WO2017053684A1 - Solid standard for calibrating turbidity measurement devices - Google Patents

Solid standard for calibrating turbidity measurement devices Download PDF

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Publication number
WO2017053684A1
WO2017053684A1 PCT/US2016/053282 US2016053282W WO2017053684A1 WO 2017053684 A1 WO2017053684 A1 WO 2017053684A1 US 2016053282 W US2016053282 W US 2016053282W WO 2017053684 A1 WO2017053684 A1 WO 2017053684A1
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standard
microspheres
turbidity
mcfarland
polymer
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PCT/US2016/053282
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French (fr)
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Ming-Hsiung Yeh
Jianjun Wang
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Becton, Dickinson And Company
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Publication of WO2017053684A1 publication Critical patent/WO2017053684A1/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47Scattering, i.e. diffuse reflection
    • G01N21/4785Standardising light scatter apparatus; Standards therefor
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08KUse of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K3/00Use of inorganic substances as compounding ingredients
    • C08K3/34Silicon-containing compounds
    • C08K3/36Silica
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08LCOMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L83/00Compositions of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing silicon with or without sulfur, nitrogen, oxygen or carbon only; Compositions of derivatives of such polymers
    • C08L83/04Polysiloxanes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06Quantitative determination
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08GMACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G77/00Macromolecular compounds obtained by reactions forming a linkage containing silicon with or without sulfur, nitrogen, oxygen or carbon in the main chain of the macromolecule
    • C08G77/04Polysiloxanes
    • C08G77/20Polysiloxanes containing silicon bound to unsaturated aliphatic groups
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/245Escherichia (G)

Definitions

  • 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.
  • turbidimetry is more advantageous, less laborious and does not necessitate time-consuming methods such as weighting a sample or preparing standard plate counts.
  • 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.
  • 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.
  • 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 compared with transmitted light. Examples of nephelometers that use light sensors to make such measurements are described in US Patent No. 5,575,481 to O'Brien and US Provisional Application Serial No. 62/056,911 filed on September 29, 2014, the disclosures of which are incorporated by reference herein.
  • McFarland is one standard unit for measuring turbidity.
  • the McFarland standard deploys solutions of barium sulfate to compare with measurements of the suspensions to determine turbidity of the suspensions. As such, McFarland determinations require measurement with reference to a standard to ascertain turbidity.
  • the first source of the variability is variability between sensors in different instruments. As noted in US Patent No. 5,575,481, a different turbidity value can be obtained for the same sample using two different sensors.
  • Another source of variability is the standard with which the measurements are compared. Comparing the measured values for the sample with the measured values for the standard can lead to variability in the measurements if the turbidity of the standard varies from instrument to instrument.
  • US Patent No. 5,757,481 describes using a solid block of material for a turbidity standard.
  • the solid block is calcium carbonate and clear acrylic.
  • the solid block 30 shown in FIG. 3 is manufactured by mixing 8.0 grams of calcium carbonate and 4.99 pounds of clear acrylic powder. The mixture is then melted and molded into the desired shape.
  • liquid standards are not stable over time as described in detail below.
  • US Patent No. 4,291,980 describes a suspension of pure styrene-divinylbenzene copolymer suspended in DI water for turbidity measurements.
  • turbidity standards are associated with McFarland values.
  • McFarland values are one way to measure turbidity by associating a McFarland value with a known concentration of E. Coli in suspension. Generating a McFarland calibration curve for an instrument based on a set of suspensions with different concentrations of E. Coli is well known to one skilled in the art and not described in detail herein.
  • the solid state McFarland standard is particularly advantageous when used in a sample cuvette, because such open cuvettes can introduce variability in McFarland readings from cuvette to cuvette that are not attributable to the suspension in the cuvette.
  • a McFarland standard with the above said features is needed to calibrate a device that measures the turbidity of samples in such cuvettes.
  • the standard described herein is a solid standard with microspheres dispersed therein.
  • the standard microspheres are dispersed into an aqueous or other liquid phase, water or liquid will be lost through the cuvette opening by evaporation or permeation via the cuvette wall.
  • the McFarland value of the standard will change over time.
  • the microspheres are dispersed into non-volatile solvent, the microspheres have to be the same density as the solvent or the size of the microspheres has to be carefully selected to prevent possible sedimentation. Sedimentation will also cause the turbidity value of the suspension to change over time. Such a precise match is difficult to obtain.
  • gas bubbles can form under certain conditions in a liquid state standard. The bubbles affect the McFarland value of the liquid. Since the size, amount and distribution of bubbles will change over time, the presence of bubbles will cause the McFarland value of a standard having such bubbles to change over time.
  • the standard described herein includes microspheres or microparticles (microspheres herein) in a cured polymeric or elastomeric matrix.
  • the polymer or elastomer when cured, is optically transparent.
  • the microspheres scatter and/or absorb light transmitted into the cured matrix. Therefore, microsphere concentration varies the turbidity value or McFarland value associated with the standard.
  • microspheres are contemplated for use in the standard described herein.
  • Silica particles preferably with a narrower particle size distribution are one example.
  • the microspheres are polystyrene.
  • the microspheres remain stable in the matrix over time. Thus, gas bubbles and materials that might be absorbed or otherwise not stable in the matrix are not suitable. However, liquids that are not miscible in the liquid polymer might be useful if such remain stable in the matrix over time.
  • the cured elastomeric matrix is a silicone elastomer.
  • the silicone elastomer itself is optically clear, when there is no microsphere presence. The addition of the microspheres reduces the clarity of the silicone elastomer and, therefore, increases its turbidity.
  • the matrix is formed from a curable liquid material.
  • the liquid material is combined with the microspheres and cured.
  • the concentration of microspheres is selected to provide a standard with a turbidity or McFarland value.
  • the polymer is cured.
  • a catalyst either alone or in combination with an inhibitor that inhibits any unwanted polymerization is used to cure the polymer.
  • the role and selection of polymerization inhibitors, catalysts and cross-linkers is well known to one skilled in the art and not described in detail herein.
  • the then cured polymer or elastomeric matrix when cured, is optically transparent at the wavelength(s) used to measure the turbidity/McFarland values of the standard. With increasing microsphere amount, the turbidity of the solid standard increases. The higher the microsphere concentration, the higher the turbidity value or McFarland value.
  • the standards described herein can be used as standards in conventional apparatus for measuring turbidity.
  • Some apparatus provide a turbidity reading as a McFarland value.
  • Discrete McFarland values correlate to discrete concentrations of E. Coli in suspension.
  • E. Coli measure about 1.5 ⁇ in diameter
  • the size of the microspheres in the matrix are largely a matter of design choice with microspheres having an average diameter in the range of hundreds of microns to sub- micron size deemed suitable.
  • One example of a suitable range of average particle sizes is 5nm to 1 mm. Variations in type of particle and particle size distribution and particle concentration will change the turbidity /McFarland value of the standard.
  • microspheres such as polystyrene-based microspheres are dispersed into a liquid vinyl-terminated polydimethylsiloxane polymer.
  • a homogenous suspension is obtained using mechanical homogenization.
  • a crosslinker is mixed with such suspension. Crosslinking is accomplished using a catalyst.
  • microspheres combined with the curable liquid polymer, are immobilized and embedded in solid clear silicone elastomer once the polymer is cured.
  • the polymer is cured by crosslinking. Since silicone elastomer is known to have a very low expansion coefficient over a wide range of temperatures, the McFarland value of the standard remains constant over a wide range of conditions. For this reason, in certain embodiments, silicone elastomer is selected as the matrix material for the standard.
  • the standard is typically formed by curing a suspension of a curable polymer to which microspheres have been added.
  • concentration of the microspheres i.e. the loading
  • the concentration of the microspheres will change depending on the target turbidity /McFarland value for the standard.
  • microspheres for the standard described herein include:
  • non-aqueous liquid materials that are not miscible in the uncured polymer and form a suspension or emulsion.
  • examples of such liquids are: lipid oils, mineral oil, alcohols & ketones with higher boiling points, etc.
  • Aqueous materials can evaporate over time, causing the turbidity /McFarland value of the standard to vary over time.
  • FIG. 1 illustrates the effect of microsphere concentration on the output of the systems
  • FIG. 2 illustrates McFarland value as in function of microsphere concentration.
  • FIG. 3 illustrates a turbidity standard according to one embodiment.
  • FIG. 1 demonstrates that the output from the calibration instrument and the microspheres concentration are linear. Such observation confirms that linear dilution of microsphere stock solution is sufficient to result in a desired calibration output value.
  • the McFarland values were calculated based on a regression of output of a serial dilution of an E. coli suspension (with density of the suspension determined by the density specifications for microbial suspensions prepared for use in the BD PhoenixTM System). PhoenixTM is a trademark of Becton Dickinson and Company of Franklin Lakes, New Jersey. There is a linear relationship between the microspheres density and estimated McFarland value. Therefore, a standard with specific McFarland value could be fabricated using known standard microsphere concentration before curing.
  • Turbidity is measured optically in devices such as a nephelometer. Nephelometers measure light transmitted through and scattered by the suspension.
  • Prior Art turbidity standards have polystyrene microspheres suspended in aqueous solution with surfactant. Such suspensions are stored in a sealed glass container to be used in a particular nephelometry instrument. Since glass material is impermeable to water, the turbidity or McFarland value of such an aqueous-based standard is constant over time.
  • the nephelometry instrument is calibrated daily. The frequency makes the liquid standard less efficient, because more liquid standard must be retrieved for each measurement.
  • the solid state standard described herein is reusable without special storage and handling requirements. Also, the reusable standard improves the instrument calibration process.
  • the solid state standard is illustrated in FIG. 3.
  • the solid state standard 15 is a polymeric or elastomeric matrix 20 in which microparticles 25 are embedded.
  • the cuvette 10 is any suitable container for the standard. Since the standard is what is being measured, the container for the standard is selected so that it will not have an effect on the measured turbidity. As such, the container is selected to be transparent to the signal being used to interrogate the turbidity of the standard.
  • a reusable turbidity standard must have a stable McFarland value over time. Liquid standards are not stable due to the loss of water through the cuvette wall, an improper cap, seal, etc. As described above, liquid turbidity standards must be stored in glass to prevent changes in the standard due to liquid loss over time.
  • the cuvette is made of polystyrene. Due to the different optical properties of glass and polystyrene, a glass cuvette is not always an option as a vessel for the standard. Water permeability through polystyrene results in water loss during turbidity standard storage time leading to an unstable standard. As noted above, the solid phase standard described herein is more stable over time, because there is no water loss over time. In a solid phase standard there are no microsphere concentration changes and therefore the optical property will not change over time for standards having a solid phase matrix.
  • the matrix is silicone elastomer.
  • Silicone elastomer is prepared by mixing vinyl functional silicone oil with a corresponding cross-linker, and cured with the aid of a catalyst. Catalytically induced curing of polymers, with and without inhibitors, is well known to one skilled in the art and not described in detail herein.
  • the viscosity of silicone oil is selected so that it is higher than that of water. This provides and maintains a homogenous concentration of microspheres once they are dispersed into the liquid phase of silicone oil. In one embodiment larger microspheres (e.g. on the order of 1 ⁇ ) with diameters commensurate with the size of microorganisms are used.
  • the cured silicone elastomer is optically clear.
  • the solid state turbidity standard uses the same principle of liquid state based standards.
  • the solid microsphere dispersed in the standard absorbs the excitation light.
  • the level of absorbance at 600nm corresponds to a McFarland value.
  • Optically clear silicone elastomer matrix does not absorb light at or around 600nm region. Silicone elastomer maintains its volume and physical properties over a wide range of temperature (typically from minus 50°C to several hundred degrees centigrade and at various pressures).
  • the standard is formed by combining the curable polymer and suitable additives for curing (e.g. catalyst, cross-linker, etc.) with the microspheres.
  • suitable additives for curing e.g. catalyst, cross-linker, etc.
  • a catalyst platinum divinyltetramethyl
  • an inhibitor cyclic vinylmethyl dimethylsiloxanes
  • dimethylvinyl terminated polydimethylsiloxane are combined to form the liquid base composition that is used to form the standard.
  • a methylhydrido polysiloxane crosslinker is added thereto to cross-link the polymer.
  • the platinum catalyst, inhibitor, and dimethylvinyl terminated polydimethylsiloxane are combined in a container. This is referred to herein as the base mixture.
  • the contents are mechanically mixed until a homogeneous mixture was obtained.
  • Polystyrene beads with an average diameter of 1 ⁇ are then added to the base mixture.
  • a cross-linker mixture is prepared by combining the cross-linker with dimethylvinyl terminated polydimethylsiloxane.
  • the cross-linker mixture is mechanically using homogenizer until a homogeneous cross linker mixture is obtained.
  • a predetermined amount of the base mixture e.g. 18g
  • An amount (3.0 + 0.3 g) of cross-linker mixture is added to the master batch weighed into the beaker and mixed manually.
  • E. Coli suspensions are prepared with varying concentrations of E. Coli. The turbidity of those suspensions is measured and a linear curve results that is McFarland values as a function of E. Coli concentration.
  • Standards are prepared with different concentrations of microspheres. The turbidity of the standards is measure.
  • the output indicates a McFarland value that corresponds to a McFarland value obtained by measuring the turbidity of an E. Coli suspension
  • the relationship between the standard and the E. Coli suspension is known. From this a linear relationship that is a function of microsphere concentration in the standard can be obtained. Such relationships are illustrated in FIG. 1 and FIG. 2.

Abstract

A standard for measuring a fixed turbidity value. The turbidity standard has microspheres or microparticles in a cured polymeric or elastomeric matrix. When analyzing liquid biological samples, the turbidity of the solution is measured as an indicator of the amount of biological sample suspended in solution. Turbidity is a relative measurement and therefore measuring turbidity requires a regular and repeatable standard against which turbidity values are compared to determine the turbidity of the suspension.

Description

SOLID STANDARD FOR CALIBRATING TURBIDITY MEASUREMENT DEVICES
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application claims the benefit of the filing date of U.S. Provisional
Application No. 62/232,767, filed September 25, 2015, the disclosure of which is hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] 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.
[0004] 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 compared with transmitted light. Examples of nephelometers that use light sensors to make such measurements are described in US Patent No. 5,575,481 to O'Brien and US Provisional Application Serial No. 62/056,911 filed on September 29, 2014, the disclosures of which are incorporated by reference herein.
[0005] McFarland is one standard unit for measuring turbidity. The McFarland standard deploys solutions of barium sulfate to compare with measurements of the suspensions to determine turbidity of the suspensions. As such, McFarland determinations require measurement with reference to a standard to ascertain turbidity.
[0006] When measuring turbidity there are two sources of variability which can affect the consistency of the measurements from instrument to instrument. The first source of the variability is variability between sensors in different instruments. As noted in US Patent No. 5,575,481, a different turbidity value can be obtained for the same sample using two different sensors. Another source of variability is the standard with which the measurements are compared. Comparing the measured values for the sample with the measured values for the standard can lead to variability in the measurements if the turbidity of the standard varies from instrument to instrument.
[0007] According to US Patent No. 5,757,481 : A booklet titled "Turbidity Standards" by
Clifford C. Hach has been published for the purpose of describing the historical development of turbidity measurements, the development of turbidity standards, the use of Formazin standards, and the use of certain secondary standards. This booklet is published by the Hach Company and identified as booklet number 12 of the Technical Information Series. This turbidity standard booklet, U.S. Pat. No. 5,444,531, U.S. Pat. No. 5,446,531, U.S. Pat. No. 5,291,626 and U.S. Pat. No. 3,892,485 are all explicitly incorporated by reference in this description of the present invention.
[0008] US Patent No. 5,757,481 describes using a solid block of material for a turbidity standard. The solid block is calcium carbonate and clear acrylic. The solid block 30 shown in FIG. 3 is manufactured by mixing 8.0 grams of calcium carbonate and 4.99 pounds of clear acrylic powder. The mixture is then melted and molded into the desired shape. However, liquid standards are not stable over time as described in detail below.
[0009] US Patent No. 4,291,980 describes a suspension of pure styrene-divinylbenzene copolymer suspended in DI water for turbidity measurements.
[0010] 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. The standard must be stable and consistent over time, simple to handle and convenient to use. For these reasons a consistent, reliable uniform standard for use with turbidity measurement continues to be sought. BRIEF SUMMARY OF THE INVENTION
[0011] Described herein is a reusable, homogeneous, solid state turbidity standard with constant turbidity value over time. In one embodiment the turbidity standards are associated with McFarland values. McFarland values are one way to measure turbidity by associating a McFarland value with a known concentration of E. Coli in suspension. Generating a McFarland calibration curve for an instrument based on a set of suspensions with different concentrations of E. Coli is well known to one skilled in the art and not described in detail herein.
[0012] The solid state McFarland standard is particularly advantageous when used in a sample cuvette, because such open cuvettes can introduce variability in McFarland readings from cuvette to cuvette that are not attributable to the suspension in the cuvette. A McFarland standard with the above said features is needed to calibrate a device that measures the turbidity of samples in such cuvettes. The standard described herein is a solid standard with microspheres dispersed therein.
[0013] If the standard microspheres are dispersed into an aqueous or other liquid phase, water or liquid will be lost through the cuvette opening by evaporation or permeation via the cuvette wall. When the volume of the solution changes over time, the McFarland value of the standard will change over time. If the microspheres are dispersed into non-volatile solvent, the microspheres have to be the same density as the solvent or the size of the microspheres has to be carefully selected to prevent possible sedimentation. Sedimentation will also cause the turbidity value of the suspension to change over time. Such a precise match is difficult to obtain. In addition, gas bubbles can form under certain conditions in a liquid state standard. The bubbles affect the McFarland value of the liquid. Since the size, amount and distribution of bubbles will change over time, the presence of bubbles will cause the McFarland value of a standard having such bubbles to change over time.
[0014] The standard described herein includes microspheres or microparticles (microspheres herein) in a cured polymeric or elastomeric matrix. The polymer or elastomer, when cured, is optically transparent. The microspheres scatter and/or absorb light transmitted into the cured matrix. Therefore, microsphere concentration varies the turbidity value or McFarland value associated with the standard.
[0015] A variety of microspheres are contemplated for use in the standard described herein.
Silica particles, preferably with a narrower particle size distribution are one example. In one embodiment, the microspheres are polystyrene. The microspheres remain stable in the matrix over time. Thus, gas bubbles and materials that might be absorbed or otherwise not stable in the matrix are not suitable. However, liquids that are not miscible in the liquid polymer might be useful if such remain stable in the matrix over time.
[0016] In one embodiment, the cured elastomeric matrix is a silicone elastomer. The silicone elastomer itself is optically clear, when there is no microsphere presence. The addition of the microspheres reduces the clarity of the silicone elastomer and, therefore, increases its turbidity.
[0017] The matrix is formed from a curable liquid material. To form the standard, the liquid material is combined with the microspheres and cured. The concentration of microspheres is selected to provide a standard with a turbidity or McFarland value. Once the microspheres are combined with the liquid polymer, the polymer is cured. A catalyst either alone or in combination with an inhibitor that inhibits any unwanted polymerization is used to cure the polymer. The role and selection of polymerization inhibitors, catalysts and cross-linkers is well known to one skilled in the art and not described in detail herein. The then cured polymer or elastomeric matrix, when cured, is optically transparent at the wavelength(s) used to measure the turbidity/McFarland values of the standard. With increasing microsphere amount, the turbidity of the solid standard increases. The higher the microsphere concentration, the higher the turbidity value or McFarland value.
[0018] The standards described herein can be used as standards in conventional apparatus for measuring turbidity. Some apparatus provide a turbidity reading as a McFarland value. Discrete McFarland values correlate to discrete concentrations of E. Coli in suspension. Although E. Coli measure about 1.5 μηι in diameter, the size of the microspheres in the matrix are largely a matter of design choice with microspheres having an average diameter in the range of hundreds of microns to sub- micron size deemed suitable. One example of a suitable range of average particle sizes is 5nm to 1 mm. Variations in type of particle and particle size distribution and particle concentration will change the turbidity /McFarland value of the standard.
[0019] In one embodiment microspheres such as polystyrene-based microspheres are dispersed into a liquid vinyl-terminated polydimethylsiloxane polymer. A homogenous suspension is obtained using mechanical homogenization. A crosslinker is mixed with such suspension. Crosslinking is accomplished using a catalyst.
[0020] In this embodiment, microspheres, combined with the curable liquid polymer, are immobilized and embedded in solid clear silicone elastomer once the polymer is cured. In certain embodiments, the polymer is cured by crosslinking. Since silicone elastomer is known to have a very low expansion coefficient over a wide range of temperatures, the McFarland value of the standard remains constant over a wide range of conditions. For this reason, in certain embodiments, silicone elastomer is selected as the matrix material for the standard.
[0021] As noted above, the standard is typically formed by curing a suspension of a curable polymer to which microspheres have been added. The concentration of the microspheres (i.e. the loading) will change depending on the target turbidity /McFarland value for the standard.
[0022] Examples of suitable microspheres for the standard described herein include:
1) silica;
2) plastic/organic polymers in powder form;
3) inorganic/metallic or non-metallic particles; and
4) non-aqueous liquid materials that are not miscible in the uncured polymer and form a suspension or emulsion. Examples of such liquids are: lipid oils, mineral oil, alcohols & ketones with higher boiling points, etc. Aqueous materials can evaporate over time, causing the turbidity /McFarland value of the standard to vary over time.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 illustrates the effect of microsphere concentration on the output of the systems, and
[0024] FIG. 2 illustrates McFarland value as in function of microsphere concentration.
[0025] FIG. 3 illustrates a turbidity standard according to one embodiment.
DETAILED DESCRIPTION
[0026] FIG. 1 demonstrates that the output from the calibration instrument and the microspheres concentration are linear. Such observation confirms that linear dilution of microsphere stock solution is sufficient to result in a desired calibration output value. In FIG. 2, the McFarland values were calculated based on a regression of output of a serial dilution of an E. coli suspension (with density of the suspension determined by the density specifications for microbial suspensions prepared for use in the BD Phoenix™ System). Phoenix™ is a trademark of Becton Dickinson and Company of Franklin Lakes, New Jersey. There is a linear relationship between the microspheres density and estimated McFarland value. Therefore, a standard with specific McFarland value could be fabricated using known standard microsphere concentration before curing.
[0027] Turbidity is measured optically in devices such as a nephelometer. Nephelometers measure light transmitted through and scattered by the suspension.
[0028] According to U.S. Patent No. 5,444,531 : "As the turbidity of the fluid in the conduit 20 increases, the magnitude of light received by the first light sensitive component will decrease and the magnitude of light received by the second light sensitive component 18 will increase. Therefore, a ratio of the signals received by the first and second light sensitive components can be used as an indicator of the degree of turbidity of the fluid within the conduit 20." U.S. Patent No. 5,444,531 is incorporated by reference herein.
[0029] Prior Art turbidity standards have polystyrene microspheres suspended in aqueous solution with surfactant. Such suspensions are stored in a sealed glass container to be used in a particular nephelometry instrument. Since glass material is impermeable to water, the turbidity or McFarland value of such an aqueous-based standard is constant over time.
[0030] For plastic cuvettes there is constant water loss due to either evaporation, permeation of the plastic container or both. Hence a constant McFarland value for such suspensions in a plastic cuvette cannot be maintained. Withdrawing an aliquot of a standard suspension from a glass container and adding it to the plastic cuvette is not a cost effective solution due to the high cost for maintaining the standard suspension.
[0031] Typically, the nephelometry instrument is calibrated daily. The frequency makes the liquid standard less efficient, because more liquid standard must be retrieved for each measurement.
[0032] The solid state standard described herein is reusable without special storage and handling requirements. Also, the reusable standard improves the instrument calibration process. The solid state standard is illustrated in FIG. 3. The solid state standard 15 is a polymeric or elastomeric matrix 20 in which microparticles 25 are embedded. The cuvette 10 is any suitable container for the standard. Since the standard is what is being measured, the container for the standard is selected so that it will not have an effect on the measured turbidity. As such, the container is selected to be transparent to the signal being used to interrogate the turbidity of the standard.
[0033] As stated above, a reusable turbidity standard must have a stable McFarland value over time. Liquid standards are not stable due to the loss of water through the cuvette wall, an improper cap, seal, etc. As described above, liquid turbidity standards must be stored in glass to prevent changes in the standard due to liquid loss over time.
[0034] In one embodiment the cuvette is made of polystyrene. Due to the different optical properties of glass and polystyrene, a glass cuvette is not always an option as a vessel for the standard. Water permeability through polystyrene results in water loss during turbidity standard storage time leading to an unstable standard. As noted above, the solid phase standard described herein is more stable over time, because there is no water loss over time. In a solid phase standard there are no microsphere concentration changes and therefore the optical property will not change over time for standards having a solid phase matrix.
[0035] In one embodiment, the matrix is silicone elastomer. Silicone elastomer is prepared by mixing vinyl functional silicone oil with a corresponding cross-linker, and cured with the aid of a catalyst. Catalytically induced curing of polymers, with and without inhibitors, is well known to one skilled in the art and not described in detail herein.
[0036] The viscosity of silicone oil is selected so that it is higher than that of water. This provides and maintains a homogenous concentration of microspheres once they are dispersed into the liquid phase of silicone oil. In one embodiment larger microspheres (e.g. on the order of 1 μιη) with diameters commensurate with the size of microorganisms are used. The cured silicone elastomer is optically clear.
[0037] The solid state turbidity standard uses the same principle of liquid state based standards.
The solid microsphere dispersed in the standard absorbs the excitation light. The level of absorbance at 600nm corresponds to a McFarland value. Optically clear silicone elastomer matrix does not absorb light at or around 600nm region. Silicone elastomer maintains its volume and physical properties over a wide range of temperature (typically from minus 50°C to several hundred degrees centigrade and at various pressures).
[0038] The standard is formed by combining the curable polymer and suitable additives for curing (e.g. catalyst, cross-linker, etc.) with the microspheres. For example, a catalyst (platinum divinyltetramethyl), an inhibitor (cyclic vinylmethyl dimethylsiloxanes) and dimethylvinyl terminated polydimethylsiloxane are combined to form the liquid base composition that is used to form the standard. A methylhydrido polysiloxane crosslinker is added thereto to cross-link the polymer.
[0039] The platinum catalyst, inhibitor, and dimethylvinyl terminated polydimethylsiloxane are combined in a container. This is referred to herein as the base mixture. The contents are mechanically mixed until a homogeneous mixture was obtained. Polystyrene beads with an average diameter of 1 μηι are then added to the base mixture.
[0040] The mixture with the polystyrene beads (average diameter of 1 μιη) added thereto was mechanically mixed using a homogenizer until a homogeneous mixture is obtained.
[0041] A cross-linker mixture is prepared by combining the cross-linker with dimethylvinyl terminated polydimethylsiloxane. The cross-linker mixture is mechanically using homogenizer until a homogeneous cross linker mixture is obtained. A predetermined amount of the base mixture (e.g. 18g) is weighed out into a small beaker. An amount (3.0 + 0.3 g) of cross-linker mixture is added to the master batch weighed into the beaker and mixed manually. The above is provided to describe one method for combining the constituents used to form the standard. Other methods are well known to those skilled in the art and are not described in detail herein.
[0042] To form the standards to conform to McFarland values, E. Coli suspensions are prepared with varying concentrations of E. Coli. The turbidity of those suspensions is measured and a linear curve results that is McFarland values as a function of E. Coli concentration. Standards are prepared with different concentrations of microspheres. The turbidity of the standards is measure. When the output indicates a McFarland value that corresponds to a McFarland value obtained by measuring the turbidity of an E. Coli suspension, the relationship between the standard and the E. Coli suspension is known. From this a linear relationship that is a function of microsphere concentration in the standard can be obtained. Such relationships are illustrated in FIG. 1 and FIG. 2.
[0043] 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 stable turbidity standard comprising:
a cured polymer;
microspheres embedded in the cured polymer or elastomer, wherein the microspheres are present in a predetermined concentration associated with a turbidity reading from an instrument;
wherein the cured polymer or elastomer is substantially optically transparent and wherein the microspheres reflect, adsorb or scatter light transmitted through the turbidity standard.
2. The standard of claim 1 wherein the microspheres are selected from silica particles and polystyrene particles.
3. The standard of claim 1 where the microspheres have an average diameter ranging from about 0.5 nm to about 1 mm.
4. The standard of claim 1 wherein the standard is a McFarland standard and the average size of the microspheres is about the average size of E. Coli which is about 0.5 μηι to about 2 μιη.
5. The standard of claim 1 wherein the cured polymer is a silicone elastomer.
6. The standard of claim 5 wherein the silicone elastomer is cured dimethyl vinyl terminated polydimethylsiloxane.
7. The standard of claim 5 wherein the microspheres are polystyrene microspheres.
8. A method for forming a turbidity standard comprising:
forming a liquid mixture of a curable polymer, a catalyst and microspheres; wherein the concentration of microspheres is predetermined to provide a value of turbidity when measured by an instrument;
curing the liquid mixture to form a solid matrix with the microspheres embedded therein, wherein the solid matrix is elastomeric and is substantially optically transparent.
9. The method of claim 8 wherein the microspheres are selected from silica particles and polystyrene particles.
10. The method of claim 8 wherein the cured polymer is a silicone elastomer.
11. The method of claim 8 wherein the curable polymer is silicone oil with a higher viscosity than water.
12. The method of claim 8 wherein the catalyst is platinum divinyltetramethyl.
13. The method of claim 8 wherein the liquid mixture further comprises a cross-linker.
14. The method of claim 13 wherein the cross-linker is methylhydrido polysiloxane.
15. The method of claim 8 where the microspheres have an average diameter ranging from about 5 nm to about 1 mm.
PCT/US2016/053282 2015-09-25 2016-09-23 Solid standard for calibrating turbidity measurement devices WO2017053684A1 (en)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
US4291980A (en) * 1978-08-14 1981-09-29 Amco Standards International Styrene-divinylbenzene copolymer and method of manufacture
EP0628604B1 (en) * 1993-06-09 1999-10-20 DOW CORNING GmbH Curable Polysiloxanes compositions
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EP0628604B1 (en) * 1993-06-09 1999-10-20 DOW CORNING GmbH Curable Polysiloxanes compositions
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