suspension is performed for 10 minutes.

71. The method of claim 67, further comprising agitating the final sample suspension.

72. The method of claim 71, wherein agitating the final sample suspension comprises vortexing the final sample suspension.

73. The method of claim 71, wherein agitating the final sample suspension is performed at at least one of prior to incubating the final sample suspension in the assay and / or concurrently with incubating the final sample suspension in the assay.

74. The method of any of claims 67-73, further comprising determining, based on the quantification of the biological contaminant, to reject a source of the liquid sample.

75. The method of any of claims 67-74, wherein the liquid sample is a fruit juice.

76. The method of claim 55, wherein the liquid sample is one or more of a pome fruit juice or a berry juice.

77. The method of claim 76, wherein the liquid sample comprises at least one of apple juice, pear juice, nashi juice, or quince juice.

78. The method of claim 76, wherein the liquid sample comprises at least one of blueberry juice, raspberry juice, goji berry juice, acai juice, aroni a berry juice, and strawberry juice, or cranberry juice.

79. The method of claim 55, wherein a total time to quantify the biologic contaminant in the liquid sample comprises one of less than 120 minutes, less than 100 minutes, less than 80 minutes, less than 60 minutes, less than 50 minutes, and less than 40 minutes.

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