APPARATUS FOR OPTICAL INSPECTION OF SMALL VOLUMES OF LIQUID SAMPLE AND CUVETTES THEREFOR

Abstract

A nephelometer that measures turbidity of low volume suspensions using measurements of light transmitted through and/or scattered by the sample. The sample suspension is placed in a tiered cuvette adapted to facilitate measuring the turbidity of low volume samples. The lower portion of the cuvette has smaller dimensions, in horizontal cross section, than the top portion. Both lower and upper portions have angled surfaces. The lower, smaller portion of the cuvette is interrogated by the nephelometer.

Claims

1. An apparatus for optical interrogation of a sample comprising: an optical source for an optical signal; optionally a first detector positioned to receive the optical signal transmitted directly through a cuvette received by the apparatus wherein the apparatus is adapted to receive a cuvette with a narrower lower portion and a wider upper portion the wider upper portion having a perimeter larger than the perimeter of the lower narrower portion the apparatus further adapted to position the cuvette such that the first detector detects the optical signal transmitted directly through the narrower, lower portion of the cuvette; a second detector positioned to receive the optical signal from the optical source scattered by contents in the lower portion of the cuvette, the second detector surface positioned approximately parallel to the optical path from the optical source to the first detector; wherein the optical source, first detector and second detector are at least partially disposed in apertures in the apparatus.

2. The apparatus of claim 1 further comprising: a channel configured to advance a linear series of cuvettes, each cuvette being advanced in series to a measurement position wherein the lower portion of the cuvette is adjacent to the optical source, first detector and second detector for measurement.

3. The apparatus of claim 2 wherein the apparatus is a nephelometer.

4. The apparatus of claim 1 wherein the apparatus is configured to receive an array of cuvettes configured as a tray from which the lower portion of the cuvette is suspended.

5. The apparatus of claim 1 further comprising a light attenuation filter positioned between the cuvette and the first detector.

6. The apparatus of claim 1 further comprising one of a focusing lens, an aperture or a series of apertures positioned intermediate between the light source and the narrower, lower portion of the cuvette.

7. The apparatus of claim 6 wherein the focusing lens, aperture or series of apertures collimates the light transmitted therethrough.

8. The apparatus of claim 1 wherein the light source is selected from the group consisting of a laser light source and an LED.

9. The apparatus of claim 1 comprising both first and second detectors and wherein the first and second detectors are positioned at a 90 degree angle from each other.

10. The apparatus of claim 1 wherein the detectors operate across the visible light spectrum from ultra-violet (UV) to infra-red (IR).

11. The apparatus of claim 10 wherein wavelength of the detected light is in the range of about 620 to about 750 nm.

12. The apparatus of claim 1 wherein the cuvette is optically transparent.

13. An apparatus for optical interrogation of a sample comprising: a base; an optical source for an optical signal positioned in the base; a first detector positioned in the base to receive the optical signal scattered by a sample disposed in a cuvette received by the apparatus wherein the apparatus is adapted to receive a cuvette with a narrower lower portion and a wider upper portion the wider upper portion having a perimeter larger than the perimeter of the lower narrower portion the apparatus further adapted to position the cuvette such that the first detector detects the optical signal scattered by the sample in the narrower, lower portion of the cuvette; wherein the optical source and first detector are at least partially disposed in apertures in the base.

14. The apparatus of claim 13 wherein the apparatus is a spectrometer.

15. The apparatus of claim 13 wherein the apparatus is a nephelometer further comprising a second detector positioned in the base to receive the optical signal from the optical source transmitted through the contents in the lower portion of the cuvette, the second detector surface positioned approximately parallel to the optical path from the optical source to the first detector.

16. The apparatus of claim 13 wherein the base is configured to receive an array of cuvettes configured as a tray from which the lower portion of the cuvette is suspended.

17. The apparatus of claim 15 further comprising a light attenuation filter positioned between the cuvette and the first detector.

18. The apparatus of claim 15 further comprising one of a focusing lens, an aperture or a series of apertures positioned intermediate between the light source and the narrower, lower portion of the cuvette.

19. The apparatus of claim 18 wherein the light transmitted to the cuvette is collimated.

20. The apparatus of claim 15 wherein the light source is selected from the group consisting of a laser light source and an LED.

21. The apparatus of claim 15 wherein the first and second detectors are positioned at a 90 degree angle from each other.

22. The apparatus of claim 15 wherein the detectors operate across the visible light spectrum from ultra-violet (UV) to infra-red (IR).

23. The apparatus of claim 22 wherein wavelength of the detected light is in the range of about 620 to about 750 nm.

24. The apparatus of claim 15 wherein the cuvette is optically transparent.

25. A method for measuring the turbidity of a sample comprising: providing a cuvette to a nephelometric apparatus, the cuvette comprising a narrower lower portion and a wider upper portion the wider upper portion having a perimeter larger than the perimeter of the lower narrower portion, the cuvette having a sample for inspection disposed in at least the lower narrower portion; receiving the cuvette in a base that positions the cuvette such that a light source emits light directed into the lower narrower portion of the cuvette; transmitting light from the light source into the lower narrower portion of the cuvette; optionally detecting, using a first detector positioned to receive the optical signal transmitted directly through a cuvette received by the apparatus, the transmitted signal; and detecting, using a second detector, the optical signal from the optical source scattered by contents in the lower portion of the cuvette, the second detector surface positioned approximately parallel to the optical path from the optical source to the first detector.

26. The method of claim 25 wherein the cuvette is positioned in an array of cuvettes and the array is placed in the nephelometric apparatus such that the turbidity of sample disposed in each cuvette is measured.

27. The method of claim 26 wherein the array of cuvettes is a tray.

28. The method of claim 24 further comprising collimating the light directed to the lower narrower portion of the cuvette.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] To assist those of ordinary skill in the relevant art in making and using the subject matter thereof, reference is made to the appended drawings.

[0019] FIG. 1A illustrates one embodiment of low volume single cuvette nephelometer.

[0020] FIG. 1B is a cut away view of the single cuvette nephelometer along line 1-1 of FIG. 1A.

[0021] FIG. 2A is a series detail view of FIG. 1B.

[0022] FIG. 2B is a perspective view of a continuous cuvette nephelometer.

[0023] FIG. 3 illustrates a linear low volume multi-cuvette array/strip design for the continuous series cuvette nephelometer.

[0024] FIG. 4A illustrates a cuvette according to one embodiment of the present invention.

[0025] FIG. 4B illustrates a cuvette according to an alternate embodiment of the present invention.

[0026] FIG. 5 is a process flow diagram illustrating one embodiment for a process to prepare a sample using the nephelometer described herein.

[0027] FIG. 6 illustrates stacked cuvettes.

[0028] FIG. 7 is a cut away view of the transmitted light detector pathway for one embodiment of the present invention.

[0029] FIG. 8 is a cut away view of the scattered light detector pathway for one embodiment of the present invention.

[0030] FIG. 9 is a cutaway view of the embodiment of FIG. 7 but illustrating the optical source and the transmitted light detector.

[0031] FIG. 10 is a perspective view of the nephelometer according to one embodiment.

DETAILED DESCRIPTION

[0032] Embodiments described herein provide for automated methods of measuring turbidity of liquid suspensions using vessels that are configured to receive and measure low sample volumes yet accommodate dilution of the suspension inside the individual vessels. The disclosed methods further allow for measuring turbidity levels in suspensions having insufficient volume to be measured using conventional vessels and apparatus. The nephelometry apparatus described herein is configured for integration into a system where suspension dilution and turbidity measurements are automated.

[0033] All numerical values within the detailed description and the claims herein are modified by “about” or “approximately” the indicated value, and take into account experimental error and variations that would be expected by a person having ordinary skill in the art.

[0034] As used herein, a “low volume” and/or a “small volume” sample refers to a sample having a volume of about 100 μL to about 500 μL and all volumes and ranges within that range (i.e., about 100 μL to about 200 μL; about 100 μL to about 300 μL; about 100 μL to about 400 μL; about 200 μL to about 500 μL; about 200 μL to about 300 μL; about 200 μL to about 400 μL about 300 μL to about 500 μL, about 300 μL to about 340 μL, about 400 μL to about 500 μL etc.)

[0035] As used herein, the term “liquid suspension” and/or “liquid sample” refer to a mixture of soluble and/or insoluble particles and/or solid materials dispersed in a liquid. In some embodiments, the liquid sample is a biological sample. Examples of a biological sample are well known to one skilled in the art and are not described in detail herein. Representative examples include biological tissue, fluid obtained in vivo, fresh blood, whole banked blood, etc.

[0036] As used herein, a “cuvette” and/or “micro-cuvette” and/or “low volume cuvette” and/or “LVC” and/or “sample vessel” or “vessel” is the container suitable for receiving a liquid suspension. The container is preferably made of optically transparent plastic or glass that is designed to hold a test sample in a specific space and orientation for testing or processing.

[0037] As used herein, “algorithms” are one or more mathematical instructions that are used to manipulate values of data to make a decision based on a mathematical value and then produce a corrected or more accurate data value representative of the desired output.

[0038] As used herein, an “amplifier” is an electronic circuit that is used to take a smaller original electronic signal and increase its amplitude to produce a proportionately larger new signal that is representative of the original signal. Suitable amplifiers are well known to those skilled in the art and are not described in detail herein.

[0039] As used herein, an “analog to digital converter” or “A/D converter” is an electronic device that is capable of taking a variable electrical signal and turning it into a number that is representative of the amplitude of the original signal.

[0040] As used herein, “dilution” means a solution or suspension produced by adding a liquid diluent to a concentrated solution or suspension resulting in a new suspension or solution with a lower uniform concentration of sample in the solution or suspension than the original.

[0041] As used herein, “laser” or “laser diode” is an electronic device that produces a concentrated and focused beam of light when an electrical current is applied.

[0042] As used herein, “light attenuation filter” is a device that is placed into a light path to absorb and reduce the amount of light as it passes through the filter resulting in the light that was passed through the filter to have proportionally lower intensity than the original light source.

[0043] As used herein, “light emitting diode” or “LED” is an electronic device that emits light of a specific type and orientation when an electrical current is applied.

[0044] As used herein, “McFarland” is a unit of measure of the amount of solid particulates dispersed in a fluid or liquid suspension.

[0045] As used herein, “nephelometer” is an instrument that is capable of measuring the amount of solid particles in a suspension. As used herein, “nephelometry” refers to a method by which the amount of suspended solids in a suspension can be measured.

[0046] As used herein, “photo-diode” and/or “detector” is an electronic device used to measure the intensity of light in a given environment.

[0047] As used herein, “saturated” and/or “saturation” is the point at which the detector has reached the maximum amount of output signal it is capable of producing. For example, adding more light to the photo-detector past saturation does not produce any further change in the detector output signal which has reached its maximum operating capability.

[0048] As used herein, “suspension” is a solution in which solids are distributed uniformly in the liquid.

[0049] As used herein, “turbidity” is the measurement of the amount of suspected solids in a solution (i.e., cloudiness of a liquid sample).

[0050] In the embodiments described below, the apparatus is described in terms of device that is configured to detect light that is both transmitted through and scattered by the sample in the cuvette. Contemplated herein are devices and methods in which only of the light scattered by the sample, transmitted through the sample, or both are measure to determine turbidity. In some embodiments, an additional photodetector may be provided to the side of the light path to one of the scattered light detector, the transmitted light detector or both. In those embodiments where the light source is an LED, the measurement made by this additional photodetector is used in a control loop to adjust power to the LED and maintain a consistent and repeatable light intensity. Use of such detectors to control LED output, address thermal drift and compensate for any degradation in signal output are well known to one skilled in the art and not described in detail herein.

[0051] FIG. 1 illustrates the system of one embodiment described herein for measuring turbidity of a liquid sample using a nephelometer and principles of nephelometry. The sample system is designed to house a single cuvette 110 that has a suspension fluid 120 placed inside a nephelometer base 100 as shown in FIG. 1A. The system also includes a light source 130, a focusing lens 170, a side scatter detector 140, a transmitted light detector 150, and a light attenuation filter 160 (FIG. 1B). The cuvette 110 with a sample 120 is positioned at the center of the apparatus and inside the nephelometer base 100. The light source 130, the scatter detector 140 and the transmitted light detector 150 are positioned at 90 degree angles from one another around the cuvette 110. Positioning the scatter detector 140 within close proximity to the cuvette containing the sample suspension 120 and parallel to the incident light source minimizes the effects of diffraction, refraction and reflection on the scattered light. The transmitted light detector 150 is positioned at 180 degrees or opposite from the light source 130. The detector 150 also may be oriented either perpendicular to the incident light beam or at a different angle to reduce reflectance effects from its surfaces. The light attenuation filter 160 is positioned between the cuvette 110 and the transmitted light detector 150. The system for measuring turbidity detects scattered and/or transmitted light that is passed through the tested sample at an angle. In this configuration, sample suspensions are individually processed inside the vessel 110.

[0052] The invention contemplates use of low volume vessels/cuvettes (or micro-cuvettes) that are designed to process relatively small quantities of biological and fluid suspensions for use with the low volume nephelometer. In the exemplary embodiments, the cuvette is molded from optically clear plastic with minimally tapered sides that have an optically smooth polish to be conveniently oriented within the nephelometer disclosed herein. The cuvettes may be configured as individual units for single use applications. In the embodiments where a series of cuvettes are used to prepare suspensions, the cuvettes can be configured for use with linear array strips for such applications. Alternatively, the cuvettes may be configured for use with a matrix array designed for processing multiple samples simultaneously. In the matrix embodiment, multiple series of suspensions are prepared in parallel. FIGS. 4A-B illustrate alternative embodiments of the low volume cuvette design for use with nephelometers described herein. The cuvette 110 has a lower portion 410 that has a small volume. The suspension is initially prepared in the small volume portion. The suspension is therefore first disposed inside the lower portion 410 of the cuvette. Next, a biological sample suspected of containing a target microorganism(s) is added to, and mixed with, the fluid suspension to provide a test sample suspension 120. The turbidity of the suspension in the lower portion 410 is measured. The light 130 is passed through the sample suspension 120 that is disposed inside the lower portion 410. The measuring apparatus is configured to measure turbidity of the sample in the lower portion 410 of the cuvette. Beneath the lower portion 410 is a “large particulate” collection area 420 which is designed to receive large particles that settle from the sample suspension that would otherwise adversely affect the accuracy of the turbidity measurements made by the nephelometer. Low volume samples otherwise have insufficient volume to allow the particulate contaminants to settle from the portion of the suspension interrogated by the nephelometer. For example, a light which passes through a low volume suspension that contains particulate impurities may not differentiate between the sample in suspension and the impurities and can yield inaccurate McFarland values that will in turn cause the sample to be processed improperly. For example, an inaccurate McFarland value may inform the wrong dilution. An inaccurate McFarland value may cause a sample to be processed downstream (either by AST or Maldi for example), when, had the true McFarland value been known, the sample would not have been further processed. That is, the true McFarland value would have informed the operator that the sample was not suited for Maldi or AST. In addition, the presence of impurities in the sample may interfere with the accurate concentration measurements of the sample being tested. The cuvettes according to the present invention provide a separate collection area 420 that is outside of the direct light path that passes through lower portion 410. Particulate contaminants settle into the collection area 420 and do not remain in the tested area of the sample suspension, which occurs in the lower portion 410. The cell length of the lower portion is in the range of about 5.5 mm and is designed to provide sufficient cell length for low volume samples to obtain adequate turbidity measurements. The lower portion is designed to provide sufficient cell length once a test sample suspension is prepared in order for the light to pass through the samples and be captured by the detectors 140 and 150. Preferably, the lower portion 410 is made of a highly polished optical material or a material having near optical clarity and other optically transmissive materials known to one skilled in the art. Such materials allow the light to pass through the walls of the lower portion of the cuvette without interference.

[0053] One skilled in the art will appreciate that there are three dimensions of design freedom to configure the small volume portion of the cuvette. The dimensions of the small volume are largely a matter of design choice. In one embodiment, the dimensions of the small volume portion are configured to receive a device (i.e. a pick tool) that will introduce the sample into the lower portion of the cuvette. For example, and not by way of limitation, the lower portion of the cuvette is dimensioned to provide adequate room for a 3 mm diameter pick tool to be submerged and rotated within the lower portion such that it does not touch the sides of the cuvette, creating scratches and surface aberrations that would degrade the optical transparency of the cuvette.

[0054] Of course, the dimensions of the lower portion must accommodate optical inspection of the sample. Specifically, the lower portion of the cuvette is dimensioned to work with the optical source and detectors of the optical inspection device. The dimensional constraints on cuvette design are therefore a function of the configuration of the device that will optically interrogate the sample.

[0055] Above the lower portion 410 is the upper portion 400 which is used to dilute the sample suspension placed inside the vessel for further processing in downstream applications. The upper portion 400 has a larger width and length than the lower portion 410. Preferably, the internal dimensions of the vessel are designed to accommodate automated mixing of the biological sample with a suspension fluid to further dilute the test sample suspension directly inside the vessel when required. In operation, the tiered vessel design allows the turbidity of the sample suspension to be measured and, if target turbidity has not been reached, to further dilute the sample and repeat the turbidity measurements. Such a configuration allows dilution of the sample in real time (i.e. as the sample is being optically interrogated). In addition, the tiered vessel design makes it possible to measure the turbidity of low volume sample suspensions (e.g. suspensions with a volume of about 200 μL to about 500 μL) yet have the benefits of a larger volume to accommodate sample dilution.

[0056] In the exemplary embodiments, the vessel is a two-tiered cuvette. The top tier has an approximately square or rectangular or round perimeter. Basically the geometric configuration of the top portion is a matter of design choice. The bottom tier also has an approximately square perimeter. The cuvette “telescopes” from top to bottom because the top tier has larger dimensions (in horizontal cross section) than the lower portion. Alternative shapes for the cuvette are also contemplated so long as the walls of the bottom portion of the cuvette are at an angle from one another (e.g., the cuvette is not cylindrical, elliptical etc.). It has been found that positioning the walls of the lower portion of the cuvette (i.e. the portion received by the nephelometer) at an angle from one another (compared to a round-shape tube) allows for less aberration to the optical signal and better mixing of the test sample. In one illustrated embodiment, the upper portion 400 has been selected to have four sides 430 that are perpendicular to one another, thereby defining a square. The lower portion 410 also has four sides 440 that are perpendicular to one another, except the dimensions of the sides 440 are narrower than the sides 430. The smaller, lower portion 410 is configured to be received by the nephelometer base and/or linear cuvette array. The top of the cuvette has an opening 450 for receiving the sample and diluent. The side walls 430 and 440 of the upper and lower portions, respectively, are configured as planar surfaces. Without being bound to any particular theory, it is believed that planar surfaces minimize diffraction and refraction of the light that passes through the surface of the cuvette. It addition, the square configuration of the cuvettes/vessels allows for the light paths to pass through and into the sample suspension and the vessel at right angles to the surface plane of the vessel. This configuration also minimizes the potential for diffraction or refraction of the light source 130 as it enters and leaves the cuvette.

[0057] Various configurations of the cuvette are contemplated. In one embodiment, the top portion of the cuvette is tapered to the lower portion. The corners of the top portion align with the corners of the lower portion as can be seen by straight edges 401 (FIG. 4A). The tapered edges 401 demark the transition between the wider upper portion 400 and the narrower lower portion 410. In another embodiment, the edges of the upper portion 400 are offset from the edges of the lower portion 410 as the offset edges 402 are illustrated in FIG. 4B. For example, the edges 402 are offset by 45 degrees from the edges of the top portion. Advantageously, this configuration allows for the light source and detectors to be arranged on either side of the cuvette when the cuvette is placed inside the nephelometer base. Placement of the cuvettes with edges 402 inside the linear array 300 allows for more efficient transport of the cuvettes through the nephelometer because they can be processed in series and received by the nephelometer and measured without additional manipulation of the cuvette.

[0058] The cuvette/nephelometer assembly for measuring turbidity operated as described in the following embodiments. The cuvette 110 is placed inside the nephelometer base 100. The cuvettes are placed inside the nephelometer base either automatically or manually. Referring to FIG. 5, the initial suspension fluid (free of microorganisms) is placed inside the cuvette 100. The fluid volume is about 200 μL to about 500 μL. Preferably, the initial suspension fluid volume is about 300 μL. Additional fluid can be added to the cuvette if dilution is needed to obtain the specified McFarland values. Next, a biological sample suspected of containing microorganisms is added to the cuvette 110 and mixed with the suspension fluid to yield a test sample suspension. The apparatus described herein measures the initial turbidity of the test sample and the McFarland value is recorded. The sample suspension is further diluted by adding additional suspension fluid if the initial turbidity readings are too high. The dilution is automated in one embodiment. The upper portion allows the volume of the suspension fluid to exceed the volume of the lower portion. The apparatus measures the turbidity of the diluted suspension. Once the predetermined McFarland value is obtained, the suspension is either processed for downstream testing, stored or discarded. The suspension may be diluted as many times as necessary in order to obtain the desired McFarland values.

[0059] A light from source 130 interrogates the suspension 120 (e.g., tested sample) disposed inside the cuvette 110. The light that impinges on a surface (e.g., flat side wall of the cuvette/vessel) is referred to herein as the incident light. The light that is scattered from the particles of the suspension 120 is referred herein as the scattered light. A portion of the incident light is reflected by the cuvette surface. The refracted or transmitted light is the portion of the incident light that is transmitted through the surface (e.g., the flat side wall of the cuvette/vessel).

[0060] In operation, the transmitted light is received by the transmitted light detector 150. In the exemplary embodiments, the transmitted light detector 150 is positioned on the incident light path to maximize the detection of the light transmitted through the suspension. In instances where the surface of the detector 150 is highly reflective, the detector 150 may be positioned such that the detector surface is located at a slight angle (not 90 degrees) in relation to the light path axis. Positioning the detector 150 at an angle optimizes detection of the transmitted light without reflecting the light back into the suspension 120 or directing the light to other portions of the nephelometer. The intensity of light collected by the detector is proportional to the turbidity of the suspension.

[0061] A light attenuation filter 160 is positioned directly in front of the transmitted light detector 150. The filter reduces the intensity of the light incident on the detector by an amount that is proportional to that of the incident beam. In the exemplary embodiments, the filter allows the detector 150 to operate without saturating and provides sufficient detector operational intensity bandwidth to detect slight variations in the intensity of the transmitted light.

[0062] The apparatus according to the present invention also measures the amount of scattered light. The scatter detector 140 is placed with its detecting surface parallel to the incident light path and along one side of the cuvette. Portions of the light that are passed through the suspension sample are scattered by the particles in suspension. The side scatter detector 140 collects some of the scattered light. The amount of scattered light that the detector 140 collects provides a signal that is proportional to the amount of particles in the tested suspension 120. One way to measure the turbidity of the suspension 120 is to process the amount of scattered light collected by the scatter detector 140 through various algorithms well known in the art. The data collected from the scatter detector 140 may be combined with the data collected from the transmission detector 150 in various ways. For example, the signals can be physically combined or the detector values mathematically manipulated to combine them in a way to further enhance the accuracy and reliability of the initial signals. The signals or data values can be combined additively, subtractively, differentially, etc. to provide a resultant signal that is representative of the combined signals. When signals of detector values are combined in this manner it is possible to enhance the resolution and accuracy of collected data for measuring turbidity. Advantageously, data collected from two separate detectors (scatter and transmittance data) may provide more accurate results for small volume samples. The dual measurement is advantageous in those embodiments where a scatter measurement does not suffice. Although applicants do not wish to be held to a particular theory, in applicants' view the measurement of both transmitted and scattered light yields is more accurate because of the limited length of the light path through the small volume of sample.

[0063] In the exemplary embodiments, scatter detector 140 and transmittance detector 150 are standard high efficiency photo diode detectors. However, other detectors having similar characteristics may also be used. Suitable detectors include those that operate across the visible light spectrum from ultra-violet (UV) to infra-red (IR). Suitable detectors may be selected based on their linear response curves, size, reproducibility of results, and the ability to operate/detect light paths within low light conditions and detect minute variations in light intensity with measurable resolution. Examples include photo diodes, photo multiplier tubes, avalanche detectors, solar cells, photo resistors, photo sensors, etc. Such detectors are commercially available, well known to one skilled in the art and not described in detail herein.

[0064] In the exemplary embodiments, the light source is a high intensity light emitting diode (LED) or diode laser. Preferably, the frequency of the LED light is about 650 nm. Preferably the wavelength of the detector light is within the red color band (i.e. about 620 to 750 nm). However the skilled person might use interrogating light at different frequencies of visible light. Optionally, a focusing lens 170 (FIG. 1B) is used to focus the light into a narrow beam (e.g., a beam that is about 3 mm in diameter). The focusing lens 170 is positioned in front of the light source 130. The use of a focusing lens 170 concentrates the light from the light source 130 inside the sample area 410 of the vessel/cuvette and minimizes the amount of light that may be scattered from the test area. One skilled in the art is aware that a light which is scattered outside of the test area (i.e., the lower portion 410 of the cuvette) renders the scatter unusable for the purposes of measuring sample turbidity due to high background signal. The focused light then passes from the focusing lens 170 (not shown) into the lower portion 410 of the cuvette at an angle perpendicular to the face of the cuvette. The perpendicular angle mitigates unwanted diffraction and refraction which occurs when a beam of light passes from one medium (e.g., air) to another medium (e.g., flat surface sides of a cuvette). The path of the focused light beam is maintained as the light transmits through the suspension towards the detectors 140 and 150. In the embodiments where the light source is a diode laser, additional lenses may not be required to focus the light beam. This is due in part to the properties of the laser which provide collimated and focused light to interrogate the suspension. A focus lens or a series of apertures is used in the embodiments where the light source is an LED and collimating or focusing of the light is desired or required.

[0065] FIG. 3 illustrates the series cuvette array/receptacle for use with one embodiment of the apparatus of the present invention. The series cuvette array is a series cuvette strip that is moved along guided channel 220. An LED light source 130 is placed on one side of the guided channel 220 that guides the strip 300. The strip 300 is slideably engaged with the channels 220. The strip 300 also include stand-offs or other structures 530 (FIG. 6) for convenient stacking, packaging and shipping. The strip 300 is advanced through nephelometer and the cuvette wells 320 are positioned between the light source 130 and detectors 140 and 150 (not shown) for processing. After processing is complete the linear strip 300 may be indexed and advanced to the next cuvette and processing continued for the subsequent samples using the same nephelometer. The cuvette strip 300 may be stored or discarded based upon individual user's needs. In this embodiment, a single nephelometer is designed to efficiently process multiple samples without the need to remove individual cuvettes and replace them with new cuvettes. The linear cuvette strip 300 may be designed with various cuvette shapes, sizes and configurations. For example, the wells 320 of the strip 300 may be designed to be more or less deep, wider, narrower, longer, shorter etc. depending upon the cuvette design. In addition, the wells may be attached to one another across individual wells or be individually inserted into the wells positioned next to one another.

[0066] In one embodiment, the cuvette strips are stackable and can be separated either into individual cuvettes or a linear strip of cuvettes, depending upon the nephelometer configuration. This embodiment is illustrated in FIG. 6. There cuvettes 500 are carried by rack 510. Rack 510 has a flat surface from which the cuvettes are suspended. The flat surface is scored (not shown) to allow the cuvettes to be separated into individual cuvettes or strips of cuvettes. The stackable cuvettes also have stand-offs 530 as described above. Note that, to facilitate stacking, the lower portion 540 of the cuvette 500 s received by the wider, upper portion 550.

[0067] FIG. 2 illustrates an embodiment where cuvettes are advanced through the nephelometer in series. The system is designed for use with a series of cuvettes that are advanced through the nephelometer in a continuous fashion. Individual cuvettes 110 may be placed directly inside a nephelometer base 100 by placing the lower portion of the cuvette into the channel 220 as shown in FIG. 2B. Alternatively, individual vessels 110 may be first placed inside the linear vessel array 300, and the linear array 300 (FIG. 3A.) housing multiple vessels can be placed inside the nephelometer via the pass through channel 220. After the vessels are placed inside the nephelometer base either individually or inside the linear array, the suspension is prepared in the cuvette and the turbidity measured as described above.

[0068] The system that houses a linear array of vessel (FIG. 2) also includes a light source 130, a focusing lens 170, a scatter detector 140, a transmitted light detector 150 and a light attenuation filter 160 (FIG. 1B as described above). The cuvette 110 with a sample 120 is positioned at the center of the apparatus and inside the nephelometer base 100. The light source 130, the scatter detector 140 and the transmitted light detector 150 are positioned at a 90 degree angle from one another around the cuvette 110 as described above. The side scatter detector surface 140 is positioned parallel to the incident beam from the light source 130. Positioning the scatter detector 140 within close proximity to the tested sample 120 and parallel to the incident light source minimizes the effects of diffraction, refraction and reflection on the scattered light. The transmitted light detector 150 is positioned opposite from the light source 130 and incident light from the light source propagates toward the transmitted light detector. The detector 150 also may be positioned either perpendicular to the incident light path or a few degrees from perpendicular to reduce reflection effects from its surfaces. The light attenuation filter 160 is positioned between the cuvette 110 and the transmitted light detector 150.

[0069] FIG. 7 is a cross section of a nephelometer showing the path for the light transmitted through the lower portion 540 of the cuvette 500. The light source (570, FIG. 9) is received by an aperture 575 on one side of cuvette receptacle 580 of the nephelometer 590. The aperture 575 receives the light source. The sensor 600 (FIG. 9) is positioned in an aperture 605 directly opposite the aperture 575, with the lower portion of the cuvette 540 positioned therebetween. The nephelometer has a lid 620.

[0070] FIG. 8 is a cross section of a nephelometer showing the path for the light scattered through the lower portion 540 of the cuvette 500. The light source (570, FIG. 9) is on one side of cuvette receptacle 580 of the nephelometer 590. The sensor 630 (FIG. 10) is positioned in an aperture 635 orthogonal to the light source 570, with the lower portion of the cuvette 540 positioned therebetween.

[0071] FIG. 9 is a cross section of a nephelometer showing the path for the light transmitted through the lower portion 540 of the cuvette 500. The light source 570 is received by an aperture 575 on one side of cuvette receptacle 580 of the nephelometer 590. Between the sensor 600 and cuvette 500 is a light attenuation filter 640 that is placed in front of the transmittance detector to decrease the light intensity to a usable level so as not to saturate sensor. The aperture 575 receives the light source 570 and the lens 650 for focusing the optical signal. The sensor 600 is positioned in an aperture 605 directly opposite the aperture 575, with the lower portion of the cuvette 540 positioned therebetween.

[0072] FIG. 10 is perspective view of the nephelometer 590 showing aperture 575 for the optical source 570, aperture 635 for the scattered light sensor and aperture 605 for the transmitted light sensor.

[0073] In one embodiment, the sample is disposed inside a cuvette and individually processed when placed into the nephelometer. After the sample is processed and McFarland values are obtained, the cuvette is removed from the nephelometer and replaced by a new cuvette. In this embodiment, the one or more nephelometers are operated independently. In an alternative embodiment, the nephelometer is configured to deliver a continuous series of cuvettes to the nephelometer for measurement. A linear cuvette channel 220 receives a strip 300 of individual cuvette wells 320 (FIG. 3B). The strip is conveyed through the nephelometer, stopping for each cuvette to be optically interrogated for measurement as described in detail elsewhere herein.

[0074] The methods of measuring the turbidity according to the present invention are automated. The data collected from the measurements may be further processed to generate meaningful results. In these embodiments, the signal from the detectors are fed to signal amplifiers. The amplifier output is communicated to an analog to digital converter circuit that outputs a digital representation of the input signal that is then processed using various algorithms to determine if the measured value is at the target value. If the measured value is higher than the target value, then the sample is diluted as described above, and the turbidity re-measured. Such re-measurement can be done manually by an operator or in an automated manner where the cuvette is transferred out of the nephelometer for dilution and transported back to the nephelometer for an additional measurement. The methods for processing the signal into a usable output are developed using varying dilutions of various biological and non-biological sample and associating McFarland values with the suspension concentrations. These data are then used to produce data sets that are further analyzed using algorithms that correct the linearity and offsets of the data curves to produce a representative output value for a turbidity value and compared with the target value. This process is repeated until the target turbidity is obtained.

[0075] Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.