Patent Publication Number: US-10316347-B2

Title: Endospore detection using hydrophobic collection material

Description:
TECHNICAL FIELD 
     This disclosure relates to bacterial endospore isolation and analysis. 
     BACKGROUND 
     Bacterial spores are generally accepted to be indicator species for validating sterility since they are the most resilient form of life against sterilization regimens. Traditional bacterial spore analysis is a labor intensive and time consuming process. For example, spore analysis may involve heat activation, serial dilution, plating on a suitable growth medium, and incubation for two to three days until enumeration can be performed. This analysis process can take several days, requiring manufacturers of product undergoing analysis to hold significant quantities of the product before receiving sterility test results that allow them to release product to the marketplace. Moreover, in instances where there is a sterility issue, the lack of real-time information can result in several days&#39; worth of production being deemed out of specification and needing to be discarded or repurposed. 
     In an attempt to provide faster analysis, designers have utilized optical analysis techniques that detect optical emission signals associated with bacterial spores and then correlate these signals with spore count. These techniques are of limited use, however, for many categories of materials desirably analyzed for bacterial spore count. For example, materials that contain a low number of bacterial spores or contain bacterial spores within a surrounding fluid that optically interferes with the emissions associated with the spores can be difficult to evaluate using optical analysis techniques. As one example, dairy production facilities monitoring bacterial spore counts in their products typically cannot use optical emission analysis techniques. This is because proteins and other molecules within the dairy products can optically interfere with emissions produced by bacterial spores. 
     SUMMARY 
     In general, this disclosure relates to techniques and systems for analyzing bacterial endospores within fluids. Depending on the application, a fluid containing the bacterial endospores may or may not be optically active such that the fluid surrounding the endospores optically interferes with optical emissions corresponding to the endospores. In either case, the fluid may be passed through a hydrophobic sieve to help isolate the endospores from the surrounding fluid. The hydrophobic sieve may be fabricated from a hydrophobic material and have a porous structure allowing substantially all of the fluid to pass through the hydrophobic sieve. As the endospores contained within the fluid come into proximity of the hydrophobic material while the fluid is passing through the sieve, the endospores may adhere to the surface of the hydrophobic sieve via hydrophobic attraction forces. The fluid surrounding the endospores may continue passing through the hydrophobic sieve. In this manner, the bacterial endospores can be substantially isolated from the carrier fluid. 
     Once isolated from the surrounding carrier fluid, the bacterial endospores captured on the surface of the hydrophobic sieve via hydrophobic attraction forces can be processed to qualitatively and/or quantitatively evaluate the characteristics of the endospores within the fluid. In some examples, the hydrophobic sieve is flushed with an optically inert flushing fluid such as water to remove residual carrier fluid from the bacterial endospores. In addition or alternatively, a germination fluid may be added to the hydrophobic sieve to germinate the captured bacterial endospores and release dipicolinic acid (DPA) from the core of the spores. When a lanthanide ion source is added to the germination fluid, the lanthanide ion can bind with the DPA to form a lanthanide-DPA complex that fluoresces when optically excited. This fluorescence can be detected and correlated to the concentration of bacterial endospores captured by the hydrophobic sieve which, in turn, can be correlated to the concentration of bacterial endospores in the original carrier fluid. 
     Using a hydrophobic material, such as a hydrophobic sieve, to help isolate bacterial endospores from a surrounding fluid can be useful for a variety of reasons. In instances where the surrounding fluid is optically active, the hydrophobic material can help separate the bacterial endospores from the optically active fluid. This can remove a source of optical emissions that may otherwise interfere with the emissions produced by the lanthanide-DPA complex during optical analysis of the bacterial endospores. As another example, the hydrophobic material can be used to increase the concentration of bacterial endospores available for analysis, which may increase the range of bacterial endospore concentrations that can be analyzed and/or reduce error effects associated with low endospore concentration fluids. For example, the number of endospores captured by the hydrophobic material may be proportional to the volume of fluid passed across the material. In such applications, the concentration of bacterial endospores available for analysis can be increased by increasing the volume of carrier fluid passed over the hydrophobic material. The concentration of bacterial endospores available for subsequent optical analysis can then be controlled by controlling the amount of germination fluid added to the hydrophobic material. Where the volume of germination fluid added to the hydrophobic material is less than the volume of carrier fluid passed across the material, the concentration of bacterial endospores may be increased for subsequent optical analysis as compared to the concentration in the original carrier fluid. This can be helpful when analyzing fluids having comparatively low concentrations of bacterial endospores. 
     In one example a method is described that includes passing a fluid containing bacterial endospores across a hydrophobic collection material and thereby collecting bacterial endospores on the hydrophobic collection material. The method also includes releasing dipicolinic acid (DPA) from the collected bacterial endospores and adding a lanthanide source to the dipicolinic acid released from the collected bacterial endospores to form a lanthanide-dipicolinic acid complex. The method further includes determining a concentration of the bacterial endospores in the fluid based on an optical response of the lanthanide-dipicolinic acid complex. 
     In another example a system is described that includes an aqueous liquid to be evaluated, a germination fluid, a lanthanide source, a hydrophobic collection material, and an optical sensor. The hydrophobic collection material is configured to receive the aqueous liquid and capture therefrom bacterial endospores, receive the germination fluid so as to release dipicolinic acid (DPA) from the captured bacterial endospores, and receive the lanthanide source so as to form a lanthanide-dipicolinic acid complex in the germination fluid. The optical sensor is configured to emit optical energy into the germination fluid and thereby generate optical emissions from the lanthanide-dipicolinic acid complex, detect the optical emissions emitted by the lanthanide-dipicolinic acid complex, and determine therefrom a concentration of bacterial endospores in the aqueous liquid. 
     In another example a method is described that includes passing an aqueous liquid through a hydrophobic sieve and thereby capturing bacterial endospores present in the aqueous liquid on a surface of the hydrophobic sieve via hydrophobic attraction between the hydrophobic sieve and the bacterial endospores. The method includes adding a germination fluid to the hydrophobic sieve so as to release dipicolinic acid (DPA) from bacterial endospores captured on the hydrophobic sieve and providing a lanthanide source to form a lanthanide-dipicolinic acid complex in the germination fluid with the dipicolinic acid released from the bacterial endospores captured on the hydrophobic sieve. The method further includes fluorometrically analyzing the germination fluid to determine a concentration of bacterial endospores in the aqueous liquid. 
     The details of one or more examples are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  is a flow diagram illustrating an example process for capturing and analyzing bacterial endospores from a fluid under evaluation. 
         FIG. 2  is a conceptual illustration of an example system that can be used to determine the bacterial endospore count in a fluid according to the example technique of  FIG. 1 . 
         FIG. 3  is a plot showing example optical responses of fluids prepared by passing milk samples across a hydrophobic material. 
         FIG. 4  is a plot showing an example number of endospores captured versus surface area of an example hydrophobic material. 
         FIG. 5  is a micrograph showing one example hydrophobic material at a resolution of 1 millimeter after passing milk through the material. 
         FIG. 6  is a 100 time magnification of the micrograph of  FIG. 5  showing endospores adhered to the surface of the material. 
     
    
    
     DETAILED DESCRIPTION 
     This disclosure generally relates to the isolation, concentration, and analysis of bacterial endospores from fluids in which the endospores are carried. In some examples, the bacterial endospores are contained within an optically active fluid that emits optical emissions within wavelengths overlapping with the wavelengths at which a lanthanide-dipicolinic acid complex liberated from the bacterial endospores emits. This can prevent direct in-situ optical analysis of the bacterial endospores within the carrier fluid because of optical interference. In some examples in accordance with the disclosure, the carrier fluid is passed across a hydrophobic collection material to help separate the bacterial endospores from the remaining carrier fluid. The hydrophobic collection material can be a porous sieve that the carrier fluid flows through, a sheet of hydrophobic material that is positioned in contact with the carrier fluid, or yet another structure that attracts and holds the bacterial endospores, e.g., via hydrophobic attraction forces. Regardless of the configuration, the hydrophobic collection material can capture and collect bacterial endospores out of the carrier fluid. 
     Bacterial endospores collected out of the surrounding carrier fluid can be analyzed using a variety of different techniques. In some examples, a germination fluid is added to the hydrophobic collection material to germinate the collected bacterial endospores and release dipicolinic acid (DPA) from the endospores. A lanthanide ion reagent can be added to the germination fluid to form a lanthanide-dipicolinic acid complex. When irradiated with light of appropriate wavelengths, this lanthanide-dipicolinic acid complex may emit fluorescent light that can be detected and quantified. The magnitude and/or wavelength of light emitted by the lanthanide-dipicolinic acid complex corresponds to the concentration of the lanthanide-dipicolinic acid complex in the germination fluid which, in turn, corresponds to the concentration of bacterial endospores in the carrier fluid. 
     The disclosed systems and techniques for isolating, concentrating, and/or analyzing bacterial endospores can be utilized with any desired fluids that may contain bacterial endospores. Example industries that may use the systems and techniques include the food industry, the beverage industry, the pharmaceutical industry, the chemical industry, the healthcare industry, the water purification industry, and other industries where material is inspected for bacterial endospores. In the case of the food and beverage industry, fluids that may be analyzed for bacterial endospores include those obtained from mammalian-consumable (e.g., human-consumable) food and beverages such as, but not limited to, dairy products (e.g., raw milk, whole and skimmed milk, condensed milk, cream, whey and whey derivatives, buttermilk), juices (e.g., fruit juices such as orange and other citrus juices, apple juice and other pomaceous juices, red berry juice, coconut milk, and tropical fruit juices, vegetable juices such as tomato juice, beetroot juice, carrot juice, and grass juice), and canned foods (e.g., canned fruits, canned vegetables, canned meat). Types of bacteria that may produce endospores desired to be analyzed include, but are not limited to,  Bacillus subtilis, Bacillus cereus, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus megaterium, Bacillus coagulans, Bacillus pumilus, Bacillus mycoides, Bacillus licheniformis, Bacillus sporothermodurans, Bacillus thuringensis, Bacillus weihenstephanensis, Geobacillus stearothermophilus, Clostridium tyrobutyricum, Alicyclobacillus, Clostridium botulinum, Clostridium difficile , and  Bacillus anthracis.    
     In general, the fluid containing bacterial endospores for analysis will be a liquid, such as an aqueous liquid (e.g., water-based liquid), although gaseous carrier fluids containing bacterial endospores can also be evaluated using the systems and techniques. The fluid may be obtained directly from a product sample under analysis or may be formed by combining the product sample with a carrier fluid to extract bacterial endospores from the sample. For example, in instances where the product sample potentially containing bacterial endospores is a solid, the solid may be ground or mixed with a carrier fluid to extract the bacterial endospores for analysis. 
     The term bacterial endospore generally refers to an endospore produced within a bacterium. The term endospore does not include conidio spores and ascospores of fungi. Endospores typically are non-reproductive structures whose primary function is to ensure survival of the bacterium through periods of environmental stress. Endospores can tolerate extreme drought, heat, and starvation. They are protected by a hardened shell of protein and carbohydrates and produced by a form of binary fission in bacteria. An endospore can comprise the DNA of its parent bacterium, ribosomes, and large amounts of dipicolinic acid. For example, dipicolinic acid may compose greater than 5 weight percent of an endospore&#39;s dry weight, such as at least 10 weight percent or at least 15 weight percent, such as from 5 weight percent to 20 weight percent, or from 7 weight percent to 16 weight percent. Dipicolinic acid is a chemical that is believed to help endospores maintain their dormancy. 
     Because endospores can survive harsh environments and are resilient to sterilization regimes, endospores can be a good indicator species for validating the sterility of a product. The absence of endospores within a product sample and/or the detection of low levels of endospores within the product can indicate that the product is suitably sterile for market release. While such information is useful for a variety of products, the information may be especially valuable for consumable products, such as ingestible foods and beverages. Indeed, governmental regulatory regimes may mandate compliance with certain sterility standards before a product can be sold to the public. The ability to measure endospore count within a product in substantially real-time in accordance with some examples of the present disclosure can enable manufacturers to monitor the quality of their products in substantially real-time and rapidly respond if a sterility issue is detected. 
       FIG. 1  is a flow diagram illustrating an example process for capturing and analyzing bacterial endospores from a fluid under evaluation. The process includes passing a fluid to be evaluated across a hydrophobic material ( 10 ) to collect bacterial endospores from the fluid, for example, by capturing the endospores on the surface of the hydrophobic material via hydrophobic attraction forces. After capturing the bacterial endospores from the fluid, the bacterial endospores are optionally flushed ( 12 ) to remove optically interfering carrier fluid and then processed to release dipicolinic acid ( 14 ). In addition, a lanthanide ion source is added to the dipicolinic acid ( 16 ) to form a lanthanide-dipicolinic complex that can then be optically analyzed ( 18 ) to determine the concentration of the bacterial endospores in the fluid under evaluation. As described in greater detail below, a variety of different processing parameters and conditions can be used to capture and analyze bacterial endospores utilizing the example process of  FIG. 1 . 
     To concentrate and/or isolate bacterial endospores from a fluid under evaluation the fluid may be passed over a hydrophobic material ( 10 ). Example hydrophobic materials that can be used to concentrate and/or isolate endospores are described in greater detail with respect to  FIG. 2 . In general, however, the hydrophobic material may be a material or combination of materials chosen to selectively bind with endospores in the fluid under analysis while allowing the surrounding fluid carrying the endospores to pass across the hydrophobic material without binding. When so configured, endospores present in the fluid under analysis may attach to the surface of the hydrophobic material while a remainder of the fluid continues to pass across the hydrophobic material without adhering. This can allow the endospores present in the fluid under analysis to collect on the surface of the hydrophobic material, concentrating and isolating the endospores from the surrounding carrier fluid. 
     The hydrophobic collection material may be hydrophobic in that it does not bind or absorb water. For example, the hydrophobic collection material may repel polar molecules, such as water and molecules soluble in water, while allowing at least some types of non-polar molecules to bind to the surface of the material. The non-polar molecules may adhere to the surface of the hydrophobic material via hydrophobic attraction. Molecules having both polar and non-polar functional groups may or may not adhere to the hydrophobic material, e.g., depending on the hydrophobicity of the material being used, the length of the carbon chain separating a polar functional group from a non-polar functional group, and/or net polarity of the molecule. 
     When used, the hydrophobic collection material can capture and collect endospores out of a fluid passing over the material via hydrophobic attraction. As a fluid containing endospores passes across the hydrophobic collection material, the endospore may pass adjacent to and, in some examples, in contact with the surface of hydrophobic collection material. Since bacterial endospores present within the fluid may be non-polar and/or contain non-polar functional groups, the endospores can attract and attach to the surface of the hydrophobic material. These endospores can bind to the surface of the hydrophobic material via hydrophobic attraction forces, such as entropic interfacial forces. The magnitude of these forces may depend on the hydrophobicity of the interacting groups (e.g., the endospore and the hydrophobic material) as well as the distance separating them. In some examples, the hydrophobic forces increase approximately exponentially with decreasing separation distance. 
     The carrier fluid surrounding the endospores may continue to pass across the hydrophobic collection material without binding to the surface of the material. For example, when the carrier fluid is an aqueous fluid, the hydrophobic material can repel the polar water molecules present in the carrier fluid, causing the carrier fluid to flow past the hydrophobic material without attaching to the surface of the material. In this manner, the hydrophobic material can capture endospores out of the fluid sample passing across the material and collect the endospores on the surface material, generating an accumulated mass of endospores for subsequent analysis. In different examples, the fluid having passed across the hydrophobic material can be disposed or recycled and again passed across the hydrophobic material. For example, the fluid may be passed across the hydrophobic material two, three, or more times to increase the number of endospores collected out of the fluid by the hydrophobic material. 
     The volume of fluid passed across the hydrophobic material can vary, e.g., depending on the size of the hydrophobic material being used and the expected concentration of endospores in the sample under analysis. In some examples, the volume of fluid passed across the hydrophobic material is equal to or greater than the volume of the hydrophobic material itself. For example, the volume of fluid containing endospores that is passed across the hydrophobic material may be at least 10 times the volume of the hydrophobic material itself, such as at least 100 times the volume of the material, at least 1000 times the volume of the material or at least 10,000 times the volume of the material. In general, increasing the volume of fluid passed across the hydrophobic material increases the number of endospores captured on the surface of the material and available for analysis. 
     A fluid under analysis may be passed across the hydrophobic collection material ( 10 ) using a variety of different techniques. In some examples, the hydrophobic collection material is dipped or immersed into a reservoir containing the fluid under analysis and then pulled out of the reservoir to provide endospores hydrophobically-bound on the surface of the material and available for analysis. In other examples, a moving stream of fluid is flowed across the surface of the hydrophobic material, allowing endospores in the stream of fluid to attach to the surface of the material while a remainder of the fluid continues flowing past and away from the hydrophobic material. In such examples, the flow of fluid under analysis may be directed generally parallel to a planar surface of the hydrophobic material and/or generally transverse (e.g., perpendicular) to the planar surface of the hydrophobic material. For example, in some configurations described in greater detail with respect to  FIG. 2 , the hydrophobic material may be a porous material with pores sized to allow substantially all of the fluid under analysis to flow through the material. When so configured, the fluid under analysis may flow generally transverse to an external face of the hydrophobic material. Endospores may collect within the pores of the material while a remainder of the fluid continues flowing through the material and discharges on an opposite side of the hydrophobic material. 
     Independent of the specific configuration of the hydrophobic material utilized to collect bacterial endospores, the example technique of  FIG. 1  includes optionally flushing the collected endospores with a flushing fluid ( 12 ). After passing the fluid to be evaluated across the hydrophobic material ( 10 ), the flow of fluid (in instance in which the fluid is flowed across the material) may be terminated to leave a hydrophobic material having collected endospores on its surface. This hydrophobic material may also contain residual fluid carrying the endospores, e.g., trapped between and around collected endospores on the surface of the collection material and/or within the pores of the material (in instances in which the material is porous). In some applications, this residual fluid may optically interfere with subsequent optical analysis of the endospores, if the fluid is not removed from the endospores before analysis. For example, the fluid carrying the endospores may emit fluorescent emissions when light within a wavelength ranging from 250 nanometers (nm) to 300 nm impinges upon the fluid. These emissions emitted by the carrier fluid may be within a wavelength ranging from 300 nm to 700 nm, which can overlap with a wavelength at which a lanthanide-dipicolinic acid complex fluoresces of, e.g., 450 nm to 650 nm. As a result, these emissions from the carrier fluid can obscure and/or distort emissions associated with the endospores, inhibiting accurate quantification of the endospores. 
     To help remove this contaminating fluid from the endospores, the endospores attached to the hydrophobic material may be flushed with a flushing fluid. The flushing fluid may be optically inert, e.g., such that residual flushing fluid remaining on the endospores does not interfere with subsequent optical analysis of the endospores. In one example, the flushing fluid is or includes liquid water (e.g., distilled water). 
     The hydrophobic material can be flushed, in different examples, by immersing the hydrophobic material containing the spores in a reservoir containing the flushing fluid and/or passing a moving stream of flushing fluid across the surface of the hydrophobic material and the endospores contained thereon. When the hydrophobic material is configured as a porous material, a pressurized stream of flushing fluid may be passed through the material (e.g., generally transversely), rinsing residual carrier fluid from around the endospores and from the pores of the material. In practice, some of the endospores captured on the surface of the hydrophobic material may be released into the flushing fluid. However, these lost endospores can be accounted for when calibrating the technique, e.g., by using a consistent amount of flushing fluid under consistent pressure conditions between a calibration run and subsequent operation. 
     When flushing is performed, the amount of flushing fluid passed over the collected endospores and/or through the hydrophobic material can vary, e.g., based on the concentration of the endospores in the sample under analysis and the optical interference impact of the carrier fluid. In some examples, the volume of flushing fluid passed across the hydrophobic material and the endospores collected thereon is at least equal to the volume of fluid under evaluation initially passed across the hydrophobic material. For example, if a volume of liquid containing endospores equal to “X” is passed across the hydrophobic collection material, the hydrophobic material may subsequently be flushed with a volume of flushing fluid greater than or equal to “X,” such as a volume of flushing fluid greater than or equal to 1.5 times “X,” greater than or equal to 2 times “X,” or between “X” and 5 times “X.” As one specific and non-limiting example, if 50 milliliters of fluid containing endospores was passed across the hydrophobic material, the material may subsequently be flushed with 100 milliliters of flushing fluid when using a flushing fluid ratio of 2 times “X.” In some examples, a volume of flushing fluid sufficient to remove substantially all (and, in other examples, all) optically interfering carrier fluid from the endospores is used. 
     After capturing the bacterial endospores from the fluid under evaluation and optionally flushing the hydrophobic material containing captured endospores to remove optically interfering carrier fluid ( 12 ), the technique of  FIG. 1  includes processing the endospores to release dipicolinic acid ( 14 ). Dipicolinic acid (DPA, 2,6-pyridinedicarboxylic acid) is typically present in high concentrations (e.g., approximately 1 molar percent or approximately 15% dry weight percent) in the core of endospores, typically as a 1:1 complex with Ca 2+ . Dipicolinic acid is also a commercially available product having the following characteristics: CAS #: 499-83-2, Synonyms: 2,6 Pyridine Dicarboxylic Acid, Molecular Formula: C7H5NO4, Molecular Weight: 167.12, Description: White crystalline powder, Sulphated Ash: 0.3% max, Moisture Content: 0.5% max, Melting Point: 242.0 to 245.0.degree Celsius. Because dipicolinic acid is an indicator uniquely associated with bacterial endospores, the concentration of dipicolinic acid in a sample can be indicative of the number of endospores in the sample. 
     To detect and quantify the dipicolinic acid present in a sample according to the example technique of  FIG. 1 , the bacterial endospores collected on the surface(s) of the hydrophobic material can be processed in situ to release the dipicolinic acid from the cores of the spores. In some examples, a germination fluid is added to the endospores collected on the surface of the hydrophobic material, e.g., causing the endospores to transform to a vegetative cell and release dipicolinic acid. Germination involves the dormant endospore starting metabolic activity and thus breaking hibernation. It commonly includes rupture or absorption of the spore coat, swelling of the endospore, an increase in metabolic activity, and loss of resistance to environmental stress. The addition of the germination fluid, alone or in combination with heating, can initiate germination of the endospores. 
     In some examples, the technique of  FIG. 1  includes adding a germination fluid to the hydrophobic material containing captured endospores to release dipicolinic acid ( 14 ). The germination fluid can be added to the endospores captured on the hydrophobic material, in different examples, by immersing the hydrophobic material containing the spores in a reservoir containing the germination fluid and/or passing a moving stream of germination fluid across the surface of the hydrophobic material and the endospores contained thereon. When the hydrophobic material is configured as a porous material, a pressurized stream of germination fluid may be passed through the material (e.g., generally transversely), exposing the endospores collected on the surface of the hydrophobic material (e.g., external surfaces and pore walls of the material) to germination fluid. Example germinating liquids that be used to release dipicolinic acid include, but are not limited to, L-alanine, L-asparagine, D-glucose, L-cysteine, dodecylamine, and the cocktail AGFK (containing D-glucose, D-fructose, KCl, and L-asparagine). 
     When used, the amount of germination fluid added to the hydrophobic material to initiate germination of the endospores captured on the material can vary. In some examples, the volume of germination fluid added to the hydrophobic material and the endospores collected thereon is less than the volume of fluid under evaluation initially passed across the hydrophobic material. For example, if a volume of liquid containing endospores equal to “X” is passed across the hydrophobic collection material, the amount of germination fluid subsequently added to the hydrophobic material and the endospores contained thereon may be less than “X,” such as a volume of flushing fluid less than 0.5 times “X,” less than 0.2 times “X,” or less than 0.05 times “X.” For example, the volume of germination fluid added to the hydrophobic material may range from 0.001 times “X” to 0.5 times “X,” such as from 0.01 time “X” to 0.2 times “X,” or from 0.025 times “X” to 0.1 times “X.” As one specific and non-limiting example, if 50 milliliters of fluid containing endospores was passed across the hydrophobic material, 5 milliliters of germination fluid may then be added to the hydrophobic material (e.g., after optionally flushing the material with any of the previously mentioned volumes of flushing fluid) when using a germination fluid ratio of 0.1 times “X.” 
     The germination fluid added to the hydrophobic material and endospores collected thereon may be captured and/or retained for subsequent optical analysis. By contrast, residual sample fluid passing over and/or through the hydrophobic material after depositing endospores and/or flushing fluid may be disposed after crossing the hydrophobic material. Accordingly, adding a smaller volume of germination fluid to the hydrophobic material can increase the concentration of endospores and/or dipicolinic acid in the germination fluid for subsequent analysis as compared to using a larger volume of germination fluid. This can be helpful when analyzing fluids having comparatively low concentrations of bacterial endospores. 
     In some examples, the germination fluid is heated prior to being added to the hydrophobic material and/or while the hydrophobic material containing endospores is immersed in the germination fluid. For example, the hydrophobic material containing captured endospores may be submerged in the germination fluid or the pore space of the hydrophobic material containing endospores may be saturated with germination fluid and then subject to heat shock. Heating the germination fluid can help initiate germination of the endospores and accelerate release of dipicolinic acid. In some examples, the germination material is heated to a temperature ranging from 75 degrees Celsius to 90 degrees Celsius, for example, for a time ranging from 15 minutes to 30 minutes. 
     In addition to or in lieu of adding a germination fluid to the bacterial endospores, the endospores may be destructively lysed by impacting the endospores with a core wall rupturing force. Example lysing methods include but are not limited to: microwaving, plasma cleaning, dry heating, autoclaving, sonicating and hydrogen chloride gassing. Lysing may destroy the core of the endospores, releasing dipicolinic acid. 
     The example technique of  FIG. 1  also includes adding a lanthanide ion source to the dipicolinic acid released from the captured endospores to form a lanthanide-dipicolinic complex (16). Dipicolinic acid is a chemistry ligand that binds metal ions with high affinity. Once bound, the dipicolinic acid complex can emit an intense green fluorescence under UV excitation. The magnitude of this fluorescence can be correlated to the concentration of dipicolinic acid which, in turn, can be correlated with the number of endospores present in the sample under analysis. 
     A lanthanide ion source can be added to the dipicolinic acid released from the endospores captured on the hydrophobic material to form a fluorescing lanthanide-dipicolinic complex. Example lanthanide ions that can be added to the dipicolinic acid include, but are not limited to: terbium (Tb 3+ ), europium (Eu 3+ ), and dysprosium. In one example, terbium (Tb 3+ ) ions are used resulting in the formation of a terbium-dipicolinic acid complex. Typically, an excess amount of lanthanide ions are added to the dipicolinic acid that is sufficient to complex all dipicolinic acid molecules present in the sample. 
     To add lanthanide ions to the dipicolinic acid released from the endospores collected on the hydrophobic material, the lanthanide ions can be introduced to the germination fluid or other fluid (e.g., when destructive lysing in performed) that contains or will contain the dipicolinic acid. In one example, the lanthanide ions are added to the germination fluid prior to introducing the germination fluid to the endospores captured on the hydrophobic material. In such an application, the lanthanide ions can complex with the dipicolinic acid as the acid is being released from the endospores during germination. In another example, the lanthanide ions are added to the germination fluid after the endospores have germinated and released their dipicolinic acid, e.g., forming a germination fluid containing released dipicolinic acid. Therefore, although  FIG. 1  illustrates the example technique as adding lanthanide ions ( 16 ) as a separate step after releasing dipicolinic acid from the captured endospores ( 14 ), it should be appreciated that the lanthanide ions may be present in the germination fluid such that the ions are added and combine with the dipicolinic acid as it is released from the endospores in a single step. 
     The technique of  FIG. 1  also includes optically analyzing a fluid containing the lanthanide-dipicolinic acid complex and determining therefrom a concentration of bacterial endospores in the fluid under evaluation ( 18 ). The fluorescence of lanthanide ions is characterized by long lifetimes (e.g., 0.1 to 1 milliseconds), small extinction coefficients, and narrow emission bands. Narrow emission bands arise because the valence f-orbitals are shielded from the environment by the outer s and p electrons, and long lifetimes/small extinction coefficients arise because the transition between the emitting excited state and ground state is prevented. Thus, direct excitation of lanthanide ions leads to weak fluorescence due to the small absorption cross section. However, coordination of organic chromophores, like dipicolinic acid, with the lanthanide ions triggers intense lanthanide fluorescence. The dipicolinic acid serves as a light-harvesting center, and strong electronic coupling with downhill energetics allows the dipicolinic acid centered excitation energy to be efficiently transferred to the lanthanide ion, which subsequently fluoresces bright green. 
     In one example, the germination fluid or other fluid containing the lanthanide-dipicolinic acid complex is optically analyzed using a fluorometer. The fluorometer emits light into the fluid under analysis and, in response to receiving the emitted light, the lanthanide-dipicolinic complex emits fluorescent emissions. The fluorescent emissions may be at a different wavelength than the wavelength(s) of light emitted by the fluorometer into the fluid containing the lanthanide-dipicolinic acid complex. The fluorometer can detect the fluorescent emissions and determine the concentration of dipicolinic acid based on the optical response. 
     In another example, the germination fluid or other fluid containing the lanthanide-dipicolinic acid complex is optically analyzed using a spectrophotometer. The spectrophotometer emits light at one or more specific wavelength(s) into the fluid containing the lanthanide-dipicolinic acid complex and detects that amount of light passing through the fluid at those one or more wavelength(s). The amount of light absorbed by the fluid sample may be proportional to the concentration of the lanthanide-dipicolinic acid complex in the sample. 
     Independent of the technique used, the optical response of the fluid containing the lanthanide-dipicolinic acid complex may be proportional to the concentration of dipicolinic acid in the fluid. In turn, this dipicolinic acid is proportional to the concentration of endospores captured on the hydrophobic material, which is further correlated to the concentration of endospores in the original fluid sample under analysis. Accordingly, the optical response of the fluid containing the lanthanide-dipicolinic acid complex can be used with calibration data to determine the concentration of endospores in the original fluid sample under analysis. 
       FIG. 2  is a conceptual illustration of an example system  20  that can be used to determine the bacterial endospore count in a fluid according to the example technique of  FIG. 1 . System  20  can be implemented as an on-line monitoring station to continuously monitor the bacterial endospore count in a fluid being processed. Alternatively, system  20  can be implemented off-line, e.g., in a laboratory environment to periodically determine bacterial endospore counts in discrete fluid samples. System  20  is illustrated as including a source of aqueous liquid to be evaluated  22 , an optional flushing fluid source  24 , a germination fluid source  26 , and a lanthanide ion source  28 . System  20  also includes a hydrophobic material  30 . Hydrophobic material  30  can receive aqueous liquid from liquid source  22 , flushing fluid from source  24 , germination fluid from source  26 , and lanthanide ions from source  28 . For example, hydrophobic material  30  and/or a housing containing the material may be fluidly connected to the different sources via fluid conduits conveying the fluids to the hydrophobic material. In other examples, hydrophobic material  30  and/or a housing containing the material may receive the fluids via manual transfer, e.g., performed by a laboratory technician. 
     In operation, hydrophobic material  30  can receive liquid from aqueous liquid source  22  and capture bacterial endospores out of the liquid. The bacterial endospores may hydrophobically bind and collect on the surfaces of the hydrophobic material  30  as the liquid flows over and/or through the material. As a result, the residual liquid having passed across hydrophobic material  30  may have a lower concentration of endospores (e.g., may be substantially devoid of endospores) than the liquid from source  22 . This residual liquid having a reduced quantity of endospores can be disposed to drain  32  after passing across hydrophobic material  30  or recycled back across the material. 
     Hydrophobic material  30  may have endospores collected and held on its surface after terminating the flow of liquid from liquid source  22 . If desired, hydrophobic material  30  can subsequently receive flushing liquid from flushing fluid source  24 . The flushing liquid can flow across the surface of hydrophobic material  30  and over the endospores captured on the surface of the material, e.g., helping to remove optically interfering liquid from the endospores in preparation for subsequent optical analysis. The flushing liquid having passed across hydrophobic material  30  and containing residual carrier liquid (e.g., optically interfering liquid) can be disposed to drain  32 . In other applications, hydrophobic material  30  is not flushed with a flushing liquid before optical analysis and, accordingly, system  20  does not include flushing fluid source  24 . 
     After terminating the flow of flushing fluid from flushing fluid source  24  (when used), hydrophobic material  30  can receive germination fluid from germination fluid source  26 . In one example, the germination fluid flows across the surface of hydrophobic material  30  and over the endospores captured on the surface of the material, e.g., collecting in a reservoir downstream of the hydrophobic material. In another example, the germination fluid is introduced into a housing containing hydrophobic material  30 , resulting in the hydrophobic material and endospores collected thereon being partially or fully immersed in the germination fluid. This can allow the endospores captured by hydrophobic material  30  to germinate in situ, while being retained on the surface of the material. As discussed above with respect to  FIG. 1 , the endospores can release dipicolinic acid during germination, increasing the concentration of dipicolinic acid in the germination fluid (e.g., from an original concentration of zero). 
     To determine the concentration of dipicolinic acid in the germination fluid added to hydrophobic material  30  and, as a result, the concentration of endospores in aqueous liquid source  22 , lanthanide ions are added to the dipicolinic-rich germination fluid from lanthanide ion source  28 . In one example, the lanthanide ions are added to the dipicolinic-rich germination fluid while hydrophobic material  30  is partially or fully immersed in the germination fluid. In another example, the germination fluid is added to the dipicolinic-rich germination fluid after removing hydrophobic material  30 , e.g., by discharging the fluid downstream of the material. In yet another example, the lanthanide ions are not added separately from germination fluid but instead are mixed with the germination fluid prior to being received by hydrophobic material  30  so as to provide a single source of both germination fluid and lanthanide ions. The lanthanide ions can combine with the dipicolinic acid released from the endospores to form an optically active lanthanide-dipicolinic acid complex. 
     System  20  in the example of  FIG. 2  also includes an optical sensor  34  and a controller  36 . Optical sensor  34  is configured to receive and optically analyze the germination fluid containing the lanthanide-dipicolinic acid complex generated from the endospores captured by hydrophobic material  30 . Optical sensor  34  may include one or more optical emitters and one or more optical detectors. In operation, the one or more optical emitters direct light into fluid containing the lanthanide-dipicolinic acid complex and the one or more optical detectors detect fluorescent emissions generated by the fluid and, in particular, the lanthanide-dipicolinic acid complex contained within the fluid. The light directed into the fluid may generate fluorescent emissions by exciting electrons of the lanthanide-dipicolinic acid complex, causing the molecules to emit energy (i.e., fluoresce) that can be detected by the optical detectors. In some examples, the one or more optical emitters direct light at one frequency (e.g., ultraviolet frequency) into the fluid and cause the lanthanide-dipicolinic acid complex to emit light energy at a different frequency (e.g., visible light frequency, a different ultraviolet frequency). 
     For example, optical sensor  34  may include one or more optical emitters that emit light within the ultraviolet (UV) spectrum, such as within wavelengths in the range from approximately 250 nm to approximately 300 nanometers. For example, the one or more optical emitters may emit within a wavelength from 270 nm to 280 nm. In response to this emitted light, the lanthanide-dipicolinic acid complexes within the fluid may generate fluorescent emissions at a wavelength from 450 nm to 650 nm. The one or more optical detectors of optical sensor  34  can detect the energy emitted by the fluorescing lanthanide-dipicolinic acid complexes at these wavelengths. 
     Controller  36  can control the one or more optical emitters to direct radiation into the fluid containing the lanthanide-dipicolinic acid complexes and also control the one or more detectors to detect fluorescent emissions emitted by the fluid. In some examples, controller  36  (or another controller within system  20 ) processes the light detection information to determine a concentration or count of endospores in the original aqueous liquid under analysis. For example, controller  36  can determine a concentration or count of the endospores in the aqueous liquid under analysis by comparing the magnitude of fluorescent emissions detected by the one or more optical detectors from a germination fluid prepared from a source liquid having an unknown concentration or count of endospores to the magnitude of the fluorescent emissions detected by the optical detectors from a germination fluid prepared having a known concentration or count of endospores. The unknown fluid may be prepared using the same or substantially same process as was used to prepare the calibration fluid. For example, the same volume of unknown source fluid may be passed across hydrophobic material  30  as was used to generate the calibration fluid, followed by use of the same volumes of flushing fluid, germination fluid, and lanthanide ion source. Calibration information can be stored in a non-transitory computer readable storage medium associated with controller  36 . 
     In some examples, controller  36  manages the overall operation of system  20 . Controller  36  may be communicatively coupled to various components within system  20 , for example via a wired or wireless connection, so as to send and receive electronic control signals and information between controller  36  and the communicatively coupled components. For example, controller  36  may electronically actuate valves and/or control pumps within system  20  to control movements of the different fluids to and from hydrophobic material  30 . Controller  36  can include a processor and memory. The memory may store software and data used or generated by controller. The memory may comprise a computer-readable medium, such as random access memory (RAM), read-only memory (ROM), non-volatile random access memory (NVRAM), electrically erasable programmable read-only memory (EEPROM), embedded dynamic random access memory (eDRAM), static random access memory (SRAM), flash memory, magnetic or optical data storage media. The memory may or may not be removable. The processor can run software stored in the memory to perform functions attributed to optical sensor  34  and controller  36  in this disclosure. The processor can include one or more processors, such as one or more microprocessors, digital signal processors (DSPs), application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), programmable logic circuitry, or the like, either alone or in any suitable combination. 
     As discussed above with respect to  FIG. 1 , hydrophobic material  30  can hydrophobically bind with endospores present in the fluid under analysis to concentrate and/or isolate the endospores from the remainder of the fluid. Hydrophobic material  30  can have a variety of different configurations and can function as a support surface on which endospores are captured and germinated. In some examples, hydrophobic material  30  is a non-porous (e.g., substantially non-porous) material. When so configured, fluid containing endospores can pass across the external surface(s) of the non-porous material and be collected on the enteral surface. In other examples, hydrophobic material  30  is a porous material having void spaces through which liquid can travel across the cross-section of the material. In these configurations, fluid containing endospores can pass across the external surface(s) of the hydrophobic material and/or internal surfaces of the material that bound and define the void spaces providing the material&#39;s porosity. 
     A porous hydrophobic material may be useful to increase the amount of surface area available for capturing endospores out of the fluid under analysis via hydrophobic attraction. In general, increasing the surface area of hydrophobic material  30  over which the fluid containing endospores passes increases the likelihood that endospores in the fluid will hydrophobically bond to the material and separate from the remaining fluid. The remainder of the fluid carrying the endospores, which may be composed of water and other polar molecules, can continue flowing through the pores or void spaces of the material and discharge on an opposite side of the material. In this way, the endospores can adhere to the wall surfaces bounding the various pores of hydrophobic material  30  while the remaining carrier fluid flows through the material, isolating and concentrating the endospores. 
     When hydrophobic material  30  is implemented using a porous material, the pores of the material may be sized large enough to allow substantially all (and, in other examples, all particles) in the fluid under analysis to flow through the pores of the material. For example, the pores of hydrophobic material  30  may be larger than the endospores being captured out of the fluid under analysis via hydrophobic attraction. In other words, instead of functioning as a filter that separates endospores via size exclusion, the pores of hydrophobic material  30  may be large enough to allow the endospores to pass through the hydrophobic material. The hydrophobic attraction forces between hydrophobic material  30  and the endospores may bind the endospores to the material and prevent the endospores from flowing out through the pores of the material, even though the endospores are smaller than the pores of the material. When so configured, hydrophobic material  30  may provide torturous (e.g., non-linear) fluid flow paths extending through the material and have a comparatively high surface area across which the fluid flows during endospore capture. 
     In some examples, hydrophobic material  30  has an average pore size greater than the average size of the bacterial endospores in the fluid under analysis, such as an average pore size at least 5 times larger than the average size of the bacterial endospores, at least 10 times larger, or at least 100 times larger, or at least 1000 times larger. In some additional examples, the distribution of pore sizes is such that at least 75% of the pores are at least 5 times larger (and in some examples at least 10 times larger) than the average size of the bacterial endospores in the fluid under analysis, such as at least 90% of the pores, at least 95% of the pores, or at least 99% of the pores. 
     The size of the pores of hydrophobic material  30  can also vary depending on the size of particles other than the endospores within the fluid under analysis, which will depend on the composition of the fluid under analysis. In the case of milk, for example, milk may contain protein micelles having a size of approximately 0.1 micrometers, fat globules ranging from 0.2 micrometers to 15 micrometers, endospores ranging from 0.6 micrometers to 1 micrometer, bacteria ranging from 1 micrometer to 5 micrometers, and somatic cells ranging from 10 micrometers to 15 micrometers, among other particles. Hydrophobic material  30  may have pores sized larger than substantially all particles in the fluid in an attempt to allow the non-endospore particles to flow through the material without blocking the pores. In some examples, hydrophobic material  30  has an average pore size greater than the average size of the particles in the fluid under analysis, such as an average pore size at least 5 times larger than the average size of the smallest particles (e.g., class of particles) in the fluid, at least 10 times larger, or at least 100 times larger, or at least 1000 times larger. In some additional examples, the distribution of pore sizes is such that at least 75% of the pores are at least 5 times larger (and in some examples at least 10 times larger) than the average size of the smallest particles in the fluid under analysis, such as at least 90% of the pores, at least 95% of the pores, or at least 99% of the pores. In one example, at least 95% of the pores range from 10 times larger to 1000 times larger than the smallest sized class of particles in the fluid under analysis. 
     While the absolute size of the pores of hydrophobic material  30  can vary based on the desired application, in some examples, the hydrophobic material has an average pore size greater than 25 micrometers, such as greater than 50 micrometers, greater than 100 micrometers, or greater than 125 micrometers. In some examples, the hydrophobic material has a porosity ranging from 20 percent of the total volume of the hydrophobic material to 70 percent of the total volume of the material. 
     Hydrophobic material  30  can capture and collect endospores out of a fluid passing over the material via hydrophobic attraction. Accordingly, hydrophobic material  30  can exhibit a sufficient hydrophobicity to selectively bind with endospores in the fluid under analysis while allowing the surrounding fluid carrying the endospores to pass across the hydrophobic material without binding. In some examples, hydrophobic material  30  is sufficiently hydrophobic such that substantially all endospores present in the fluid under analysis bind to the hydrophobic material while passing across the material. In different configurations, hydrophobic material  30  may capture greater than 30% of the endospores present in the fluid under analysis on the surface of the material, such as greater than 50% of the endospores, greater than 70% of the endospores, greater than 90% of the endospores, or greater than 95% of the endospores. The resulting fluid having passed across hydrophobic material  30  may contain less than 50% of the endospores present in the original fluid initially contacting the material, such as less than 40% of the endospores, less than 30% of the endospores, or less than 10% of the endospores. The percentage of endospores captured by the hydrophobic material may be modified by adjusting the hydrophobicity of the material and/or the amount of hydrophobic material the fluid encounters. 
     Because hydrophobic materials repel water, the hydrophobicity of a material can be characterized by the contact angle between a water droplet placed on the material and the surface of the material. For example, the water contact angle of hydrophobic material  30  can be determined by placing a droplet of water on the surface of the material and taking measurements of the drop and/or measuring the wetting angle that forms at the liquid-surface interface. One standard method used to measure the water contact angle is by measuring the maximum height and width of a sessile drop. Based on a ratio of the drop height to the drop width, the contact angle between the drop and the surface is calculated according to known equations. In some examples, hydrophobic material  30  exhibits a contact angle with water greater than 80 degrees, such as a contact angle greater than 90 degrees. 
     Hydrophobic material  30  can be constructed from a variety of different materials. Example materials that can be used to fabricate hydrophobic material  30  include, but are not limited to, polyethylene, polypropylene, polyvinylchloride, polyamide, polystyrene, polytetrafluoroethylene (e.g., Teflon®), ethylene propylene diene (EPDM), nitrile rubber (e.g., Buna-N), polyethylene terephthalate, aliphatic polyamides (e.g., Nylon), aramid fabric (e.g., Kevlar®), and stainless steel. For example, hydrophobic material  30  may be formed from a sintered metal powder or porous plastic. Hydrophobic material  30  may or may not be surface treated to increase the hydrophobicity of the material. 
     Hydrophobic material  30  can have any desired size and shape. Typically, the size of hydrophobic material  30  will vary based on the amount of fluid intended to be analyzed, with larger volumes of fluid using larger volumes of hydrophobic material. Hydrophobic material  30  can be implemented as a planar sheet of material (e.g., a disc), a sphere of material, a cylinder of material (e.g., an annulus), or any other desired shape. In some examples, hydrophobic material  30  is implemented as a single structure (e.g., sheet) that fluid containing endospores passes over and/or through. In other examples, a plurality of hydrophobic structures may be used in close proximity, e.g., to provide a packed bed, column, or cartridge of hydrophobic material that fluid containing endospores flows through. 
     In general, the foregoing examples describe the use of a hydrophobic material to concentrate and/or isolate bacterial endospores from a fluid sample followed by germination and optical analysis to determine the concentration or count of endospores in the fluid sample. Hydrophobic materials described as being suitable for capturing and collecting endospores out of a fluid may be used in other applications beyond quantifying the endospores via optical analysis. For example, the hydrophobic materials can be used in a manufacturing facility to purify a fluid containing endospores by passing the fluid across the hydrophobic material. The hydrophobic material can capture the endospores in the fluid and purify the fluid for downstream processing or sale. For example, the hydrophobic material may remove greater than 80% of the endospores present in the fluid, such as greater than 90% of the endospores, or greater than 99% of the endospores. In such applications, the hydrophobic material may be periodically flushed to remove captured endospores and to reopen surface area for endospore bonding. In still other examples, the hydrophobic material may be used in a laboratory setting to concentrate and/or isolate bacterial endospores from a fluid sample without subsequently germinating and/or optically analyzing the captured endospores. Rather, the concentrated and/or isolated endospores can be used to perform other types of laboratory analyses. 
     Further, although the foregoing examples describe the use of a hydrophobic material to capture and collect endospores out of a fluid passing over the material via hydrophobic attraction, in other examples, endospores can be isolated from a fluid sample without relying on hydrophobic attraction forces. For example, a mechanical filter may be used to separate endospores from a surrounding fluid based on size exclusion. In these examples, the mechanical filter may or may not be fabricated from a hydrophobic material. 
     The following examples may provide additional details about systems and techniques in accordance with this disclosure. 
     EXAMPLE 1 
     A variety of milk solutions containing different bacterial endospores counts were passed across a hydrophobic material having a porosity of approximately 50 percent (e.g., ranging from 25% void space to 75% void space). Fifty milliliters of milk were passed across the hydrophobic material followed by two successive flushes of 50 milliliters each of water. Subsequently, 2 milliliters of L-alanine (10 mM) type germination fluid were added to the hydrophobic material and heated at a temperature of 75 degrees Celsius for 15 minutes. A terbium reagent was then added to the germinate fluid to form a terbium-dipicolinic acid complex. The germinate fluid containing the terbium-dipicolinic acid complex was fluorometrically analyzed by emitting light into the fluid samples and generating and detecting fluorescent emissions from the fluids. 
       FIG. 3  is a plot showing the optical response of the example milk samples. The X-axis of the plot is the endospore level in the milk samples in counts. The Y-axis is the optical response of the germinant fluid containing terbium-dipicolinic acid complex that was generated from the milk samples, in relative fluorescence units. 
     EXAMPLE 2 
     To evaluate the effect of hydrophobic material pore size on endospore capture, hydrophobic materials were fabricated having different average pore sizes, including one having an average pore size of 65 micrometers and one having an average pore size of 125 micrometers. Subsequently, 50 milliliters of milk having an endospore count of 1×10 2  was passed through each hydrophobic collection material. The endospores were believed to have ranged in size from approximately 0.7 micrometers to approximately 1 micrometer. The residual milk discharged from the hydrophobic collection material was recycled back through the material such that the milk passed through hydrophobic material three times.  FIG. 4  is a plot showing the number of endospores captured following the process versus the surface area of the hydrophobic material. The larger surface areas correspond to the hydrophobic materials having the small average pore sizes. The testing showed the hydrophobic material captured from about 50% of the spores to about 70% of the spores following the third pass of the milk. 
       FIG. 5  is a micrograph of one of the hydrophobic materials at a resolution of 1 millimeter after passing the milk through the material three times. The figure shows the overall structure of the material and the relative size of an example pore structure.  FIG. 6  is a 100 time magnification of the micrograph of  FIG. 5  showing an endospore  50  adhered to the surface of material. The images indicate the endospores were captured via hydrophobic attraction and not mechanical size exclusion (e.g., filtering). 
     In the example, the hydrophobic materials were subsequently flushed with 50 milliliters of water. Testing showed that greater than from approximately 99% to approximately 99.9% of the endospores remained adhered to the surface of the hydrophobic material after flushing.