METHOD OF DETECTION OF A BIOLOGICAL ACTIVITY

专利摘要:
method of detecting a biological activity the present invention features a method of detecting a predetermined biological activity. the method includes using an aqueous mixture comprising a first indicator reagent with a first absorption spectrum and a second indicator reagent. the second indicator reagent is converted by the predetermined biological activity into a second biological derivative with a second emission spectrum. the first absorbance spectrum includes absorbance detectable in at least a portion of wavelengths present in the second emission spectrum. the first indicator reagent is received and concentrated from an aqueous liquid by a substrate, facilitating the detection of the second biological derivative.
公开号:BR112013010096B1
申请号:R112013010096-6
申请日:2011-10-28
公开日:2020-03-10
发明作者:Sailaja Chandrapati；Heather M. Webb；Jeffrey C. Pederson；Kurt J. Halverson；Brian J. Engel
申请人:3M Innovative Properties Company；
IPC主号:

专利说明:

“METHOD OF DETECTION OF A BIOLOGICAL ACTIVITY” Reference to related filing requests This application claims the benefit of provisional patent applications US No. 61 / 408,966 and US 61 / 408,977, both filed on November 1, 2010, the descriptions of which are hereby incorporated by reference in their entirety.
Background Methods for detecting a cell (for example, a pathogenic microorganism or cancer cell) in a sample often involve the detection of a biological activity (for example, an enzymatic activity or a biochemical pathway) known to be associated with the particular cell. Often, biological activity is detected using an indicator system that is changed through biological activity to a biological derivative.
Some methods employ two indicator systems to detect a specific cell type. For example, methods for detecting E. coli may include a first indicator system that includes lactose in combination with a pH indicator. The fermentation of lactose to organic acids indicates the presence of a member of coliform bacteria (which includes E. coli and other enteric microorganisms). The methods also include a second indicator system, such as 4-methylumbelliferyl-8-D-glucuronic acid, which is used to detect the enzyme β-glucuronidase, an enzyme found in most E. coli. Thus, in a method that employs both indicator systems, the accumulation of acidic end products from lactose, together with the accumulation of a fluorescent compound (4-methyl umbeliferone) may indicate the presence of E. coli in a sample. The detection of a particular biological activity in a sample can be indicative of viable cells in the sample. Bacterial spores, for example, include biological activities (for example, enzymatic activities such as α-glycopyranosidase or β-glycopyranosidase) that can be used in methods (for example, including rapid methods) to detect the presence of viable spores in a sample. The destruction of one of these other biological activities can be used to verify and / or validate the effectiveness of a sterilization process.
Summary of the Invention The present disclosure relates generally to methods for detecting biological activity in a sample. The methods of the invention provide a means to detect biological activity with at least two indicator reagents (for example, "first" and "second"). The methods provide sensitive and rapid detection of a biological derivative of the second indicator reagent in a reaction mixture that initially includes a high enough concentration of a first indicator reagent to interfere with the detection of the biological derivative. The present In one aspect, the present description presents a method of detecting a biological activity. The method may comprise providing a sample which may comprise a source of one or more predetermined biological activities, a first indicator system comprising a first indicator reagent with a first absorbance spectrum, a second indicator system comprising a second indicator reagent which is converted by a second predetermined biological activity for a second biological derivative with a second emission spectrum, and a substrate that receives and concentrates the first indicator reagent from an aqueous mixture. The first indicator reagent can be converted by a first predetermined biological activity to a first biological derivative. The first absorbance spectrum may include absorbance detectable in at least a portion of the wavelengths present in the second emission spectrum. The method may further comprise forming a first aqueous mixture comprising the sample, the first indicator reagent and the second indicator reagent. The method may further comprise placing the first aqueous mixture in fluid communication with the substrate to form a second aqueous mixture in which the concentration of the first indicator reagent is less than the concentration of the first indicator reagent in the first aqueous mixture. The method may further comprise detecting a presence or absence of fluorescence from the second biological derivative.
In some embodiments, detecting the presence or absence of fluorescence in the second biological derivative may comprise detecting the presence or absence of fluorescence in the second aqueous mixture. In some embodiments, the method may further comprise observing the substrate to detect the first indicator reagent or the first biological derivative. In any of the above embodiments, a concentration of first indicator reagent in the first aqueous mixture may be sufficient to prevent the detection of an otherwise detectable amount of the second biological derivative. In any of the above embodiments, the method may further comprise providing a nutrient to facilitate the growth of a biological cell, wherein the formation of the first aqueous mixture comprises the formation of a mixture that includes the nutrient. In any of the above embodiments, the method may further comprise exposing biological activity to a sterilizer. The sterilizer can be selected from the group consisting of water vapor, ethylene oxide, hydrogen peroxide, formaldehyde and ozone.
In any of the above embodiments, the first indicator reagent may comprise a chromophore, wherein the detection of a biological derivative of the first reagent comprises detecting a color. In any of the above embodiments, the first indicator reagent may comprise a chromogenic indicator. In any of the above embodiments, the first indicator reagent may comprise a pH indicator or an enzyme substrate. In some embodiments, the first indicator reagent may comprise purple bromocresol.
In any of the above embodiments, the second indicator reagent may comprise a fluorogenic compound. The fluorogenic compound can comprise a fluorogenic enzyme substrate.
In any of the above embodiments, detecting the presence or absence of the second biological derivative may further comprise measuring an amount of the second biological derivative. In any of the above embodiments, detecting the presence or absence of the first biological derivative may further comprise measuring an amount of the first biological derivative.
In any of the above embodiments, the method may further comprise providing an instrument that detects the first indicator reagent or the biological derivative of the second indicator reagent and using the instrument to detect the first indicator reagent or the biological derivative of the second indicator reagent.
In some embodiments, the method may further comprise providing an instrument that detects the first indicator reagent or the second biological derivative and using the instrument to detect the first indicator reagent or the second biological derivative. In some embodiments, the method may further comprise providing an instrument that detects the first indicator reagent and the second biological derivative and using the instrument to detect the first indicator reagent and the second biological derivative.
In another aspect, the present description presents a method of detecting a biological activity. The method may comprise providing a compartment, a container, a source of a second predetermined biological activity, and a substrate. The compartment can comprise first and second chambers. The container can contain a first aqueous liquid. The container can be arranged in the first chamber. At least a portion of the container may be frangible. The first aqueous liquid can comprise a first indicator system comprising a first indicator reagent with a first absorbance spectrum and a second indicator system comprising a second indicator reagent that is converted by a predetermined biological activity to a second biological derivative with a second spectrum emission, in which the first absorbance spectrum includes absorbance detectable in at least a portion of wavelengths present in the second emission spectrum. The first indicator reagent can be converted by a first predetermined biological activity to a first biological derivative. The source of the predetermined biological activity can be arranged in the second chamber. The substrate can be arranged in the compartment and can receive and concentrate the first reagent indicating the first aqueous liquid. The method may further comprise placing the first aqueous mixture in fluid communication with the substrate to form a second aqueous mixture in which the concentration of the first indicator reagent is less than the concentration of the first indicator reagent in the first aqueous mixture. The method may further comprise detecting a presence or absence of fluorescence from the second biological derivative. In some embodiments, detecting the presence or absence of fluorescence in the second biological derivative may comprise detecting the presence or absence of fluorescence in the second aqueous mixture. In some embodiments, placing the first aqueous mixture in fluid communication with the substrate to form a second aqueous liquid may comprise fracturing at least a portion of the frangible container. In some embodiments, the biological sterilization indicator may additionally comprise a fracturing device arranged in the compartment, where fracturing of the frangible container comprises inducing the container and fracturing against each other. In some embodiments, the sterilization biological indicator compartment may include a first portion and a second portion. The second portion can be adapted to be coupled to the first portion, the second portion is movable with respect to the first portion, when coupled to the first portion, between a first position and a second position. The method may further comprise moving the second portion of the compartment from the first position to the second position.
In another aspect, the present description presents a system for detecting a predetermined biological activity. The system may comprise a first indicator system comprising a first indicator reagent with a first absorbance spectrum, a second indicator system comprising a second indicator reagent that is converted by a predetermined biological activity to a second biological derivative with a second emission spectrum , a vessel configured to contain a liquid medium, a substrate that receives and concentrates the first indicator reagent from an aqueous mixture, and an instrument configured to receive the vessel and to detect the first indicator reagent or a biological derivative of the second indicator reagent. The first indicator reagent can be converted by a first predetermined biological activity to a first biological derivative. The first absorbance spectrum includes absorbance detectable in at least a portion of the wavelengths present in the second emission spectrum. In some embodiments, the instrument can be configured to detect the first biological derivative. In some embodiments, the system may additionally comprise a processor. In any of the above modes of the system, the instrument can be additionally configured to regulate a temperature of a liquid medium. In any of the above modalities of the system, the instrument can be configured to detect both the first indicator reagent and the second biological derivative.
The words "preferential" and "preferably" refer to the modalities of the invention that can provide certain benefits, under certain circumstances. However, other modalities may also be preferred under the same or other circumstances. In addition, the recitation of one or more preferred modalities does not imply the disuse of other modalities and is not intended to exclude other modalities from the scope of the invention.
The terms "understands" and variations of it do not have a limiting meaning, these terms appearing in the description and in the claims.
For use in the present invention, "one", "one", "o", "a", "at least one", "at least one", "one or more" and "one or more" are used interchangeably . In this way, for example, a substrate can be interpreted as "one or more" substrates. The term "and / or" means one or all of the elements mentioned or a combination of any two or more of the elements listed. "Biological activity", as used here, refers to any specific catalytic process or groups of processes associated with a biological cell. Some non-limiting examples of biological activities include catabolic enzymatic activities (for example, carbohydrate fermentation routes), anabolic enzymatic activities (for example, synthetic pathways for nucleic acids, amino acids or proteins), coupled reactions (for example, a metabolic pathway) biomolecule-mediated redox reactions (eg electron transport systems) and bio-invisible reactions. “Predetermined” biological activity means that the method is directed towards the detection of a specific biological process (eg, an enzyme reaction) or group of biological processes (eg, a biochemical pathway). It will be noted by an individual of ordinary skill in the art that certain predetermined biological activities may be associated with a particular type of cell (for example, a cancer cell or a microorganism) or a pathological process. “Biological derivative”, as used here, refers to a product of a biological activity. This includes, for example, enzyme reaction products and biological electron transport systems. "Biomolecules", as used here, can be any chemical compound that occurs naturally in living organisms, as well as derivatives or fragments of such naturally occurring compounds. Biomolecules consist mainly of carbon and hydrogen, along with nitrogen, oxygen, phosphorus and sulfur. Other elements are sometimes incorporated, but are much less common. Biomolecules include, but are not limited to, proteins, polypeptides, carbohydrates, polysaccharides, lipids, fatty acids, steroids, prostaglandins, prostacyclins, vitamins, cofactors, cytokines and nucleic acids (including DNA, RNA, nucleosides, nucleotides, purines and pyrimidines ), metabolic products that are produced by living organisms including, for example, antibiotics and toxins. Biomolecules can also include derivatives of naturally occurring biomolecules, such as a protein or antibody that has been modified with chemicals (for example, oxidized with sodium periodate). Biomolecules can also include cross-linked naturally occurring biomolecules, or a cross-linked product of a naturally occurring biomolecule with a chemical. Thus, "biomolecule" includes, but is not limited to, both unmodified and modified molecules (eg, glycosylated proteins, oxidized antibodies) and fragments thereof (eg, protein fragments). Fragments of biomolecules may include those resulting from hydrolysis due to chemical, enzymatic or irradiation treatments, for example.
For use in the present invention, recitations of numeric ranges with extremes include all numbers contained in this range (for example, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). The foregoing summary of the present invention is not intended to describe each of the embodiments presented or all implementations of the present invention. The following description more particularly illustrates the illustrative modalities. In several places, during application, guidance is provided through lists of examples, in which examples can be used in various ways. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
Brief description of the figures Figure 1A is a top perspective view of a substrate and a vessel containing a liquid medium comprising an indicator reagent. Figure 1B is a top perspective view of the Figure 1A vessel immediately after immersing the Figure 1A substrate in the liquid medium. Figure 1C is a top perspective view of the Figure 2A vessel after a period of time. Figure 2 is a drawing of a UV visible absorbance spectrum of an aqueous solution of purple bromocresol and a fluorescence emission spectrum of a solution of 4-methyl umbeliferone. Figure 3 is a block diagram of a modality of a method for detecting a biological activity in accordance with the present description. Figure 4 is a front perspective view of a biological sterilization indicator according to an embodiment of the present description, 1 being that the biological sterilization indicator includes a compartment that includes a first portion and a second portion. Figure 5 is an exploded rear perspective view of the biological sterilization indicator of Figure 4. Figure 6 is an exploded front perspective view of the biological sterilization indicator of Figures 4 and 5. Figure 7 is a side cross-sectional view. of the biological sterilization indicator in Figures 4 to 6, taken along line 4-4 of Figure 4, where the biological sterilization indicator is shown in a first state, and the second portion of the biological sterilization indicator compartment shown in a first position. Figure 8 is a top cross-sectional view of the biological sterilization indicator in Figures 4 to 6, taken along line 5-5 of Figure 6. Figure 9 is a side cross-sectional view of the biological sterilization indicator in Figures 4 to 8, in which the second biological sterilization indicator is shown in a first state, and the second portion of the compartment of the second biological sterilization indicator is shown in a second position. Figure 10 is a top cross-sectional view of the biological sterilization indicator in figures 4 to 9, with portions removed for clarity.
Detailed description The present disclosure relates to a rapid method for detecting a biological activity. The method includes the use of two or more indicator reagents. The method includes providing a liquid mixture comprising a first and a second indicator reagent, where the first indicator reagent is present in the mixture at a concentration sufficient to interfere with the detection (for example, optical detection) of an otherwise detectable amount. a biological derivative of the second indicator reagent. The method of the invention provides sensitive and rapid detection of a biological activity by sequestering at least a portion of the interfering quantity of the first indicator reagent from the batch of the liquid mixture in order to facilitate the detection of the biological derivative of the second indicator reagent. The method additionally provides a means to more easily observe the first indicator reagent or a biological derivative thereof. The method of the invention can be used in a system for the automated detection of a biological activity. The system and / or method of the invention of the present disclosure can be used to detect a biological activity (for example, an activity associated with an enzyme, a cell or a microorganism). In some embodiments, the system and / or method of the invention can be used, for example, to detect a biological activity associated with a particular type of microorganism (for example, a vegetative cell or spore) that has survived exposure to a process (for example, a disinfection process, a food or drink preparation process, a sterilization process). The method of the invention relates to the detection of a biological activity in a sample. The sample can be any sample that includes a biological activity as defined here. Some non-limiting examples of suitable samples include cell suspensions or cultures (eg mammalian cells, insect cells, yeast cells, filamentous fungi, bacterial cells), environmental samples (eg surface wads), food (for example, raw materials, in-process samples and finished product samples), beverages, clinical samples (for example, blood, urine, sputum, tissue, mucus, feces, wound exudate, pus) and water (for example, drinking water surface, drinking water, process water).
Microorganisms (eg bacteria, fungi, viruses) are a source of biological activity and can be analyzed in a test sample that can be derived from any source, such as a physiological fluid, eg blood, saliva, fluid ocular lens, synovial fluid, cerebral spine fluid, pus, sweat, exudate, urine, mucus, lactation milk or the like. In addition, the test sample can be derived from a location on the body, for example, wound, skin, nostrils, scalp, nails, etc.
Samples of particular interest include mucus-containing samples, such as nasal samples (for example, from anterior nostrils, nasopharyngeal cavities, nasal cavities, anterior nasal vestibule, etc.), as well as samples from the outer ear, middle ear, mouth, rectum, vagina or other similar tissue. Examples of specific mucosal tissues include buccal, gingival, nasal, ocular, tracheal, bronchial, gastrointestinal, rectal, urethral, ureteral, vaginal, cervical and uterine mucous membranes.
In addition to physiological fluids, other test samples may include other liquids as well as solids (s) dissolved in a liquid medium. The samples of interest may include process flows, water, soil, plants or other vegetation, air, surfaces (for example, contaminated surfaces) and the like. Samples can also include cultured cells. Samples may also include samples on or in a device comprising cells, spores or enzymes (for example, a biological indicator device).
Solid samples can be disintegrated (for example, by mixing, sonication, homogenization) and can be suspended in a liquid (for example, water, buffer, broth). In some embodiments, a sample collection device (for example, a pad, a sponge) that contains the sample material can be used in the method. Alternatively, the sample material can be eluted (e.g., rinsed, scraped, expressed) from the sample collection device prior to using the sample material in the method. In some embodiments, liquid or solid samples can be diluted in a liquid (for example, water, buffer, broth).
Suitable samples are also liquid and / or solid samples that have been exposed to a sterilizer. Some non-limiting examples of these samples include spore suspensions, spore strips and coupons from various materials on which a spore suspension or vegetative microbial cells have been applied.
Suitable samples also include cell suspension media (e.g., culture broth, semi-solid cell culture media, and tissue culture media, filtrate) that contain cells or previously contained cells. Suitable samples also include cell lysates. Cell lysates can be produced by chemical means (for example, detergents, enzymes), mechanical means (sonic vibration, homogenization, French Press), or by other cell lytic means known in the art.
Figures 1A to 1C illustrate the process of receiving and concentrating a liquid medium from an indicator reagent (or a biological derivative thereof) on or in a substrate according to the present description. Figure 1A shows a top perspective view of an embodiment of a substrate 30 and a vessel 10 containing a liquid mixture 20 comprising a colored indicator reagent. Figure 1B shows a top perspective view of vessel 10 of Figure 1A immediately after immersing substrate 30 in liquid mixture 20. Figure 1C shows a top perspective view of vessel 10 of Figure 1B after a period of time sufficient to allow that substrate 30 receives and concentrates the colored indicator reagent from the liquid mixture 20. It can be seen in Figure 1C that the color of the liquid mixture becomes less intense, while substrate 30 received and retained the colored indicator reagent and, thus, changed its natural colorless state to a colored state.
In some embodiments, the substrate can passively receive and concentrate the indicator reagent or biological derivative thereof (for example, by simply diffusing the reagent or derivative through the liquid medium). Alternatively or additionally (not shown), the substrate can receive and actively concentrate the indicator reagent and / or biological derivative (for example, the substrate can be moved relative to the liquid through mixing or revolution and / or the liquid medium can be moved in relation to the substrate through the fluid flow that is generally lateral, tangential or orthogonal to a main surface of the substrate).
Indicating Reagents The prior art includes several chromic and fluorogenic enzyme substrates from various sources that are known, available for sale, that have been used in methods to detect predetermined biological activities, and are suitable for use as the first or second indicator reagent according to this description. Among these are a variety of 4-methylumbelliferyl derivatives (hydrolyzable to 4-methyl umbeliferone); derivatives of 7-starch-4-methyl-coumarin, for example, as presented in GB patent No. 1,547,747 and European patent No. 0.000.063, each of which is incorporated herein by reference in its entirety; diacetylfluorescein derivatives; and fluorescamine. The first indicator reagent, according to the present description, comprises a reagent that has a first absorption spectrum and, in this way, absorbs light in the ultraviolet and / or visible wavelengths of the electromagnetic spectrum.
In some embodiments, the first indicator reagent may be an indicator dye (for example, a pH indicator dye, an oxidation dye). The specific indicator dye used to detect any particular biological activity will be selected according to criteria that are known in the art, including, for example, compatibility (eg, preferably non-inhibitory) with the biological activity to be detected, solubility, system detection (for example, visual and / or automated).
In any of the method modalities, the indicator dye can be a suitable pH indicator to detect biological activity. The indicator dye can be selected according to criteria known in the art, such as pH range, compatibility with biological activity and solubility. In some embodiments, a salt form of the pH indicator can be used, for example, to increase the solubility of the pH indicator in an aqueous mixture. Some non-limiting examples of pH dye indicators include, for example, thymol blue, tropeolin 00, methyl yellow, methyl orange, bromphenol blue, bromocresol green, methyl red, bromtimol blue, phenol red, neutral red, phenolphthalein, thymolphthalein, alizarin yellow, tropeolin O, nitramine, trinitrobenzoic acid, thymol blue, bromphenol blue, tetrabromophenol blue, bromocresol green, bromocresol purple, methyl red, bromtimol red, phenol red , Congo red and cresol red.
In either method, the dye indicator can be an oxidation-reduction indicator (also called a redox indicator) suitable for detecting biological activity. Oxidation reduction dyes can be pH dependent or pH independent. Some non-limiting examples of redox indicator dyes include 2,2'-Bipyridine (Ru complex), Nitrophenanthroline (Fe complex), N-phenylanthranilic acid, 1,10-phenanthroline (Fe complex), N-ethoxycrisoidine, 2 , 2'-bipyridine (Fe complex), 5,6-dimethylphenanthroline (Fe complex), o-dianisidine, sodium diphenylamine sulfonate, diphenylbenzidine, diphenylamine, Viologen, 2,6-Dibromophenol-indophenol sodium, 2,6 -Dichlorophenol-indophenol sodium, o-Cresol indophenol sodium, thionine (synonym: Lauth violet), methylene blue, sulfonic indigotetraacid, sulfonic indigotriacid, sulfonic indigodiac acid, sulfonic indigomonoacid, phenosafranin, Safranin T, and neutral red.
In some embodiments, the first indicator reagent may be a sulfophthalein pH indicator (for example, bromocresol purple), as shown in Example 4. The sulfophthalein pH indicator (for example, bromocresol purple) may be present in the mixture water at a concentration of about 0.03 g per liter. The sulfophthalein pH indicator can be received and concentrated by a substrate (for example, a loaded nylon substrate such as a 0.45 micron MAGNAPROBE-loaded nylon membrane, part number NP0HY00010, available from GE Osmonics Labstore, Minnetonka, MN, USA). The substrate can be configured as a generally flat strip (for example, a strip that is about 3 mm to about 10 mm). The second indicator reagent, according to the present description, can be converted to a second biological derivative. The second biological derivative comprises a reagent that has a second absorption spectrum. In addition, the second biological derivative has a characteristic second emission spectrum (for example, a fluorescent emission spectrum). In some embodiments, the second biological derivative has a characteristic second absorption spectrum that includes wavelengths in the ultraviolet portion of the electromagnetic energy spectrum. The second emission spectrum of the second biological derivative can include wavelengths in the visible portion of the electromagnetic energy spectrum.
Compounds suitable for use as a second indicator reagent include fluorogenic compounds (for example, fluorogenic enzyme substrates). Enzymatic substrates include 4-methylumbelliferyl derivatives, 7-starch-4-methylcoumarin derivatives and diacetylfluorescein derivatives.
Derivatives of 4-methylumbelliferyl include, for example: 4-methylumbeliferyl-2-acetamido-4,6-O-benzylidene-2-deoxy-p-D-glycopyranoside; 4-methylumbelliferyl acetate; 4-methylumbelliferyl-N-acetyl-p-D-galactosaminide; 4-methylumbelliferyl-N-acetyl-a-D-glycosaminide; 4-methylumbelliferyl-N-acetyl-3-D-glycosaminide acid; 2, - (4-methylumbelliferyl) -a-D-N-acetyl neuramine; 4-methylumbeliferyl α-L-arabinofuranoside; 4-methylumbelliferyl a-L-arabinoside; 4-methylumbelliferyl butyrate; 4-methylumbeliferyl β-D-cellobioside; methylumbeliferyl β-D-N, N'diacetyl chitobioside; 4-methylumbeliferyl elaidate; 4-methylumbeliferyl β-D-fucoside; 4-methylumbelliferyl a-L-fucoside; 4-methylumbeliferyl β-L-fucoside; 4-methylumbelliferyl a-D-galactoside; 4-methylumbeliferyl β-D-galactoside; 4-methylumbelliferyl a-D-glycoside; 4-methylumbeliferyl β-D-glycoside; 4-methylumbelliferyl β-D-glucuronide; 4-methylumbelliferyl p-guanidinobenzoate; 4-methylumbeliferyl heptanoate; 4-methylumbeliferyl α-D-mannopyranoside; 4-methylumbelliferyl β-D-mannopyranoside; 4-methylumbeliferyl oleate; 4-methylumbeliferyl palmitate; 4-methylumbelliferyl phosphate; 4-methylumbeliferyl propionate; 4-methylumbeliferyl stearate; 4-methylumbelliferyl sulfate; 4-methylumbelliferyl β-DN, Ν ', N "-triacetylquitotriose; 4-methylumbeliferyl 2,3,5-tri-o-benzoyl-α-arabinofuranoside; 4-methylumbeliferyl-p-trimethylammonium chloride; and 4-methylumbeliferyl β -D-xyloside.
Suitable 7-starch-4-methylcoumarin derivatives include, for example: L-alanine-7-starch-4-methylcoumarin; L-proline 7-starch-4-methylcoumarin; L-tyrosine-7-starch-4-methylcoumarin; L-leucine-7-starch-4-methylcoumarin; L-phenylalanine-7-starch-4-methylcoumarin; and 7-glutarylphenylalanine-7-starch-4-methylcoumarin.
Suitable peptide derivatives of 7-starch-4-methyl coumarin include, for example: N-t-BOC-1-Glu-Gly-Arg 7-starch-4-methylcoumarin; N-t-BOC-Leu-Ser-Thr-Arg 7-starch-4-methylcoumarin; N-CBZ-Phe-Arg 7-starch-4-methyl-coumarin; Pro-Phe-Arg 7-starch-4-methylcoumarin; N-t-BOC-Val-Pro-Arg 7-starch-4-methylcoumarin; and N-glutaryl-Gly-Arg 7-starch-4-methylcoumarin.
Suitable diacetylfluorescein derivatives include, for example, fluorescein diacetate (di-β-D-galactopyranoside) and fluorescein dilaurate.
Where the biological activity to be detected is alpha-D-glycosidase, chymotrypsin or fatty acid esterase, for example, from Geobacillus stearothermophilus, the preferred fluorogenic enzymatic substrates are 4-methylumbelliferyl-alpha-D-glycoside, 7-glutarylphenylalanine-7- 4 methyl coumarin starch, or 4-methylumbelliferyl heptanoate, respectively. Where the biological activity to be detected is alpha-L-arabinofuranosidase, for example, derived from Bacillus subtilis, a preferred fluorogenic enzyme substrate is 4-methylumbelliferyl-alpha-L-arabinofuranoside. Where the biological activity to be detected is beta-D-glycosidase, for example, derived from Bacillus subtilis, a preferred fluorogenic enzyme substrate is 4-methylumbelliferyl-beta-D-glycoside. In order to carry out the method of the present invention in the detection of a biological activity that comprises an enzyme, in which the operator must have knowledge about the enzymatic activity to be detected and about the enzymatic substrates that will react with the enzyme in order to produce a product that can be detected by its fluorescence, color, etc. (see M. Roth, Methods of Biochemical Analysis, Volume 7, D. Glock, Ed., Interscience Publishers, New York, NY, USA, 1969, which is incorporated herein in its entirety, by reference). The appropriate enzyme substrate to be used will depend on the biological activity to be detected.
The methods of the present description include a first indicator reagent with a first absorption spectrum and a second indicator reagent that is converted by a biological activity to a second biological derivative with a second emission spectrum, where the first absorption spectrum overlaps at least partially the second emission spectrum. Thus, when both the first indicator reagent and the second biological derivative are present in a liquid mixture, the first indicator reagent can absorb at least a portion of the light emitted by the second indicator reagent, thereby decreasing the ability to detect the second biological derivative.
A drawing can illustrate the relationship between a first indicator reagent and a second biological derivative according to the present description. Figure 2 shows the absorbance spectrum of bromocresol purple (from this point on in this document, called “BCP”), a first exemplifying indicator reagent and the fluorescence emission spectrum of 4-methylumbelliferone (from this point on in present document, called “4MU”), a possible biological derivative of 4-methylumbelliferyl β-D-glycoside, a second exemplifying indicator reagent. The spectra were obtained as described in Examples 1 and 2. The “A” strain, which shows the BCP absorbance spectrum, indicates a maximum absorbance in the visible range around 600 nm, with relatively less absorbance by BCP at wavelengths. from 425 to 550 nm. The data show an absorbance peak at visible wavelengths around 600 nm and an absorbance peak at ultraviolet wavelengths at <330 nm. The “B” strain, which shows the fluorescence emission spectrum of 4MU, indicates a maximum emission around 450 nm, with relatively less emission in the 375 to 425 nm and 475 to 525 nm ranges. It can be seen in Figure 2 that the BCP absorbance spectrum substantially overlaps the entire peak of fluorescence emission (centered around 450 nm) of 4MU.
A person of ordinary skill in the art will recognize that the amount of absorbance of any particular wavelength of light by a solution containing a first indicator reagent will be influenced by the concentration of the first indicator reagent in the solution and by the molar extinction coefficient of the indicator reagent at the selected wavelength. The person skilled in the art will also recognize that the amount of light emission of any particular wavelength by a solution containing a biological derivative of a second indicator reagent will be influenced by the concentration of the second biological derivative in the solution and by the quantum yield of fluorescence of the biological derivative. Therefore, the concentration of the first indicator reagent in the liquid mixture can be selected in conjunction with an appropriate substrate to allow i) the substrate to remove sufficient first substrate indicator from the liquid mixture to allow more sensitive detection of the second biological derivative and ii) the first reagent indicator (or biological derivative thereof) is easily detected in the substrate material. The combination of bromocresol purple and 4-methylumbelliferyl-a-D-glycoside represents an example of first and second suitable indicator reagents, respectively, in accordance with the present description. This combination can be used to detect a first biological activity such as fermentation of a carbohydrate for acidic end products and a second biological activity such as -a-D-glycosidase enzyme activity, for example. These activities may indicate the presence or absence of a viable spore after exposure of a biological indicator of sterilization to a sterilization process, for example. Bromocresol purple can be used at a concentration of about 0.03 g / l in the aqueous mixture, for example. 4-methylumbeliferyl-aD-glycoside can be used, for example, at a concentration of about 0.05 to about 0.5 g / l (for example, about 0.05 g / l, about 0, 06 g / l, about 0.07 g / l, about 0.08 g / l, about 0.09 g / l, about 0.1 g / l, about 0.15 g / l, about 0.2 g / l, about 0.25 g / l, about 0.3 g / l, about 0.35 g / l, about 0.4 g / l, about 0.45 g / l, about 0.5 g / l) in the aqueous mixture.
Thus, according to the present description, the first indicator reagent can interfere with the detection of an otherwise detectable amount of the biological derivative of the second indicator reagent. The spectral interference between any proposed first and second indicator reagents can be demonstrated by an individual of ordinary skill in the art by performing the following simple experiment.
First, the operator produces a relatively diluted but fluorescently detectable aqueous solution of the expected biological derivative of the proposed second indicator reagent. For example, if the second indicator reagent is a 4-methylumbelliferyl compound, the expected biological derivative is 4MU. The solution may contain, for example, about 0.05 to 0.2 micrograms per milliliter of 4MU. The operator then adds an effective amount of the first proposed indicator reagent. For example, if BCP is the first proposed indicator reagent, it can be added to a concentration (for example, 0.04 milligrams per milliliter) that is used in microbiological growth media for the detection of fermentative microorganisms. By comparing the fluorescence of the 4MU solutions with and without the BCP, it can be determined whether the first indicator reagent (in this example, the BCP) can interfere with the detection of the biological derivative of the second indicator reagent (in this case, the 4MU). The operator can then test whether the addition of reduced amounts of BCP to the 4MU solution improves the detection of relatively low concentrations of 4MU. This type of experiment can be easily performed with any combination of first and second indicator reagents. An example of this procedure is shown in example 3.
Substrate Suitable substrates according to the present description are configured to receive and concentrate the indicator reagent. The ability of the substrate to concentrate the indicator reagent or biological derivative thereof can be affected by one or more of a variety of forces known in the art and discussed in the present invention. Thus, an individual of ordinary skill in the art can select a substrate that is known to be positively charged to concentrate an indicator reagent (or biological derivative thereof) that is known to be negatively charged, for example. Adversely, an individual of ordinary skill in the art may select a substrate that is known to be negatively charged to concentrate an indicator reagent (or biological derivative thereof) that is known to be positively charged, for example. A person of ordinary skill in the art can select a substrate that is known to have hydrophobic properties to concentrate an indicator reagent (or biological derivative thereof) that is known to comprise hydrophobic portions that would be retained by a hydrophobic substrate. In addition, a person of ordinary skill in the art can easily select a suitable substrate material by contacting a candidate substrate material with a liquid over a period of time that comprises the indicator reagent or biological derivative thereof and analyzing the substrate to determine whether a detectable amount of the indicator reagent or derivative thereof accumulates on or in the substrate.
It will be apparent to a person of ordinary skill in the art that the substrate material can be selected according to known properties of the indicator reagent or the biological derivative thereof. For example, a positively charged substrate can be selected for use in the method when the biological derivative of the indicator reagent is a negatively charged molecule. In addition, a negatively charged substrate can be selected for use in the method when the biological derivative of the indicator reagent is a positively charged molecule.
Alternatively, the suitability of any substrate material determined for use with a given first indicator reagent in the method of the invention can be readily determined using the following experimental approach. In a suitable vessel (for example, a test tube), a source of predetermined biological activity (for example, microbial cells capable of fermenting a carbohydrate for acidic end products) can be added with a first indicator reagent (for example, an indicator pH) to a liquid medium selected to facilitate biological activity (for example, a broth medium comprising fermentable carbohydrate). The liquid medium can be brought into contact with a candidate substrate under conditions to facilitate predetermined biological activity and the substrate can be removed from the medium, optionally rinsed and / or stained to remove excess liquid, and observed visually or instrumentally (for example , with a spectrophotometer or a fluorometer) to determine if the substrate material concentrated the first indicator reagent and / or a biological derivative of it during contact with the liquid medium. In the illustrative example, a suitable substrate / indicator combination would show evidence that the first indicator reagent or biological derivative of the same concentrated on or in the substrate material (outside the liquid medium) during the contact period. A control reaction without the substrate material can be operated to confirm the presence of biological activity in the mixture. The substrate can be manufactured in a generally flat blade form (for example, a membrane strip, as shown in Figure 1A). The effective size and / or surface area of the substrate can also affect the ability of the substrate to concentrate the indicator reagent (or biological derivative of it). Preferred materials for the substrate include porous materials (for example, woven materials, non-woven materials, porous membranes, microporous membranes, filter paper). In some embodiments, particularly preferred substrate materials include loaded membranes such as loaded nylon membranes (e.g. 0.45 micron loaded nylon membrane MAGNAPROBE, part number NP0HY00010, available from GE Osmonics Labstore, Minnetonka , MN, USA). The substrates used in the present disclosure can be manufactured from a variety of materials. US patent No. 6,562,297, which is incorporated herein in its entirety by reference, describes membranes for immobilizing pH indicators. Some non-limiting examples of suitable substrate materials include, for example, natural materials (for example, cellulose), synthetic materials (for example, nylon) and combinations and / or derivatives thereof. Method of Detecting a Biological Activity: Figure 3 shows a block diagram of a modality of a method for detecting one among a plurality of biological activities according to the present description. The method includes step 40 of providing a sample that can include one of a plurality of predetermined biological activities, first and second indicator reagents, and a substrate that receives and concentrates from the aqueous medium the first indicator reagent and, optionally, a biological derivative of it.
In some embodiments, the method may include the optional step 45 of exposing biological activity to a disinfectant, antibiotic or sterilizer. This optional step can be included to determine the effectiveness of a sterilization process or to detect a predetermined biological activity (or micro-organism) subsequent to a selective enrichment culture process. Exposing biological activity to a sterilizer may include exposing biological activity to a sterilization process. Sterilization processes include exposing the sample, for example, to sterilants such as water vapor, dry heat, ethylene oxide, formaldehyde, peroxides, hydrogen peroxide, peracetic acid, ozone or mixtures thereof (for example, a mixture of ozone and hydrogen peroxide). The method includes step 50 of forming a first aqueous mixture comprising the sample and the first and second indicator reagents. The first aqueous mixture is formed in an aqueous medium. The source of biological activity in the method can be any sample that comprises, or is suspected of understanding, one or more biological activities, as described here. "Aqueous medium", as used here, refers to an aqueous liquid in which the first and second indicator reagents are or can be dissolved or suspended. Preferably, the medium does not substantially interfere with the detection of a predetermined biological activity to be detected. In some embodiments, the aqueous medium may comprise a component (i.e., a buffering agent) for adjusting the pH of the medium. The aqueous medium can additionally comprise a reagent (for example, a detergent, a cofactor, a cell lysis agent) which is known in the art for facilitating the detection of one or more biological activities.
In some embodiments, the sample comprises water and, thus, the sample itself can be considered an aqueous medium. In either embodiment, the sample can optionally be mixed with a second liquid (for example, an aqueous medium, a diluent, a buffer, a solution to neutralize a disinfectant) before mixing the sample with the first and second indicator reagents.
In some embodiments, the aqueous medium can be combined with the first and / or second indicator reagents before the medium is mixed with the sample. In some embodiments, the first and second indicator reagents and the sample can be added sequentially to the aqueous medium to form the first aqueous mixture.
In some embodiments, the first and second indicator reagents can be combined with an aqueous medium and the sample simultaneously to form the first aqueous mixture. In either embodiment, one or both of the first and second indicator reagents may initially be in the form of a dry reagent, a liquid, a gel or a film before the reagent is combined with an aqueous medium and / or a sample for form the first aqueous mixture. The first indicator reagent can be any suitable reagent described herein. Due to the fact that the first indicator reagent is selected to detect a predetermined biological activity, the chemical nature of the first indicator reagent and its biological derivatives is known and, therefore, suitable substrate materials can be identified as described here. The second indicator reagent can be any suitable reagent described herein.
In any embodiment of the method, the formation of the first aqueous mixture may comprise forming a first aqueous mixture that includes a nutrient. The nutrient can be provided to facilitate the growth of a microorganism or target cell, for example, and can be supplied as a mixture of nutrients. Nutrients and nutrients to facilitate the growth of microorganisms are known in the art and can be found, for example, in Ronald Atlas's “Handbook of Microbiological Media”, published by CRC Press, Boca Raton, FL, USA. Matner et al. (US Patent No. 5,073,488) describes a nutrient medium for the growth and detection of bacterial spores in a biological sterilization indicator. Nutrients and nutrients to facilitate the growth of eukaryotic cells (e.g., mammalian cells, insect cells) are also known in the art and include, for example, sugars (e.g., glucose), amino acids, vitamins (e.g. , thiamine, niacin), choline, inositol, serum and mixtures thereof.
The methods of the present description further include step 60 of placing the first aqueous mixture in fluid communication with the substrate to form a second aqueous mixture. Typically, the process of placing the first aqueous mixture in fluid communication with the substrate takes place in a vessel (for example, a tube, a bottle, a flask, a well). In either mode, the vessel can be sealed to minimize evaporation and / or to prevent contamination by exogenous biological activity, for example. In either embodiment, placing the first aqueous mixture in fluid communication with the substrate may include contacting the liquid mixture and the substrate under conditions that facilitate predetermined biological activity. An individual of ordinary skill in the art will recognize the conditions that facilitate predetermined biological activity. Conditions may include, for example, pH, ionic intensity or buffering capacity of the mixture; the concentration of first and / or second indicator reagents; presence of cofactors in the mixture or in the vessel; and / or temperature of the mixture.
In either method, placing the first aqueous mixture in fluid communication with the substrate may include controlling the temperature of the mixture. In some embodiments, the temperature can be controlled at a temperature higher than the ambient temperature (for example, a temperature that facilitates a reaction, such as a catalytic reaction or a binding reaction, which involves biological activity) with the use of a block of heating, an incubator or some other suitable heating medium known in the art. In some embodiments, the temperature of the mixture can be controlled at a temperature below room temperature. In some embodiments, the mixture may be subjected to a temporary temperature shift (for example, a heat shock or a cold shock) to facilitate the detection of predetermined biological activity. Placing the first aqueous mixture in fluid communication with the substrate according to the present description comprises concentrating the first indicator reagent and; optionally, a biological derivative thereof; on and / or on the substrate. As a result, the concentration of the first indicator reagent in the second aqueous mixture is less than the concentration of the first indicator reagent in the first aqueous mixture. As discussed above, the substrate is selected to receive and concentrate the first indicator reagent. The substrate receives the first indicator reagent through contact with the aqueous medium. The substrate retains the first indicator reagent or biological derivative of the same through various means. Without sticking to the theory, the accumulation of the first indicator reagent or biological derivative of the same on and / or in the substrate material can occur through one or more of a variety of chemical attraction forces including, but not limited to, ionic interaction , hydrophobic interaction, van der Waal forces and hydrogen bonding, for example. The process of reception and concentration of the first indicator reagent or biological derivative of the same by the substrate occurs during the period of fluid communication between the aqueous medium and the substrate. During this period of fluid communication, the first indicator reagent or biological derivative of the same accumulates on the substrate at a rate that can be dependent on several factors including, for example, the concentration of the first indicator reagent (or biological derivative of the same), the surface area of the substrate material that comes in contact with the liquid medium, the porosity of the substrate, a charge density associated with the substrate material and / or other substances in the liquid medium that can interact with the substrate and / or the first indicator reagent (or biological derivative thereof) in a way that interferes with the reception or concentration of the first indicator reagent or biological derivative thereof by the substrate. The reception and concentration of at least a portion of the first indicator reagent or biological derivative of the same on the substrate can occur within a relatively short contact period (for example, within several minutes) and can continue for a longer contact period (for example, up to 1 hour, up to 2 hours, up to 4 hours, up to 18 hours, up to 24 hours, up to 7 days, up to two weeks). In some embodiments, the first indicator reagent can concentrate on or on the substrate within a relatively short period of time (for example, minutes, hours), while the first biological derivative, if present, may not be detectably concentrated on or on the substrate for a relatively longer period of time (for example, hours, days).
During any of the periods of fluid communication between the aqueous mixture and the substrate described above, the substrate can receive and concentrate all or a portion of the first indicator reagent (or biological derivative thereof). In some embodiments, the substrate receives and concentrates at least 5 percent of the first indicator reagent (or biological derivative). In some embodiments, the substrate receives and concentrates at least 10 percent of the first indicator reagent (or biological derivative). In some embodiments, the substrate receives and concentrates at least 20 percent of the first indicator reagent (or biological derivative). In some embodiments, the substrate receives and concentrates at least 30 percent of the first indicator reagent (or biological derivative). In some embodiments, the substrate receives and concentrates at least 40 percent of the first indicator reagent (or biological derivative). In some embodiments, the substrate receives and concentrates at least 50 percent of the first indicator reagent (or biological derivative). In some embodiments, the substrate receives and concentrates at least 75 percent of the first indicator reagent (or biological derivative). In some embodiments, the substrate receives and concentrates at least 80 percent of the first indicator reagent (or biological derivative). In some embodiments, the substrate receives and concentrates at least 90 percent of the first indicator reagent (or biological derivative). In some embodiments, the substrate receives and concentrates more than 90 percent of the first indicator reagent (or biological derivative). In some embodiments, the substrate receives and concentrates more than 95 percent of the first indicator reagent (or biological derivative). The determination that the substrate receives and concentrates the first indicator reagent (or biological derivative of it) can be easily carried out by placing a liquid medium that comprises the first indicator reagent (or biological derivative of the same) in fluid communication with the substrate over a period of time and analyzing the substrate for the presence of the reagent (or biological derivative thereof), as shown in Example 1. Preferably, any excess liquid medium is removed from the substrate (for example, by staining or by centrifugation) before analyzing the substrate so that the amount of reagent or biological derivative associated with the substrate indicates the amount retained by the substrate. Appropriate methods of analysis will be apparent to an individual of ordinary skill in the art. For example, a substrate that receives and concentrates a first colored indicator reagent (for example, a pH indicator) can be analyzed by reflectance spectroscopy using, for example, a portable X-Rite model 530P spectrodensitometer.
Thus, when a liquid medium comprising a sample and the first indicator reagent (or biological derivative thereof) is placed in fluid communication with a suitable substrate, the concentration of the first indicator reagent (or biological derivative thereof) in the liquid medium in question. volume decreases as the first indicator reagent (or biological derivative thereof) is received and concentrated by the substrate. This feature of the invention facilitates the detection of relatively small concentrations of the biological derivative of the second indicator reagent due to the fact that at least a portion of the interference (i.e., fluorescence absorption) by the first indicator reagent is removed as the first indicator reagent is concentrated. on the aqueous mixture substrate. In some embodiments, the first indicator reagent and / or a biological derivative thereof, when in a freely diffusable form (that is, in the liquid medium by volume) can inhibit biological activity. In these embodiments, a further advantage of the invention is that the substrate can effectively sequester at least a portion of the first indicator reagent, thereby reducing the inhibition of biological activity by the first indicator reagent.
Referring again to Figure 3, the method may additionally include optional step 65 of facilitating cell growth. Facilitating cell growth is widely used to include providing conditions (eg, nutrients, germinants, buffers, oxidation-reduction potential, gases) to facilitate, for example, spore germination, energy metabolism, biosynthesis and / or cell division. Facilitating cell growth can result in the amplification of one or more predetermined biological activities from the original sample and, thus, can improve sensitivity to detect predetermined biological activities.
The methods of the present description further include step 70 of detecting a biological derivative of the second indicator reagent (in the present invention, called "second biological derivative"). In some embodiments, the second biological derivative can be detected in an aqueous medium. The detection of the presence or absence of the second biological derivative is indicative of the presence or absence, respectively, of the corresponding predetermined biological activity in the sample. The second biological derivative can be detected by several means. In some embodiments, the second biological derivative can be detected optically. In some embodiments, the detection of the second biological derivative may comprise the detection of the biological derivative visually. In some embodiments, the detection of the second biological derivative may comprise the detection of the biological derivative using an instrument. For example, the second biological derivative can be detected using an optical instrument such as a fluorometer.
In any of the above embodiments, detecting the presence or absence of the second biological derivative thereof may further comprise measuring the amount of the second biological derivative. The measurement of the quantity can be done by any means known in the art including, for example measuring the quantity using an instrument (for example, a fluorometer). In some embodiments, measuring the amount of second biological derivative may comprise comparing the fluorescence in the aqueous mixture with a fluorescent standard.
In either embodiment, the methods of the present disclosure optionally include step 75 of detecting the first indicator reagent or a first biological derivative thereof. The means for detecting the first indicator reagent or the first biological derivative depends on the nature of the first indicator reagent or the first biological derivative, as will be seen by one of ordinary skill in the art. For example, if the first indicator reagent is a chromium (colored) and / or the first biological derivative is a chromium compound, then the first indicator reagent and / or the first biological derivative can be detected optically (either visually or by an instrument ( for example, a spectrophotometer)). In some embodiments, the detection of the first indicator reagent or the first biological derivative may further comprise the detection of the first indicator reagent or the first biological derivative in a portion of the aqueous mixture that is not associated with the substrate (for example, in the liquid in question). volume). For example, if the first indicator reagent or the first biological derivative is detected using an optical instrument such as a spectrophotometer, the optical path does not cross any portion of the substrate.
In either embodiment, detecting the presence or absence of the first indicator reagent or the first biological derivative may further comprise measuring the amount of the first indicator reagent or the first biological derivative. Measurement of quantity can be made by any means known in the art including, for example, measurement of quantity using an instrument (for example, a spectrophotometer, a spectrodensitometer).
US patents No. 5,252,484 and 5,418,167 each of which is incorporated herein, by reference in its entirety, describe a modality of a quick-read biological indicator in which the biological indicator comprises an enzyme carrier ( spore strip) and an ampoule contain a solution with 4-methylumbelliferyl-aD-glycoside (“MUG”, a fluorogenic enzyme substrate) and purple bromocresol (“BCP”, a pH indicator). MUG is known to be hydrolyzed by enzymatic activity to 4-methylumbelliferone (4MU), a fluorescent derivative of MUG. As shown in examples 1 and 2 of US patent No. 5,252,484, the 4MU produced by enzymatic hydrolysis of the MUG can be detected visually by fluorescence within minutes after the enzyme carrier is placed in fluid communication with the solution containing MUG and BCP.
The present investigators concluded that the concentration of BCP used in a solution similar to that described in example 1 of US patent No. 5,252,484 is sufficient to interfere with the detection of low concentrations of 4MU in an aqueous solution. The removal of at least a portion of the BCP from the solution according to the present description will allow the detection of smaller amounts of 4MU in a biological indicator, thereby allowing the early detection of biological activity (for example, spores, enzymes ) that were exposed to a sterilization process and were not thereby inactivated and / or exterminated.
System for Detecting a Biological Activity The present disclosure includes a system for detecting a predetermined biological activity in a sample. The system can be used according to the method of the invention to detect one or more biological activities in a sample. The system includes a first indicator system that comprises a first indicator reagent that can be converted by a first predetermined biological activity to a first biological derivative. The first indicator reagent has a first absorption spectrum and, optionally, a first emission spectrum. The system further includes a second indicator system comprising a second indicator reagent that can be converted by a second predetermined biological activity to a second biological derivative. The second biological derivative has a first absorption spectrum and a second emission spectrum. The system additionally includes an instrument configured to receive a liquid sample that can comprise the first indicator reagent, the second indicator reagent, the first biological derivative, the second biological derivative or any combination of two or more of the aforementioned items. The instrument can be configured to remove the liquid sample from an external container using a “sorter” medium, as known in the analytical instrument technique. Alternatively, the instrument can be configured to receive a vessel (for example, a tube, a microcavity plate, or the like) that contains the liquid sample. The instrument is configured to detect the second biological derivative. Optionally, the instrument can be further configured to detect the first indicator reagent, the second indicator reagent, the first biological derivative or any combination of two or more of the above items. The indicator reagent of the system can be any suitable indicator reagent, as described here, to detect particular predetermined biological activity. The first and second indicator reagents can be provided in a kit, for example, which can optionally include an aqueous medium (for example, a buffer, a suspension medium, a diluent) in which the indicator reagent and the sample are mixed. As discussed in this document, the sample can comprise water and, thus, can constitute the aqueous medium. Optionally, the kit may also include a vessel (for example, a tube, a crucible or the like) in which an aqueous mixture is formed which comprises the sample and the first and second indicator reagents. In some embodiments, the system can be used with a biological sterilization indicator such as, for example, the biological indicators in US patent applications No. 61 / 408,977 and 61 / 408,988, filed on November 1, 2010, and the indicators described in US Patent No. 5,252,484, each of which is incorporated herein by reference in its entirety.
Instruments for detecting the absorption spectra of chromic compounds are known in the art and include, for example, a variety of commercially available spectrophotometers and spectrodensitometers. Instruments for detecting the emission spectra of fluorescent compounds are also known in the art and include, for example, a variety of commercially available fluorometers. Such instruments can be readily adapted to detect an indicator reagent (or biological derivative thereof) associated with a liquid sample and / or a substrate positioned in a predetermined location.
In some embodiments, the substrate can be removed from the aqueous mixture and positioned (for example, on a surface or in a crucible) so that the indicator reagent (or biological derivative of it) can be detected by the instrument. US patent No. 6,025,189, which is incorporated herein by reference, describes an instrument configured to detect, at a predetermined location in a one-piece biological indicator, a fluorescent signal associated with a biological activity. Modification of such an instrument to detect a chromic signal is common in the art.
In some embodiments, the system may additionally comprise a processor. In some embodiments, the instrument may comprise a microprocessor capable of controlling the instrument and collecting and / or transmitting data associated with the detection of the indicator reagent or biological derivative thereof. In some embodiments of the system, the processor may comprise an external processor. The external computer may comprise a personal computer (PC), type computer, laptop type computer, portable computer, workstation or the like. For example, software programs can be loaded on an external computer to control the instrument and / or to facilitate the collection, transfer and / or analysis of instrument data.
In some embodiments, the system may additionally comprise means for regulating the temperature of a liquid. The means for temperature control can include any means known in the art, for example, thermocouples and heat exchangers. Advantageously, these modalities provide a system that can facilitate biological activity through temperature control and can detect the product of biological activity.
Biological sterilization indicators: Figures 4 to 10 illustrate a biological sterilization indicator system 100 according to an embodiment of the present description. Other suitable modalities of biological sterilization indicators are described in copying PCT publication No. WO 2011/011189 entitled “Biological Sterilization Indicator and Method of Using Same”; US patent application No. 61 / 409,042 entitled “Biological Sterilization Indicator System and Method”; US patent application No. 61 / 408,997 entitled “Biological Sterilization Indicator System and Method”; and in US patent application No. 61 / 408,977 entitled “Biological Sterilization Indicator and Method of Using Same”; each of which is incorporated herein, as a reference in its entirety. The biological sterilization indicator 100 may include a compartment 102, which may include a first portion 104 and a second portion 106 (e.g., a lid) adapted to be coupled thereto to provide the one-piece biological sterilization indicator. In some embodiments, the first portion 104 and the second portion 106 may be formed from the same materials, and in some embodiments, the first portion 104 and the second portion 106 may be formed from different materials. The compartment 102 can define a reservoir 103 of the biological sterilization indicator 100 in which other components can be positioned and in which a sterilizer can be directed during a sterilization process. The compartment 102 can be defined by at least one liquid impermeable wall, such as the wall 108 of the first portion 104 and / or a wall 110 of the second portion 106. It should be understood that a single piece unit 102 can also be employed or that the first and second portions 104 and 106 may adopt other formats, dimensions or relative structures without deviating from the character and scope of the present disclosure. Suitable materials for compartment 102 (for example, walls 108 and 110) may include, but are not limited to, a glass, a metal (for example, foil), a polymer (for example, polycarbonate (PC), polypropylene (PP), polyethylene, polystyrene (PS), polyester (for example, polyethylene terephthalate (PET)), polymethyl methacrylate (PMMA or acrylic), acrylonitrile-butadiene-styrene (ABS), cycloolefin polymer (COP ), cycle olefin copolymer (COC), polysulfone (PSU), polyether sulfone (PES), polyetherimide (PEI), polybutylene terephthalate (PBT)), ceramic, porcelain, or combinations thereof.
In some embodiments, the biological sterilization indicator 100 may also include a frangible container 120 that contains a liquid 122, and that is sized to be received within the biological sterilization indicator 100, for example, within at least a portion of the compartment 102 (for example, at least within the first portion 104 of compartment 102). The frangible container 120 for being formed by a variety of materials, including, but not limited to, one or more of metal (for example, foil, a polymer (for example, any of the polymers mentioned above in relation to compartment 102 ), glass (for example, a glass ampoule), and combinations thereof. In some embodiments, only a portion of container 120 is frangible, for example, container 120 may include a frangible portion or cover (for example, a film , frangible membrane or barrier or the like). The frangible container 120 may have a first state in which it is intact and the liquid 122 is contained therein, and a second state in which at least a portion of the container 120 is fractured. container 120, the liquid 122 may be in fluid communication with the reservoir 103 of the biological sterilization indicator 100, for example, when the container 120 is positioned in the biological indicator of sterilization terilization 100.
As shown in the illustrated embodiment, the container 120 can be held in place within the biological sterilization indicator 100 and / or fractured by an insert 130, which is described in more detail below. The container 120 can be fractured, for example, by inciting the container 120 against the insertion element 130 (for example, an insertion element that functions as a fracture) or by inciting the insertion element 130 against the container 120. The first portion 104 of compartment 102 can be adapted to house most components of the biological sterilization indicator 100, and can be called "tube", "tubular body", "base" or the like. Compartment 102 may include a reservoir 103 that can be defined by one or both of the first portion 104 and the second portion 106 of compartment 102. The biological sterilization indicator 100 may further include spores or other source (s) of biological activity 115 (or a spore site) positioned in fluid communication with reservoir 103. As shown in Figures 4 to 6, the second portion 106 of compartment 102 may include one or more openings 107 to provide fluid communication between the interior of the compartment 102 (for example, reservoir 103) and the environment. For example, one or more openings 107 can provide fluid communication between spores 115 and the environment during the sterilization process, and can serve as an input to the biological sterilization indicator 100 and as an input to a sterilizer path 164 (described in more details below). In some embodiments, the second portion 106 of compartment 102 can be coupled to a first end (for example, open) 101 of the first portion 104 of compartment 102, and spores 115 can be positioned on a second end (for example, closed) 105, opposite the first end 101, of the first portion 104 of compartment 102.
In some embodiments, a barrier or filter (for example, a sterile barrier; not shown) can be positioned on the sterilizing path 164 (for example, at the entrance formed by opening 107) to inhibit the entry of contamination or foreign, objective or materials in the biological sterilization indicator 100. This barrier can include a gas transmissive material and is impermeable to microorganisms, and can be coupled to compartment 102 and a variety of coupling means, including, but not limited to, an adhesive, a seal heat, sonic welding, or the like. Alternatively, the barrier can be coupled to the sterilizing path 164 via a support structure (such as a second portion 106) that is coupled to the first portion 104 of compartment 102 (for example, in a quick-fit coupling, a coupling by screw, pressure coupling, or a combination thereof). During exposure to a sterilizer, it can cross the barrier in the trajectory of sterilizer 164 and come in contact with spores 115.
In some embodiments, as shown in the illustrated embodiment, compartment 102 may include a lower portion 114 and an upper portion 116, which may be at least partially separated by an inner wall (or partial wall) 118, protrusion, partition, flange or the like , in which an opening 117 can be formed which provides fluid communication between the lower portion 114 and the upper portion 116. In some embodiments, the lower portion 114 of the first portion 104 of compartment 102 (sometimes simply called "lower portion 114" or "lower portion 114 of compartment 102") can be adapted to house spores 115 or a spore site. In some embodiments, the lower portion 114 may be called the "detection portion" or "detection region" of compartment 102, because at least a portion of the lower portion 114 may be interrogated for signs of spore growth. In addition, in some embodiments, the upper portion 116 of the first portion 104 of compartment 102 (sometimes called “upper portion 116” or “upper portion 116 of compartment 102” for the sake of simplicity) can be adapted to accommodate at least a portion of the frangible container 120, particularly prior to activation.
In some embodiments, the portion of reservoir 103 that is defined at least partially by the upper portion 116 of compartment 102 may be called the first chamber (or reservoir, zone, region or volume) 109 and the portion of reservoir 103 that is defined at least partially through the bottom 114 of compartment 102 can be called the second chamber (or reservoir, zone, region or volume) 111. In some embodiments, the second chamber 111 can be called a “spore growth chamber” or a “spore growth chamber” detection ”, and may include a volume to be questioned about spore viability to determine the effectiveness of a sterilization process. The first chamber 109 and the second chamber 111 can be positioned in fluid communication with each other to allow a sterilizer and liquid 122 to move from (i.e., through) the first chamber 109 to the second chamber 111. In some modalities, the degree of fluid connection between the first chamber 109 and the second chamber 111 (for example, the size of an opening, such as opening 117, which connects the first chamber 109 and the second chamber 111) may increase afterwards, simultaneously with and / or in response to the activation step (i.e., the liquid 122 that is released from the container 120). In some embodiments, control of fluid communication (or extension of fluid connection) between the first chamber 109 (for example, in the upper portion 116) and the second chamber 111 (for example, in the lower portion 114) can be provided by at least a portion of the insert 130. The container 120 can be positioned and maintained in the first chamber 109 during sterilization and when the container 120 is in a first non-fractured state. The spores 115 can be housed in the second chamber 111 and in fluid communication with the environment when the container 120 is in the first state. The first chamber 109 and the second chamber 111 can be configured so that the container 120 is not present in the second chamber 111, and particularly, not when the container 120 is in its first non-fractured state. A sterilizer can move to the second chamber 111 (for example, through the first chamber 109) during sterilization and the liquid 122 can move to the second chamber 111 (for example, the first chamber 109) during activation, when the container 120 is fractured and liquid 122 is released into compartment 102.
As a result, when the container 120 is in the first state, the first chamber 109 and the second chamber 111 can be in fluid communication with each other, and with the environment (for example, during sterilization). For example, the first chamber 109 and the second chamber 111 may be in fluid communication with the environment through one or more openings 107. In some embodiments, the first chamber 109 and the second chamber 111 may be in fluid communication with the environment. such that the first chamber 109 is positioned upstream of the second chamber 111 when a sterilizer is entering the biological sterilization indicator 100. That is, the first chamber 109 can be positioned between the inlet of the sterilizer (for example, the one or more openings 107) and the second chamber 111, and the sterilizer inlet can be positioned on the opposite side of the first chamber 109 with respect to the second chamber 111.
As shown in Figures 7 and 9, in some embodiments, the first chamber 109 can be defined by one or both of the first portion 104 and the second portion 106, particularly when the container 120 is in the first state. In addition, in some embodiments, the first chamber 109 may include a first end 112 positioned adjacent the open end 101 of the first portion 104 of compartment 102, adjacent to the second portion 106 of compartment 102, and / or at least partially defined by the second portion 106. The first chamber 109 may additionally include a second end 13 positioned adjacent and in fluid communication with the second chamber 111 and positioned towards the closed end 105 of compartment 102. The first end 112 of the first chamber 109 can be defined by the first portion 104 and / or the second portion 106 of compartment 102.
As further shown in Figures 7 and 9, in some embodiments, the second chamber 111 may include a first end 124 positioned adjacent and in fluid communication with the first chamber 109 and positioned towards the open end 101 of compartment 102, and a second end 125 at least partially defined by, including or positioned adjacent to the closed end 105 of compartment 102.
Otherwise, as shown in Figures 7 and 9, the biological sterilization indicator 100 can include a longitudinal direction DL, and in some embodiments, the first chamber 109 can be positioned longitudinally above the second chamber 111.
In some embodiments, the second chamber 111 may be at least partially defined by, may include or may be positioned adjacent to the closed end 105 of the biological sterilization indicator 100. In addition, in some embodiments, the second chamber 111 may be smaller (for example, example, in volume and / or cross-sectional area) than at least one of the first chamber 109 and the volume of liquid 122 in container 120 that will be released when the sterilization biological indicator 100 is activated. As a result, in such embodiments, the second chamber 111 may exhibit an air locking effect in which the gas (for example, air) that is present in the second chamber 111 can inhibit fluid movement to the second chamber 111. In some embodiments , as described in greater detail below, a fluid path that allows the second chamber 111 to vent another portion of the sterilizing biological indicator 100 can facilitate fluid movement in the second chamber 111.
In some embodiments, wall 118 (sometimes called a “separation wall”) can be angled or tilted, for example, oriented at a non-right or non-zero angle to the longitudinal direction DL of compartment 102 (for example, where the longitudinal direction DL extends along the length of compartment 102). This angulation or inclination of the wall 118 can facilitate the movement of the liquid 122 from the upper portion 116 to the lower portion 114 after sterilization and after the container 120 has been ruptured to release the liquid 122.
As shown in Figures 4 to 6, in some embodiments, wall 118 can be at least partially formed by a change in the internal dimension of compartment 102. For example, as shown, wall 118 can be formed by a decrease in an area of the cross section from a first longitudinal position in the first chamber 109 to a second longitudinal position in the second chamber 111. Furthermore, just as an example, the shape of the internal cross section of compartment 102 may change in the transition from the first chamber 109 to the second chamber 111 from substantially round (for example, with a flat side that produces less than 50% of the perimeter) in the first chamber 109 to substantially parallelepipedal (for example, substantially square) in the second chamber 111.
Additionally, in some embodiments, wall 118 may also be at least partially formed by a change in the external dimension of compartment 102. As shown in Figures 4 to 6, in some embodiments, compartment 102 includes a step (or protrusion, suspension, transition or similar) 123 which is consistently angled with wall 118 (if wall 118 is angled), and which includes a change in the external shape and dimension of compartment 102. However, it should be understood that in some modalities, even if internal dimension of compartment 102 change to create a second chamber 111 that has a shape or cross-sectional dimension different from the first chamber 109, the external shape and dimension of compartment 102 do not need to change, or change consistently with the change in internal format and / or dimension. For example, in some embodiments, step 123 can be oriented substantially and perpendicularly to the longitudinal direction DL.
In some embodiments, reservoir 103 has a volume of at least about 0.5 millimeters (ml), in some embodiments, at least about 1 ml, and in some embodiments, at least about 1.5 ml. In some embodiments, reservoir 103 has a volume of no more than about 5 ml, in some embodiments no more than about 3 ml, and in some embodiments, no more than about 2 ml.
In some embodiments, the frangible container 120 has a volume of at least about 0.25 ml, in some embodiments, at least about 0.5 ml, and in some embodiments, at least about 1 ml. In some embodiments, the frangible container 120 has a volume of no more than about 5 ml, in some embodiments, no more than about 3 ml, and in some embodiments, no more than about 2 ml.
In some embodiments, the volume of liquid 122 contained in the frangible container 120 is at least about 50 microliters, in some embodiments at least about 75 microliters, and in some embodiments at least about 100 microliters. In some embodiments, the volume of liquid 122 contained in the frangible container 120 is no greater than about 5 ml, in some embodiments no greater than about 3 ml, and in some embodiments, no greater than about 2 ml.
In some embodiments, the first chamber 109 (that is, formed by the upper portion 116 of the first portion 104 of compartment 102) has a volume of at least about 500 microliters (or cubic mm), in some embodiments, at least about 1000 microliters, in some modalities, at least about 2000 microliters, and in some modalities, at least about 2500 microliters. In some embodiments, the first chamber 109 has a volume not greater than about 5000 microliters, in some embodiments not greater than about 4000 microliters, and in some embodiments, not greater than about 3000 microliters. In some embodiments, the first chamber 109 has a volume of about 2790 microliters or 2800 microliters.
In some embodiments, the second chamber 111 (i.e., formed by the lower portion 114 of the first portion 104 of compartment 102) has a volume of at least about 5 microliters, in some embodiments, at least about 20 microliters, and in some modalities, at least about 35 microliters. In some embodiments, the second chamber 111 has a volume of no more than about 250 microliters, in some embodiments, no more than about 200 microliters, in some embodiments, no more than about 175 microliters, and in some embodiments; no more than about 100 microliters. In some embodiments, the second chamber 111 has a volume of about 208 microliters or 210 microliters. 'In some embodiments, the volume of the second chamber'111 is at least about 5% of the volume of the first chamber 109, and in some embodiments, at least about 7%. In some embodiments, the volume of the second chamber 111 is no more than about 20% of the volume of the first chamber 109, in some embodiments; no more than about 15%, in some modalities, no more than about 12%, and in some modalities, no more than about 10%. In some embodiments, the volume of the second chamber 111 is about 7.5% of the volume of the first chamber 109.
In some embodiments, the volume of the second chamber 111 is no more than about 60% of the volume of the liquid 122 housed in the container 120, in some embodiments, no more than about 50%, and in some embodiments, no more than about 25%. In some embodiments, the design of the second chamber 111 has a volume that is substantially less than that of the liquid 122 housed in the container) 20 can ensure that the volume of additional liquid can compensate for unintended evaporation.
In some embodiments, the first chamber 109 (i.e., formed by the upper portion 116 of the first portion 104 of compartment 102) has a cross-sectional area (or average cross-sectional area) in the transition between the first chamber 109 and the second chamber 111, or in the position adjacent to the second chamber 111, of at least about 25 mm2; in some modalities, at least about 30 mm2; and in some modalities, at least about 40 mm2. In some embodiments, the first chamber 109 has a cross-sectional area at the transition between the first chamber 109 and the second chamber 111, or in the position adjacent to the second chamber 111, of no more than about 100 mm2, in some embodiments, no more than about 75 mm2, and in some modalities, no more than about 50 mm2.
In some embodiments, the second chamber 111 (i.e., formed by the lower portion 114 of the first portion 104 of compartment 102) has a cross-sectional area at the transition between the first chamber 109 and the second chamber 111, or in the position adjacent to the first chamber 109, of at least about 5 mm2, in some embodiments, at least about 10 mm2, and in some embodiments, at least about 15 mm2. In some embodiments, the second chamber 111 has a cross-sectional area (or average cross-sectional area) of no more than about 30 mm2, in some modalities, no more than about 25 mm2, and in some modalities, no more than about mm2.
In some embodiments, the cross-sectional area of the second chamber 111 in the transition between the first chamber 109 and the second chamber 111 may be no more than about 60% of the cross-sectional area of the first chamber 109 in the transition, in some embodiments, no more than about 50%, in some modalities, no more than about 40%, and in some modalities, no more than about 30%.
In some embodiments, the biological sterilization indicator 100 may additionally include a substrate 119. In some embodiments, as shown in Figures 4 to 7 and 9, substrate 119 can be sized to be positioned adjacent to wall 118, and particularly, the rest above wall 118. Substrate 119 can be positioned between the upper portion 116 (i.e., the first chamber 109) and the lower portion 114 (that is, the second chamber 111) of the sterilization biological indicator 100 and, in some embodiments , you can at least partially define the first chamber 109 and the second chamber 111. As such, in some embodiments, substrate 119 can be positioned between container 120 and spores 115. In some embodiments, substrate 119 can be positioned in the first chamber 109, or on one side of the first chamber of the wall 118, so that the substrate 119 is not positioned in the second chamber 111.
In addition, substrate 119 can be positioned to minimize diffusion of a test signal (e.g., fluorescence) out of second chamber 111. In some embodiments, depending on the material composition of substrate 119, substrate 119 can also absorb dyes, indicator reagents or other solution materials that can inhibit accurate reading of a signal from the sterilization biological indicator 100 (ie, “inhibitors”). In some embodiments, as shown in Figures 4 to 7, 9 and 10, substrate 119 can include one or more openings 121, which can be configured to control (i.e., facilitate and / or limit, depending on the number, size, shape and / or local) the movement of fluid between the first chamber 109 and the second chamber 111 of the biological sterilization indicator 100, and particularly, which can facilitate the movement of the liquid 122 towards the spores 115 when the container 120 is fractured. Only by way of example, the particular benefits or advantages were observed when opening 121 was positioned in front of (or "ahead of") the center of substrate 119, as shown. In the embodiment illustrated in Figures 4 to 10, the “front” of the sterilization biological indicator 100 or components therein can generally be described as being towards a face plane 126. In general, the “front” of the sterilization biological indicator 100 can refer to the portion of the biological sterilization indicator 100 that will be interrogated by a reading device.
Furthermore, for example only, aperture 121 is illustrated as being circular or round; however, other opening formats in cross section are possible and included in the scope of the present disclosure. Additionally, for example only, and as shown in Figure 6, substrate 119 is shaped to substantially fill the cross-sectional area of the first chamber in the transition between the first chamber 109 and the second chamber 111. However, other shapes of the substrate 119 are possible and can be adapted to accommodate compartment 102, first chamber 109, second chamber 111, wall 118 or another component of the sterilization biological indicator 100.
As mentioned above, the second chamber 111 can include a volume to be interrogated. Such volume can be tested for spore viability to determine the lethality or effectiveness of a sterilization procedure. In some embodiments, the volume to be interrogated can be all or a portion of the second chamber 111. In some embodiments, substrate 119 can be positioned outside the volume to be interrogated, which can minimize the number of structures in the volume that can interfere in the testing processes. For example, in some embodiments, substrate 119 can be positioned so that substrate 119 is not in direct contact with at least one of the spores 115, the spore carrier 135 and the spore reservoir 136. In some embodiments, the substrate 119 can be positioned so that substrate 119 is not located between a detection system (for example, an optical detection system, such as a source of fluorescence excitation and an emission detector) and at least one among the spores 115 , the spore carrier 135 and the spore reservoir 136. The substrate 119 can have the above positions when the container 120 is in the first state and / or the second state, but particularly when the container 120 is in the second state.
In some embodiments, the position of the substrate in the biological sterilization indicator 100 may affect the correlation of a rapid detection system for spore viability (for example, fluorescence detection) with a slower (for example, one-day detection system) to the other or 24 h) (for example, a pH indicator that may exhibit a color change (for example, in 24 h) in response to spore growth). For example, in some embodiments, substrate 119 can optimize the correlation of fluorescence readings at various time points with growth results after 24 h. Particularly, when substrate 119 is positioned in a "first" position - as described here and as shown in Figures 1, 2 and 4 to 7 - fluorescence can be precisely correlated with growth. Such a correlation can be an improvement over the other substrate positions and biological indicators of sterilization without substrate.
In addition, substrate 119 can be positioned on the biological sterilization indicator 100 so that substrate 119 is not in direct contact with container 120 when container 120 is in the first state. For example, in some embodiments, substrate 119 may be positioned in the first chamber 109 (e.g., adjacent to a lower end (e.g., second end 113) of the first chamber 109), but still in such embodiments, substrate 119 may be positioned so that the substrate 119 does not come into contact with the container 120. For example, as shown in Figures 4 and 5 and 7 to 9, in some embodiments, the insert 130 can be positioned between the container 120 and the substrate 119 when the container 120 is in the first state, so that the insert 130 keeps the container 120 in the first state. The insert 130, or a portion thereof, can be positioned adjacent to the substrate 119. For example, as shown in the illustrated embodiment, the substrate 119 can be positioned between (e.g. sandwiched between) the insert 130 and the wall 118. As such, in some embodiments, substrate 119 can be positioned between insertion element 130 and second chamber 111.
As mentioned above, in some embodiments, substrate 119 can be positioned and configured to control or affect the flow of fluid in the biological sterilization indicator 100, and particularly, to control the flow of fluid between the first chamber 109 and the second chamber 111 For example, in some embodiments, substrate 119 can be configured (for example, sized, shaped, oriented and / or constructed from certain materials) to control the rate at which a sterilizer is released into the second chamber 111 (and the spores 115). For example, the sterilant release rate may be lower than it would otherwise be if substrate 119 was not present between the first chamber 109 and the second chamber 111.
In addition, in some embodiments, substrate 119 can be configured (e.g., sized, shaped, positioned, oriented and / or constructed from certain materials) to control the rate at which detectable products diffuse out of the volume to be interrogated. In some embodiments, the detectable product may include a signal (e.g., a fluorescent signal) that indicates spore viability, and in some embodiments, the detectable product may be the spore (s) 115 itself. Controlling the diffusion of detectable products out of the volume to be interrogated can be particularly useful in modalities where the volume of liquid 122 is greater than the volume of the second chamber 111 (or the volume to be interrogated), due to the fact that the liquid 112 in such modalities can extend in the biological sterilization indicator 100 to a level higher than the second chamber 111 (or the volume to be interrogated) when the container 120 is in its second fractured state. In such embodiments, the detectable products may be free to move through the entire volume of liquid 122 (that is, to a volume outside the volume to be interrogated), unless there is some barrier or means to control such diffusion, such as the substrate 119. For example, in some embodiments, substrate 119 can be positioned at a level just above the volume to be interrogated (ie, below the level of liquid 122), to inhibit the movement of detectable products to the portion of liquid 122 which is positioned above substrate 119.
In some embodiments, substrate 119 can control the rate of sterilizing release (for example, in the second chamber 111) and / or the rate of diffusion of detectable products (for example, outside the second chamber 111) by providing a barrier physical or blockage for the sterilizer and / or detectable products. Such a physical barrier may also work to collect broken portions of container 120 when container 120 is in the second fractured state to inhibit movement of the broken portions to the volume to be interrogated where the broken portions could block, refract, reflect or otherwise interfere detection processes (for example, optical detection processes).
In addition, in some embodiments, liquid 122, before or after entering fluid communication with spores 115, may include one or more inhibitors, or other components, which may interfere with an accurate detection or testing process. In some embodiments, examples of inhibitors may include at least one among dyes, indicator reagents, other materials or substances that may inhibit a reaction (eg, an enzymatic reaction) necessary for the detection of spore viability (eg, salts, etc.), other materials or substances that may interfere with the detection process, or combinations thereof. In such embodiments, substrate 119 can be configured to selectively absorb and / or concentrate one or more inhibitors of liquid 122, or at least the volume of liquid 122 to be interrogated.
For example, in some embodiments, more than one indicator reagent may be present in liquid 122, prior to spore contact 115 or as a result of contact with spore 115. In such embodiments, although a first indicator reagent (for example, used for fluorescence detection) may be necessary to detect spore viability, a second indicator reagent (for example, a pH indicator) may actually interfere with the detection of the first indicator reagent. Only as an example, in modalities in which the second indicator reagent is a pH indicator (for example, one or more among bromocresol purple, methyl red or a combination thereof), the pH indicator may conflict or interfere with fluorescence reading of the first indicator reagent, for example, in modes where the pH indicator emits electromagnetic radiation at a wavelength that is similar to the fluorescence spectral band of the first indicator reagent (for example, when the pH indicator displays a color shift). In such embodiments, substrate 119 can be configured (for example, formed of a suitable material) to selectively absorb and / or concentrate the second indicator reagent when positioned in contact with liquid 122 to reduce the concentration of the second indicator reagent in liquid 122 , or at least in the volume of the liquid 122 to be interrogated.
In addition, in some embodiments (for example, in which the wall 118 is tilted and the substrate 119 is positioned adjacent to the wall 118), the substrate 119 can be angled or tilted, for example, oriented at a non-right angle or different from zero in relation to the longitudinal direction DL of compartment 102. This angulation or inclination of substrate 119 can facilitate the movement of liquid 122 from the first chamber 109 to the second chamber 111 after sterilization and after the container 120 has been broken to release the liquid 122.
In some embodiments, substrate 119 may be formed from a variety of materials to perform one or more of the above functions. Examples of substrate materials may include, but are not limited to, cotton, glass wool, cloth, non-woven polypropylene, non-woven rayon, non-woven rayon / polypropylene blend, non-woven nylon, non-woven fiberglass or others non-woven fibers, filter papers, microporous hydrophobic and hydrophilic films, glass fibers, open cell polymeric foams and semipermeable plastic films (for example, particle-filled films, thermally induced phase separation membranes (TIPS), etc.) , and combinations thereof. For example, in modalities in which substrate 119 can be used to seitively concentrate one or more indicator reagents (for example, bromocresol purple (BCP)), substrate 119 can be formed of a loaded nylon (like transfer membranes loaded with probing available from GE Osmonics (under the trade name “MAGNAPROBE” (eg 0.45 micron, catalog no. NP0HY00010, material no. 1226566)).
An example of a method and system that can employ substrate 119 is also described in copending US patent application No. 61 / 408,887, filed on November 1, 2010, entitled “Method of Detecting a Biological Activity”, which is incorporated herein in full by reference.
In some embodiments, at least a portion of one or more within insertion element 130, wall 118, and / or substrate 119, or an opening therein, can provide fluid communication between the first chamber 109 (for example, in the upper portion 116 ) and the second chamber 111 (for example, in the lower portion 114), and / or can control the fluid communication between the first chamber 109 and the second chamber 111 (for example, by controlling the extent of the fluid connection between the first chamber 109 and the second chamber 111). The biological sterilization indicator 100 may include a first fluid path 160 that can be positioned to fluidly couple the first chamber 109 and the second chamber 111, and which may allow the sterilizer (for example, during sterilization, when the container 120 is in a first, non-fractured state) and / or liquid 122 (for example, after sterilization and during activation, when container 120 is in a second fractured state) to reach spores 115. In the illustrated embodiment, the first fluid path 160 can generally be defined by one or more of the following: (1) the insertion element 130, for example, through an opening 177 described below, an opening formed in the insertion element 130, and / or any spaces opened around the insertion element 130, as between the insertion element 130 (for example, a front portion thereof) and the housing 102; (2) wall 118, for example, opening 117 defined by wall 118; (3) substrate 119, for example, opening 121 formed there, or any open spaces around substrate 119, such as between substrate 119 (e.g., a front portion thereof) and compartment 102; (4) compartment 102, for example, any openings or spaces formed there; and combinations thereof. As a result, the first fluid path 160 is generally represented in the embodiment illustrated by an arrow in Figures 7 and 10. The biological sterilization indicator 100 may additionally include a second fluid path 162 positioned to fluidly couple the second chamber 111 to another chamber or portion of the sterilization biological indicator 100, such as the first chamber 109. The second fluid path 162 can be additionally positioned to allow gas that was previously present in the second chamber 111 to be displaced and out of the second chamber 111, for example, when the sterilizer and / or liquid 122 is moved to the second chamber 111. As such, the second fluid path 162, which is described in more detail below, can serve as an internal vent in the sterilization biological indicator 100.
In some embodiments, substrate 119 may provide a physical barrier or blockage between the first chamber 109 and the second chamber 111 that may allow at least one of the following: to control the sterilant release rate / extermination rate at which the sterilizer is released in the second chamber 111; controlling the diffusion of spores 115 and / or detectable products out of the second chamber 111; controlling the rate of release of liquid 122 into the second chamber 111 (and the spores 115) when the container 120 is in the second fractured state; or a combination of them.
Due to the fact that, in some embodiments, substrate 119 can provide a physical barrier to release liquid 122 into the second chamber 111 during activation (i.e., when container 120 is in the second state), opening 121 in the substrate 119 and / or the angle of the substrate 119 can be controlled to effect a desired liquid release rate. In addition, or alternatively, the second fluid path 162 can provide a vent for any gas or air that is trapped in the second chamber 111 to facilitate movement of the liquid 122 through or beyond substrate 119 and to the second chamber 111 when desired.
In addition, or alternatively, compartment 102 can be configured (for example, formed from a suitable material and / or configured with microstructured grooves and other physical surface modifications) to facilitate the movement of liquid 122 to the second chamber 111 when desired.
In some embodiments, liquid 122 may include a nutrient medium for the spores, such as a germination medium that will promote the germination of the surviving spores. In some embodiments, liquid 122 may include water (or another solvent) which can be combined with nutrients to form a nutrient medium. Suitable nutrients may include nutrients needed to promote the germination and / or growth of surviving spores in a dry form (eg, powdered form, tablet form, caplet form, capsule form, film or coating, trapped in a microsphere or other support, another suitable shape or configuration, or a combination thereof) in reservoir 103, for example, in a region of the sterilization biological indicator 100 next to spores 115. The nutrient medium can generally be selected to induce germination and initial spore growth, if feasible. The nutrient medium can include one or more sugars, including, but not limited to, glucose, fructose, celibiose, or the like, or a combination thereof. The nutrient medium may also include a salt, including, but not limited to, potassium chloride, calcium chloride, or the like, or a combination thereof. In some embodiments, the nutrient may also include at least one amino acid, including, but not limited to, at least one of methionine, phenylalanine, and tryptophan.
In some embodiments, the nutrient medium may include indicator molecules, for example, indicator molecules that have optical properties that are altered in response to germination or spore growth. Suitable indicator molecules may include, but are not limited to, pH indicator molecules, enzyme substrates, DNA-binding dyes, RNA-binding dyes, other suitable indicator molecules, or a combination thereof.
As shown in Figures 4 to 10, the biological sterilization indicator 100 can additionally include an insertion element 130. In some embodiments, the insertion element 130 can be adapted to hold or transport container 120, so that container 120 is kept intact in a location separate from spores 115 during sterilization. That is, in some embodiments, the insert 130 may include (or function as) a carrier 132 (see Figure 4) for the container 120, particularly, before the container 120 is fractured during the activation step (i.e., the step in which liquid 122 is released from container 120 and introduced to spores 115, which can occur after a sterilization process). In some embodiments, the insert 130 may be additionally adapted to allow the container 120 to move at least a little in the compartment 102, for example, longitudinally relative to the compartment 102. The insert 130 of the illustrated embodiment is described in further details below. Examples of other suitable insertion elements and carriers are described in copying PCT publication No. WO 2011/011189.
In some embodiments, the biological sterilization indicator 100 may additionally include a spore carrier 135, as shown in Figures 4 to 7 and 9. However, in some embodiments, the insert 130 may be modified to include a portion adapted to accommodate spores 115. For example, in some embodiments, the insert 130 and the spore carrier 135 can be integrally formed as an insert element comprising a first portion adapted to contain and eventually break container 120, when desired, and a second portion adapted to house the spores 115 in a region of the biological sterilization indicator 100 that is separated from the container 120 during sterilization (i.e., before breaking).
As shown in Figures 4 to 7 and 9, the spore carrier 135 can include a spore reservoir 136 (which can also be called a depression, hole, cavity, recess or the like), in which the spores 115 can be positioned directly or on a substrate. In embodiments that employ a nutrient medium that is positioned to be mixed with liquid 122 when it is released from container 120, the nutrient medium can be positioned close to or within the spore reservoir 136, and the nutrient medium can be mixed with (for example, dissolved in) water when water is released from container 120. Only as an example, in modalities in which the nutrient medium is supplied in a dry form, the dry form may be present within the reservoir 103 , the spore reservoir 136, on a substrate for the spores, or a combination thereof. In some embodiments, a combination of liquid and dry nutrient media can be employed.
In some embodiments, the spore reservoir 136 has a volume of at least about 1 microliter, in some embodiments, at least about 5 microliters, and in some embodiments, at least about 10 microliters. In some embodiments, the spore reservoir 136 has a volume no greater than about 250 microliters, in some embodiments no greater than about 175 microliters, and in some embodiments, no greater than about 100 microliters.
As shown in Figures 7 and 9, in some embodiments, the biological sterilization indicator 100 may additionally include a rib or protuberance 165 that can be coupled to or integrally formed with a wall 108 of compartment 102, which can be positioned to hold the carrier of spores 135 at a desired location in compartment 102 and / or at a desired angle or orientation, for example, in relation to the detection systems (for example, optical detection systems) of the reading apparatus 12.
As shown in Figures 4 to 7 and 9, the second portion 106 of compartment 102 can be adapted to be coupled to the first portion 104. For example, as shown, the second portion 106 can be adapted to be coupled to the upper portion 116 (for example, example, the first end 101) of the first portion 104 of compartment 102. In some embodiments, as shown in Figures 4 to 7, the second portion 106 may be in the form of a lid that can be sized to receive at least a portion of the first portion 104 of compartment 102.
As shown in Figures 4 to 5 and 7 to 8, during sterilization and before activation, the second portion 106 can be in a first "not activated" position 148 relative to the first portion 104, and the container 120 can be in a first state intact. As shown in Figure 9, the second portion 106 of compartment 102 can be moved to a second "activated" position 150 (for example, where the second portion 106 is completely lowered) relative to the first portion 104, and the container 120 can be in a second fractured state. For example, after sterilization, the biological sterilization indicator 100 can be activated by moving the second portion 106 from the first position 148 to the second position 150 (that is, a sufficient amount) to cause the container 120 to break and release the liquid 122 from the container 120, to allow the liquid 122 to be in fluid communication with the spores 115. The biological sterilization indicator 100 can be activated before the positioning of the biological sterilization indicator 100 in a cavity of a reading device, after the positioning of the biological sterilization indicator 100 in the cavity, or according to the biological sterilization indicator 100 is positioned in the cavity (that is, the biological sterilization indicator 100 can be slid in place on the reading device, and the second portion 106 can continue to be pressed until it is in its second position 150, for example, in which the bottom of the vity provides sufficient strength to move the second portion 106 to its second position 150). The second position 150 can be located closer to the closed end 105 of the first portion 104 of the sterilization biological indicator 100 than the first position 148.
As shown in the illustrated embodiment, in some embodiments, the first portion 104 of compartment 102 may include a step, suspension or transition from round to plane 152. Step 152 is shown to be exposed when the second portion 106 is in its first position 148 and as being hidden or covered when the second portion 106 is in its second position 150. As such, step 152 can be detected to determine whether the second portion 106 is in the first position 148 (i.e., the sterilization biological indicator 100 is disabled), or is in the second position 150 (that is, the biological sterilization indicator 100 is activated). The use of such features of the biological sterilization indicator 100 to determine a status of the biological sterilization indicator 100, for example, to confirm that the biological sterilization indicator 100 has been activated, is described in more detail in US Copending Order No. 61 /409,042. The longitudinal position of step 152 is shown by way of example only; however, it should be understood that step 152 may instead be located in a different longitudinal position (for example, closer to the closed end 105 of the biological sterilization indicator 100), or, in some embodiments, the transition of a portion round to a flat face can be gradual, tapered or highlighted.
A variety of coupling means can be employed between the first portion 104 and the second portion 106 of compartment 102 to allow the first portion 104 and the second portion 106 to be removably coupled to each other, including, but not limited to , gravity (for example, a component can be fixed on top of another component, or a joined portion of them), thread threads, press fit couplings (sometimes called “friction fit coupling” or “friction coupling”) forced adjustment ”), snap-fit coupling, magnets, adhesives, thermal bonding, other suitable removable coupling means, and combinations thereof. In some embodiments, the biological sterilization indicator 100 does not need to be reopened and the first portion 104 and the second portion 106 do not need to be coupled to each other, but instead can be coupled permanently or semi-permanently to each other. These permanent or semi-permanent coupling means may include, but are not limited to, adhesives, sutures, staples, threads, nails, rivets, headless nails, crimping, welding (eg, sonic welding (eg, ultrasonic)), any heat seal technique (for example, heat and / or pressure applied to one or both components to be coupled), press fit coupling, press fit coupling, heat sealing, other permanent or semi-permanent coupling means and combinations of the same. The person skilled in the art will recognize that some of the semi-permanent or permanent coupling means can also be adapted to be removable and vice versa, and are categorized in this way for example only.
As shown in Figures 7 and 9, the second portion 106 can be movable between a first longitudinal position 148 with respect to the first portion 104 and a second longitudinal position 150 with respect to the first portion 104; however, it should be understood that the biological sterilization indicator 100 could instead be configured differently, so that the first and second positions 148 and 150 are not necessarily longitudinal positions with respect to one or both of the first portion 104 and the second portion 106 of compartment 102. The second portion 106 may additionally include a seal 156 (for example, a projection, protrusion, flap, flange, seal ring or the like, or combinations thereof) which may be to come into contact with contact with the first end 101 of the first portion 104, and in particular, an open upper end 157 of the first portion 104 to close or seal (e.g., hermetically seal) the biological sterilization indicator 100 after the second portion 106 has been moved to the second position 150 and liquid 122 has been released from container 120 (that is, when container 120 is in a second and fractured condition). That is, the spores 115 can be sealed from the environment when the container 120 is in the second state. The seal 156 can take a variety of shapes and is shown in Figures 7 and 9 by way of example as forming a ring or internal cavity which together with the wall 110 of the second portion 106 is dimensioned to receive the upper end 157 of the first portion 104 of compartment 102 to seal the biological sterilization indicator 100.
In some embodiments, one or both of the seal 156 and the upper end 157 may additionally include a structure (for example, a protuberance) configured to engage the other between the upper end 157 and the seal 156, respectively, in order to couple the second portion 106 of compartment 102 to first portion 104 of compartment 102.
In addition, in some embodiments, the second portion 106 of compartment 102 can be coupled to the first portion 104 of compartment 102 to seal the biological sterilization indicator 100 from the environment after activation. Such a seal can inhibit contamination, evaporation or spillage of liquid 122 after it has been released from container 120, and / or can inhibit contamination inside the biological sterilization indicator 100. Insert element 130 will now be described in more detail.
As shown in Figures 4 to 5 and 7, during sterilization and before activation, the second portion 106 can be in a first position 148 in relation to the first portion 104. In the first position 148, the container 120 can be kept intact in a separate position from lower portion 114, second chamber 111 or spores 115, and liquid 122 may be contained in container 120.
As shown in Figure 9, after sterilization, the biological sterilization indicator 100 can be activated to release liquid 122 from container 120 to move liquid 122 to the second chamber 111. That is, the second portion 106 of compartment 102 can be moved to a second position 150 with respect to the first portion 104. When the second portion 106 is moved from the first position 148 to the second position 150, the seal 156 of the second portion 106 of compartment 102 can engage the upper end 157 of the first portion 104 to seal the reservoir 103 of the sterilization biological indicator 100 from the environment. In these embodiments, the second portion 106 can be reversibly engaged with the first portion 104 in the second position 150, and in some modalities, the second portion 106 may be irreversibly engaged with the first portion 104. However, it must be understood that the structures and coupling means for the first portion 104 and the second portion 106 are shown in the embodiment illustrated by way of example only and any of the coupling means described above may instead be employed between the first portion 104 and the second portion 106 of compartment 102. Insertion element 130 can be adapted to hold or transport container 120, so that container 120 is kept intact in a location separate from spores 115 during sterilization. That is, as mentioned above, the insert 130 may include (or function as) a carrier 132 for the container 120, particularly, before the container 120 is fractured during the activation step (i.e., the step where the liquid 122 is released from container 120 and introduced to spores 115, which typically after a sterilization process).
In addition, the insert 130 can be adapted to keep the container 120 intact in a position in the compartment 102 that maintains at least minimum spacing (for example, a minimum cross-sectional area of space) between the container 120 and the compartment 102 and / or between container 120 and any other components or structures in compartment 102 (for example, at least a portion of the insert 130, such as carrier 132, etc.), for example, to maintain a substantially sterilizing path constant 164 in the biological sterilization indicator 100. In some embodiments, the insert 130 may be adapted to hold the container 120 in a substantially consistent location in compartment 102.
In some embodiments, as shown in Figure 6, at least a portion of compartment 102 may include a tapered portion 146 where compartment 102 (for example, wall 108 and / or an internal surface thereof) generally tapers in the direction longitudinal DL of compartment 102. As a result, the cross-sectional area in compartment 102 can generally decrease along the longitudinal direction DL.
In some cases, without providing the means to maintain at least minimal spacing around the container 120 (for example, between the container 120 and the surrounding structure), there may be a possibility that the container 120 will be positioned in the compartment 102 (for example, example, in the tapered portion 146) so that it obstructs or blocks the sterilizing path 164. However, the biological sterilization indicator 100 of the present description is designed to prevent this from occurring. For example, in the illustrated embodiment, the insert 130 (and particularly, the carrier 132) can be configured to keep the container 120 out of the tapered portion 146 of compartment 102, so that at least one area of the minimum cross section is maintained around container 120 in any orientation of the sterilization biological indicator 100 prior to activation. For example, in the embodiment illustrated in Figures 4 to 8, even if the biological sterilization indicator 100 is turned upside down, the container 120 may fall out of contact with the insert 130, but in no orientation, the container 120 is moved closer to the tapered portion 146 or spores 115 until activation of the sterilization biological indicator 100. In addition, until activation, at least a minimum spacing (and particularly, an area of the cross section of that spacing) between the container 120 and the compartment 102 and / or the insert 130 can be maintained to provide a substantially constant sterilizing path 164, for example, around the container 120, through the first fluid path 160 and into the second chamber 111.
In some embodiments, the sizing and relative positioning of the components of the biological sterilization indicator 100 can be configured so that, prior to activation, the container 120 is kept intact in a location substantially consistent with the biological sterilization indicator 100. This configuration can provide a sterilizing path 164 substantially constant and can hold the container 120 in a position so that the container 120 is not able to move substantially, or in any way, on the biological sterilization indicator 100 prior to activation.
In some embodiments, at least a portion of the insert 130 may be adapted to allow the container 120 to move in compartment 102, for example, longitudinally with respect to compartment 102, between a first (longitudinal) position where container 120 it is intact and a second position (longitudinal) in which at least a portion of the container 120 is fractured. For example only, the insert 130 may include one or more projections or arms 158 (two projections 158 spaced on the container 120 are shown by way of example only) adapted to contain and support the container 120 prior to activation and to allow container 120 to move in compartment 102 during activation, for example, when the second portion 106 is moved relative to the first portion 104 of compartment 102. The projections 158 can also be adapted (for example, shaped and / or positioned ) to break container 120 in a desired manner when the sterilization biological indicator is activated. As a result, the insert 130 can sometimes function to keep the container 120 intact before activation, and can function to break the container 120 during activation. As a result, the insert 130, or a portion thereof, may sometimes be called a "carrier" (for example, carrier 132) and / or a "breaker".
As an example only, the projections 158 are shown in Figures 4 and 6 to 10 as being coupled to a base or support 127 adapted to be in a boundary position with the partition wall 118. For example, the base 127 can be dimensioned for be received in reservoir 103 and sized to sit on top, be in a borderline position or otherwise cooperate with or be coupled to the separation wall 118. Such coupling with an internal structure of the sterilization biological indicator 100 can provide strength and strength necessary for the rupture of the container 120 when desired. In some embodiments, however, the insert 130 does not include the base 127, and the projections 158 may be coupled to or form a portion of the compartment 102. In some embodiments, the insert 130 is integrally formed with or provided by the compartment 102.
As shown, the insert 130 may additionally include a side wall 131 that connects the projections 158 and is shaped to accommodate an internal surface of the housing 102 and / or an external surface of the container 120. Such a side wall 131 can provide support and rigidity for projections 158 for safe fracture assistance of container 120 in a consistent manner. The side wall 131 can also be shaped and dimensioned to guide the container 120 in a desired manner as it is moved in the compartment 102 during activation, for example, to bring the projections 158 into contact in a desired manner to safely fracture the container 120 Side wall 131 and / or wall 108 of compartment 102 (or an internal surface thereof) can also be shaped to define at least a portion of the second fluid path 162 of the sterilization biological indicator 100, for example, between a external surface of the insert 130 and an internal surface of the compartment 102. For example, in some embodiments, as shown in Figures 4 to 5, 8 and 9, the side wall 131 of the insert 130 may include a groove (or groove) , indentation or the like) 169 configured to form at least a portion of the second fluid path 162. The second fluid path 162 may function ionize as an “internal breather” or a “breather channel” within the biological sterilization indicator 100 to allow gas (for example, displaced gas, such as air that has been trapped in the second chamber 111 (for example, near the closed end) 105 of the biological sterilization indicator 100) exhaust from the second chamber 111 of the biological sterilization indicator 100. In some embodiments, the second fluid path 162 may provide an exhaust, or internal breather, for a gas present in the second chamber 111 during activation to facilitate the movement of liquid 122 into the second chamber 111 of the first chamber 109 as it is released from container 120. In addition or alternatively, in some embodiments, the second fluid path 162 may provide an exhaust, or internal breather, for a gas present in the second chamber 111 during sterilization to facilitate the movement of a sterilizer to the second chamber 111 of the biological sterilization indicator 100 and for spores 115, with more efficient sterilization penetration in the second chamber 111.
As an example only, as shown in Figures 5 and 10, the second fluid path 162 can be at least partially defined by a portion of the insert 130 (for example, channel 169) and a channel (or groove, recess or the like) 163 formed on the wall 108 of the compartment 102 (for example, on an internal surface of the wall 108). However, it should be understood that in some embodiments, the second fluid path 162 may be formed entirely from compartment 102 or from various combinations of other components of the sterilizing biological indicator 100 so that the second fluid path 162 provides fluid connection between the second chamber 111 and another internal portion or region of the biological sterilization indicator 100. For example, the second fluid path 162 does not need to be formed by both compartment 102 and insertion element 130, but it can be formed by one of these components , or other components. In addition, as shown in Figures 5 and 10, channel 163 that defines at least a portion of the second fluid path 162 is molded on an outer surface and an inner surface of compartment 102, so that channel 163 is visible inside and outside of compartment 102. However, the outer surface of compartment 102 need not include such a shape, and preferably, in some embodiments, the outer surface of compartment 102 may remain substantially uniform or unchanged, and the inner surface of compartment 102 ( for example, a wall 108 of compartment 102) can include channel 163.
In addition, in some embodiments, neither the insertion element 130 nor the compartment 102 includes the channel 169 or the channel 163, respectively, but preferably the insertion element 130 and the compartment 102 are shaped and dimensioned so that a space either the gap is provided between the insert 130 and the compartment 102 which is in fluid communication with the second chamber 111, and such a gap or gap works with the second fluid path 162.
As additionally shown in Figures 7 and 9, in some embodiments, the first fluid path 160 and / or the second fluid path 162 can be at least partially defined by one or more of the wall 118, the substrate 119, the insert 130 and compartment 102. In addition, at least one of the first fluid path 160 and the second fluid path 162 can be defined at least partially by the spore carrier 135, or a portion thereof.
In some embodiments, the biological sterilization indicator 100 may include the following components arranged in the following order when the container 120 is in a first non-fractured state: the closed end 105 of compartment 102 of the biological sterilization indicator 100, the second chamber 111, the substrate 119, the insert 130, the first chamber 109, the container 120, the open end 101 of compartment 102 (or the second portion 106 of compartment 102).
As shown in the illustrated embodiment, the second fluid path 162 may allow the second chamber 111 to vent another portion of the biological sterilization indicator 100, such as the first chamber 109. In some embodiments, the second fluid path 162 may come out of the second chamber 111 in a position located above (for example, vertically above) the position where the first fluid path 160 enters the second chamber 111, particularly in embodiments where the second fluid path 162 ventilates the second chamber 111 back to the first chamber 109. Otherwise, in some embodiments, the second fluid path 162 may extend from the second chamber 111 to a position (for example, a fourth L4 level, described below) on the sterilization biological indicator 100 that is above the position (for example, a first U level or a second L2 level, described below) where the first fluid 160 enters the second chamber 111. Additionally, in some embodiments, the position in which the second fluid path 162 enters the first chamber 109 can be located above (for example, vertically above) the position in which the first fluid path 160 enters the second chamber 111.
In some embodiments, the first fluid path 160 can be positioned to fluidly couple the second chamber 111 to a proximal portion of the biological sterilization indicator 100 (for example, a portion of the first chamber 109 that is located proximally or adjacent to the second chamber 111, for example, at the first level L1 and / or at the second level L2), and the second fluid path 162 can be positioned to fluidly couple the second chamber 111 to a distal portion of the sterilization biological indicator 100 (i.e., a portion of the first chamber 109 which is additionally located from the second chamber 111, for example, on a third level L3, described below, and / or on a fourth level L4). As a result, the position at which the second fluid path 162 enters the first chamber 109 can be further positioned from the second chamber 111 relative to the position at which the first fluid path 160 enters the second chamber 111.
More specifically and for example only, with reference to Figures 7 and 9, in some embodiments, the fluid can enter the second chamber 111 in a variety of locations, such as the first level, height or position (for example, longitudinal position) Q generally located in front of the insert 130, substrate 119, compartment 102 and / or the second chamber 111, as well as on the second level, height or position (for example, longitudinal position) L2 located approximately at the level of the opening 121 on substrate 119. As described above, it should be understood that the variety of openings and spaces between the first chamber 109 and the second chamber 111 that allow the fluid to move into the second chamber 111 can collectively be called the first fluid path 160. As further illustrated in Figure 7, in some embodiments, gas (for example, displaced gas) can leave the second chamber 111 through the second path fluid 162 (i.e., as the fluid moves to the second chamber 111 through the first fluid path 160) at the third level, height or position (for example, longitudinal position) L3 generally located at the rear of the insert 130, substrate 119, compartment 102 and / or second chamber 111.
In the vertical orientation of the biological sterilization indicator 100 shown in Figures 7 and 9, the third level L3 is located at or above both the first level U and the second level L2. In addition, in some modalities, the third level L3 can still be located at or above both the first level U and the second level L2 in operation of the biological sterilization indicator 100 (for example, when seated in a cavity of a reading device , during sterilization, and / or during activation). That is, in some modalities, the biological sterilization indicator 100 can be tilted in operation (for example, towards the left side of Figure 7 or 9, towards the direct side of Figure 4 or 6, on the page of Figure 4 or 6, and / or off the page of Figure 7 or 9). The first, second and third levels L1; L2 and L3 are shown as an example only; however, it should be understood that the exact location at which the first fluid path 160 enters the second chamber 111 and / or the exact location at which the second fluid path 162 exits the second chamber 111 may be different from that illustrated in Figures 7 and 9.
As shown in Figures 7 and 9, the second fluid path 162 is at least partially defined by channel 169 of insertion element 130 and / or channel 163 of compartment 102, which will be generically referred to simply as "the channel" in the following discussion , which can be interpreted as referring to at least a portion of channel 163 and / or channel 169 of the illustrated modality. In the illustrated embodiment, the channel has an entrance that can be described as being located anywhere in the second chamber 111, or in the third level L3, and an exit that is usually positioned in the fourth level, height or position (for example, longitudinal position ) L4. As shown in Figures 7 and 9, the channel outlet position (that is, the fourth level L4) is generally located above the position where the first fluid path 160 connects with the second chamber 111 (that is, the first level U and / or the second level L2), for example, in operation of the biological sterilization indicator 100.
Otherwise, the first fluid path 160 can be positioned to fluidly couple the second (lower) end 113 of the first chamber 109 to the first (upper) end 124 of the second chamber 111. The second fluid path 162, on the other hand, it can be positioned to fluidly couple the second chamber 111 (e.g., the first (upper) end 124 of the second chamber 111) to an upper portion (e.g., the first (upper) end 112) of the first chamber 109.
Additionally, in some embodiments, the position or level at which the second fluid path 162 (or the channel) connects with the second chamber 111 can be described as being located in the portion of the second chamber 111 that is the last to be filled with the liquid 122 when container 120 is in its second fractured state.
In some embodiments, when the container 120 is in the second fractured state, and the second chamber 111 is at least partially filled with liquid 122, the liquid 122 may have a level, height or position (e.g., longitudinal position) L, and the second fluid path 162 may extend between a position below level L and a position above level L. As a result, as the second chamber 111 is filled with liquid 122 when the container is in the second state, the second chamber 111 it can be continuously ventilated by the second fluid path 162.
In some embodiments, the first fluid path 160 can function as the main fluid communication path between the first chamber 109 and the second chamber 111, and the second fluid path 162 can serve as an accessory or secondary fluid communication path between the second chamber 111 and first chamber 109 (for example, when the second fluid path 162 exits in the first chamber 109 and not in another portion of the biological sterilization indicator 100). In such embodiments, the space, volume and / or collective area of the second fluid path 162 may be substantially smaller than that of the first fluid path 160. In some embodiments, at least a portion of the first fluid path 160 and the second path of fluid 162 can be described as being substantially isolated from each other or as being substantially parallel and not intersecting. In some embodiments, the first fluid path 160 and the second fluid path 162 may extend substantially and longitudinally (for example, substantially parallel to the longitudinal direction DL) between the first chamber 109 and the second chamber 111.
That is, in general, the biological sterilization indicator 100 which includes (1) a first fluid path, such as the first fluid path 160, configured to accommodate at least a major part of the fluid movement from the first chamber 109 to the second chamber 111, and (2) a second fluid path, such as the second fluid path 162, configured to vent gas from the second chamber 111 would have advantages over a biological sterilization indicator 100 that included only an internal chamber or only a fluid path connecting the first chamber 109 and the second chamber 111, so that the gas would have to leave the second chamber 111 through the same fluid path as the fluid enters the second chamber 111.
By configuring the first fluid path 160 and the second fluid path 162 as shown in the illustrated embodiment, in some embodiments, the biological sterilization indicator 100 can at least partially eliminate any air jamming effects that may occur as a result of attempt to move a sterilizer and / or liquid 122 into the second chamber 111. In addition, in some embodiments, the second fluid path 162 may allow the biological sterilization indicator 100 to be activated, and liquid 122 to be moved to the second chamber 111 due to gravity, while the biological sterilization indicator 100 remains in the same orientation (for example, a substantially vertical orientation, as shown in Figures 4 to 5, 7 and 9), without requiring the biological sterilization indicator 100 to be turned upside down or otherwise reoriented to move liquid 122 to the second chamber 111.
In continuous reference to the insert 130, the projections 158 of the insert 130 are shown to be relatively rigid and stationary. That is, in some embodiments, the projections 158 may not be adapted to substantially flex, distort, deform or otherwise orient container 120 as it is moved in compartment 102. Preferably, in some embodiments, as shown in Figures 4 to 7 and 9, the projections 158 can be configured to have an upper end 159 at the top where the container 120 can be positioned and kept intact before activation. As shown in Figures 4 to 5 and 7, in some embodiments, the projections 158 can be positioned to fracture the container 120 at its radius end, for example, when an oblong-shaped container or capsule 120 is employed.
A potential advantage of having projections 158 of at least a portion of carrier 132 is that the bottom of container 120 can be unrestricted when container 120 is fractured, so that liquid 122 can be released from container 120 and moved to the spores 115 with relative ease and reliability.
In such embodiments, the insert 130 can be used to fracture the container 120 in a direction that is substantially perpendicular to a flat side of the container 120, for example, when an oblong or capsule-shaped container 120 is employed. In such embodiments, the fracturing of the container 120 along its side can be achieved, along with the maintenance of some open spaces around the lower end of the container 120 to facilitate the movement of the liquid 122 of the container 120 towards the proximity of the spores 115 when container 120 is fractured.
As mentioned above, the projections 158 can be adapted to fracture the container 120 as the container 120 is moved in relation to the compartment 102 (for example, along the longitudinal direction DL), for example, in response to the movement of the second portion 106 of the compartment 102 in relation to the first portion 104 of compartment 102 (for example, from the first position 148 to the second position 150).
In some embodiments, the projections 158 may include one or more edges (for example, tapered edges) or points or otherwise be configured to concentrate the crushing force to increase the pressure in the container 120 in the regions adjacent to the projections 158, and to facilitate the fracturing of container 120 more easily and in one or more desired regions. In some embodiments, such a concentration of force may reduce the total effort or force required to move the second portion 106 relative to the first portion 104 and to fracture the container 120 (or a portion thereof).
As shown in Figures 4 to 7 and 9, the projections 158 are integrally formed with the base 127 of the insert 130; however, it should be understood that projections 158 can instead be integrally formed with wall 108 of compartment 102. In addition, in some embodiments, projections 158 can be coupled to compartment 102, or projections 158 and the base 127 can be provided by separate inserts. In such embodiments, the projections 158 can be a separate insertion element, or multiple projections 158 can be provided by one or more insertion elements. In addition, the insert 130 can be configured to be in a position bordering the wall 118 to inhibit movement of the first portion of the insert 130 towards the proximity of spores 115 (e.g., the bottom 114 of compartment 102) .
In addition, in some embodiments, as shown in Figures 4 to 7 and 9, the projections 158 may extend a distance along the longitudinal direction DL, and the length and / or thickness (for example, which may vary over time). length) of the projections 158 can be customized to control the fracturing of the container 120 in a desired position in compartment 102 and in a desired manner. The configuration of the projections 158 is shown in Figures 4 to 10 only as an example.
In general, each of the projections 158 is shown only as an example as increasing in thickness (for example, inward towards container 120 or center of compartment 102) along the longitudinal direction DL towards spores 115. Such a configuration it can decrease the area of the cross section that is available for the container 120, as the container 120 is moved towards the spores 115, for example, in response to the movement of the second portion 106 to the second position 150.
Additionally, the biological sterilization indicator 100 is shown in Figures 3 to 10 including two projections 158 and a side wall 131 just as an example, but it should be understood that one projection 158 or as many as are structurally possible, and other configurations, can be employed. In addition, the projections 158 can be shaped and dimensioned as desired, depending on the shape and dimensions of the compartment 102, the shape and dimensions of the container 120, the shape and dimensions of the insert 130 and / or the way and shape of the container. desired position to fracture the container 120.
As mentioned above, in some embodiments, at least a portion of compartment 102 can be tapered (see, for example, tapered portion 146 in Figure 6). As a result, the cross-sectional area in compartment 102 can generally decrease along the longitudinal direction DL. However, it must be understood that the internal dimensions of compartment 102 can generally decrease in the tapered portion along the longitudinal direction Di without changing the external dimensions of compartment 102. In some embodiments, the outer dimensions of compartment 102 may be uniform across its length, even though the inner portion of compartment 102 tapers along its length. In some embodiments, the one or more projections 158 alone may vary in thickness (that is, towards the container 120, for example, in a radial direction) along the longitudinal direction DL, so that the cross-sectional area available for container 120 generally decreases as container 120 is moved in compartment 102 during activation, even if the dimensions of compartment 102 do not change (for example, even if compartment 102 does not include any tapered portion 146, internally or externally).
As shown in Figures 4 to 10, the upper end 159 of each of the projections 158 includes a round, curved or arched surface, which can facilitate the movement of the container 120 from the first position 148 in which the container 120 is at least partially seated above from the upper end 159 of the projection 158 to a position where the container 120 is forced, at least partially, into the region of the smaller cross-sectional area between the projections 158 (or between the wall 108 of the compartment 102 and one or more projections 158 ). In addition, the rounded top end 159 can inhibit premature rupture of container 120, which can inhibit premature activation of the biological sterilization indicator 100 (i.e., premature release of liquid 122).
In some embodiments, as shown in figure 6, the insert 130 can be sized and shaped to allow the container 120 to be kept above the projections 158 and outside the adjacent region of any portion of a surface facing inwards of one or more from projections 158 to inhibit accidental or premature activation of the biological sterilization indicator 100. This configuration can also inhibit inadvertent rupture due to shock or expansion of the material (for example, due to exposure to heat during a sterilization process). Carrier 132, which can be formed at least partially by the upper ends 159 of projections 158, can be configured to hold a lower portion of container 120, and projections 158 can be positioned to fracture container 120 at a location near the bottom of the container. container 120 as it is positioned in compartment 102. This configuration can allow container 120 to rupture near its bottom and can facilitate the removal of liquid 122 from container 120, which can improve the availability of liquid 122 to spores 115, and can improve the reliability of the release of liquid 122 in fluid communication with spores 115 (for example, with spore reservoir 136). This configuration is shown by way of example only, however, it should be understood that the projections 158 can be configured and positioned to fracture the container 120 in any desired manner.
Some embodiments of the present disclosure provide an ideal and safe rupture of a frangible container 120 with relatively weak force, while accentuating the transfer of liquid 122 to the spore region (for example, the second chamber 111 of compartment 102) of the sterilization biological indicator 100 , and / or accentuates the confinement of liquid 122 in the spore region of the biological sterilization indicator 100. In addition, some embodiments of the present disclosure operate to conduct a liquid to a particular area of the biological sterilization indicator 100, such as a detection chamber (e.g., second chamber 111) of the biological sterilization indicator 100.
In the embodiment illustrated in Figures 4 to 10, the insert 130 is illustrated as including two projections 158 that are approximate and equally spaced on the container 120 and / or on the side wall 131. However, in some embodiments, the side wall 131 it may include a solid projection (for example, substantially annular or semi-annular) 158 that extends radially into the side wall 131. Additionally, in some embodiments, the side wall 131 may extend further around the inner surface of compartment 102 that has been illustrated. However, the use of one or more narrower projections 158 (for example, in an angular dimension), such as those shown in figures 4 to 10, can provide a substantially constant or substantially unobstructed path of sterilizer 164 around the container 120.
If the insert 130 includes one or more projections 158 or side walls 131, the insert 130 can be configured to hold the container 120 in the compartment 102 in a consistent location to provide a substantially constant sterilizing path 164 during sterilization. For example, instead of allowing the container 120 to move or reach (for example, radially and / or longitudinally) in compartment 102 before activation (for example, during sterilization), the insert 130 can hold the container 120 in a substantially consistent position, which allows a sterilizer to have a substantially consistent and relatively unobstructed path between an outer surface of compartment 120 and an inner surface of compartment 102, with little or no opportunity for inadvertent blocking.
As shown in the illustrated embodiment, the insert 130 may additionally include one or more projections 161 positioned horizontally or perpendicularly substantially with respect to the longitudinal direction DL of a biological sterilization indicator (for example, when the insert 130 is positioned in a biological sterilization indicator). Projections 161 can be called "second projections" or "horizontal projections", while projections 158 used to contain and / or break container 120 can be called "first projections" or "vertical projections". Second projections 161 are not angled down like base 127. As a result, second projections 161 can be used for a variety of purposes. For example, the second projections 161 can stabilize the insert 130 (for example, aid in retaining the insert 130 in a desired position in compartment 102 of the sterilizing biological indicator 100) under the fracturing force of the container 120. In addition In addition, the second projections 161 can work to retain and / or collect the fractured portions of the container 120 after it has been fractured to inhibit the movement of these portions in the vicinity of the spores in the biological sterilization indicator, which could negatively affect spore growth. and / or detection of spore growth. Other shapes and configurations of the second projections 161 can be employed that will still allow fluid movement downward to spores 115 while inhibiting solid downward movement to spores 115.
In some embodiments, the insert 130 (for example, the base 127) can be adapted to one or more of facilitating or allowing movement of fluid (e.g., movement of liquid 122) into the second chamber 111 (i.e., the lower portion 114) of compartment 102; minimizing the movement of fractions or portions (e.g., solids) from the fractured container 120 to the second chamber 111 of compartment 102, that is, collecting and / or retaining the portions of the fractured container 120; and / or to minimize the diffusion of spores 115 and / or signals out of the second chamber 111 of compartment 102. For example, in some embodiments, base 127 can be configured to function as a grid or filter. In some embodiments, spore growth is determined by fluorescent indicators / molecules (eg, fluorophores) or other markers. In some embodiments, if the liquid level after activation of the sterilization biological indicator 100 is above the spore 115 site, these molecules or markers, or the spores 115 themselves, can move or diffuse away or out from the spore reservoir 136 and potentially out of the second chamber 111 of compartment 102. As a result, the portions of the sterilization biological indicator 100 (for example, the insert 130) can be configured to inhibit unwanted diffusion of various indicators, molecules and / or markers outside the second chamber 111 of the biological sterilization indicator 100. In some embodiments, as described above, substrate 119 can also inhibit such undesirable diffusion.
In the embodiment illustrated in Figures 4 to 7, the base 127 of the insert 130 is generally U-shaped or horseshoe-shaped and includes a central opening 177 (see Figure 5) that facilitates the movement of the sterilizer towards the spores 115 during sterilization and the movement of liquid 122 towards spores 115 during activation. The horseshoe shape of the base 127 can increase the gap between the upper portion 116 (i.e., the first chamber 109) and the lower portion 114 (i.e., the second chamber 111) of compartment 102; however, this format is shown as an example only, and other formats can be used.
In some embodiments, the insert 130 may be described as including one or more downwardly extending projections 127 adapted to be in a boundary position or otherwise coupled to the wall 118 or another internal structure of the sterilizing biological indicator 100 for providing a base or support for the insert 130, to inhibit the movement of the insert 130 and the container 120 relative to the compartment 102 prior to activation, and / or to provide resistance or strength to aid in the rupture of the container 120 during activation. As a result, in some modalities, base 127 may instead be called “third projections” 127.
As shown in the illustrated embodiment, in some embodiments, the insert 130 may be configured to reside entirely in the first chamber 109 of the sterilization biological indicator 100, so that the insert 130 does not extend into the second chamber 111 where it would potentially interfere with interrogation or detection processes. In addition, the insert 130 can be configured to inhibit movement of other portions of the biological sterilization indicator 100 (for example, the fractured container 120) to the second chamber 111. The insert 130 of the illustrated embodiment is generally symmetrical about a longitudinal center line of symmetry, so that there are two identical first projections 158, two identical second projections 161 and two identical third projections 127. However, the insert 130 does not need to include any lines of symmetry, and the first projections 158 do not they need to be equal to each other, the second projections 161 need not be equal to each other, and the third projections 127 need not be equal to each other. The insertion elements 130 and the projections 158, 161 and 127 can be dimensioned and positioned to control the sterilizing path 164, for example, to adjust the death / survival rate of the biological sterilization indicator 100, to inhibit the inadvertent fracture of the container 120, to facilitate movement of container 120 in compartment 120, to join with or engage with compartment 102, and / or to control rupture of container 120.
For example only, the insert 130 is shown as a unitary device that includes at least the following: means for holding the container 120, before activation, for fracturing the container 120, during activation; to allow movement of container 120 in compartment 102; to provide a substantially constant sterilizing path 164; to collect and / or retain portions of the fractured container 120 after activation (or at least partially inhibit movement of portions of the fractured container 120 to the second chamber 111 of compartment 102); and / or to minimize spore diffusion 115 and / or signals from the second chamber 111 to the first chamber 109 of compartment 102 after activation. However, it should be understood that in some embodiments, the insert 130 may include multiple portions that may not be part of a single unitary device, and each portion may be adapted to perform one or more of the above functions. The insertion element 130 is called the "insertion element" due to the fact that, in the illustrated embodiment, the device that performs the above functions is a device that can be inserted in the reservoir 103 (and, particularly, in the first chamber 109) of compartment 102. However, it should be understood that insertion element 130 may instead be provided by compartment 102 itself or another component of the biological sterilization indicator 100 and is not necessarily amenable to insertion into compartment 102. The term “Insertion element” will be described throughout this description for the sake of simplicity, but it should be understood that this term is not intended to limit, and it should be considered that other equivalent structures that perform one or more of the above functions can be used instead, or in combination with, the insert 130. In addition, in the illustrated embodiment, the insert 130 is liable to be inserted into, and removable from, compartment 102, and particularly, into and out of first portion 104 (and first chamber 109) of compartment 102. However, it should be understood that even if the insertion element 130 is insertable in compartment 102, the insert 130 may not be removable from compartment 102, but can instead be fixedly attached to compartment 102 in a way that inhibits the removal of insert 130 from compartment 102 after positioning the insert 130 in a desired location.
In some embodiments, at least a portion of compartment 102, for example, the lower portion 114 of compartment 102, may be transparent to a wavelength of electromagnetic radiation or a range of wavelengths (for example, transparent to visible light when they are optical detection methods), which can facilitate the detection of spore growth. That is, in some embodiments, as shown in Figures 6, 7 and 9, at least a portion of compartment 102 can include or form a detection window 167.
In addition, in some embodiments, as shown in Figure 6, at least a portion of compartment 102, for example, the lower portion 114 may include one or more planar walls 168. These planar walls 168 can facilitate detection, (for example, optical detection) of spore growth. In addition, as shown and described above, the wall 108 of the first portion 104 of compartment 102 may include one or more graduated or tapered regions, such as step 152, step 123, and a tapered wall, or step 170. The wall tapered 170 can work to reduce the thickness and total size of the lower portion, or detection portion, 114 of compartment 102, so that the outer dimensions of compartment 102 are reduced beyond the inner dimensions. Such a reduction in size and / or thickness of the lower portion 114 of the sterilization biological indicator 100 can facilitate detection. In addition, the fact of having one or more features, such as steps and / or tapered walls 123, 152, 170 can allow the biological sterilization indicator 100 to be coupled to a reader or detection device in just one orientation, so that the biological sterilization indicator 100 is “switched” in relation to a reading device, which can minimize user error and enhance the reliability of a detection process. In some embodiments, one or more portions of the biological sterilization indicator 100 can be switched in relation to a reading device. The biological sterilization indicator of the present description generally keeps the liquid 122 and the spores 115 separated but in relatively close proximity (for example, within the biological sterilization indicator 100 one-piece) during sterilization, so that the liquid 122 and spores 115 can be readily combined after exposure to a sterilization process. Liquid 122 and spores 115 can be incubated during a detection process (for example, the reading device 12 can incubate the biological sterilization indicator 100), or the biological sterilization indicator 100 can be incubated before a detection process . In some embodiments, during the spore incubation with liquid 122, an incubation temperature above room temperature can be used. For example, in some embodiments, the incubation temperature is at least about 37 ° C, in some embodiments, the incubation temperature is at least about 50 ° C (for example, 56 ° C), and in some embodiments, at least about 60 ° C. In some embodiments, the incubation temperature is not greater than about 60 ° C, in some embodiments, not greater than about 50 ° C, and in some embodiments, not greater than about 40 ° C.
A detection process can be adapted to detect a detectable change in the spores 115 (for example, from within the spore reservoir 136) of the liquid 122 surrounding the spores 115. That is, a detection process can be adapted to detect a variety of characteristics, including, but not limited to, electromagnetic radiation (for example, in the bands of ultraviolet, visible, and / or infrared light), fluorescence, luminescence, light scattering, electronic properties (for example, conductance, impedance, or similar , or combinations thereof), turbidity, absorption, Raman spectroscopy, ellipsometry, or the like, or a combination thereof. The detection of these characteristics can be performed by one or more of a fluorimeter, spectrophotometer, colorimeter, or similar, or combinations thereof. In some modalities, such as those that measure fluorescence, visible light, etc., the detectable change is measured by detection at a particular wavelength.
Spores and / or liquid 122 may be adapted (for example, labeled) to produce one or more of the following characteristics as a result of a biochemical reaction that is a sign of spore viability. As a result, no detectable change (for example, when compared to a baseline or antecedent reading) can mean an effective sterilization process, while a detectable change can mean an ineffective sterilization process. In some embodiments, the detectable change may include a rate at which one or more of the above characteristics are being changed (for example, increased fluorescence, decreased turbidity, etc.).
In some modalities, the spore viability can be determined by exploiting the enzymatic activity. As described in Matner et al., US Patent No. 5,073,488, entitled “Rapid Method for Determining Efficacy of a Sterilization Cycle and Rapid Read-out Biological Indicator”, which is incorporated here by way of reference, enzymes can be identified for a particular type of spore in which the enzyme has particularly useful characteristics that can be exploited to determine the effectiveness of a sterilization process. These characteristics may include the following: (1) the enzyme, when subjected to sterilization conditions that would be sufficient to decrease a population of 1 X 106 test microorganisms by about 6 records (that is, to a population of about zero when measured by the lack of flowering of microorganisms), has a residual activity that is equal to the "antecedent" as measured by the reaction with a substrate system for the enzyme; and (2) the enzyme, when subjected to sufficient sterilization conditions just to decrease the population of 1 X 106 test microorganisms in at least 1 record, but less than 6 records, has greater enzyme activity than the "antecedent" as measured by reaction with the enzyme substrate system. The enzyme substrate system can include a substance, or mixture of substances, that is influenced by the enzyme to produce a product modified by a detectable enzyme, as evidenced by a detectable change.
In some embodiments, the biological sterilization indicator 100 can be tested in a unilateral mode, in which the biological sterilization indicator 100 includes only a detection window (for example, detection window 167 of figure 6) that is positioned, for example , next to spores 115. In some embodiments, however, the biological sterilization indicator 100 may include more than one detection window (for example, a window formed by all or a portion of both parallel walls 168 of the lower portion 114 of the compartment 102), so that the sterilization biological indicator 100 can be tested through more than one detection window. In modalities that employ multiple detection windows, the detection windows can be positioned side by side (similar to the one-sided mode), or the detection windows can be oriented at an angle (for example, 90 degrees, 180 degrees, etc.) in relation to each other.
In general, spores 115 are positioned within spore reservoir 136 which is in fluid communication with reservoir 103. In some embodiments, spore reservoir 136 forms a portion of reservoir 103 (for example, a portion of second chamber 111) . As shown in figure 7, reservoir 103 is in fluid communication with the environment (for example, through opening 107) during sterilization to allow the sterilizer to enter reservoir 103 during the sterilization process to sterilize spores 115. The container 120 can be configured to contain liquid 122 during sterilization to inhibit fluid communication of liquid 122 with spores 115, reservoir 103, and sterilizer during sterilization. Various details of spores 115 and / or spore reservoir 136 will now be described in greater detail.
In some embodiments, spores 115 can be positioned directly in the lower portion 114 of compartment 102, or spores 115 can be positioned in a spore reservoir, such as spore reservoir 136 (for example, provided by spore carrier 135). If spores 115 are positioned directly in the lower portion 114 of compartment 102 or in a spore reservoir, such as the spore reservoir, spores 115 can be provided in a variety of ways. In some embodiments, the spores 115 can be a spore suspension that can be positioned at a desired location on the biological sterilization indicator 100 and subjected to drying. In some embodiments, the spores 115 can be supplied on a substrate (not shown) that can be positioned and / or secured at a desired location on the biological sterilization indicator 100. Some modalities may include a combination of 115 spores supplied in a dry form and spores 115 provided on a substrate.
In some embodiments, the substrate can be positioned to support spores 115 and / or help to keep spores 115 in a desired location. Such substrate can include a variety of materials, including, but not limited to, paper, a polymer (for example, any of the polymers mentioned above in relation to compartment 102), an adhesive (for example, acrylate, synthetic or natural rubber) , silicone, polyurea silicone, isocyanate, epoxy, or combinations thereof), a woven cloth, a non-woven cloth, a microporous material (for example, a microporous polymeric material), a reflective material (for example, a foil), a glass, a porcelain, a ceramic, a gel-forming material (for example, guar gum), or combinations thereof. In addition, or alternatively, that substrate may include or be coupled to a hydrophilic coating to facilitate intimate contact of the liquid 122 with the spores 115 (for example, when the liquid 122 employed is aqueous). In addition, or alternatively, this hydrophilic coating can be applied to any fluid path positioned to fluidly couple liquid 122 and spores 115. In some embodiments, in addition to, or in place of, a hydrophilic coating, a coating hydrophobic can be applied to other portions of compartment 102 (for example, the lower portion 114 of compartment 102) and / or of the spore reservoir 136, so that the liquid 122 is preferably moved to come in contact with the spores 115.
Some modalities of the biological sterilization indicator 100 do not include spore carrier 135. Preferably, the spore reservoir 136 is provided by the lower portion 114 of compartment 102 itself, and the spores 115 can be positioned in the lower portion 114, adsorbed on an inner surface of the lower portion wall 114, or combinations thereof. In some embodiments, the spores 115 may be provided on a substrate that is positioned in the lower portion 114 of compartment 102.
In some embodiments, spores 115 can be positioned in a spore site or in a plurality of spore sites, all of which can be positioned in reservoir 103, in the lower 114 portion of compartment 102, and / or in spore reservoir 136 In some embodiments, the fact that there are multiple spore sites can maximize spore exposure to sterilizer and liquid 122, can optimize preparation (for example, spore disposition can be facilitated by placing each spore site in a depression within the biological sterilization indicator 100), and can optimize the detection characteristics (for example, due to the fact that spores may not be so easily detected in the middle of a large spore site). In embodiments employing a plurality of spore sites, each spore site may include a different known number of spores, and / or each spore site may include different spores, so that a plurality of types of spores can be tested. When employing multiple types of spores, the biological sterilization indicator 100 can be used for a variety of sterilization processes and a specific spore location can be analyzed for a specific sterilization process, or multiple types of spores can be used to test additionally the effectiveness, or reliability, of a sterilization process.
In addition, in some embodiments, the biological sterilization indicator 100 may include a plurality of spore reservoirs 136, and each spore reservoir 136 may include one or more spore sites 115. In some embodiments, the use of a plurality of reservoirs spore 136, the plurality of spore reservoirs 136 can be positioned in fluid communication with reservoir 103.
In some embodiments, the spores 115 can be covered with a cover (not shown) adapted to fit on or over the spores 115 and / or the spore reservoir 136. This cover can help to keep the spores within a desired region of the biological sterilization indicator 100 during preparation, sterilization and / or use. The cover, if used, may be formed of a material that does not substantially impede a detection process, and / or that is at least partially transmissible to the wavelengths of electromagnetic radiation of interest. In addition, depending on the composition of the covering material, in some embodiments, the covering may facilitate absorption by capillary effect of liquid 122 (for example, the nutrient medium) next to the spores 115. In some embodiments, the covering may also contain features for facilitating the flow of fluids within the spore reservoir 136 (or for spores 115), such as capillary channels, hydrophilic microporous fibers or membranes, or the like, or a combination thereof. In addition, in some embodiments, coverage can isolate a signal, or improve the signal, which can facilitate detection. This cover can be used if the spores 115 are positioned inside the spore reservoir 136 or directly in the lower portion 114 of compartment 102. Furthermore, this cover can be used in modalities that employ a plurality of spore sites. The cover can include a variety of materials, including, but not limited to, paper, a polymer (for example, any of the polymers mentioned above in relation to compartment 102), an adhesive (for example, acrylate, natural or synthetic rubber , silicone, silicone polyurea, isocyanate, epoxy, or combinations thereof), a woven cloth, a non-woven cloth, a microporous material (for example, a microporous polymeric material), a glass, a porcelain, a ceramic, a forming material gel (for example, guar gum), or combinations thereof.
In some embodiments, the biological sterilization indicator 100 may further include a modified inner surface, such as a reflective surface, a white surface, a black surface, or other suitable surface modification to optimize the optical properties of the surface. A reflective surface (for example, provided by a foil) can be positioned to reflect a signal sent to the spore reservoir 136 from a test or detection device and / or to reflect any signal generated within the spore reservoir 136 back to the test device. As a result, the reflective surface can work to optimize (for example, optimize the intensity of) a signal from the biological sterilization indicator 100. This reflective surface can be provided by an internal surface of compartment 102; a material coupled to the internal surface of compartment 102; an internal surface of the spore reservoir 136; a material coupled to the internal surface of the spore reservoir 136; or similar; the reflective surface can form a portion of or be coupled to a spore substrate; or a combination of them.
Similarly, in some embodiments, the biological sterilization indicator 100 may also include a white and / or black surface positioned to increase and / or decrease a particular signal sent to the spore reservoir 136 from a test device and / or increase and / or decrease a particular signal generated within the spore reservoir 136. By way of example only, a white surface can be used to improve the signal, and a black surface can be used to reduce the signal (i.e., noise).
In some embodiments, spores 115 can be positioned on a functionalized surface to promote immobilization of spores 115 on the desired surface. For example, such a functionalized surface may be provided by an internal surface of compartment 102, an internal surface of spore reservoir 136, may form a portion of or if coupled to a spore substrate, or the like, or a combination thereof.
In some embodiments, the spores 115 are positioned (for example, applied by coating or an application method) on a microstructured or micro-replicated surface (for example, as the microstructured surfaces presented in Halverson et al., PCT publication No. WO 2007 / 070310, Hanschen et al., US publication US No. 2003/0235677, and Graham et al., PCT publication No. WO 2004/000569, all of which are incorporated herein by reference). For example, this microstructured surface can be provided by an internal surface of compartment 102, it can be provided by an internal surface of spore reservoir 136, it can form a portion of or if coupled to a spore substrate, or the like, or a combination of the same.
In some embodiments, the biological sterilization indicator 100 may further include a gel-forming material positioned to be combined with spores 115 and liquid 122 when liquid 122 is released from container 120. For example, the gel-forming material it can be positioned close to spores 115 (for example, in spore reservoir 136), in the lower portion 114 of compartment 102, it can form a portion to be coupled to a spore substrate, or the like, or a combination thereof. Such a gel-forming material can form a gel (for example, a hydrogel) or a matrix comprising spores and nutrients when the liquid 122 comes in contact with the spores. A gel-forming material (for example, guar gum) can be particularly useful because it has the ability to form a gel upon hydration, and can assist in locating a signal (for example, fluorescence), can anchor spores 115 in place , can help minimize spore diffusion 115 and / or a signal from spore reservoir 136 and / or can improve detection.
In some embodiments, the biological sterilization indicator 100 may further include an absorbent or material for absorption by capillary effect. For example, the material for absorption by capillary effect can be positioned close to spores 115 (for example, in spore reservoir 136), can form at least a portion of, or be coupled to, a spore substrate, or the like, or combination thereof. This capillary absorption material includes a porous capillary absorption block, an immersion block, or the like, or a combination thereof, to facilitate placing the liquid 122 in close contact with the spores.
In some embodiments, the frangible container 120 can be configured to facilitate fracturing of the frangible container 120 in the desired manner. For example, in some embodiments, a lower portion of the frangible container 120 may be formed of a thinner and / or weaker material, so that the lower portion fractures, preferably, in another portion of the frangible container 120. In addition , in some embodiments, the frangible container 120 may include a variety of features positioned to facilitate fracturing of the frangible container 120 in a desired manner, including, but not limited to, a thin and / or weakened area, a cut line, perforation, or the like, or combinations thereof. The frangible container 120 may have a first closed state in which liquid 122 is contained within frangible container 120 and a second open state in which frangible container 120 has been fractured and liquid 122 is released into reservoir 103 and / or spore reservoir 136, and in fluid communication with the spores 115.
In some embodiments, the biological sterilization indicator 100 can be activated (for example, the second portion 106 can be moved to the second position 150) manually. In some embodiments, the biological sterilization indicator 100 can be activated by a reading device (for example, how the biological sterilization indicator 100 is positioned on the reading device). In some embodiments, the biological sterilization indicator 100 can be activated with a device (for example, an activation device) independent of such a reading device, for example, by positioning the biological sterilization indicator 100 on the device before positioning the device. biological indicator of sterilization 100 in a cavity of a reading device. In some embodiments, the biological sterilization indicator 100 can be activated by a combination of two or more of the reading device, a device independent of the reading device and manual activation.
One or both, the biological sterilization indicator 100 and another device, such as a reading device, can be additionally configured to inhibit premature or accidental fracture of the frangible container 120. For example, in some embodiments, the biological sterilization indicator, the activation device or reading apparatus 100 may include a lock or locking mechanism which is positioned to prevent the second portion 106 of compartment 102 from moving to the second position 150 as far as desired. In these modalities, the biological sterilization indicator 100 cannot be activated until the lock is moved, removed or unlocked. In addition, or alternatively, in some embodiments, the biological sterilization indicator, the activation device and / or the reading apparatus 100 may include a lock or locking mechanism which is positioned to prevent the second portion 106 of compartment 102 from becoming move from second position 150 back to first position 148 after activation.
In some embodiments, as shown in the illustrated embodiment, at least a portion of the compartment may be flat (for example, the parallel walls 168), and may be substantially planar with respect to the spore reservoir 136, and one or both of the parallel walls 168 or a portion thereof (e.g., the detection window 167) substantially matches at least one dimension of the spore reservoir 136 and / or the location of the spores 115. In other words, wall 168 or a portion of the same (for example, the detection window 167) can include a cross-sectional view that is substantially the same size as the cross-sectional area of the spore reservoir 136 and / or the spore location 115. This test for equal size between the wall 168 / detection window 167 and the spore reservoir 136 and / or the spore site 115 can maximize the signal detected during a detection or testing process. Alternatively, or in addition, the wall 168 or detection window 167 can be dimensioned to match the reservoir 103 (for example, at least one dimension or the cross-sectional areas can be dimensioned to be equalized). This test for equal size between the detection zones can improve the spore detection test. The biological sterilization indicator 100 shown in Figures 4 to 10, at least the portion of the biological sterilization indicator 100 where the spores 115 are positioned, is relatively thin (i.e., the “z dimension” is minimized), so that the optical path of the spores to wall 168 (or detection window 167) is minimized and / or any effect of interference of substances in liquid 122 (or nutrient medium) is minimized.
In use, the biological sterilization indicator 100 can be placed together with a sterilization batch during a sterilization process. During sterilization, a sterilizer is in fluid communication with the reservoir 103 (that is, the first chamber 109 and the second chamber 111), the spore reservoir 136, and the spores 115 mainly through the sterilizing path 164, so that the sterilizer can reach the spores to produce sterile spores. As described above, the cooperation of the first fluid path 160 and the second fluid path 162 can facilitate the movement of the sterilizer to the second chamber 111, and particularly, to the closed end 105 of the biological sterilization indicator 100. In addition, during sterilization, the frangible container 120 is in a closed state, kept intact at least partially by the carrier 132 of the insert 130. When the frangible container 120 is in a closed state, the liquid 122 is protected from the sterilizer and is not in communication fluid with reservoir 103 (particularly, the second reservoir 111 formed at least partially by the lower portion 114 of compartment 102), the spore reservoir 136, the spores 115 or the sterilizing path 164. Sterilization may additionally include moving a sterilizer from the first chamber 109 to the second chamber 111 through the first path fluid 160 when the container 120 is in the first state, and moving the displaced gas (e.g., trapped air) out of the second chamber 111 through the second fluid path 162 in response to, or to facilitate, the movement of the sterilizer from the first chamber 109 to the second chamber 111.
After sterilization, the effectiveness of the sterilization process can be determined using the biological sterilization indicator 100. The second portion 106 of compartment 102 can be unlocked, if previously locked in the first position 148, and moved from the first position 148 (see Figure 6) to the second position 150 (see Figure 7) to cause the activation of the biological sterilization indicator 100. Such movement of the second portion 106 can cause the frangible container 120 to move in compartment 102, for example, along the longitudinal direction DL from a position above the upper ends 159 of the projections 158 to a position within the projections 158, which can cause the frangible container 120 to fracture. Fracturing frangible container 120 can change frangible container 120 from its closed state to its open state and release liquid 122 in reservoir 103, and in liquid communication with spore reservoir 136 and spores 115. Liquid 122 may include a nutrient medium (for example, germination medium) for spores, or liquid 122 may contact the nutrient medium in a dry form (for example, in a powdered or tablet form) to form the nutrient medium, so that the mixture that includes the sterile spores and the nutrient medium is formed. The mixture can then be incubated before or during a detection or testing process, and the biological sterilization indicator 100 can be interrogated for signs of spore growth. Activation may additionally include moving liquid 122 from the first chamber 109 to the second chamber 111 through the first fluid path 160 when the container 120 is in the second state, and moving the displaced gas (e.g., trapped air) out of the second chamber 111 through the second fluid path 162 in response to, or to facilitate, the movement of liquid 122 from the first chamber 109 to the second chamber 111 through the first fluid path 160.
To detect a detectable change in spores 115, the sterilization biological indicator 100 can be tested immediately after liquid 122 and after spores 115 have been combined to achieve a baseline reading. After that, any detectable change in the baseline reading can be detected. The biological sterilization indicator 100 can be monitored and measured continuously or intermittently. In some embodiments, a portion of the incubation step, or the entire incubation step, can be performed before measuring the detectable change. In some embodiments, incubation can be performed at a temperature (for example, at 37 ° C, between 50 and 60 ° C, etc.), and the measurement of the detectable change can be performed at a different temperature (for example, at room temperature, 25 ° C, or 37 ° C). The reading time of the biological sterilization indicator 100 (that is, the time to determine the effectiveness of the sterilization process) can be, in some modalities, less than 8 hours, in some modalities less than 1 hour, in some modalities less than 30 minutes, in some modalities less than 15 minutes, in some modalities less than 5 minutes, and in some modalities less than 1 minute.
Modalities Modality 1 is a method of detecting a biological activity that comprises: providing a sample that can comprise a source of one or more predetermined biological activities; a first indicator system comprising a first indicator reagent with a first absorbance spectrum, wherein the first indicator reagent can be converted by a first predetermined biological activity to a first biological derivative; a second indicator system comprising a second indicator reagent that is converted by a predetermined biological activity to a second biological derivative with a second emission spectrum; and a substrate that receives and concentrates the first indicator reagent from an aqueous mixture; forming a first aqueous mixture comprising the sample, the first indicator reagent and the second indicator reagent; placing the first aqueous mixture in fluid communication with the substrate to form a second aqueous mixture in which the concentration of the first indicator reagent is less than the concentration of the first indicator reagent in the first aqueous mixture; and detecting a presence or absence of fluorescence from the second biological derivative; wherein the first absorbance spectrum includes absorbance detectable in at least a portion of wavelengths present in the second emission spectrum. Mode 2 is the method of mode 1, in which the detection of the presence or absence of fluorescence of the second biological derivative comprises detecting the presence or absence of fluorescence in the second aqueous mixture. Mode 3 is the method of mode 1 or mode 2, which further comprises looking at the substrate to detect the first indicator reagent or the first biological derivative. Mode 4 is the method according to any of the previous modalities, wherein a concentration of the first indicator reagent in the first aqueous mixture is sufficient to prevent the detection of an otherwise detectable amount of the second biological derivative. Mode 5 is the method as defined in any of the modalities which further comprises providing a nutrient to facilitate the growth of a biological cell, wherein the formation of the first aqueous mixture comprises the formation of a mixture which includes the nutrient. Mode 6 is the method as defined in any of the modalities that further comprises exposing biological activity to a sterilizer. Modality 7 is the method of modality 6, in which the sterilizer is selected from a group consisting of water vapor, ethylene oxide, hydrogen peroxide, formaldehyde and ozone. Mode 8 is the method as defined in any of the previous modalities which further comprises exposing biological activity to a temperature shift for a period of time. Mode 9 is the method as defined in any of the modalities, wherein the first indicator reagent comprises a chromophore, where the detection of the first biological derivative comprises the detection of a color. Mode 10 is the method of mode 9, in which the first indicator reagent comprises a chromogenic indicator. Mode 11 is the method of mode 9 or mode 10, wherein the first indicator reagent comprises a pH indicator or an enzyme substrate. Modality 12 is the method of modality 11, in which the first indicator reagent is selected from a group consisting of bromocresol purple, bromocresol green, Congo red and methyl orange. Mode 13 is the method as defined in either mode, wherein the second indicator reagent comprises a fluorogenic compound. Modality 14 is the method of modality 13, in which the fluorogenic compound comprises a fluorogenic enzymatic substrate. Mode 15 is the method as defined in any of the modalities, wherein detecting the presence or absence of the second biological derivative further comprises measuring an amount of the second biological derivative. Mode 16 is the method as defined in any of the modalities, wherein detecting the presence or absence of the first biological derivative further comprises measuring an amount of the first biological derivative. Mode 17 is the method of mode 16, in which measuring the amount of the first biological derivative comprises comparing an amount of color measured in a portion of the second aqueous mixture not associated with the substrate with a color standard. Mode 18 is the method as defined in any of the modalities which further comprises: providing an instrument that detects the first indicator reagent or the second biological derivative; and use the instrument to detect the first indicator reagent or the second biological derivative. Mode 19 is the method as defined in any of the modalities which further comprises: providing an instrument that detects the first indicator reagent and the second biological derivative; and use the instrument to detect the first indicator reagent and the second biological derivative. Mode 20 is a method of detecting a biological activity which comprises: providing a biological indicator of sterilization which comprises; a compartment comprising first and second chambers; a container containing a first aqueous liquid, in which the container is disposed in a first chamber, in which at least a portion of the container is frangible, the liquid comprises a first indicator system comprising a first indicator reagent with a first absorbance spectrum and a second indicator system comprising a second indicator reagent that is converted by a second predetermined biological activity to a second biological derivative with a second emission spectrum, wherein the first indicator reagent can be converted by a first predetermined biological activity to a first biological derivative, in which the first absorbance spectrum includes absorbance detectable in at least a portion of wavelengths of the second emission spectrum; a source of the second predetermined biological activity arranged in a second chamber; and a substrate that receives and concentrates the first reagent indicating the first aqueous liquid, in which the substrate is disposed in the compartment; placing the first aqueous liquid in fluid communication with the substrate to form a second aqueous liquid in which the concentration of the first indicator reagent is less than the concentration of the first indicator reagent in the first aqueous liquid; and detecting a presence or absence of fluorescence from the second biological derivative in the second aqueous mixture. Mode 21 is the method of mode 20, in which placing the first aqueous liquid in fluid communication with the substrate comprises fracturing at least a portion of the frangible container. Mode 22 is the method of mode 21, in which the biological sterilization indicator additionally comprises a fracturing device arranged in the compartment and in which fracturing the frangible container comprises inciting the container and the fracturing against each other. Mode 23 is the method as defined in any of modalities 20 to 21, in which the compartment of the biological sterilization indicator includes: a first portion, and a second portion adapted to be coupled to the first portion, the second portion is movable in in relation to the first portion, when coupled to the first portion, between a first position and a second position; wherein the method further comprises moving the second portion of the compartment from the first position to the second position. Mode 24 is the method of mode 23, wherein the compartment includes a longitudinal direction, and where the movement of the second portion of the compartment includes moving the second portion of the compartment in the longitudinal direction. Mode 25 is the method of mode 23 which further comprises moving the container in the compartment in response to the movement of the second portion of the compartment from the first position to the second position. Mode 26 is the method of mode 25, in which the movement of the container in the compartment causes the container to fracture. Modality 27 is a system for detecting a predetermined biological activity comprising: a first indicator system comprising a first indicator reagent with a first absorbance spectrum, in which the first indicator reagent can be converted by a first predetermined biological activity into a first biological derivative; a second indicator system comprising a second indicator reagent that is converted by a predetermined biological activity to a second biological derivative with a second emission spectrum; a vessel configured to contain a liquid medium; a substrate that receives and concentrates the first indicator reagent in an aqueous mixture; and an instrument configured to receive the vessel and to detect the first indicator reagent or the second biological derivative in which the first absorbance spectrum includes absorbance detectable in at least a portion of wavelengths present in the second emission spectrum. Mode 28 is the system of mode 27 which additionally comprises a processor. Mode 29 is the system of mode 27 or mode 28, in which the instrument is additionally configured to regulate a temperature of the liquid medium. Modality 30 is the system as defined in any of modalities 27 to 29, in which the instrument is configured to detect both the first indicator reagent and the second biological derivative. The present invention is illustrated by means of the following examples. It should be understood that the examples, materials, quantities and specific procedures must be interpreted in an open manner, according to the scope and spirit of the invention, as described herein.
Examples Reference example 1 - Bromocresol Purple Absorbance Spectrum (BCP).
This reference example shows the absorbance spectrum of purple bromcresol. The purple bromocresol obtained from Sigma Chemical Co., St Louis, MO, USA, (catalog number B-5880), was dissolved in phosphate buffered saline, pH 7.3, at a concentration of 0.004. The solution was placed in a quartz crucible and the visible UV absorbance spectrum was scanned using the 1 cm crucible adapter equipped with the TECAN INFINITE M200 Plate Reader (Tecan US, Durham, NC, USA). The scanning parameters are shown in Table 1.
The results are shown in the graph shown in Figure 2. The absorbance peaks can be seen at wavelengths of about 300 nm, about 380 nm and about 600 nm.
Table 1. Scanning parameters for BCP absorbance spectrum.
Reference example 2 Emission spectrum of 7-hydroxy-4-methylcoumarin (4-methylumbelliferone).
This reference example shows the emission spectrum of 4-methylumbelliferone. 4-methyl umbeliferone (4MU), catalog number m1381, obtained from Sigma Chemical Co., St Louis, MO, USA, was dissolved in phosphate buffered saline, pH 7.3, at a concentration of 0.004 mg / ml. The solution was placed in a quartz crucible and the emission spectrum was noted using the 1 cm crucible adapter equipped with the TECAN INFINITE M200 Plate Reader. The scanning parameters are shown in Table 2.
The results are shown in the graph illustrated in Figure 2. An emission peak can be seen at a wavelength of about 450 nm.
Table 2. Scanning parameters for 4-methylumbelliferone emission spectrum.
Reference example 3 - Effect of BCP on 4MU detection
This reference example shows the effect of BCP on fluorescence detection of 4MU when the two compounds are present in the same solution.
A stock solution of 4-methylumbelliferone (4MU), prepared as described in Example 2, was serially diluted in phosphate buffered saline to the concentrations shown in Table 3. A stock solution of purple bromocresol (BCP) was prepared in phosphate-buffered saline, as described in example 1. Bromocresol purple (0.03 mg / ml final concentration) was mixed with the respective 4MU solutions shown in Table 3. The triplicate aliquots (100 microliters / well) of each respective solution was loaded onto a 96 well plate and the fluorescence in each well was measured using a TECAN INFINITE M200 Plate Reader. The excitation wavelength was 350 nm and the detection was at 420 nm. The results, mentioned as relative fluorescence units (RFU), are shown in Table 3. The data shows that, at each 4MU concentration tested, the presence of bromocresol purple in the solution resulted in a decrease in measurable fluorescence.
Table 3 - Fluorescent detection of 4MU in the presence or absence of BCP
The results are an average of three replicates. All values are reset to the Relative Fluorescence Units (RFUs).
Reference example 4. - BCP adsorption from a liquid medium This reference example shows the BCP adsorption from a growth medium on a substrate material.
A spore growth medium solution was prepared consisting of 17 g of a bacteriological peptone, 0.17 g of L-alanine and 0.03 g of purple bromocresol pH indicator dye, per liter of water. The pH of the nutrient solution was adjusted to 7.6 with 0.1 N sodium hydroxide. To each of the borosilicate glass tubes 60 (12 ml, VWR catalog number 53283-802) was added 1.0 ml of the prepared growth medium and finished with unlined cap closures (VWR catalog number 66010-680 ).
Two different substrate materials were evaluated: GE loaded nylon (MAGNAPROBE 0.45 micron loaded nylon membrane, part number ΝΡ0ΗΥ00010, available from GE Osmonics Labstore, Minnetonka, MN, USA) and paper (Whatman chromatography paper from grade 1 Chr cellulose, available from Whatman Inc. USA, Piscataway, NJ, USA).
Twenty strips of each of the two substrate materials were cut to size, 4 mm x 10 mm. All strips were pre-sterilized by placing them in a Propper CHEX-ALL II Instant Sealing Containment Bag (Propper, Manufacturing Inc., Long tsland City, NY, USA) and sterilizing them for 30 minutes in a water vapor liquid at 121 ° C in an AMSCO sterilizer (Steris, Mentor, OH, USA).
The sterile substrate strips were aseptically removed from the containment bag and transferred to the glass tubes, five strips of nylon substrate for each of 20 tubes and five strips of paper substrate for each of 20 different tubes.
The spore strips were captured from the disassembled ATTEST 1292 Rapid Reading Biological Indicator Water Steam Sterilizers (3M, St. Paul, MN, USA), containing G. stearothermophilus spores (ATCC 7953). The spore strips were cut into equal squares, each approximately 6.4 mm x 6.4 mm, and added to the glass tubes according to Table 4 and further described below. A piece (6.4 mm x 6.4 mm) of a 1292 ATTEST spore strip was added to each of the 10 glass tubes, each containing 5 pieces of the nylon substrate and the growth medium. A piece of the spore strip was added to each of the 10 glass tubes, each tube containing 5 pieces of Whatman's paper and the growth medium. A piece of spore strip was added to each of the 10 glass tubes, each tube containing only growth medium, without substrate. No spore strip was added to the remaining 30 tubes: 10 tubes containing 5 pieces of nylon substrate, 10 tubes containing 5 pieces of paper substrate and 10 tubes without substrate.
Table 4 Sample Preparation for Example 4 Two tubes from each of the samples above were selected for the following observations and analyzes at the time of 1 minute. The color of the nylon or paper substrate material while in the tube was compared to the color of the surrounding liquid growth medium in relation to whether the substrate is darker or lighter than the medium. The color of the substrate materials was observed and noted when read from the glass tubes containing growth medium.
The strips of nylon and paper substrate were removed from the tubes and placed on a KIMWIPE (Kimberly-Clark) before densitometry readings were taken using an X-Rite 530P densitometer (X-Rite, Grand Rapids, Ml, USA). The optical density (DO) setting on the X-Rite 530P densitometer was set to “color” to provide the V filter results. The X-Rite densitometer was set to “compare” to the ΔΕ substrate results with Pantone 2665U and 102U selected. The CIE76 formula was used to calculate ο ΔΕ in each Pantone. The value of ΔΕ is the distance in color space L * A * B from a measured point to a reference value, a Pantone color. A lower ΔΕ indicates that a measured color is closer to the reference value. A value of about 2.5 ΔΕ is about the minimum limit for a human eye to differentiate color. The two reference values used were Pantone 2665U (a light purple) and Pantone 102U (bright yellow). It is observed that, due to the fact that the two values are not diametrically opposed in the “color wheel”, an increase in ΔΕ in 2665U does not necessarily mean an exact decrease in ΔΕ at 102U. In other words, ο ΔΕ in 2665U only indicates whether or not there is more correlation with "purple", no longer with "yellow". The color of the medium in each tube was also observed and noted (Table 5). In triplicate, an amount of 200 µl of medium was removed from each tube and placed on a 96-well plate (COSTAR CLS-3603-48EA 96-well plate treated with black tissue culture) and the optical density (OD) at 590 nm and 430 nm was measured with a SYNERGY 4 spectrophotometer with Gen 5 software. Optical density (DO) measurements were taken using Monochromater, (BioTek, Winooski, VT, USA).
The remaining tubes were incubated at 56 ° C. At each of the following times: 30 minutes, 1 hour, 4 hours and 24 hours of incubation; 2 tubes from each sample were removed from the incubator, visually observed and instrumentally measured as described above.
Table 5 Observations of Media Color The media in all vials remained purple until after 4 hours of reading. The spore-free samples remained purple after 24 hours. All spore samples changed to a visually yellow color for 24 hours due to the growth of cells that lead to a decrease in the pH of the media, indicated by the BCP pH dye.
At each time interval, before the substrate was removed from the media, the color of the substrate was compared to that of the media (see Table 6). If there is any difference between the two colors the difference has been documented. In all cases where the nylon substrate was used as the substrate, the substrate appeared to be a darker shade of color compared to the surrounding media. In all cases where the paper was used as the substrate, the substrate appeared to be a lighter shade of color compared to the surrounding media. These results show that the nylon substrate is superior to the paper substrate in the reception and concentration of the indicator reagent.
Table 6 Substrate vs. color. Media color In most cases of "darker", the substrate showed a noticeably darker purple color than the media, with the exception of 24 h of nylon with spores, which showed a darker yellow color. In most cases of “lighter”, the substrate had a purple color visibly lighter than the media, with the exception of 24 h of paper with spores, which showed a lighter yellow color.
For spore samples, measuring OD at 590 nm in 24 hours will not show differences in yellow intensity. Therefore, only the DO values taken at 430 nm in 24 hours were evaluated.
Table 7 Optical Density (OD) Average of Media at 430 nm in 24 hours The 24-hour readings at 430 nm of the samples of the medium with spores in the presence of paper substrate and the sample of the medium without substrate (control) with spores, had similar OD values of 0.827 and 0.835 respectively, as shown in Table 7. However, the sample of the medium with spores in the presence of nylon had an OD of only 0.271; which is 0.5 units of OD less than the control or sample with the paper substrate. This shows that the intensity of the yellow color of the medium in the presence of nylon has been reduced due to the fact that the nylon substrate receives and concentrates the indicator reagent.
Table 8 Average Optical Density of the Medium at 590 nm All values represent n = 6 (3 readings x 2 tubes). * n = 5 readings: 3 readings for tube 1 and 2 readings for tube 2. *** samples are yellow in color and therefore the OD at 590 nm does not accurately measure the color of the medium. The absorbance of the control without substrate (with and without spores) in 1 minute was considered the baseline OD measurement for the medium. Table 8 shows that even in 1 minute, the OD at 590 nm of the sample medium, with spores, in the presence of nylon, (1.124) was lower than the OD of the sample medium in the presence of the spore paper (1.404) or the control with spores (1.402). This difference indicates that the purple color intensity of the medium has already been reduced due to the fact that the nylon substrate quickly receives and concentrates the BCP indicator reagent. In 24 hours, the OD at 590 nm of the sample medium without spores in the presence of nylon was 1.122, which is much less than the OD of the medium in the presence of paper (1.708) or that of the control sample without spores, 1.812 .
Table 9 Pantone Color of Substrate in 24 hours Table 10 Reading Average Densitometry of Substrate Using V Filter *** Spore substrate samples in 24 hours are yellow in color and the V filter does not accurately measure the color of the substrate .
Table 11 Reading of Average Densitometry of the Substrate: Δ E of Pantone 2665U (purple) *** The samples of substrate with spores in 24 hours are yellow in color and the filter V does not accurately measure the color of the substrate.
Table 12 Substrate Average Densitometry Reading: Pantone 102U Δ E (yellow) The tables above show the substrate densitometry readings after exposure to the media (with and without spores) for varying lengths of time. The time reading 0 for each substrate is the initial densitometry reading before the substrate sample is placed in the middle. When evaluating the substrates that are purple, the filter V and ο Δ E (Pantone 2665U) showed the greatest contrast. The average densitometry readings with the V filter for the nylon substrate as shown in Table 10 increased and remained throughout the experiment (with only the exception when the substrate was yellow at the time of 24 hours for the “spore” sample) ). On the contrary, the densitometry readings for the paper substrate remained reasonably constant over the points in time. Similarly, the Δ E value of the nylon substrate (Pantone 2665U) shown in Table 11 generally decreased throughout the experiment (with the only exception being when the substrate was yellow at the 24 hour time point for the “spore” sample). ”). This indicates that the nylon substrate was receiving and concentrating the BCP indicator reagent. In contrast, the value of Δ E (Pantone 2665U) for the paper substrate has remained reasonably constant. Table 12 illustrates that at the 24-hour time point the value of Δ E (Pantone 102U) for the nylon substrate was considerably lower than the value of E (Pantone 102U) for the paper substrate, indicating that the substrate of Nylon was closer to the Pantone 10211 color (brighter yellow) than the paper substrate.
Reference example 5 Adsorption of BCP Nylon Substrate from a Liquid Medium After Two 24-Hour Incubations This reference example shows the adsorption of BCP from a liquid growth medium onto a nylon substrate. The same medium and components used in example 4 were used in example 5. To each of the 4 glass tubes, 1.0 ml of the prepared growth medium was added. A piece of a 1292 ATTEST spore strip cut in approximately 6.4 mm x 6.4 mm was added to each glass tube. The tubes were placed in an incubator at 56 ° C for 24 hours to promote the growth of G. stearothermophilus cells. After the 24-hour incubation, five (5) strips (each cut to 4 mm x 10 mm) of the nylon substrate were added to two (2) of the tubes. The tubes were placed in an incubator at 56 ° C for another 24 hours. After the second 24-hour incubation period (24 hours after adding the nylon substrate) to the tubes, the following analyzes were performed. The pieces of nylon substrate were removed from the tubes, placed in a KIMWIPE and the densitometry readings of the substrate strips were taken. From each tube, three 200 pl aliquots were removed and placed in a 96-well plate. The optical density (OD) at 430 nm of the media was measured.
Table 13 DO Average of Media at 430 nm in 48 hours; 24 hours after the nylon substrate n = 12 (3 readings from each of the 4 tubes) n = 3 (1 read from a control tube) Table 13 shows the decrease in OD at 430 nm of the media 24 hours after the substrate of nylon have been added to the tubes, compared to the control, where no substrate has been added. The difference in OD measurement between the two samples indicates the difference in the amount of yellow present in the sample medium. This shows that the intensity of the yellow color of the media in the presence of nylon has been reduced due to the fact that the nylon substrate receives and concentrates the indicator reagent.
Table 14 Substrate Average Densitometry Reading: Pantone Δ E 102U (yellow) n = 10 (5 strips of nylon substrate x 2 tubes) Table 14 shows the value of ΔΕ (102U) of the nylon substrate 24 hours after being added to a media tube with spores that have already been incubated for 24 hours. This was compared with the nylon substrate that was not placed in the media. The difference in the ΔΕ measurements between the two samples indicates that the substrate exposed to media (yellow) with growth is closer in color to pantone 102U (bright yellow) than the substrate not exposed to the media.
Reference example 6 - BCP absorption from a liquid medium by several substrates This reference example shows the adsorption of BCP from a liquid growth medium on various substrate materials.
A spore growth medium solution was prepared consisting of 17 grams of a bacteriological peptone C, 0.17 grams of L-alanine and 0.03 grams of bromocresol purple pH indicator dye, per liter of Water. The pH of the nutrient solution was adjusted to 7.6 with 0.1 N sodium hydroxide. To each of the borosilicate glass tubes (12 ml, VWR catalog number 53283-802) was added 1.0 ml of the prepared growth medium and finished with unlined cap closures (VWR catalog number 66010-680 ).
Four different substrate materials were evaluated: (1) GE loaded nylon (MAGNAPROBE 0.45 micron loaded nylon membrane, part number NP0HY00010, available from GE Osmonics Labstore, Minnetonka, MN, USA); (2) BIO-RAD high strength nylon membrane positively charged with quaternary amine groups (ZETA-PROBE GT Genomics, catalog number 162-0196, available from BIO-RAD LifeSciences, Hercules, CA, USA); (3) 0.2 μΜ of nitro celluloses (catalog number LC-2000, available from Invitrogen Corporation Carlsbad, CA, USA), and (4) 0.2 μΜ polyvinylidene difluoride (PVDF) membrane (n LC-2002 catalog number, available from Invitrogen Corporation Carlsbad, CA, USA). Several strips of each substrate material were cut to size: 4 mm x 10 mm, enough for one (1) strip for each glass tube.
All strips were pre-sterilized by placing them in a Propper CHEX-ALL II Instant Sealing Containment Bag (Propper, Manufacturing Inc., Long Island City, NY, USA) and sterilizing them for 30) minutes in a cycle of liquid water vapor (at 121 ° C) in an AMSCO sterilizer (Steris, Mentor, OH, USA).
The strips were then aseptically transferred to each tube. Two tubes of each substrate were evaluated together with two control tubes that did not contain substrate.
The following observations and analyzes were performed at 0, 30 minutes, 1 hour, 4 hours and 24 hours: (1) the color of the substrate material in each tube was compared with the color of the surrounding media of the same tube, (darker or lighter), (2) the substrate material was removed from the tube, placed in a KIMWIPE for drying, and then the densitometry readings were taken with the V filter as described above, (3) the 200 pl removed from the media from each tube and transferred in triplicate to a 96-well plate (96-well plate treated with COSTAR CLS-3603-48EA black tissue culture with a light background) and the optical density of the media at 590 nm was measured with a SYNERGY 4 spectrophotometer with Gen 5 software. DO measurements were taken using Monochromater, (BioTek, Winooski, VT).
The remaining tubes were incubated at 56 ° C. At each of the following times: 30 minutes, 1 hour, 4 hours and 24 hours of incubation; 2 tubes from each sample were removed from the incubator, visually observed and instrumentally measured as described above.
Table 15. Substrate vs. color. Media color for Various Substrates Darker = substrate was visibly darker purple than media Lighter = substrate was visibly lighter purple than media In each reading before the substrate was removed from the media the color of the substrate was visually compared to the media see Table. The difference between the color of the substrate and the color of the media was observed and reported in Table 15. After 30 minutes in contact with the media, both the nylon substrate materials were noticeably darker than the media and remained darker throughout. experiment.
Table 16 Average Densitometry Reading of Various Substrates after Media with BCP Table 16 shows the densitometry readings of the substrate materials after exposure to the media for varying lengths of time. The reading of time 0 for each substrate is the initial densitometry reading within 30 seconds of placing the substrate in the media. In all cases, the densitometry of nylon substrates increased within 30 minutes and remained high throughout the experiment.
Table 17 OD at 590 nm of Media in the Presence of Various Substrate Materials Table 17 shows the average optical density (OD) reading of the media removed from the tube containing each substrate material at the specified time. It is notable that at a time point, the OD for the media that was in the presence of nylon substrate was lower than the OD reading for the media containing nitrocellulose or PVDF. Additionally, nitrocellulose or PVDF show very little change in OD reading and are quite similar to the control OD values.
Reference example 7 - Absorption of Methyl Red Substrate (MR) from a Liquid Medium This reference example shows the adsorption of BCP from a liquid growth medium on various substrate materials.
A spore growth medium solution was prepared consisting of 17 grams of a bacteriological peptone, 0.17 grams of L-alanine and 0.03 grams of methyl red pH indicator dye, per liter of water. The pH of the nutrient solution was adjusted to 4.2 with 0.1 N hydrochloric acid. To each of the borosilicate glass tubes (12 ml, VWR catalog number 53283-802) was added 1.0 ml of the prepared growth medium and finished with unlined cap closures (VWR catalog number 66010-680 ).
Two different substrate materials were evaluated: GE loaded nylon (0.45 micron MAGNAPROBE loaded nylon membrane, part number NP0HY00010, available from GE Osmonics Labstore, Minnetonka, MN, USA), and BIO high strength nylon membrane -RAD positively charged with quaternary amine groups (Zeta-Probe GT Genomics, catalog number 162-0196, available from Bio-Rad LifeSciences, Hercules, CA, USA). Several strips of each substrate material were cut to size: 4 mm x 10 mm, enough for one (1) strip for each glass tube.
All strips were pre-sterilized by placing them in a Propper CHEX-ALL II Instant Sealing Containment Bag (Propper, Manufacturing Inc., Long Island City, NY, USA) and sterilizing them for 30) minutes in a cycle of liquid water vapor (at 121 ° C) in an AMSCO sterilizer (Steris, Mentor, OH, USA).
The strips were then aseptically transferred to each tube.
The following observations and analyzes were performed for the two tubes of each substrate at time 0, 30 minutes, 1 hour, 4 hours and 24 hours: (1) the substrate material was removed from the tube, placed in a KIMWIPE for drying and, then, densitometry readings were taken with the V filter as performed above, (2) the color of the substrate material in each tube was compared with the color of the surrounding media of the same tube, (darker or lighter).
The remaining tubes were incubated at 56 ° C. At each of the following times: 30 minutes, 1 hour, 4 hours and 24 hours of incubation; 2 tubes from each sample were removed from the incubator, visually observed and instrumentally measured as described above.
Table 18. Substrate Mean Densitometry Reading after Methyl Red, Filter V
The tables above show the densitometry readings of the substrates after exposure to the media for a varying length of time. The reading of time 0 for each substrate is the initial densitometry reading within 30 seconds of placing the substrate in the media. In all cases, the densitometry of nylon substrates increased within 30 minutes and remained high throughout the experiment.
Table 19. Substrate vs. color. Media color after Methyl Red Darker = substrate was noticeably darker than media Lighter = substrate was visibly lighter than media In each reading before substrate materials were removed from media, media color was compared with that of the substrate (see Table 19). The difference between the color of the substrate and the color of the media was observed and reported. After 30 minutes in contact with the media, both the nylon substrate materials were noticeably darker than the media and remained darker throughout the experiment.
Reference example 8 Inhibition of Acridine Orange Detection (AO) with BCP and Methyl Red This reference example shows the effect of BCP and methyl red on the detection of acridine orange fluorescence when one of the pH indicators (ie , BCP or MR) is present in a solution with acridine orange.
A spore growth medium solution was prepared consisting of 17 grams of a bacteriological peptone and 0.17 grams of L-alanine. A 200 µl volume of growth medium was added to each well in two (2) 96 well plates.
A dilution series of pH indicator solutions was made for both methyl red (MR) and purple bromocresol (BCP) starting at 4.8 g / l and diluted at 0.75 g / l. An acridine orange dilution series was done starting at 1:50 and diluting to 1: 800.
In plate No. 1, 20 pl the appropriate dilution of BCP was added to each row of the plate and 20 μΙ of the appropriate dilution of acridine orange (AO) were added to each column in plate No. 1. In plate No. 2, 20 μΙ of the appropriate dilution of methyl red was added to each row of the plate and 20 μΙ of the appropriate dilution of acridine orange (AO) were added to each column of plate No. 2. See Table 20 for the configuration of plate No. 1 and plate No. 2.
Table 20 Configuration for BCP 96-well Plate No. 1 and MR for Plate No. 2 Plate No. 1 and No. 2 were placed on the SYNERGY 4 spectrophotometer and the absorbance readings taken at 590 nm. In addition, fluorescence excitation / emission readings at 435 nm / 530 nm were also collected (see Tables 21A-B).
Table 21A: Acridine Orange Detection Inhibition with BCP ** Signal above instrument detection threshold Table 21B: Acridine Orange Detection Inhibition with Signal above instrument detection threshold For all orange concentrations of acridine, as the amount of purple bromocresol in solution decreases the signal generated by the increased acridine orange. In other words, the presence of BCP masked the acridine orange signal. For example, for the row with an initial BCP concentration of 0.3 g / l, between about 27 to 36% of the acridine orange fluorescence signal is lost, compared to the row with 0 BCP.
Table 22A: Acridine Orange Detection Inhibition with Methyl Red ** Sign above Instrument Detection Threshold Table 22B: Acridine Orange Detection Inhibition with Signal Above Instrument Detection Threshold As the BCP, methyl red also masked the acridine orange fluorescence signal. The higher the concentration of methyl red the lower the detected fluorescence signal of acridine orange. For example, for the row with an initial methyl red concentration of 0.3 g / l, between about 3 to 27% of the acridine orange fluorescence signal is lost, compared to the row with no methyl red ( see Tables 22A-B).
Examples 1 to 3: Detection of a Biological Activity The 3M ATTEST 1291 quick-read biological indicators are obtained from the 3M Company, St. Paul, MN, USA. The loaded nylon membrane (0.45 micron MAGNAPROBE loaded nylon membrane, part number NPOHY00010) is obtained from GE Osmonics Labstore (Minnetonka, MN, USA).
The covers of the biological indicators are removed and the glass ampoules are removed by inverting the biological indicator tube. The ampoules are set aside for later use. The nylon membrane is cut into small strips (0.5 cm x 2 cm). A strip is placed (longitudinally) adjacent to the wall at the bottom of the biological indicator tubes and the glass ampoule is replaced in each tube. The caps are carefully replaced on each tube.
The modified biological indicators are subjected to exposure to water vapor for varying lengths of time (shown in Table 23). Exposure to water vapor is conducted at 132 ° C / 270 ° F Gravity Water Vapor in a H&W Water Vapor Resistometer (available from H&W Technology LLC, Rochester, NY, USA). After exposure to water vapor, the biological indicators are cooled naturally and the ampoules are crushed in the biological indicators according to the manufacturer's instructions.
The ampoules are placed in an incubator at 56 ° C and periodically (for example, after 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours , 18 hours, 24 hours, 36 hours, 48 hours and / or 72 hours) removed to observe the color of the liquid medium, the color of the nylon membrane and the fluorescence of the liquid medium. Fluorescence can be detected visually by lighting the tubes with a portable ultraviolet light source, or alternatively, the tubes (or the liquid thereof) can be placed on a suitable fluorometer to measure fluorescence.
Biological indicators that are subjected to little or no exposure to water vapor (for example, 0 to 1 minutes of water vapor exposure) will show conversion of the pH indicator (purple from bromcresol) from purple to yellow in the broth and on the nylon membrane. These biological indicators will also show the substantial conversion of the fluorogenic enzyme substrate into a final fluorescent product (4-methylumbelliferone).
Biological indicators that are subjected to a lethal exposure to water vapor (for example,> 15 minutes) will show accumulation of purple bromcresol on the nylon membrane, but will not show substantial conversion of the purple indicator to yellow. These biological indicators also will not show the substantial conversion of the fluorogenic enzyme substrate to a final fluorescent product.
Table 23. Substrate in the first position - fluorescence Preparatory example 1 - Preparation of a biological sterilization indicator (BI) To exemplify the present disclosure, several biological sterilization indicators (Bis) were prepared, according to the descriptions provided above and as shown in Figures 4 to 7. The particular details of the Bis used in the examples are provided below.
As shown in Figures 4 to 7, the sterilization biological indicator 100 included a compartment 102, which contained a first portion 104 (for example, a hollow tube) and a second portion 106 (for example, a cap) that were attached to provide a one-piece biological sterilization indicator. The cover was molded polypropylene with general dimensions of approximately 21 mm in length and 14 mm in diameter. The first portion 104 (hollow tube) was molded polycarbonate, with the general dimensions of about 52 mm in length and 12 mm in diameter at the top, with the shape shown in Figures 4 to 6. The total volume of the first portion 104 (by example, a hollow tube) was approximately 3 ml.
As shown in Figures 4 to 6, the second portion (lid) 106 of compartment 102 included 6 openings or openings 107, which provided fluid communication between the interior of compartment 102 (e.g., reservoir 103) and the environment. A filter paper material (not shown in Figures 4 to 6) that acted as a barrier; it was positioned on the sterilizing path over the openings 107 and kept in place with a paper label with a pressure sensitive adhesive backing. The filter paper material was the same as the material present in the cover of quick-read biological indicators currently available from 3M ATTEST 1291 For Water Steam Sterilizer, available from 3M Company of St. Paul, MN, USA. The biological sterilization indicator 100 additionally included a frangible container 120 which contained liquid growth medium 122. Frangible container 120 was produced from borosilicate glass and contained spore growth medium. The media consisted of a modified soybean tryptcasein broth (TSB) containing a purple bromocresol pH indicator, and an enzymatic substrate of 4-methylumbelliferyl-alpha-D-glycoside. The ampoule was approximately 40 mm long and approximately 4 mm in diameter and kept approximately 500 pl of liquid from the media. The liquid growth medium 122 was the same as the medium used in the product currently available from the 3M Company of St. Paul, MN, USA as 3M ATTEST 1291 quick-read biological indicators for Water Steam Sterilizers.
As shown in Figures 4 to 7, the liquid medium container 120 was held in place within the biological sterilization indicator 100 by an insertion element 130. The insertion element (also called a fracturing device) 130 served both to hold the container 120 in place as well as to function to facilitate controlled rupture of container 120, which occurs during an activation step of the BI, when the second portion 106 is pushed down to break the liquid medium container 120. The insert 130 was a frame molded polycarbonate with an approximate dimension of 22 mm long by 9 mm wide. The second portion 106 had a sealing projection 156 positioned to contact the first end 101 of the first portion 104, at the open top end 157 of the first portion 104 to close or seal (for example, hermetically seal) the biological sterilization indicator 100 after activation. The biological sterilization indicator 100 additionally included spores of G. stearothermophilus (ATCC 7953) 115 positioned in fluid communication with the first portion 104. Spores 115 were deposited in a spore reservoir 136 of a polypropylene spore carrier 135 (9 mm x 4 mm). Spores 115 were deposited directly on the polypropylene surface, and spore reservoir 136 had a volume of approximately 15 pl. The compartment 102 included a lower portion 114 (which at least partially defined a first chamber 109) and an upper portion 116 (which at least partially defined a second chamber 111), which were partially separated by a wall or partial internal projection 118, in that an opening 117 was formed which provided fluid communication between the first chamber 109 and the second chamber 111. The second chamber 111 was adapted to accommodate the spores 115. The first chamber 109 was adapted to accommodate the frangible container 120, particularly prior to activation . Wall 118 was angled or tilted, at an angle other than zero and not straight in relation to the longitudinal direction of compartment 102, as shown in Figures 4 to 7. The second chamber 111, which can also be called the “growth chamber of spores ”or“ detection chamber ”, included a volume to be interrogated regarding the spore viability to determine the effectiveness of a sterilization process. The liquid medium container 120 was positioned and maintained in the first chamber 109 during sterilization and when the container 120 was not fractured. Spores 115 were housed in the second chamber 111 and in fluid communication with the environment during sterilization. The sterilizer moved to the second chamber 111 (for example, through the first chamber 109) during sterilization. Subsequently, the liquid medium 122 moved to the second chamber 111 (for example, from the first chamber 109) during activation, when the container 120 was fractured and the liquid 122 was released into the compartment 102. The first chamber 109 had a volume of about 2800 microliters (without all internal components). The cross-sectional area of the first chamber 109, immediately above the wall 118 was approximately 50 mm2. The second chamber 111 had a volume of about 210 microliters. The cross-sectional area of the second chamber 111, immediately below the wall 118, was approximately 20 mm2. The biological sterilization indicator 100 additionally included a substrate 119. Substrate 119 was approximately 9 mm x 8 mm in size and was sized to rest on top of wall 118. Substrate 119 was positioned between the first chamber 109 and the second chamber 111 of the biological sterilization indicator 100. Substrate 119 included an opening 121 formed through it about 3.2 mm (0.125 inch) in diameter, the hole was approximately centered on the substrate. Substrate 119 was positioned between (for example, sandwiched between) insertion element 130 and wall 118. Substrate 119 was formed from loaded nylon, and in particular, was a resurfacing loaded transfer membrane available from GE Water & Process Technologies, Trevose, PA, USA, under the trade name "MAGNAPROBE" (pore size 0.45 micron, 30 cm X 3 m cylinder, catalog no. NP0HY00010, material no. 1226566). The biological sterilization indicator 100 had a ventilation feature 162 as shown in Figure 7, positioned to fluidly couple the second chamber 111 to the first chamber 109. In addition, as shown in Figure 7, the biological sterilization indicator 100 had a rib or protuberance 165 which was integrally formed with a wall 108 of compartment 102, which was positioned to hold spore carrier 135 in a desired location in compartment 102. Compartment 102 was tapered (see, for example, tapered portion 146 in Figure 6) so that the cross-sectional area in compartment 102 generally decreases along the longitudinal direction DL.
Example 1 - Correlation of Fluorescence Readings with Growth after 24 hours The biological indicators (BI) of the project shown in Figures 4 to 7 and described above in Preparatory Example 1 were constructed with ~ 1 X 107 CFU from a G spore collection stearothermophilus ATCC 7953. Some of the Bl's (results shown in Tables 26 and 27 and discussed below) were made without the substrate material. The liquid growth medium 122 was the same as that used in 3M ATTEST 1292 quick-read biological indicators for Water Steam Sterilizers, available from the 3M Company in St. Paul. Each BI was then operated through a water vapor sterilization cycle of different lengths of 1 minute, 1 minute and 45 seconds, 2 minutes, 2 minutes and 15 seconds, 2 minutes and 30 seconds, and 3 minutes at 132 ° C / 270 ° F Gravity Water Vapor on a H&W Water Vapor Resistometer (available from H&W Technology LLC, Rochester, NY, USA). After sterilization, the Bl’s were naturally cooled and activated in a 490 AUTOREADER reading device, available from 3M Company, St. Paul, MN, USA, similar to the 290 AUTOREADER reading device, available from 3M Company; certain features of the 490 AUTOREADER reading device are described in copendent orders US No. 61/409042 (Summary No. 66175US002) and 61/408997 (Summary No. 66176US002). The fluorescent readings in excitation / emission of 365/460 nm were taken every 1 minute for 60 minutes. If fluorescence is detected, it is reported as “YES”; if no fluorescence is detected, it is reported as "NF" (ie, no fluorescence). In addition, after 24 hours of incubation in the reading device, the Bis were removed and evaluated for growth, based on a color change (from the pH indicator) in the purple to yellow media. If the color change is observed, it is reported as “YES”; if no color changes are observed, this is reported as "NO".
The results shown in Table 24 and Table 25, below, indicate a good correlation between fluorescence results and 24-hour growth confirmation is achieved when the substrate is positioned in the first location for all Bis exposed to all extensions of sterilization cycles.
The results shown in Table 26 and Table 27, below, indicate that inconsistent results were observed for Bis when no substrate was present, particularly at cycle times of 2 minutes and 15 seconds, 2 minutes and 30 seconds and 3 minutes.
Table 24. Observations of fluorescence Table 25. Observations of growth (color change after 24 h) Table 26. Without substrate - fluorescence Table 27. Without substrate - growth after 24 h Even if approximations are the ranges and numerical parameters that establish the broad scope of the invention, the numerical values set out in the specific examples are reported as accurately as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective test measurements.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the headings, unless specified.
The complete descriptions of all patents, patent applications, publications and nucleic acid and protein database entries that are cited in the present invention are hereby incorporated by reference as if individually incorporated. Various modifications and alterations of the present invention will become evident to those skilled in the art without departing from the scope and intention of this invention and it should be understood that the present invention should not be unduly limited by the illustrative modalities presented herein. CLAIMS:

权利要求:
Claims (6)
[1]
1. Method of detecting a biological activity, CHARACTERIZED by the fact that it comprises: providing a sample comprising a source of one or more predetermined biological activities; a first indicator system comprising a pH indicator with a first absorbance spectrum, in which the pH indicator can be converted by a first predetermined biological activity into a first biological derivative; a second indicator system comprising a fluorogenic reagent that is converted by a predetermined biological activity to a fluorescent derivative with a second emission spectrum; a substrate that receives and concentrates the pH indicator of an aqueous mixture; and an instrument that detects the pH indicator or the fluorescent derivative, wherein the instrument comprises an optical path; forming a first aqueous mixture comprising the sample, the pH indicator and the fluorescent reagent; placing the first aqueous mixture in fluid communication with the substrate to form a second aqueous mixture in which a concentration of the pH indicator is less than the concentration of the pH indicator in the first aqueous mixture; and detecting a presence or absence of fluorescence of the fluorescent derivative, wherein detecting a presence or absence of fluorescence of the fluorescent derivative comprises using the instrument to detect the fluorescent derivative, wherein the optical path does not intersect any portion of the substrate; observe the substrate to detect the pH indicator or the first biological derivative; in which the first absorbance spectrum includes absorbance detectable in at least a portion of wavelengths present in the second emission spectrum.
[2]
2. Method according to claim 1, CHARACTERIZED by the fact that the detection of the presence or absence of fluorescence of the fluorescent derivative comprises detecting the presence or absence of fluorescence in the second aqueous mixture.
[3]
3. Method according to claim 1 or 2, CHARACTERIZED by the fact that a concentration of the pH indicator in the first aqueous mixture is sufficient to prevent the detection of an otherwise detectable amount of the fluorescent derivative.
[4]
4. Method, according to any one of claims 1 to 3, CHARACTERIZED by the fact that it also includes exposing biological activity to a sterilizer.
[5]
Method according to any one of claims 1 to 4, CHARACTERIZED by the fact that the pH indicator comprises a chromophore, wherein the detection of the first biological derivative comprises the detection of a color.
[6]
6. Method according to any one of claims 1 to 5, CHARACTERIZED by the fact that it further comprises: providing an instrument that detects the pH indicator and the fluorescent derivative; and use the instrument to detect the pH indicator and the fluorescent derivative.

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法律状态:
2018-05-22| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

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2019-06-04| B06T| Formal requirements before examination|

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2020-01-28| B09A| Decision: intention to grant|

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2020-03-10| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 28/10/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US40896610P| true| 2010-11-01|2010-11-01|

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US40897710P| true| 2010-11-01|2010-11-01|

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US61/408,966|2010-11-01|

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US61/408,977|2010-11-01|

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PCT/US2011/058212|WO2012061213A1|2010-11-01|2011-10-28|Method of detecting a biological activity|

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