WO2014137333A1

WO2014137333A1 - Method and apparatus for determining sample turbidity

Info

Publication number: WO2014137333A1
Application number: PCT/US2013/029270
Authority: WO
Inventors: Tuan-Linh Ngoc NGUYEN; William B. BRASSO; Ming-Hsiung Yeh
Original assignee: Becton, Dickinson And Company
Priority date: 2013-03-06
Filing date: 2013-03-06
Publication date: 2014-09-12

Abstract

Various embodiments disclosed are directed to a detection apparatus and methods to estimate turbidity for a microbial suspension of volumes of about 0.25 ml to about 100 ml. The disclosed apparatus and methods utilize uniform color bands and/or a graduated color band as a basis for a turbidity determination. The visual detection apparatus has a holding receptacle, a channel configured to receive a suspension tube, and color bands disposed over the surface of said channel. The suspension tube is placed so that the suspension overlies a uniform color band or the graduated color band. The disclosed methods allow for simple and accurate estimate of turbidity based on the visual comparison of the relative color intensities between the uniform color bands or a uniform appearance of the graduated color band.

Description

METHOD AND APPARATUS FOR DETERMINING SAMPLE TURBIDITY

BACKGROUND OF THE INVENTION

[ 0001 ] In microbiological, clinical and other similar laboratories, it is necessary to estimate or determine the number of bacterial cells (i.e., bacterial enumeration) in a broth culture or liquid medium. Bacterial cell numbers can be determined using various direct and indirect methods, including for example, standard plate counts, turbidimetric measurements, visual comparison of turbidity with a known standard, direct microscopic counts, cell mass determination, as well as measurement of cellular activity. One indirect method of estimating the number of bacteria in a medium is to measure the turbidity or "cloudiness" of a culture and then translate this measurement into cell numbers. Compared to the direct methods for estimating the number and/or weight of cells in a microbial suspension, turbidimetry is more advantageous, less laborious and does not necessitate time- consuming methods such as weighting a sample or preparing standard plate counts.

[ 0002 ] For example, methods that require measuring standard plate counts are based on first determining the turbidity of different concentrations of microorganisms and then utilizing the standard plate count to determine the number of viable organisms per milliliter of a sample. In contrast, only a measure of turbidity is needed to determine cell numbers in a medium. Accordingly, estimating the number of cells in a liquid medium by measuring the turbidity of the medium is a fast and efficient method of obtaining microbial concentration/density relative to the alternative methods set forth above .

[ 0003 ] Turbidity of microbial suspensions is usually determined by an instrument such as a nephelometer or a densitometer and/or by utilizing a visual comparison tool such as a Wickerham card. These methods are based on physical principles of light scattering which result from the interaction of light with particle (s) in a suspension. Turbidity of the samples effects the transmission and scattering of the light, and allows for a measure of intensity of light transmitted through a sample. A nephelometer measures the intensity of scattered light, whereas a visual detection tool such as a Wickerham card may be based, in part, on the visual observation of the intensity of an unscattered light in a sample to be tested compared to a standard. The standard microbiological method of estimating turbidity of a sample is based on obtaining a quantitative value known as a McFarland value. McFarland values are well known to those skilled in the art and are not described in detail herein. McFarland standards are solutions of a known turbidity that are used to standardize culture density in microbiological, clinical and other similar laboratories.

[ 0004 ] A nephelometer is an automatic instrument used to measure turbidity. One disadvantage of using a nephelometer is that it requires relatively large volume samples in order to provide an adequate reading of McFarland value.

Similarly, most densitometers, such as the PhoenixSpec™

(Becton Dickinson), require a cell length of at least one (1) centimeter (cm) in their associated tubes, and are therefore not for use with very small volume of microbial suspensions, such as, for example, suspensions of about 250 or about 500 microliters (μΙ_) . In standard test tubes (e.g., a 15 ml test tube), a suspension of as low as about 500 may yield cell lengths of less than 1 cm, which would not be adequate to measure McFarland value using a nephelometer. A narrower test tube, for example, may be used to increase the height of the sample to a detectable level. However, such test tubes are often not utilized with nephelometers and/or densitometers because these instruments are designed to house test tubes of specific sizes and configurations. Therefore, an apparatus and method are needed that can allow for a rapid and accurate measure of turbidity for suspensions having a volume of less than about 500 μΙ_, and preferably as low as about 250 μΙ_ . In addition, an apparatus and methods are needed to estimate turbidity using a visual detection system that is simple to use and can accommodate various test tube sizes and shapes that can house a wide range of sample volumes .

[ 0005 ] McFarland value of a suspension can also be qualitatively measured using a manual visual comparison tool such as a Wickerham card. This method requires comparing a microbial suspension to be tested to a McFarland turbidity standard (standard suspension) against a background that is comprised of a white card with printed black lines (i.e., a Wickerham card) . A Wickerham card method is largely subjective and is not standardized. Therefore, the measure of turbidity may be highly variable among different users and different conditions such as light source, tube size, suspension volume, etc. The subjective nature of this method lends itself to certain limitations. For example, it is possible that cultures of similar turbidity to the standard suspension will blur the black lines to about the same extent. Therefore, subtle differences in sample turbidity among samples are often difficult to perceive.

[ 0006 ] In addition, using a Wickerham card method to estimate McFarland value may not provide accurate results. A

Wickerham card method relies on a use of suspension standards that cannot differentiate between light diffraction and light absorbance when compared to a microbial suspension to be tested. The measure of turbidity therefore may not be accurate because the inability to differentiate may lead to circumstances where the microbial suspension may appear visually darker or lighter than the standard suspension.

Alternatively, a standard suspension may appear visually darker or lighter than the microbial suspension. The same microbial suspension, therefore, can provide two different McFarland values, where the value is otherwise the same when measured using an automated instrument such as a nephelometer .

[0007] In addition, a Wickerham card method is difficult to perform for small volume suspensions and has limited applications. For example, a small volume sample dispensed in a standard test tube with a curved bottom measuring 18x150mm usually yields low cell lengths. As the small volume sample is confined to the bottom curved portion of the tube, it is both awkward to position the tube relative to the card and difficult to estimate the McFarland value by holding both the test tube and a suspension tube against a card. For ease of use, a Wickerham card method requires a sample volume of at least 2 ml. Similarly, it may be difficult to handle samples of larger volume to estimate McFarland values, particularly when using standard test tubes. Accordingly, an apparatus and method are needed for measuring turbidity where an accurate, consistent and reliable McFarland value can be obtained for a microbial suspension that is simple to handle and convenient to use.

BRIEF SUMMARY OF THE INVENTION

[0008] The apparatus and methods according to the present invention provide a simple and accurate estimate of McFarland values for a microbial suspension of volumes from less than

500 μΐ and up to volumes as high as 100 ml by utilizing uniform color bands and/or a graduated color band as a basis for a turbidity determination. The methods described herein utilize the principles of turbidimetry and physics of visual and optical relationship of a light transmitted through the sample. In the described methods, a diameter of the tube, sample volume and angle at which the sample is positioned within a sample tube are selected to provide a sample cell length for convenient visual observation. According to the embodiments described herein, color bands are printed directly on a channel of a visual detection system or apparatus configured to receive the tube containing a microbial suspension to be subjected to a turbidity determination. The apparatus and methods described herein further provide an adjustable holding receptacle configured to receive tubes of various sizes, volumes and cell lengths. Because the apparatus is adjustable, it is convenient for use in various microbiological, clinical and laboratory settings.

[ 0009 ] The present invention is directed to methods and apparatus for making qualitative turbidity estimates of a liquid suspension. In the embodiments described herein, a visual detection apparatus having a holding receptacle, a channel configured to receive a suspension tube, and color bands disposed on the surface of said channel is provided. In one embodiment, two uniform color bands are disposed on the channel configured to receive a suspension tube. In yet another embodiment, a graduated color band is disposed on the surface of the channel. The graduated color band is configured such that a single color transitions from a darker, more intense bottom portion of the band, to a lighter, less intense top portion, with varying degrees of intensity of the color between the top and bottom portions. The size and shape of the suspension tube may vary. The tubes can accommodate liquid samples ranging from about 0.25 ml to about 100 ml.

[ 0010 ] In one embodiment, the uniform color bands comprise a lower color band and an upper color band disposed on the surface of the channel configured to receive a suspension tube. The uniform color bands are positioned on the surface of the channel such that the distance from one another will permit turbidity measurement of samples in tubes of various lengths and widths. In one embodiment, position of the uniform color bands is such that only the lower color band is behind the liquid sample in the suspension tube when the tube is placed in the channel. The uniform color of the bands is selected to provide a visually perceptible contrast between the color intensity of the lower and upper bands when a suspension tube having a sample therein is placed inside the channel for observation. The placement of the suspension tube with a liquid sample into the channel is such that only the lower uniform color band is behind the liquid sample while the upper uniform color band is not behind a portion of the tube with liquid. That is, the upper color band is above the liquid level in the tube. The sample turbidity is estimated based on the visual comparison of the color intensities between the lower uniform color band below the top of the liquid sample and the upper uniform color band. In some embodiments, the observed color intensity of the color band below the liquid level is then compared to a color associated with a standard suspension of a known McFarland value to make a turbidity measurement. In some embodiments, the observed color of the lower uniform color band is compared to the color of the upper uniform color band, wherein the upper uniform color band is disposed on the channel above the biological sample, and wherein the upper uniform color band is associated with a predetermined McFarland value.

[ 0011 ] In other embodiments, the channel length and/or the distance between the color bands is adjustable. This allows the apparatus to receive suspension tubes of varying lengths and with varying volumes of sample disposed therein. In one embodiment, the apparatus further comprises an adjustable slide with color bands disposed thereon. The adjustable slide forms the base of the channel that receives the suspension tube. The adjustable slide is retractable and/or extendable to decrease or increase the distance between the lower and upper uniform color bands disposed on the slide.

[ 0012 ] In another embodiment, the slide is bidirectional which allows the position of one or both of the uniform color bands to be adjusted in the channel. The adjustable slide may also be placed on the surface of the channel such that it forms an extendable channel. The extendible channel has a stationary portion and an adjustable portion. In one configuration, the stationary portion has the lower uniform color band disposed thereon, and the adjustable portion has the upper uniform color band disposed thereon. The channel length may be adjusted to receive suspension tubes of various lengths using the adjustable slide.

[ 0013 ] In another embodiment, the upper uniform color band is disposed on the surface of the adjustable slide, and the lower uniform color band is disposed on the surface of the channel. In this embodiment, the position of the lower color band is fixed and the position of the upper band is adjustable. The length of the channel is adjusted by extending the adjustable slide from the channel to receive a larger test tube or a tube in which the liquid level is higher. The adjustable portion of the channel has the upper uniform band disposed thereon. Preferably the slide is adjusted such that the upper uniform band is above the surface of the liquid sample in the tube being integrated.

[ 0014 ] In yet another embodiment, the holding receptacle comprises a channel with a graduated color band imprinted thereon. The graduated color band comprises a darker bottom portion and a lighter top portion, wherein the band is disposed on the proximal end of said channel. The position of the graduated color band is to ensure that it is behind the liquid sample when the sample-filled tube is placed thereover. In an alternative embodiment, the graduated color band is disposed on an adjustable slide overlaying the channel .

[ 0015 ] The graduated color band is designed such that the colors transition from a darker bottom portion to a lighter top portion, with varying degrees of color intensity between the top and bottom portions. In one embodiment, the graduated color band is designed such that each of the color intensities within the variable-intensity color band corresponds to a known McFarland standard.

[ 0016 ] In other embodiments, the channel is configured to receive one or more inserts. The inserts may have the features of the previously described adjustable slide. Each insert is provided for its own purpose. For example, one insert may have two color bands disposed at a stationary distance from each other. Another insert may have two color bands disposed on a slide, yet another insert may have one graduated color band disposed on a slide. By configuring the channel to receive different inserts, the holding receptacle described herein can be adapted for many different turbidity determinations .

[ 0017 ] In yet another embodiment, the visual detection apparatus comprises a plurality of holding receptacles and channels having either two uniform color bands or a graduated color band disposed thereon. The plurality of holding receptacles and the plurality of channels inside said holding receptacles are positioned parallel to one another and are configured to house a plurality of suspension tubes . In one embodiment, the plurality of channels may be designed of equal size. In an alternative embodiment, the channels may be made of varying widths and may further comprise an adjustable slide to accommodate test tubes of varying lengths. In an alternative embodiment, the plurality of channels are further configured to receive a plurality of different inserts (e.g., one having at least two uniform color bands; another having a graduated color band disposed on its surface, etc.), and wherein the plurality of inserts are capable of being removed from the plurality of channels.

[ 0018 ] In the embodiments described herein, the visual detection apparatus is configured such that the holding receptacle and the channel configured to receive a suspension tube are positioned at about 0.1 degree to about 89 degrees angle. In another embodiment, the holding receptacle and the channel are positioned at about 30 degree to about 60 degree angle, and preferably at about a 45 degree angle relative to the base of the apparatus. In one embodiment, the suspension tube with a liquid sample is placed inside a channel positioned at a predetermined angle to increase the surface area of the liquid, wherein the channel is imprinted with a graduated color band, and wherein the liquid is disposed over the entire length of the graduated color band. In another embodiment, the suspension tube with a liquid sample is placed inside the channel positioned at a predetermined angle to increase the surface area of the liquid, wherein the channel is imprinted with an upper and lower uniform color bands, and wherein the liquid overlies the lower uniform color band.

[ 0019 ] Various embodiments described herein further provide a simple and reliable method for estimating turbidity of a suspension based on observed relative intensities of color bands compared to a standard suspension of a known McFarland value. The methods described herein contemplate estimating sample turbidity using color bands disposed on the surface of the channel. When deploying uniform color bands, the methods provide for estimating turbidity based on the visual appearance of the sample and relative intensities between the covered and uncovered portions of the color bands . When deploying a graduated color band, the methods provide for estimating turbidity based on observing a uniform color appearance of the graduated color band disposed behind the sample overlaying the entire length of the graduated color band, wherein said uniform color is indicative of a predetermined McFarland value.

[ 0020 ] The methods contemplate placing a suspension tube with a liquid sample into the channel of the visual detection apparatus positioned at about a 0.1 degree to about a 89 degree angle, preferably between about 30 to 60 degree angle, and more preferably at about a 45 degree angle relative to the base of the apparatus. Placing a suspension tube at an angle displaces the liquid sample such that the cell length (i.e., the distance between the bottom of the tube and the uppermost portion of the sample surface in the tube) is increased to allow for accurate visual observation of the relative intensities between the color bands. Because the test tube is at an angle, the distance between the bottom portion of the tube and the top surface of the liquid varies. As is readily understood, the sample surface remains horizontal to the ground as the tube is tilted. When the tube is tilted, one side of the tube is closer to the ground than the other side of the tube. Since the plane of the sample surface is no longer parallel to the plane of the tube bottom or tube top, there is variable distance of the sample surface from the top and bottom of the tube.

[ 0021 ] In some embodiments described herein, the methods allow for observing the sample at an angle by looking vertically through the sample tube toward the highest point of the sample (relative to the top of the tube), and in some embodiments, toward the upper color band that is positioned above the liquid sample when the tube is resting in the channel. In some embodiments of the apparatus having two uniform color bands, the observed color of the covered band relative to the uncovered band is then compared to a standard suspension of a known McFarland value. In some embodiments of the apparatus having the graduated color band, the observed color is uniform and reflects a predetermined

McFarland value. As is understood by the skilled person, with the sample surface at an angle relative to the planes of the top and bottom of the tube, the optical path length from the top surface of the sample to the back of the tube varies.

This causes variation in appearance of the color band disposed behind the sample. Preferably, the graduated color band is configured to complement this variation in optical path. Specifically, the variation in optical path length causes the graduated color band to appear to be of almost uniform color . When the graduated color band has the uniform color appearance, the observer or sensor will "know" that the sample has certain turbidity.

[ 0022 ] An alternative embodiment of the methods described herein comprises placing a suspension tube into an adjustable channel having an adjustable slide, wherein the adjustable slide is positioned at a 45 degree angle relative to the base of the channel, observing the sample, recording the observed relative intensities between the covered and uncovered uniform color bands or uniform appearance of the sample disposed over a graduated color band, and determining whether a desired turbidity has been reached. The channel may be adjusted by retracting or extending the adjustable slide thereby decreasing or increasing the length of the channel to accommodate the subject sample tube and the sample volume to be tested.

[ 0023 ] Various embodiments described herein further provide a reliable and accurate method of measuring the turbidity of a suspension where said suspension has insufficient volume to be read in devices designed to measure turbidity, such as nephelometers, densitometers or visual detection methods such as a Wickerham card. The methods described herein obtain turbidity estimates of a liquid sample having a volume of about 0.25 ml to about 100 ml, as well as low volume samples of about 0.25 ml to about 6 ml.

The methods described herein further provide for turbidity estimates of volume of less than 0.5 ml, and preferably as low as 0.25 ml. The methods include a visual detection system comprising uniform color or graduated color bands that reflect a standard, and that are designed to indicate whether target turbidity has been reached. The methods of various embodiments enable measuring McFarland levels of samples for use with methods such as Mass Spectrometry (e.g., matrix- assisted laser desorption/ionization - time-of-light mass spectrometer, MALDI-TOF) .

BRIEF DESCRIPTION OF THE DRAWINGS

[ 0024 ] 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 .

[ 0025 ] Figure 1 illustrates one embodiment of the apparatus described herein that has a suspension tube and a holding receptacle for use with the uniform color bands .

[ 0026 ] Figures 2A and 2B illustrate one embodiment of the method described herein for measuring a turbidity of liquid samples using uniform color bands.

[ 0027 ] Figures 3A and 3B illustrate an alternative embodiment of the methods described herein for measuring turbidity of liquid samples using the uniform color bands illustrated in Figures 2A and 2B, respectively.

[ 0028 ] Figure 4 illustrates another embodiment of the apparatus and method described herein that deploys a holding receptacle with an adjustable slide for use with uniform color bands that receives a suspension tube for testing.

[ 0029 ] Figures 5A, 5B and 5C illustrate an alternative view of the apparatus with the suspension tube disposed inside the holding receptacle and an adjustable slide for use with uniform color bands .

[ 0030 ] Figures 6A-6C illustrates another embodiment of the apparatus described herein that has a suspension tube and a holding receptacle for use with a graduated color band.

[ 0031 ] Figures 7A and 7B illustrates one embodiment of the method described herein for measuring a turbidity of liquid samples using the graduated color band.

[ 0032 ] Figures 8A and 8B illustrate an alternative embodiment of the methods described herein for measuring turbidity of liquid samples using the graduated color band illustrated in Figures 7A and 7B, respectively. [ 0033 ] Figure 9 illustrates an alternative embodiment of the apparatus described herein having a plurality of channels .

DETAILED DESCRIPTION

[ 0034 ] Embodiments described herein provide for apparatus and methods of measuring turbidity of liquid samples using uniform color bands and/or a graduated color band. The disclosed apparatus and methods further allow for measuring turbidity levels in samples having insufficient volume to be detected by instruments such as nephelometers , densitometers or alternative visual comparison tools such as a Wickerham card, used to obtain McFarland values for integration with various manual microbiology testing systems .

[ 0035 ] 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.

[ 0036 ] As used herein, a "low volume" and/or a "small volume" sample refers to a sample having a volume of about 0.25 ml to about 6 ml.

[ 0037 ] As used herein, the term "liquid suspension" and/or "liquid sample" refers 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.

[ 0038 ] As used herein, a "suspension tube" is any tube suitable for receiving a suspension. The tubes are preferably transparent to permit accurate inspection. Suitable suspension tubes are well known to those skilled in the art and are not described in detail herein. [ 0039 ] In one embodiment, a method of measuring turbidity of a liquid sample is deployed using uniform color bands. Figure 1 illustrates the system of one embodiment described herein for measuring turbidity of liquid samples based on relative intensities of the uniform color bands disposed on the surface of channel 140 configured to receive a suspension tube 100. The upper uniform color band 110 is disposed at a sufficient distance from the lower band 120 on the surface of channel 140. The distance between the lower band 120 and the upper band 110 may be adjusted depending upon the size of the suspension tube 100 and the volume of liquid dispensed therein. The McFarland standard may be prepared according to methods well known in the art such as, for example, a solution of latex particles, etc.

[ 0040 ] Any uniform color bands may be designed using an arbitrary color. The uniform color bands disclosed in the present invention are designed such that the selected color corresponds to a suspension standard of a known McFarland value. Accordingly, various bands may be designed to correspond to various McFarland values. When uniform color bands are deployed, the visual appearance of the sample is observed based on the relative intensities of the covered and uncovered bands. This visual appearance is then compared to a standard suspension and/or the color of the upper uniform color band to estimate turbidity of the sample. The turbidity may also be measured by comparing the color of the upper color band, which is associated with a known McFarland value and serves as a control, to the color of the lower uniform color band over which the sample is disposed. When a graduated color band is deployed, the visual appearance of the band as observed through the sample in the tube overlaying the length of the graduated color band preferably appears to have a uniform color, which reflects a predetermined McFarland value. As such, the graduated band serves as a test and a control strip. [ 0041 ] Referring still to Figure 1, the tube 100 has body

150, configured to receive and hold the suspension for which turbidity is to be measured, and cap 160. The holding receptacle 130 is configured to receive the suspension tube 100 at approximately a 45 degree angle. The angle of the holding receptacle 130 and the channel 140 configured to receive the suspension tube 100 can be positioned from about 0.1 degree to about 89 degrees depending on the sample size and the volume of the sample in the suspension tube for measuring the turbidity. Advantageously, the holding receptacle 130 may be adjusted such that the suspension tube 100 can be positioned at a desired angle for enhanced visual observation and for determining the McFarland value of a sample .

[ 0042 ] The methods according to the disclosed embodiments allow for use of suspension tubes having lengths of less than one centimeter (< 1cm) which can house a sample having a volume as low as about 250 microliters (250 or 0.25 ml) .

The disclosed methods can be deployed by dispensing samples into Phoenix™ ID Broth tubes (Becton Dickinson) . Such tubes can receive a sample volume of less than 8.0 milliliters. Various test tubes may be deployed depending upon the volume of the sample, the angle of the holding receptacle 130, the angle at which the sample is observed, as well as the color bands reflecting a standard of a known McFarland turbidity. Advantageously, the bottom portion of the body 150 is flat thereby increasing the diameter of the tube at the bottom of the tube 100 (compared to a curved bottom tube) to house a small sample volume for convenient visual observation when positioned in channel 140 of the holding receptacle 130. Other volume tubes having similar features that are suitable for use with manual and/or automated microbiology systems can also be used. Alternatively, round bottom test tubes as well as conical test tubes may also be used. [ 0043 ] Without being bound to any particular theory, it is believed that the diameter of the tube, the tube transparency, the sample volume and the angle of the holding receptacle, all affect the ability to visually determine the sample turbidity. The visual determination of sample turbidity is based, in part, on the optical properties of the sample disposed in tube 100. The chemical and physical properties of the sample modify the light such that the light would go through, for example, a transparent sample or it would reflect off of an opaque sample. Optical phenomena that result from such interaction include absorption, refraction, transmission, scattering and reflection.

[ 0044 ] The visual appearance of the sample may depend on the nature of light, the composition and density of the sample, and other factors such as the geometrical disposition of the light source, the angle at which the sample is observed, as well as on the optical properties of the surrounding surfaces. The visual appearance of the sample may also vary depending on the vision capacity of the observer and other similar factors.

[ 0045 ] The methods described herein are also based, in part, on the change in intensity of the light when interacting with the sample. The observed color of the sample are attributed to the hue, saturation (or purity), and lightness of the sample based on changes in the light path and/or light quality. For example, the turbidity of the sample is inversely related to the lightness. Accordingly, as the lightness of the sample decreases, the turbidity increases .

[ 0046 ] Preferably, a holding receptacle designed to house the sample tube 100 at an angle of about 45 degrees or greater (from vertical) provides for a greater contrast and facilitates better visual estimates of turbidity.

Accordingly, the ability to accurately determine whether the sample has sufficient or insufficient turbidity (the turbidity being consolidated with concentrations of bacteria in the samples), is influenced by the angle of the holding receptacle 130 and the channel 140, which houses the sample to be visualized.

[0047] Figures 2A and 2B illustrate the method of one embodiment described herein wherein the turbidity of liquid suspensions is measured using bands of uniform color. Figures 2A and 2B illustrate sample tubes having a body 250a and 250b, with respective caps 260a and 260b thereon. To illustrate the distribution of the sample in the tubes when placed in the channel 140 of device 130, the tubes with liquid therein are illustrated with the tubes substantially vertical (200a and 200b), tilted as a result of being placed in channel 140 (210a and 210b) and placed over uniform color band 120 (220a and 220b) . In this embodiment, a clear sample is compared to a cloudy (i.e., turbid) sample dispensed in Phoenix™ ID broth tubes. The suspension tube 200a illustrated in Figure 2A is filled with about 500 microliters ( L) of clear sample. The suspension tube 200b illustrated in Figure 2B is filled with about 500 microliters (μΙ_) of a turbid sample. The turbid sample is shown with dashes. The tubes 200a and 200b are placed into channels 140 configured to receive the tubes. As illustrated in Figures 230a and 230b, the holding receptacle 130 is positioned to receive sample tubes 200a and 200b, respectively, at about a 45 degree angle. When so placed, the liquid in the slanted tubes is distributed differently, the surface area of the liquid increasing compared to the tube in a vertical position (shown in Figs. 200a and 200b) . The increase in the surface area of the liquid is shown as 270a and 270b for clear and turbid samples, respectively. As such, the distance of the liquid surface varies from the front of the tube to the back of the tube.

[0048] For the clear sample, the surface of the liquid inside the tube 210a is shown in the side view 230a. For the cloudy sample, the surface of the liquid inside the tube 210b is shown in the side view 230b. The front view for the samples 210a and 210b is illustrated as 240a and 240b, respectively. The placement of the tubes into the channel

140 of the holding receptacle 130 redistributes the liquid.

The upper uniform color band 110 and the lower uniform color band 120 are disposed on the channel 140 designed to receive the samples. The bottom portion of the sample overlies the lower uniform color band 120 shown as 220a and 220b for clear and turbid samples, respectively. When the tube 200a is placed in the channel 140, the lower band 120 is behind a portion of the liquid sample. The upper band 110 is placed such that it is not behind the portion of the tube with liquid. The position of the lower band 120 and upper band

110 is configured such that the liquid sample overlies only the lower color band 120. The upper color band 110 is disposed above the liquid in the test tube to serve as a control strip over which no sample is disposed. Accordingly, the turbidity of the sample is measured based on the visual comparison of the relative intensities of the two uniform color bands, one of which is covered by the sample (120) and the other one is not (110), to a standard solution.

[ 0049 ] Figures 3A and 3B illustrate more detailed perspective views of sample tubes disposed in the device for determining sample turbidity described herein. As in Figures

2A & 2B, sample tubes with both clear and turbid liquid are illustrated. The holding receptacles 330a and 330b have slanted channels 140 which receive the tubes 300a and 300b.

Again, the surface area, 310a and 310b, of the clear liquid

305a and turbid liquid 305b is increased compared with the surface area of the liquid in the tube when in a vertical position (see 200a and 200b in Figure 2A) . Referring to 330a and 330b, the liquid (305a and 305b, respectively) overlies uniform color band 120 but not uniform color band 110. The lower color band 120 is behind the liquid, while the upper color band 110 is not. The top of the sample disposed in a tube is shown as 306a and 306b for clear and turbid samples, respectively, lies below the upper color band 110. The turbidity of the test sample is measured based on the visual comparison of the relative intensities between the uniform color band 120 over which the 500 turbid sample is disposed and the uniform color band 110 over which no liquid is disposed. The observed relative intensities are then compared to a McFarland standard of a known value. The McFarland standard most commonly used in a clinical microbiology laboratory for routine antimicrobial susceptibility tests is 0.5 McFarland, which represents about 1.5 x 10⁸ bacteria per milliliter of the suspension. Preparation of Routine Media and Reagents Used in Antimicrobial Susceptibility Testing, Ch. 5.14 McFarland Standards ("McFarland Standards 5.14") The methods described herein are also used to determine the turbidity of solutions in various applications, such as nucleic acid amplification, fibrin formation, etc.

[ 0050 ] In the embodiments described herein, the microbial suspensions are prepared in accordance with and compared to various McFarland standards. McFarland turbidity standards are used to standardize the approximate number of bacteria in a liquid suspension by visually comparing the turbidity of a test suspension with the turbidity of a McFarland standard.

McFarland Standards 5.14. McFarland standards can be prepared using barium sulfate or latex particles suspensions that are used to perform visual or spectrophotometric comparisons of bacterial densities in water, saline, or liquid growth medium. A McFarland standard is prepared by measuring the optical density of the standard at wavelength of 625 nanometers (nm) and results are recorded. An acceptable range for a 0.5 McFarland standard is an optical density of about 0.08 to 0.10. Other optical density ranges may be established based on the desired McFarland standard for various bacterial concentrations, for example, McFarland 1 through 10. McFarland Standards 5.14. McFarland standards may be prepared according to standard protocols utilized in various clinical and laboratory settings . Table 1 illustrates approximate bacterial concentrations in a McFarland standard prepared using a 1.175% barium chloride (BaCl₂- 2H₂0) and 1% sulfuric acid (H2S0₄) medium to prepare different grades of McFarland standards . Other suitable standards, such as latex suspensions may also be used.

[ 0051 ] TABLE 1: MCFARLAND STANDARDS USING 1.175% BARIUM

(BaCl₂- 2H₂0) AND 1% SULFURIC ACID (H₂S0 )

[ 0052 ] Various methods are contemplated where the holding receptacle can accommodate tubes of various heights and widths. Figure 4 illustrates a holding receptacle 130 where the uniform color bands are disposed on the channel with an adjustable slide 450. Adjustable slide 450 is configured to be retracted or extended thus decreasing or increasing the distance between bands 110 and 120. This allows the holding receptacle to adapt to sample tubes 100 of different lengths and different volumes of sample therein. The channel 140 with an adjustable slide 450 provides a bidirectional adjustable slide (depicted by the arrows) to allow the position of one or both of the uniform color bands to be adjusted in the channel (or holding receptacle) .

[ 0053 ] In yet another embodiment, the uniform and/or the graduated color bands can be of different widths for use with test samples of various volumes and tube sizes. Preferably, the bands are designed to accommodate sample volumes of about 0.25 ml to about 100 ml, and more preferably low volume samples of about 0.25 ml to about 5-6 ml. The adjustable slide 450 and color bands 110 and 120 according to the embodiments described herein are adaptable and may be configured to accommodate various types and sizes of test tubes as well as a wide range of sample volumes .

[ 0054 ] Figures 5A, 5B and 5C illustrate a more detailed view of the adjustable slide 450 illustrated in Figure 4. The distance between the color bands 110 and 120 on the slide is fixed. The slide 450 can be extended if there is more sample in the tube 100 and retracted if there is less volume in the tube 100. The holding receptacle has a channel 140 positioned at about a 45 degree angle and the channel 140 is adjusted in the direction of the arrow to accommodate tubes of varying lengths. The slide 450 with a tube thereon is illustrated in a perspective view in Figure 5C. The lower 120 and upper 110 uniform color bands are printed onto the adjustable slide 450 disposed in the channel 140 of the holding receptacle 130. The microbial suspension in the test tube is disposed over the lower uniform color band 110 such that the lower band 110 is behind the proximal portion of the tube with liquid sample. The distance between the bottom of the tube shown as B1-B3, and the uppermost portion of the sample surface in the tube shown as T1-T2, illustrate the cell length. In this configuration, placing the suspension tube into channel 140 at a 45 degree angle displaces the liquid sample such that the cell length is increased to allow for accurate visual observation of the relative intensities between the color bands .

[0055] According to one embodiment, the methods of measuring turbidity are based on inspecting the front horizontal view 500 of the sample. In this embodiment, the microbial sample tube 100 is disposed in the channel 140 when the position of the slide 450 is adjusted in the direction of the arrow. In order to measure the McFarland value of the suspension, the position of the slide 450 is adjusted so that the position of the top band 110 is above the sample 305b. The turbidity is measured based on the relative color intensities of the lower uniform color band 120 over which the liquid is disposed compared to the upper uniform color band 110. Sufficient turbidity is determined when relative intensities of the lower uniform color band 120 compared to the upper uniform color band 110 correlate to the relative intensities observed for a McFarland standard.

[0056] In one embodiment, the holding receptacle 130 can be configured to house a plurality of suspension tubes with liquid samples therein positioned side-by-side inside at least two channels 140. Figure 9 illustrates a holding receptacle 130 having two channels 140 with adjustable slides

450 positioned adjacent to one another. The bands of uniform color are disposed on the adjustable slide 450. In this embodiment, a suspension tube 100 with a turbid sample therein can be placed next to standard suspension of a known

McFarland value for convenient visualization and visual comparison. The holding receptacle 130 having a plurality of channels 140 is also adaptable for use with graduated color bands 610 disposed on the proximal end of each channel or on the adjustable slides 450 of said channels.

[0057] In yet another embodiment of the visual detection apparatus described herein, a turbidity of liquid samples is measured using a graduated color band disposed on the channel of a visual detection apparatus configured to receive a microbial suspension tube. Figure 6 (which is similar to Figure 1) illustrates a system for measuring turbidity of liquid samples using a single band 610 having a graduated color. The band 610 is designed to provide a color transition from a more intense darker bottom portion to a less intense lighter top portion. A grey color graduated band 610 is shown. The graduated color transitions from a darker to a lighter shade of color grey, with varying degrees of color intensity between the top and bottom portions. The band 610 is disposed on the channel 640 that receives a suspension tube 600. The suspension tube 600 has a body portion 650 that contains a microbial suspension. After a suspension is placed inside the tube body 650, the tube 600 is covered using cap 660 before the tube is placed into the channel 640. The visual detection apparatus 630 is designed to receive the suspension tube at approximately a 45 degree angle relative to the base of the apparatus . The angle of the visual detection system 630 and the channel 640 can be positioned from about 0.1 degree to about 89 degrees depending on the sample size and the volume of the sample in the suspension tube. In this embodiment, the graduated color band 610 serves as a test strip and a control strip. The graduated color band eliminates the need for and does not rely on a standard suspension for measuring turbidity of a sample. The graduated color band permits turbidity measurements of a liquid sample without the use of a second reference band based on observing the color of the liquid sample disposed over the entire band 610.

[ 0058 ] Figures 7A and 7B (similar to Figs. 2A-B) illustrate another embodiment of the methods described herein wherein the turbidity of liquid samples is measured using a graduated color band. Figures 7A and 7B illustrate sample tubes having a body 750a and 750b, with respective caps 760a and 760b thereon. To illustrate the distribution of the sample in the tubes when placed in the channel 640 of device 630, the tubes with liquid therein are illustrated with the tubes substantially vertical (700a and 700b), tilted as a result of being placed in channel 640 (710a and 710b) and placed over the graduated color band 610 (720a and 720b) . A clear sample is compared to a cloudy (i.e., turbid) sample dispensed in Phoenix ID broth tubes. The suspension tube 700a illustrated in Figure 7A is filled with about 500 microliters ( L) of clear sample. The suspension tube 700b illustrated in Figure 7B is filled with about 500 microliters (μΙ_) of a turbid sample. The tubes 700a and 700b are placed into channels 640 designed to receive the tubes. As illustrated in Figures 730a and 730b, the holding receptacle 630 is positioned to receive sample tubes 700a and 700b, respectively, at about a 45 degree angle. The turbid sample is shown in dashes. When so placed, the liquid in the slanted tubes is distributed differently, the surface area of the liquid increasing compared to the tube in a vertical position (shown in Figs. 700a and 700b) . The increase in the surface area of the liquid is shown as 770a and 770b for clear and turbid samples, respectively. As such, the distance of the liquid surface varies from the front of the tube to the back of the tube. For the clear sample, the surface of the liquid inside the tube 710a is shown in the side view 730a. For the cloudy sample, the surface of the liquid inside the tube 710b is shown in the side view 730b. The front view for the samples 710a and 710b is illustrated as 740a and 740b, respectively. The placement of the tubes into the channel 640 of the holding receptacle 630 redistributes the liquid. The graduated color band 610 is disposed on the channel 640 designed to receive the samples. The sample overlies the graduated color band 610 as shown in Figs. 720a and 720b for clear and turbid samples, respectively. When the suspension tube is placed inside the channel 640 of a holding receptacle 630 at an angle, the sample redistributes such that the sample overlies the entire length of the graduated color band 610. The top portion of the sample, when redistributed at a tilt, is positioned above the lightest top portion of the graduated color band, as shown in Figures 720a and 720b for clear and turbid samples, respectively. The graduated color has a variable intensity of color transitioning from a darker more intense bottom portion to a lighter less intense top portion of the band.

[0059] In one embodiment, the graduated color band is designed using various intensities of color grey. Each degree of color intensity reflects a predetermined McFarland value. Any color can be selected to design a graduated color band, so long as there are varying intensities transitioning from dark to light, and each degree of color intensity preferably corresponds to a known McFarland standard. Accordingly, turbidity is measured based on the visual appearance and color of the graduated color band as observed through the sample in a tube disposed over the band at a preset angle. As shown in Figure 720b, the entire graduated color band preferably has a uniform color appearance that corresponds to a degree of color intensity present in the graduated band. In this embodiment, the graduated color band is designed to provide a visual indication of turbidity when the graduated color band, disposed behind the sample in the tube, is observed to have a uniform color that corresponds to a predetermined McFarland value. Advantageously, the graduated color band has a dual purpose, as it provides the McFarland estimate of the sample and serves a control. Therefore, the observed color need not be compared to a suspension standard as the observed color itself reflects a known McFarland standard.

[0060] In the embodiments described herein, the visual detection apparatus configured with a graduated color band 610 may have an adjustable slide disposed on channel 640 to accommodate tubes of varying lengths and widths. The graduated color band 610 may be imprinted on the surface of the adjustable slide disposed on channel 640.

[ 0061 ] Figures 8A and 8B are similar to Figs. 3A-B, and illustrate more detailed perspective views of sample tubes disposed in the device 630 for determining sample turbidity described herein using the graduated color band 610. As in Figures 7A & 7B, sample tubes with both clear and turbid liquid are illustrated. The holding receptacles 830a and 830b have slanted channels 640 which receive the tubes 800a and 800b. Again, the surface area, 810a and 810b, of the clear liquid 805a and turbid liquid 805b is increased compared with the surface area of the liquid in the tube when in a vertical position (see 700a and 700b in Figures 7A-B, respectively). Referring to 830a and 830b, the liquid (805a and 805b, respectively) fully overlies the entire length of the graduated color band 610. The graduated color band 610 is behind the portion of the tube with a liquid sample such that the liquid covers the band. Preferably, the height of the liquid sample in a slanted tube extends above the top portion of the graduated color band 610. The turbidity of the test sample is estimated by observing the color of the graduated band 610 behind the sample. The observed color of the graduated band disposed behind the liquid sample preferably appears uniform over the entire surface of the sample. The observed color reflects a known McFarland standard. The color intensities of the band 610, the method of observing the sample, as well as the observed uniform color may vary among different users.

[ 0062 ] According to some embodiments described herein, the graduated color band 610 is used to measure the turbidity of liquid samples ranging from as low as about 0.25 ml to about 100 ml. The described embodiments further provide an apparatus and methods for measuring the turbidity of low volume samples, ranging from about 0.25 ml to about 6 ml.

Accordingly, the graduated color band is designed to accommodate various sample volumes so that the length of the sample tube when placed in the apparatus 630 does not interfere with a user ' s ability to observe the graduated color band 610 through a sample. In one embodiment, the graduated color band 610 is made of different sizes in order to accommodate various test tubes, tube diameters, and liquid volumes. The embodiments according to the described methods are also used to determine the turbidity of solutions in various applications, such as nucleic acid amplification, fibrin formation, etc.

[ 0063 ] 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 .

Claims

1. A detection apparatus for measuring turbidity of a liquid suspension comprising a holding receptacle having at least one channel configured to receive a suspension tube with a liquid suspension therein, and at least one color band disposed over the surface of said at least one channel, and positioned such that, when the detection apparatus receives the suspension tube, the at least one color band is capable of being at least partially disposed behind the suspension in the tube .

2. The apparatus of claim 1, wherein the at least one color band is selected from a group consisting of at least two uniform color bands and a single graduated color band.

3. The apparatus of claim 2, wherein the first of the at least two uniform color bands is capable of being positioned behind the suspension in the suspension tube and the second uniform color band is capable of being positioned behind the suspension tube but not the suspension in the tube .

4. The apparatus of claim 2, wherein the graduated color band is configured using a single color, the single color transitioning from a darker bottom portion of the band to a lighter top portion of the band with varying degrees of color intensity between the top and bottom portions.

5. The apparatus of claim 1, wherein the apparatus is configured for a visual turbidity determination.

6. The apparatus of claim 3, wherein the first and second uniform color bands are disposed at a fixed distance over the surface of the channel.

7. The apparatus of claim 3, wherein the distance between the first and second color bands is adjustable.

8. The apparatus of claim 4, wherein the graduated color band is disposed over the proximal portion of the surface of the channel .

9. The apparatus of claim 1, wherein the liquid suspension has a volume of about 0.25 ml to about 100 ml.

10. The apparatus of claim 9, wherein the liquid suspension has a volume of about 0.5 ml to about 6 ml.

11. The apparatus of claim 7, further comprising an adjustable slide with at least one of the first and second color bands disposed thereon.

12. The apparatus of claim 11, wherein the adjustable slide is retractable and/or extendable.

13. The apparatus of claim 1, wherein the holding receptacle and the channel configured to receive a suspension tube are positioned at about 0.1 degree to about 89 degree angle relative to the base of the apparatus .

14. The apparatus of claim 13, wherein the holding receptacle and the channel configured to receive a suspension tube are positioned at about 30 degree to about 60 degree angle relative to the base of the apparatus .

15. The apparatus of claim 14, wherein the holding receptacle and the channel configured to receive a suspension tube are positioned at about a 45 degree angle relative to the base of the apparatus.

16. The apparatus of claim 1, wherein the at least one channel is adapted to receive a removable insert, and wherein the at least one color band is disposed on the removable insert .

17. The apparatus of claim 1, wherein the holding receptacle has a plurality of channels.

18. A method for measuring turbidity of a liquid suspension, said method comprising:

receiving at least a portion of a liquid suspension into a suspension tube;

placing said suspension tube inside a holding receptacle of a visual detection apparatus comprising at least one channel configured to receive the suspension tube, a lower uniform color band and an upper uniform color band disposed on the surface of said at least one channel;

observing the color of the lower uniform color band through the liquid suspension overlaying the lower uniform color band, wherein the uppermost portion of the suspension in the tube falls below the upper uniform color band;

observing the relative intensities between the lower and the upper uniform color bands;

recording the observed relative intensities;

comparing the observed relative intensities to a McFarland standard of a known value; and

determining whether a desired turbidity has been reached .

19. The method of claim 18, wherein the liquid suspension is a biological sample.

20. The method of claim 18, wherein the uniform color bands are designed using a preselected color to reflect a known McFarland value.

21. A method for measuring turbidity of a liquid suspension, said method comprising:

receiving at least a portion of a liquid suspension into a suspension tube;

placing said suspension tube inside a holding receptacle of a visual detection apparatus comprising at least one channel configured to receive the suspension tube, and a graduated color band disposed on a proximate end of said at least one channel;

observing the graduated color band disposed behind the liquid suspension overlaying the length of the graduated color band;

evaluating the color appearance of the graduated color band as observed through the liquid suspension, wherein the color appearance is uniform;

recording the uniform color appearance of the graduated color band; and

determining whether a desired turbidity has been reached .

22. The method of claim 21, wherein the liquid suspension is a biological sample.

23. A method for measuring turbidity of a liquid suspension, said method comprising:

receiving at least a portion of a liquid suspension into a suspension tube;

observing the color of the lower uniform color band over which the liquid suspension is disposed;

comparing the observed color of the lower uniform color band to the color of the upper uniform color band, wherein the upper uniform color band is disposed on the channel above the liquid suspension, and wherein the upper uniform color band reflects a predetermined McFarland value;

observing a relative intensity between the lower uniform color band and the upper uniform color band; and

determining whether a desired turbidity has been reached .

24. A detection apparatus for measuring turbidity of a liquid suspension comprising a holding receptacle having at least one channel configured to receive a suspension tube with a liquid suspension therein, at least one adjustable slide disposed on the surface of said at least one channel, and at least one color band disposed over the surface of said at least one adjustable slide, and configured such that, when the detection apparatus receives the suspension tube, the at least one color band is capable of being at least partially disposed behind the suspension in the tube.

25. The apparatus of claim 24, wherein the at least channel is further configured t o receive the at least adjustable slide having at least one color band disposed the surface of said at least one adjustable slide.

26. The apparatus of cl; im 25, wherein the at least one color band is selected from a group consisting of at least two uniform color bands and a single graduated color band.

27. The apparatus of claim 24, further comprising a plurality of holding receptacles having a plurality of channels configured to receive a plurality of suspension tubes with a liquid suspension therein, and at least one color band disposed over the surface of each of the plurality of channels .

28. The apparatus of claim 27, wherein the at least one color band is selected from a group consisting of at least two uniform color bands and a single graduated color band.

29. The apparatus of claim 26, wherein the first of the at least two uniform color bands is capable of being positioned behind the suspension in the plurality of suspension tubes and the second uniform color band is capable of being positioned behind the suspension but not the suspension in the plurality of suspension tubes.

30. The apparatus of claim 26, wherein the graduated color band is configured using a single color, the single color transitioning from a darker bottom portion of the band to a lighter top portion of the band with varying degrees of color intensity between the top and bottom portions.

31. The apparatus of claim 27, wherein first and second uniform color bands are disposed at a fixed distance on the surface of the plurality of channels.

32. The apparatus of claim 27, wherein a graduated color band is disposed over the proximal portion of the surface of the plurality of channels.

33. The apparatus of claim 27, further comprising a plurality of adjustable slides with at least one of the first and second color bands disposed thereon.

34. The apparatus of claim 33, wherein the plurality of adjustable slides are retractable and/or extendable.

35. The apparatus of claim 33, wherein the plurality of adjustable slides are removable from their respective channels .