Methods and systems for analyzing biological activity of a fluid sample

Abstract

A method and system for analyzing the bioactivity of fluid samples. The method includes: providing a filter unit comprising a membrane having a front and a back side, allowing a fluid sample to pass through the membrane from the front side; applying the filter unit to a container; adding a culture medium to the container; and performing a scanning and image analysis procedure on the filter membrane using at least one selected scanning wavelength. The scanning is an optical 3D scan and includes acquiring multiple images along at least one scanning path.

Description

Technical Field

[0001] This invention relates to systems and methods for analyzing the biological activity of fluid samples, such as for determining the presence, concentration, and / or activity of microorganisms and / or for determining one or more characteristics of microorganisms. Background Technology

[0002] Microbiological and bioactivity testing is used for many different fluid or solid extracts. For example, it is commonly used to check for parasites in water, for disease-related microorganisms in bodily fluids, for fungal growth in building elements, and for microbial growth in food. Furthermore, in industrial processes, various microbiological tests (including qualitative and quantitative tests) are standard practice.

[0003] Many of these tests are performed in a laboratory by collecting samples and culturing them on or in a culture medium until microorganisms can be identified. Such tests typically take several days and are very labor-intensive. Furthermore, the concentration of microorganisms can be very low, necessitating sample concentration before testing.

[0004] In many cases, it is crucial that inspections be both quick and accurate. For example, if drinking water is contaminated with high levels of microorganisms such as E. coli, it is essential to immediately stop the supply to avoid contaminating the pipes and preventing people from experiencing discomfort from the water.

[0005] US2014342397 discloses an apparatus and method for detecting particles, particularly parasites, in water for online applications. The method includes passing at least a portion of water through a filter; indirectly sonicating the filter using ultrasound to release parasites already collected in the filter without damaging them; collecting the parasites for detection; and detecting the collected parasites. This is used to collect parasites in the filter and / or increase the concentration of parasites before filtration and / or disrupt aggregates without damaging the parasites themselves.

[0006] US2011315625 discloses a method and apparatus for detecting microorganisms, such as yeast and bacteria in a mixture. The method includes passing a sample mixture through a filtration device pretreated with a detergent, resuspending the filter residue from the filtration membrane, and detecting the presence of microorganisms in the filter residue.

[0007] US3741877 discloses a structure for collecting and culturing microorganisms obtained from an aqueous solution, comprising a filter sealed to a surface of an absorbent pad containing microbial nutrients. Microorganisms in the aqueous solution are filtered through the filter by capillary action on the pad. The microorganisms deposit on the filter, and the aqueous solution flowing into the pad provides a carrier for contact with the nutrients and organisms. Summary of the Invention

[0008] One object of the present invention is to provide a system and method for analyzing the bioactivity of fluid samples that is rapid, simple to operate, and has high precision and high sensitivity.

[0009] In one embodiment, an objective is to provide a system and method for analyzing the bioactivity of fluid samples, enabling highly accurate analysis even at very low microbial concentrations.

[0010] In one embodiment, an objective is to provide a system and method for analyzing the bioactivity of fluid samples, by which one or more characteristics of microorganisms can be determined.

[0011] These and other objectives have been achieved by the invention as defined in the claims and as described below.

[0012] It has been discovered that the present invention or its embodiments have many additional advantages, which will be apparent to those skilled in the art from the following description.

[0013] A method for analyzing the bioactivity of fluid samples has been discovered that is both rapid and accurate.

[0014] The method includes

[0015] • Provides a filter unit comprising a membrane having a front and a back side.

[0016] • Allow the fluid sample to pass through the filter membrane from the front side.

[0017] • Apply the filter unit to the container.

[0018] • Add the culture medium to the container, and

[0019] • Perform a scanning and image analysis procedure on the filter membrane using at least one of the selected scanning wavelengths.

[0020] The scan is an optical 3D scan and includes acquiring multiple images along at least one scan path.

[0021] This method can be used to determine biological activity even when the concentration of microorganisms in a fluid sample is very low. Samples can be large as needed, ranging from a few microliters to several liters, such as from 0.1 mL to about 100 liters, or from 0.5 mL to 500 mL. Typical sample volumes will be from about 1 mL to about 100 mL.

[0022] Fluid samples can include fluid liquids and / or gases.

[0023] Fluid samples can be passed through a filter membrane, for example, by squeezing the fluid sample through the filter membrane, by applying a pressure reduction (e.g., vacuum) to one side of the back of the filter membrane, by suction, by pumping, and / or by centrifugation.

[0024] Microorganisms in a fluid sample are collected on and / or in a filter membrane, and by placing the microorganisms collected by the filter membrane in a culture medium and then scanning the filter membrane, it is very rapid to determine whether microorganisms have been collected on and / or in the filter membrane. Advantageously, the quantity and / or quality (live or dead) of the collected microorganisms can be determined, and thus the concentration of microorganisms in the liquid sample can be determined.

[0025] The front side of the filter is the filter surface from which the fluid enters the filter, and the back side of the filter is the side opposite to the front side.

[0026] Typically, the main part of the filtrate is collected at the front of the filter, while small particles such as microorganisms may enter the filter membrane and be captured within it.

[0027] It should be emphasized that the term “including / comprises” as used herein should be interpreted as an open term, that is, it should be regarded as specifically indicating the presence of features specifically stated, such as elements, units, wholes, steps, components and combinations thereof, but does not exclude the presence or addition of one or more other stated features.

[0028] References to "some embodiments" or "one embodiment" mean that a particular feature, structure, or characteristic described in connection with such embodiments is included in at least one embodiment of the disclosed subject matter. Therefore, the phrases "some embodiments" or "one embodiment" appearing in various places throughout the specification do not necessarily refer to the same embodiment. Furthermore, those skilled in the art will understand that particular features, structures, or characteristics can be combined in any suitable manner within the scope of the invention as defined in the claims.

[0029] The term “basically” should be understood in this document to include common product variations and tolerances.

[0030] Throughout the specification or claims, unless the context otherwise requires, the singular includes the plural.

[0031] All features of the invention and embodiments thereof as described herein, including the scope and preferred scope, may be combined in various ways within the scope of the invention unless there is a specific reason for not combining these features.

[0032] Samples may include liquids and / or gases. Water-based samples are advantageous. Samples may advantageously be selected from wastewater, surface water, drinking water, wash water, liquid food, human or animal bodily fluids (e.g., saliva or urine), or any sample containing any of these components. Other examples of samples include exudates, such as wound exudates, and samples scraped from the skin and mixed with a liquid (e.g., water).

[0033] The scanning and image analysis procedure can be performed using any scanning and analysis procedure suitable for the method of the present invention. Examples of applicable scanning and image analysis procedures are described, for example, in US2016069786A, WO11072698A1, US2011261164A, US2017350800A and / or US2003138139A, wherein the scanning and image analysis procedure is modified to perform a 3D scan of the filter membrane, and preferably such that the 3D scan includes at least a portion of the thickness of the filter membrane and advantageously includes at least one layer of culture medium on the front side of the filter membrane.

[0034] In one embodiment, the scan is or includes a time-delayed series of scans. A time-delayed series of scans may, for example, include performing consecutive scans with a first time delay between each scan, and then running the images of the respective scans or processed images with a second time delay, which is advantageously shorter than the first time delay. For example, the corresponding consecutive scans may be performed with approximately 1 minute to 2 hours between each scan, and the images of the respective scans or processed images may be run, for example, by displaying them immediately after each other, for example, with a second time delay of 1 to 30 seconds.

[0035] In one embodiment, a delayed series of scans includes comparing each of a plurality of subsequent scans with one or more scans that are previous scans that are the corresponding subsequent scans.

[0036] In one embodiment, time-lapse serial scanning includes comparing historical images with later-acquired images.

[0037] It has been found that a surprisingly rapid determination of biological activity can be obtained by sequentially comparing the most recently acquired scan with one or more previous scans (e.g., including immediately adjacent previous scans).

[0038] Advantageously, the filter membrane is at least partially transparent to at least one scanning wavelength. The filter membrane does not need to be completely transparent. Advantageously, when immersed in water or a culture medium, the filter membrane is at least about 2% transparent to the selected scanning wavelength. Immersion in water or culture medium should advantageously be just sufficient to cover the filter membrane.

[0039] The filter membrane can be advantageously embedded in the culture medium.

[0040] Transparency can be determined by emitting a light beam containing a selected scanning wavelength toward the front of the membrane at an angle of approximately 90 degrees (normal incidence) and by determining the selected scanning wavelength as a percentage of the membrane.

[0041] In cases of very low transparency, another scanning wavelength or several scanning wavelengths can be selected. Alternatively, the intensity of at least one of the selected scanning wavelengths can be increased.

[0042] Preferably, when immersed in water or a culture medium, the filter membrane is at least about 10% transparent for the selected scanning wavelength, for example, at least about 50% transparent.

[0043] It has been found that, at the selected scanning wavelength, the analysis of biological activity is particularly accurate, efficient, and rapid when the refractive index of the filter membrane material is relatively close to that of the culture medium. When the filter membrane is not immersed in the culture medium, light waves may be scattered in multiple directions due to the small openings in the membrane. However, when the filter membrane is immersed in the culture medium, and at the selected scanning wavelength, the refractive index of the culture medium is relatively close to that of the filter membrane, the filter membrane may become substantially invisible, at least at that or these scanning wavelengths.

[0044] In one embodiment, the filter membrane is a material having a refractive index of less than about 1.5 at the selected scanning wavelength, for example, between 1.25 and 1.38, or between 1.3 and 1.35.

[0045] In one embodiment, the filter membrane has a refractive index of less than about 1.5 at a wavelength of 589.29 nm, for example, between 1.25 and 1.38, or between 1.3 and 1.35.

[0046] Preferably, the filter membrane is made of a material having the filter membrane refractive index and the culture medium has the culture medium refractive index, wherein the refractive index difference (RID) between the filter membrane refractive index and the culture medium refractive index is less than about 0.35, for example less than about 0.1, for example less than about 0.05, for example less than about 0.025, for example less than about 0.01, at the selected scanning wavelength and / or at a wavelength of 589.29 nm. It has been found that a low RID is particularly advantageous for rapid detection of biological activity. For example, the scan can begin immediately after the filter membrane contacts and embeds into the culture medium, i.e., intermediate culture is omitted.

[0047] In one embodiment, biological activity can be determined within several hours. Advantageously, the filter membrane has transparency and refractive index, which are selected such that when the filter membrane is immersed in a culture medium, a scanning and image analysis procedure will reveal the presence of microorganisms with a thickness of 0.6-0.7 μm on the membrane, said scanning and image analysis procedure comprising wave characteristic scans selected from at least one wavelength of phase velocity, group velocity dispersion, wave dispersion, wavefront, wave phase, group delay dispersion, or time of flight.

[0048] Therefore, if the microorganisms change at least one of phase velocity, group velocity dispersion, wave dispersion, wavefront, wave phase, group delay dispersion, or time of flight for the selected scanning wavelength, the filter membrane can be used to perform the method of the present invention.

[0049] This can be determined by comparing scans of phase velocity, group velocity dispersion, wave dispersion, wavefront, wave phase, group delay dispersion, or time of flight at the selected scan wavelength with the presence of microorganisms to the corresponding scans without microorganisms. Microorganisms can be, for example, *Escherichia coli*.

[0050] Therefore, in one embodiment, the method includes using scans of phase velocity, group velocity dispersion, wave dispersion, wavefront, wave phase, group delay dispersion, or time of flight to analyze biological activity.

[0051] Advantageously, the at least one selected scanning wavelength includes multiple scanning wavelengths, such as a wavelength range and / or two or more discrete wavelengths. Therefore, performing morphological analysis on microorganisms becomes simpler.

[0052] Preferably, the scan is performed at a scan depth of at least about 0.05 μm, for example from about 1 μm to about 5 mm, for example from about 5 μm to about 3 mm.

[0053] Therefore, it is possible to analyze microorganisms trapped within the filter membrane. To date, no one has suggested analyzing microorganisms trapped within filter membranes. Because microorganisms in a sample can range in size, they are highly likely to be trapped within the filter membrane. Therefore, methods that include analyzing microorganisms trapped within the filter membrane are more accurate. Furthermore, a quantitative determination of the number of microorganisms can be obtained with very high precision.

[0054] In one embodiment, the microorganisms include fungi and / or viruses and / or bacteriophages.

[0055] In one embodiment, the method includes performing a assay for detecting or quantifying a virus.

[0056] A) The potential viral content in the liquid sample is retained on the filter membrane by filtration. The filter with the captured virus is placed on a preferred confluent monolayer of host cells and covered with an immobilized culture medium. The host cells are, for example, bacteria used for bacteriophages or viruses.

[0057] B) Mix the potentially virus-carrying liquid sample with host cells and retain it on the filter membrane via filtration. Place the filter containing the captured virus and cells on a solidified culture medium or directly on a transparent window and cover it with solidified culture medium.

[0058] C) Potentially virus-infected cells are retained on the filter membrane by filtration. The filter with the captured cells is placed on a solidified culture medium or directly on a transparent window and covered with solidified culture medium.

[0059] Continuous monitoring of cells at the onset of their lysis cycle involves the formation of infection units. Infectious viruses migrate from the initially infected host cell to surrounding cells, causing nearby cells to lyse and form plaques. These are called plaque-forming units (PFUs) and are typically well-defined because the area contains damaged cells.

[0060] In some cases, the virus does not kill the host cell, but may form a visible population of infected cells (represented as lesions) and be counted as lesion-forming units (FFUs).

[0061] Advantageously, the 3D scan includes scanning at least a portion of the filter membrane thickness. Preferably, the scan includes scanning the entire filter membrane thickness.

[0062] In one embodiment, the 3D scan includes scanning the entire thickness of the filter membrane and includes at least a volume of culture medium located on the front side adjacent to the membrane, and optionally a volume of culture medium located on the back side adjacent to the culture medium.

[0063] Filtrate containing live microorganisms can be trapped within the thickness of the filter membrane, specifically between its front and back sides. By matching the refractive index of the filter membrane material and the culture medium, optical access can now be made to the filtrate trapped within the membrane, and even to objects located on the back side of the membrane. Therefore, the detection of bioactivity becomes insensitive to the orientation (up / down) of the filter surface.

[0064] However, orientation can be important when the method involves determining the growth of microbial colonies. When the filter membrane faces the bottom of the container, a limited volume of culture medium can be located between the bottom of the container and the filter. Colonies may be confined to this volume because they may have difficulty growing into the filter membrane. Although microorganisms may grow inside the filter membrane, the limited space within the membrane may result in limited growth.

[0065] Therefore, in cases where the biological activity includes the activity of one or more microbial colonies, it may be advantageous for the front of the filter membrane to face away from the bottom of the container.

[0066] In one embodiment, the filter membrane faces the bottom of the container, and the growth of microorganisms on the front side is confined to a thin culture medium layer geometrically defined by the bottom of the container and the front side of the filter. The volume to be scanned will be quite small, allowing for rapid identification of any growth and biological activity. This starting point may be good for CFU determination, as colonies may already be measurable after a few cell divisions.

[0067] In certain types of colony determination, allowing growth to occur in thicker media may be advantageous, enabling colonies to freely form 3D structures that can be used for microbial typing. However, it cannot be ruled out that thin-layer growth may produce colony structures (two-dimensional) suitable for type differentiation and determination.

[0068] Furthermore, it may be advantageous to place the bioactive material as close as possible to the window (e.g., the bottom of the container or the lid from which the scanned image is obtained): for typical imaging systems, point distortion increases with the lengthening of the light path through the window and the culture medium between the bioactive material and the window. That is, by making this light path smaller, distortion can be minimized.

[0069] In one embodiment, the entire filtration membrane, including the filtrate and a surrounding thin layer of culture medium, is subjected to 3D scanning microscopy and image analysis. This culture medium is advantageously included, particularly when it is functionalized with an indicative substance, which, for example, causes a color change for the selected activity. For example, a chromogenic culture medium may induce a localized color reaction to specific enzymes secreted by bacteria, archaea (bacteria), and fungi.

[0070] A scanner comprising a light emitter and an image acquisition device, wherein the image acquisition device has an optical axis, can be used to perform the scanning.

[0071] The scanning path can be at any angle to the optical axis, but preferably, the scanning path is substantially orthogonal to the optical axis.

[0072] In one embodiment, the scan path may coincide with the optical axis. In one embodiment, the angle between the scan path and the optical axis is up to about 45 degrees, for example up to about 30 degrees, for example up to about 15 degrees, for example up to about 10 degrees, for example up to about 5 degrees, for example up to about 2.5 degrees, for example up to about 1 degree.

[0073] Advantageously, the light emitter has a central axis arranged at an angle to the front / back surface of the filter membrane, such angle being up to about 45 degrees, for example up to about 30 degrees, for example up to about 15 degrees, for example up to about 10 degrees, for example up to about 5 degrees, for example up to about 2.5 degrees, for example up to about 1 degree. The image acquisition device can advantageously be tilted at the corresponding angle relative to the front / back surface of the filter membrane.

[0074] The scanning and image analysis procedure advantageously involves performing scans continuously along the scan path and generating a set of acquired images for each scan. Scans can be performed immediately, one after another, or there may be a time delay between scans. This time delay can, for example, depend on any differences between previous scans, thus increasing the scan frequency in areas of higher biological activity compared to areas where less biological activity was observed.

[0075] Advantageously, the scanning and image analysis procedure includes performing a background scan along the scan path and generating a set of background images. These background images, or one or more composite images, include data derived from these background images.

[0076] The background images are preferably generated as soon as possible after the culture medium has been added to the container. Preferably within 20 minutes, for example within 10 minutes, for example within 5 minutes, for example within 1 minute.

[0077] In one embodiment, the scanning and image analysis procedure includes processing each group of acquired images by a method comprising subtracting the corresponding pixel value of the corresponding background image from the corresponding pixel value of the corresponding acquired image in the group of acquired images.

[0078] Therefore, any changes caused by biological activity will be detected very quickly and displayed with high precision. Noise caused by minute defects in the filter membrane and minute refractive index differences between the filter membrane material and the culture medium can be suppressed.

[0079] The scanning and image analysis process may include synthesizing a result image from images acquired from a corresponding set. Synthesis may include generating an image seen from another viewpoint (e.g., at a 90-degree angle to the front of the filter), merging partial or entire images, for example, such that a portion of the filter membrane and all biological activity throughout the entire depth of the filter membrane can be seen in a single image. In one embodiment, each result image is primarily synthesized from a set of acquired images. Preferably, each result image is synthesized from a set of acquired images and, optionally, up to 10% of previously acquired images as replacement images.

[0080] The images acquired in each group can be advantageously associated with a time attribute that indicates the time at which a selected image was acquired, such as the time at which the first image of the scan was acquired or the time at which the last or any other pre-selected image of the scan was acquired.

[0081] Therefore, it is possible to provide a representation of the acquired image set as a function of time from the image set generated by continuous scanning.

[0082] In one embodiment, each of the resulting images is associated with a temporal attribute of the acquired image set primarily used for image synthesis. Therefore, each image and each synthesized image is associated with a temporal attribute, which simplifies the sorting of images to determine bioactivity.

[0083] Scanning and image analysis procedures can, in principle, be performed using any type of microscopy technique. For example, scanning and image analysis procedures may include bright-field microscopy, dark-field microscopy, phase-contrast microscopy, and / or fluorescence microscopy.

[0084] In one embodiment, the scanning and image analysis procedure includes tilted illumination, off-axis illumination, multi-axis illumination (e.g., biaxial illumination), and / or inline illumination, optionally with the illumination varying from one scan to the next.

[0085] In one embodiment, the scanning and image analysis procedure includes light transmission scanning and / or light reflection scanning.

[0086] In one embodiment, the scanning and image analysis procedure includes hyperspectral scanning and imaging.

[0087] Hyperspectral scanning has been found to be highly beneficial in this method. Hyperspectral scanning involves scanning the scene at multiple selected wavelengths. The sensor can collect information as a set of wavelength images, where each image represents a narrow wavelength range of the electromagnetic spectrum, also known as a spectral band. These "images" are combined to form a three-dimensional (x,y,λ) hyperspectral data cube for processing and analysis, where x and y represent the two spatial dimensions of the scene, and λ represents the spectral dimension (including a series of wavelengths).

[0088] The accuracy of these sensors can be measured by spectral resolution, which is the width of each spectral band captured. If a scanner detects a large number of fairly narrow frequency bands, it can identify objects even if they are captured in only a few pixels.

[0089] In one embodiment, the scanning and image analysis procedure includes a wave characteristic scan of at least one wavelength, wherein the wave characteristic includes a characteristic of at least one wavelength that depends on the material through which it passes. Examples of wave characteristics include phase velocity, group velocity dispersion, wave dispersion, wavefront, wave phase, group delay dispersion, time of flight, or any combination thereof.

[0090] Wavelength characterization scanning can be used to very quickly detect whether any particles or microorganisms have been captured by the filter membrane. For microbial characterization, a selected scanning wavelength in the visible light range can be preferably used.

[0091] Time-of-flight is a measure of the time it takes for an object, particle, or wave to travel a distance in a culture medium or to be reflected. A time-of-flight scanner will detect this if the filter is at least partially transparent to the wavelength and the captured particles / microorganisms are opaque to the wavelength or have other degrees of transparency.

[0092] In cases where wavefront scanning is involved, a wavefront sensor can be applied to measure any wavefront aberrations in the detected optical signal from light having a selected scanning wavelength after passing through a filter membrane. If the wavefront aberration increases from one scan to subsequent scans, it indicates the presence of biological activity.

[0093] In one embodiment, baud rate scanning includes acquiring multiple baud rate images along a scanning path (e.g., at least one scanning path).

[0094] Advantageously, the scanning and image analysis procedure includes performing a boolean background scan along the scan path, generating a set of background boolean images, and comparing at least one of the acquired boolean images with a corresponding image of the background boolean image.

[0095] At least one selected scanning wavelength may, in principle, include any wavelength that does not impede biological activity (e.g., by killing microorganisms). At least one selected wavelength may, for example, include one or more wavelengths in the range of 200 nm to 1200 nm, such as at least one wavelength in the visible light range of 380 nm to 740 nm, such as from 450 nm to 700 nm.

[0096] The desired wavelength depends on the type of microorganisms expected to be found in / on the filter membrane. A combination of selected scanning wavelengths may be necessary, such as one wavelength at approximately 700 nm and another at approximately 400 nm.

[0097] Where microorganisms can produce a color reaction, at least one of the selected wavelengths preferably includes the wavelength of the color in question. For example, the culture medium can be selected to allow live microorganisms to produce or change color, for example, due to changes in pH.

[0098] Wavelengths in the ultraviolet (UV) range can be destructive to microorganisms, thus limiting the intensity or duration of such exposures. However, some microorganisms are more resistant to UV wavelengths than others, and this factor can be used, for example, to characterize microorganisms.

[0099] At least one selected wavelength can be advantageously chosen depending on the culture medium used.

[0100] The culture medium is usually pale yellow and may have high absorbance at wavelengths below 500 nm.

[0101] The culture medium may or may not absorb some of the scanning light, including at least one of the selected scanning wavelengths. In cases where the culture medium and / or the bottom of the container and / or the filter absorb at least one of the selected scanning wavelengths, it is advantageous to adjust the intensity of the selected scanning wavelength to ensure that the acquired image is sufficiently clear, for example by increasing the intensity to compensate for light loss due to absorbance.

[0102] In one embodiment, the at least one selected wavelength includes one or more excitation wavelengths for exciting fluorophores, such as fluorophores present in a culture medium, fluorophores expected to be generated in the culture medium upon microbial activation, and / or fluorophores of microorganisms expected to be biofluorescently active in the sample.

[0103] In one embodiment, the scanning and image analysis procedure includes hyperspectral microscopy and / or Raman spectroscopy.

[0104] Scanning and image analysis procedures may advantageously include determining at least one morphological parameter, such as size, shape, and / or texture, of one or more microorganisms and / or colonies. To determine at least one morphological parameter, it may be necessary to apply at least one selected scanning wavelength in the visible light range, for example, from about 450 nm to about 750 nm.

[0105] In one embodiment, the scanning and image analysis procedure includes determining at least one time parameter, such as the temporal variation of number, size, shape, and / or texture of one or more microorganisms and / or colonies.

[0106] 3D scanning can be advantageously performed using two or more selected scanning wavelengths, and the image analysis procedure includes spectral analysis, such as multiplexing between two or more wavelengths.

[0107] The steps of applying the filter unit to the container and adding the culture medium to the container can be performed in any order. In one embodiment, the culture medium is added both before and after applying the filter unit to the container.

[0108] In one embodiment, at least a portion of the culture medium is added before the filter unit is applied to the container.

[0109] In one embodiment, the culture medium is pre-prepared in a container, and a filter unit is placed on the pre-prepared culture medium in the container with its front facing away from the culture medium. The culture medium permeates the filter membrane and can therefore be brought into contact with microorganisms captured on and within the filter membrane.

[0110] The filter can be embedded in the culture medium.

[0111] In one embodiment, at least a portion of the culture medium is added after the filter unit is applied to the container.

[0112] The container includes a bottom and, preferably, a wall defining the top of the opening. Preferably, the 3D scan is performed from the top of the opening. This avoids any absorption, reflection, or scattering of light due to the material of the container.

[0113] Even if the filter membrane is facing down, a scan can be performed from the top. A scan can include scanning the entire thickness of the filter membrane.

[0114] In one embodiment, applying the filter unit to the container includes applying the filter unit to the container such that the front side of the filter membrane faces the bottom of the container.

[0115] In one embodiment, applying the filter unit to the container includes applying the filter unit to the container such that the back side of the filter membrane faces the bottom of the container.

[0116] When scanning is performed from the bottom, the bottom of the container is at least partially transparent to the selected scanning wavelength. In one embodiment, the bottom of the container is at least 50% transparent to the selected scanning wavelength.

[0117] In one embodiment, scanning is performed from the front of the filter membrane.

[0118] In one embodiment, scanning is performed from the back of the filter membrane.

[0119] Advantageously, the culture medium is added in liquid form.

[0120] The culture medium can be any kind of culture medium suitable for growing and / or testing microorganisms.

[0121] In one embodiment, the culture medium is nutrient broth and / or agar.

[0122] Examples of culture media include the following:

[0123] Nutrient media – amino acids and nitrogen sources (e.g., beef, yeast extract). This is an undefined medium because the amino acid source contains a variety of compounds whose exact composition is unknown. These media contain all the elements required for the growth of most bacteria and are non-selective, therefore they are used for the general culture and maintenance of bacteria preserved in laboratory culture collections.

[0124] Basic culture media—mediums containing the minimum nutrients necessary for colony growth, typically lacking amino acids—are frequently used by microbiologists and geneticists to grow "wild-type" microorganisms. These media can also be used to selectively support or inhibit the growth of specific microorganisms. Generally, a wealth of information about the microorganism is required to determine its basic culture medium requirements.

[0125] Selective culture media – used only for the growth of selected microorganisms. For example, if microorganisms develop resistance to a certain antibiotic (such as ampicillin or tetracycline), this antibiotic can be added to the culture medium to prevent the growth of other non-resistant microorganisms.

[0126] Differential media—also known as indicator media—are used to distinguish one type of microorganism from another growing on the same medium. This type of medium utilizes the biochemical characteristics of microorganisms growing in the presence of specific nutrients or indicators (such as Neutral Red, Phenolic Red, Eosin Y, or Methylene Blue) added to the medium to clearly indicate the defining characteristics of the microorganisms. This type of medium is used for the detection and identification of microorganisms.

[0127] In one embodiment, the culture medium is a culture medium in which optical changes occur due to microbial activity.

[0128] In one embodiment, the culture medium is a chromogenic medium, such as a chromogenic medium whose color is locally altered depending on whether it contains coliform bacteria and Escherichia coli. They secrete different enzymes. Coliform bacteria secrete β-d-galactosidase, turning the medium red, while Escherichia coli secretes β-d-galactosidase and β-d-glucuronidase, turning the medium blue.

[0129] The method may further include adding an additive to the container, preferably a biocide and / or antibiotic. This allows for the determination of the response of microorganisms or colonies to the additive, which can provide additional information about the microorganisms.

[0130] In one embodiment, the culture medium may include one or more subcultures, which may be added to the culture medium and / or a filter membrane. The subcultures may advantageously be microorganisms that promote the growth of the target microorganism or microorganisms that inhibit the growth of the target microorganism, for example, by competing with the target microorganism or by producing substances that inhibit the growth of the target microorganism, such as substances toxic to the target microorganism.

[0131] The target microorganism is used to refer to the microorganism tested in the fluid sample.

[0132] Subcultures can be added to fluid samples, for example, before filtration.

[0133] In one embodiment, the subculture is added immediately after the sample passes through the filter membrane, for example by passing a fluid volume containing the subculture through the filter membrane.

[0134] In one embodiment, the subculture is added to the filter membrane immediately after the filter membrane has come into contact with the culture medium.

[0135] In one embodiment, after the filter membrane has been in contact with the culture medium and the first signs of biological activity on the filter membrane have been observed, a subculture is added to the filter membrane.

[0136] In one embodiment, the method includes testing whether a therapeutic agent has a desired effect in treating an infection or microbiome imbalance at a patient's body site, wherein the sample includes a sample from said body site, such as a fluid sample, urine, saliva, wound secretions, etc., and / or cells suspended in a fluid. The therapeutic agent is added to the sample, culture medium, and / or filter as described above regarding the addition of subcultures. Any effect of the therapeutic agent can be observed as a change in bioactivity relative to a corresponding procedure omitting said therapeutic agent. The therapeutic agent may be, for example, one or more probiotics, such as lactic acid bacteria.

[0137] Therefore, therapeutic agents can be tested to determine if they have the desired effect before use. For example, probiotic skincare products and / or wound care agents can be tested to determine if they have the desired effect before being applied to a patient.

[0138] In one embodiment, the method includes determining the number of live microorganisms and / or colonies on and in the filter membrane, and correlating that number with the volume of the liquid sample to determine the number of microorganisms per volume unit.

[0139] The method of the present invention has been found to be very useful for determining the characteristics of filtrate on the front side of a filter membrane as a function of time. Since changes caused by biological activity can be determined with high precision as a function of time, the method can include determining the characteristics of one or more microorganisms, for example, as a response to a culture medium and / or additional additives.

[0140] In one embodiment, the scanning and image analysis procedure includes performing continuous scanning and generating a set of acquired images, wherein the first scan is a reference scan, and after each subsequent scan, a subsequent set of acquired images is processed by methods including: extracting corresponding pixel values ​​of corresponding background images from corresponding pixel values ​​of corresponding images in the subsequent set of acquired images, synthesizing at least one result image, and analyzing the result image to indicate the characteristics of live microorganisms and / or detected live microorganisms.

[0141] In one embodiment, the method includes analyzing multiple fluid samples obtained from the same parent sample, wherein at least two of the liquid samples have undergone different culture media and / or additives. The method may include comparing the resulting images of the analyses from the individual fluid samples. Preferably, the method includes timely comparison of corresponding resulting images of the analyses from the individual fluid samples.

[0142] Culture media and / or added subcultures can aid in the identification of detected live microorganisms. Furthermore, detected microorganisms can be identified directly from their growth pattern, shape, or size. In one embodiment, one or more detected microorganisms can be further identified. In one embodiment, the method includes picking microorganisms from one or more colonies, for example using a picking method known as picking from conventional agar plates. Further identification steps can then be performed on the selected microorganisms.

[0143] In one embodiment, the method includes extracting the entire filter membrane or a portion thereof and subjecting it to an additional step of identifying microorganisms located thereon. The additional step of identifying microorganisms may include, for example, polymerase chain reaction (PCR) and / or matrix-assisted laser desorption / ionization (MALDI) analysis, such as using a time-of-flight mass spectrometer (MALDI-TOF). Pre-filtration of the sample may be necessary when it contains large particles or other solid or semi-solid materials.

[0144] In one embodiment, the method includes a two-step filtration process, comprising providing a pre-filter unit having a pre-filter membrane including a front and a back side, wherein a fluid sample is squeezed through the pre-filter membrane from the front side before the sample is squeezed through the filter membrane, wherein the pre-filter membrane has a larger cutoff particle size than the filter membrane.

[0145] After pre-filtration, the pre-filter unit can be discarded, or the method can include analyzing the pre-filter unit after the filtration process, wherein the analysis includes...

[0146] • Apply the pre-filter unit to the container.

[0147] • Add the culture medium to the container, and

[0148] • Perform scanning and image analysis procedures on the filter membrane of the pre-filtering unit using at least one selected scan.

[0149] Furthermore, the scan described is an optical 3D scan and includes acquiring multiple images along the scan path.

[0150] The pre-filter unit's filter membrane can be analyzed using the methods described herein for analyzing the filter unit's filter membrane.

[0151] This method can be advantageously implemented using the system described herein.

[0152] The present invention also includes a system for analyzing the bioactivity of fluid samples. This system includes...

[0153] • A filter unit, comprising a membrane having a front and a back side, and a filter ring assembly surrounding the membrane.

[0154] • Filter housing, suitable for holding the filter unit in a temporary fixed position, and

[0155] • Containers suitable for filter units

[0156] The filter housing includes an inlet for feeding a fluid sample into the housing and through a filter membrane when the filter unit is in its temporary fixed position, and wherein the filter unit is adapted to be released from its temporary fixed position within the housing and transferred to a container.

[0157] The filter unit, filter membrane, and container can be as described above.

[0158] In one embodiment, when immersed in water and / or in a selected culture medium, the filter membrane is at least about 2% transparent to the selected scanning wavelength, preferably at least about 10% transparent to at least one scanning wavelength, for example, at least about 50% transparent. The selected culture medium can be as described above.

[0159] In one embodiment, the filter membrane is made of a material having a refractive index of less than about 1.5 under dry conditions, for example, between 1.25 and 1.38, or for example, between 1.3 and 1.35, at a selected scanning wavelength, preferably in the visible light range and / or at a wavelength of 589.29 nm.

[0160] Filter membranes can be made from a single material or a combination of materials.

[0161] The filter membrane advantageously possesses transparency and refractive index, selected such that when the filter membrane is immersed in a culture medium, scanning and image analysis procedures, including wave characteristic scans selected from at least one wavelength for phase velocity, group velocity dispersion, wave dispersion, wavefront, wave phase, group delay dispersion, or time of flight, will reveal the presence of microorganisms on the membrane with a cell size such as 0.6–0.7 μm in length. The microorganisms can be as described above.

[0162] The filter membrane may be made of, for example, one or more glass, one or more ceramics, one or more semiconductor materials, one or more metals, or any combination thereof.

[0163] The filter membrane is preferably made of a polymer material. Examples of suitable materials for the filter membrane include polytetrafluoroethylene (PTFE), copolymers of tetrafluoroethylene (TFE), perfluorocyclic polymers (Cytop), perfluorodimethyldioxolane (PDD), and any combination thereof comprising at least one of these.

[0164] It has been found that inorganic material filter membranes may not provide the desired transparency for effective optical scanning. Although inorganic material filter membranes can be provided as very thin and thin-walled membranes, making them appear to have a relatively low refractive index under wetting conditions, the actual refractive index (i.e., under non-wetting conditions) may still be high and may result in less clear scans compared to using polymer filter membranes.

[0165] The filter membrane advantageously has a thickness of about 0.05 μm to about 0.5 cm, for example about 1 μm to about 1 mm, for example about 10 μm to about 500 μm, for example about 30 μm to about 100 μm. In one embodiment, the thickness is 50 micrometers or less.

[0166] The cutoff size of the filter membrane is advantageously selected based on the desired biological activity to be determined.

[0167] In one embodiment, the filter membrane has a cut-off size of about 0.01 μm to about 500 μm, for example about 0.05 μm to about 300 μm, for example about 0.1 μm to about 100 μm.

[0168] The membrane may advantageously have a substantially flat front side and preferably a substantially flat back side.

[0169] The filter membrane can be produced, for example, by a method comprising biaxially stretching a polymer sheet, such as by the method described in US 4,049,589.

[0170] To ensure the filter membrane remains in a stable position, the ring is advantageously rigid at 20°. The ring is preferably made of a polymer material, such as a polymer material compatible with the filter membrane material. The ring can be mechanically fixed, glued, or welded to the filter membrane, for example.

[0171] In one embodiment, the filter housing includes a front portion and a rear portion. The front and rear portions can be releasably mounted to each other to form a housing. Advantageously, the rear portion is adapted to hold the filter unit in a temporary fixed position. Preferably, the front and rear portions are adapted to be mounted to each other to hold the filter unit in a temporary fixed position within the housing.

[0172] The rear portion of the filter housing advantageously includes a release device for releasing the filter unit from its temporary fixed position. Therefore, the filter unit can be transferred into the container in a very simple manner, while reducing the risk of contamination of the filter unit. Preferably, the rear portion is adapted to mechanically hold the filter unit in the temporary fixed position, and preferably, the release device is adapted to mechanically release the filter unit from its temporary fixed position.

[0173] In a variant thereof, the front portion of the filter housing is adapted to hold the filter unit in a temporary fixed position, and the front portion is adapted to mechanically hold the filter unit in the temporary fixed position, and the release device is adapted to mechanically release the filter unit from its temporary fixed position at the front.

[0174] The inlet for feeding the fluid sample into the filter housing is preferably located at the front of the filter housing. Preferably, the outlet for the filtered sample is located at the rear of the housing. Therefore, the filtered sample can be discharged in a simple manner. Alternatively, the rear of the housing includes an outlet collector for collecting the filtered sample. The sample can then be disposed of later, or the entire housing can be placed in its single-use location.

[0175] However, it is desirable that the casing be made of a material that can be sterilized by autoclaving, dry heat sterilization or ethylene oxide sterilization, electron beam sterilization and / or gamma radiation sterilization, such as sterilization-compatible polymers, ceramics, glass, metals or any combination thereof.

[0176] Advantageously, the outer shell is made of or comprises a serializable polymer material, such as polyglycolic acid (PGA), ethylene propylene (diene-) terpolymer (EPDM), silicone, polyvinylidene fluoride (PVF2), polyvinyl fluoride (PVF), ethylene tetrafluoroethylene (ETFE), ethylene trifluorochloroethylene (ECTFE), fluorinated ethylene propylene (FEP), perfluoroalkoxy (PFA), polysulfone (PSU), polyphenylene sulfide (PPS), liquid crystal polymer (LCP), polyether ether ketone (PEEK), high-temperature polycarbonate (PC), acetal (POM), polypropylene copolymer (PPC), polypropylene (PP), aromatic polyamide (AP), or any combination thereof.

[0177] In one embodiment, the release device is a spring-loaded release device comprising at least one spring. Preferably, when the filter unit is held in its temporary fixed position, at least one spring is in an unloaded position.

[0178] Therefore, the filter unit can be easily removed from the housing by separating the rear from the front. The rear of the housing can then be used to place the filter unit into the container, and when the filter unit reaches the container, the spring can be compressed to release the filter unit from the rear of the housing.

[0179] The container preferably has a substantially flat bottom or a convex bottom. A flat bottom may be preferred because there is only a small volume of culture medium between the filter membrane and the bottom of the container.

[0180] The container advantageously has inner wall dimensions that are suitable for the outer periphery of the filter unit's ring. Therefore, the filter unit can be placed with high precision.

[0181] In one embodiment, when the filter is arranged in a container, the bottom of the container has an inner surface that aligns with the front side of the filter membrane.

[0182] Advantageously, the container bottom is at least 2% transparent to the selected scanning wavelength, and preferably the container bottom is made of an optical grade polymer, such as polycarbonate (PC), PMMA, polystyrene (PS), polyethylene (PE), ionomer resin, cyclic olefin copolymer, amorphous copolyester (PETG), and / or polyethylene terephthalate (PET). In one embodiment, the container bottom is made of glass and / or ceramic material.

[0183] The container can be advantageously formed into a multi-container plate, such as part of a multi-grooved plate. This allows for the continuous scanning of several filter membranes.

[0184] In one embodiment, the membrane filter comprises two or more laterally positioned filter membranes. For example, each laterally positioned membrane may be surrounded and separated by a ring device, and preferably separated from each other by the ring device. Thus, a single filter unit can be used to perform multiple determinations. The system may further include, for example, a container separator adapted to divide a container into laterally positioned portions associated with the respective filter membranes of the laterally positioned membranes. Thus, one of the laterally positioned filter membranes can be subjected to one culture medium and / or additional substance, while another of the laterally positioned filter membranes can be subjected to another culture medium and / or additional substance.

[0185] In one embodiment, the laterally positioned filter membranes are identical.

[0186] In one embodiment, the laterally positioned filter membranes differ in at least one property, such as in thickness, structural cutoff dimensions, or refractive index.

[0187] The filter membrane may advantageously include optically detectable markers located at predefined positions, such as positions defined in a coordinate system.

[0188] This can help the 3D scanning system find the focus of the filter.

[0189] For example, a preview can be used to create a scan profile to ensure optimal focusing in each subsequent hourly interval measurement.

[0190] The markers in the filter can also be used to align images recorded at different times, especially when the filter may have shifted due to evaporation of the culture medium.

[0191] The markers can be further used as a "coordinate system" to locate colonies for further analysis. Color calibration markers can be used to correct the color of the culture medium, thereby improving spectral analysis.

[0192] In one embodiment, the system further includes a pre-filtration unit comprising a pre-filtration membrane having a cutoff size larger than that of the filter unit. This pre-filtration unit can be used when the fluid sample contains a large number of particles, such as both large and small particles, for example, as described above. The housing may be adapted to hold the pre-filtration unit in front of the filter unit. The pre-filtration unit may, for example, be held in a temporary fixed position by the front portion of the housing. Preferably, the front portion is adapted to mechanically hold the pre-filtration unit in a temporary fixed position and includes a spring-activated release mechanism, for example, as described above for the rear portion.

[0193] The system may also include at least one culture medium. Preferably, the at least one culture medium has a culture medium refractive index, wherein the refractive index difference (RID) between the filter refractive index and the culture medium refractive index is less than about 0.25, for example less than about 0.1, for example less than about 0.05, for example less than about 0.025, for example less than about 0.01, at the selected scanning wavelength and / or at a wavelength of 589.29 nm.

[0194] The system may also include a scanning and image analysis system adapted to perform a 3D reflective scanning and image analysis procedure on the front side of the filter membrane using at least one selected scanning wavelength in the visible light range, including acquiring multiple images along the scanning path. The scanning and image analysis system may be, for example, as described above.

[0195] All features of the invention, including the scope and preferred scope, can be combined in various ways within the scope of the invention unless there is a specific reason for not combining these features. Attached Figure Description

[0196] The invention will now be further described with reference to preferred embodiments and the accompanying drawings. These drawings are schematic and may not be drawn to scale.

[0197] Figure 1a This is a top view of a filter unit applicable to the method of the present invention.

[0198] Figure 1b and 1c A filter unit including a laterally positioned filter membrane is shown.

[0199] Figure 2 This is a perspective view of the system of the present invention, including a filter housing holding a filter unit (not shown), a syringe, and a container suitable for the filter unit.

[0200] Figure 3 This is a cross-sectional view of the filter housing and filter unit.

[0201] Figure 4 This is a perspective view of the filter housing and filter unit.

[0202] Figure 5-8 The steps for applying a filter to a container are shown.

[0203] Figure 9 An image of the time-delay sequence is shown.

[0204] Figures 10a-10c An example of the scanning results of aquatic bacteria collected and cultured on a PTFE filter is shown.

[0205] Figures 11a-11b An example of a scanning image of aquatic bacteria collected and cultured on a PTFE filter is shown.

[0206] Figures 12a-12d A series of steps of an example of the method of the present invention are shown. Detailed Implementation

[0207] The filter unit shown in Figure 1 includes a filter membrane 1 and a ring device 2 surrounding the filter membrane. As mentioned above, the filter membrane is advantageously substantially flat and includes a front and a back side.

[0208] Figure 1b and 1c The filter unit shown includes laterally positioned filter membranes 1a-1d. Figure 1b The filter unit includes two laterally positioned filter membranes 1a and 1b. The laterally positioned filter membranes 1a and 1b are surrounded by ring devices 2a and 2b, wherein a first portion 2a of the ring device defines the periphery of the filter unit, and a second portion 2b of the ring device defines the cross spacing between the two laterally positioned filter membranes 1a and 1b.

[0209] Figure 1c The filter unit includes four laterally positioned filter membranes 1a, 1b, 1c, and 1d. The laterally positioned filter membranes 1a, 1b, 1c, and 1d are surrounded by ring devices 2a and 2b, wherein the first part 2a of the ring device defines the periphery of the filter unit, and the second part 2b of the ring device defines two intersecting intervals between the four laterally positioned filter membranes 1a, 1b, 1c, and 1d.

[0210] Laterally positioned filter membranes can be arranged in any desired configuration.

[0211] To ensure uniform filtration across the laterally positioned filter membranes, it may be desirable that the flow resistance on each laterally positioned filter membrane be substantially equal. A filter unit with laterally positioned filter membranes ensures that several determinations can be performed using a single filter unit (e.g., as described above).

[0212] Figure 2The system shown discloses an example of a filter housing 5 (not shown) holding a filter unit, a syringe 3, and a container 4 suitable for the filter unit.

[0213] The filter housing 5 includes a front portion 5a and a rear portion 5b. The filter unit, not shown, is held in a temporary fixed position between the front portion 5a and the rear portion 5b of the filter housing.

[0214] The syringe 3 is arranged to feed a fluid sample to the inlet 5a1 of the front portion 5a of the filter housing 5. As mentioned above, the sample can be driven through the filter membrane in any way, therefore the syringe is an optional part of the system. The container 4 is part of the container plate 4a, which includes multiple containers, such as recesses.

[0215] exist Figure 3 Examples of filter housings 15a and 15b and an example of filter unit 16 are shown in more detail in the figure.

[0216] The filter unit includes a filter membrane 17 having a front side F and a back side R, and an annular device 18 surrounding the filter membrane 17. The annular device 18 extends beyond the back side R of the filter membrane 17 to form a mounting device 19 for mechanically holding it in a temporary fixed position via the rear portion 15b of the filter housing.

[0217] The filter housing includes a front portion 15a and a rear portion 15b. The front portion 15a includes an inlet 11, for example, having a shape suitable for a syringe or other device for injecting fluid samples using a Luer interface (LUER) connector. Inside the front portion 15a of the filter housing, a gasket 12 is provided such that when the filter housing is assembled with the filter unit 16, the ring assembly 18 of the filter unit abuts against the gasket 12 of the front portion 15a for sealing. The front portion also includes a plurality of sliding frames 13 (only two shown) for locking the front portion 15a to the rear portion 15b.

[0218] The rear portion 15b of the filter housing includes a first movable portion 15c and a second movable portion 15d, as well as a spring device 21 arranged to hold the first movable portion 15c and the second movable portion 15d and thereby provide a release mechanism suitable for mechanically releasing the filter unit from its temporary fixed position at the rear.

[0219] The rear portion 15b of the filter housing includes a gasket 19 located at the first portion 15c, which seals against the inside of the ring assembly 18 when the filter housing is assembled with the filter unit 16. A support structure 22 is arranged to support the filter membrane and ensure that the membrane does not deform or burst when a fluid sample is squeezed through it. A snap-fit ​​and / or friction structure 20 is provided at the second portion 15d, adapted to engage with the mounting device 19 to mechanically hold the filter unit in a temporary fixed position.

[0220] The first part 15c includes the rearmost flange 15c', and when the second part 15d is pulled toward the rearmost flange 15c', the filter unit, which was held in its temporary fixed position by the rear part 15b, is released from that position. Simultaneously, the spring device 21 is pressed down. The filter unit 16 is not released until the spring device 21 is pressed down. Therefore, the spring device 21 ensures that the filter unit 16 is securely held in its temporary fixed position until actively released from there.

[0221] The second portion 15d of the rear portion 15b of the filter housing also includes a plurality of protruding locking elements 14 of a locking device, adapted to engage with the same number of sliding frames 13 of the front portion 15a for locking the front portion 15a to the rear portion 15b. The locking elements 14 can be twisted into the sliding frames 13 and held in that position for a desired time. When the locking device is in its locked position, the first movable portion 15c and the second movable portion 15d are held in a fixed relationship to each other.

[0222] The rear section 15b also includes an outlet 23 through which the filtered sample can escape. As mentioned, the rear section 15b may alternatively include a collection chamber for collecting the filtered sample, or an additional disposable collection chamber may be temporarily installed to the rear section 15b to collect the filtered sample.

[0223] exist Figure 4 The filter housing and filter unit are shown in perspective view. Reference numerals are... Figure 3 same.

[0224] Figure 5-8 The steps for applying a filter to a container are shown. Figure 5 In this configuration, the rear portion 15b of the filter housing has been removed from the front portion (not shown) of the filter housing. The filter 16 remains in its temporary fixed position relative to the rear portion 15b, and the user will apply the filter unit into the container 4.

[0225] exist Figure 6 In this process, the user has inserted the filter unit along with a portion of the rear 15b into the container 4 and pressed down the spring device to release the filter unit 16 from the rear 15b.

[0226] exist Figure 7 As can be seen, filter unit 16 has been released and the user is removing the rear part 15b.

[0227] exist Figure 8 In the middle, filter unit 16 is now ready to add culture medium.

[0228] Figure 9This is an example of a series of time-lapse images showing a single bacterium forming a colony on a membrane filter over 15 hours.

[0229] The bacteria depicted are one of many collected from fluid samples on a PTFE filter (pore size cutoff: 0.2 μm) and embedded in PCA (principal component analysis) medium.

[0230] The image series clearly illustrates the transformation from a single bacterium to a microcolony-forming unit through proliferation.

[0231] Figure 10a An example of aquatic bacteria collected and cultured on a PTFE filter after 5 hours of growth is shown. The bacteria were collected on a PTFE filter and embedded in WPCA (Horizontal Plate Counting Agar) medium. Filter parameters were: pore size cutoff of 0.2 μm and diameter of 13 mm.

[0232] Figure 10a The background scan subtraction (or correction) image of the filtered image scanned in 10a is shown. The corrected image enhances the temporal changes that have occurred since the recording of time-delayed image 1 (the first time-delayed scan image). The first time-delayed image was used as the background subtraction image.

[0233] Figure 10c The graph shows the CFU concentration detected by bacterial growth on the filter as a function of incubation time. Note the slight decrease in CFU after 8 hours. This decrease is due to the fission of some colonies.

[0234] Figure 11a An example is shown: scanning images of aquatic bacteria collected and cultured on a PTFE filter after 6 hours of growth. The bacteria were collected on a PTFE filter and embedded in WPCA medium. Filter parameters were: pore size cutoff of 0.2 μm and diameter of 13 mm.

[0235] Figure 11a Details of the scanned image are shown, revealing the heterogeneity of cell-forming units on the filter.

[0236] Figures 12a-12d A series of steps illustrating an example of the method of the present invention are shown. In Figure 12, a filter unit is installed in a filter housing 15, and a fluid sample enters the housing 12 via inlet 11 and exits the housing 15 via outlet 23.

[0237] exist Figure 12bIn this process, filter unit 16 has been removed from the filter housing. A layer of culture medium M has been applied to container 4, and filter unit 16 has been placed inside container 4, facing the bottom of container 4. A scanning and image analysis system, including an illumination device (light emitter) 24 and an image acquisition device 25, has been arranged to perform a scanning and image analysis procedure on the filter membrane using at least one selected scanning wavelength.

[0238] Figure 12c Images from two subsequent scans are shown.

[0239] Figure 12d The results of the determined bioactivity are shown, showing the change in colony-forming units (CFU) per milliliter of sample over time (in hours).

Claims

1. A method for analyzing the bioactivity of fluid samples, the method comprising: • Provide a filter unit comprising a filter membrane having a thickness, a front side, and a back side. • Pass the fluid sample through the filter membrane from the front side. • Apply the filter unit to the container. • Add the culture medium to the container, and • Perform a scanning and image analysis procedure on the filter membrane using at least one selected scanning wavelength. The scan is an optical 3D scan and includes acquiring multiple images along at least one scan path, and the optical 3D scan includes scanning at least a portion of the filter membrane thickness.

2. The method of claim 1, wherein, The scan includes a delayed series of scans, wherein the delayed series of scans includes comparing each of a plurality of subsequent scans with one or more scans that are previous scans as the corresponding subsequent scans.

3. The method according to claim 1, wherein, The filter membrane is at least 2% transparent to the selected scanning wavelength.

4. The method according to any one of claims 1-3, wherein, The filter membrane is made of a material having a refractive index of less than 1.5 at the selected scanning wavelength.

5. The method according to any one of claims 1-3, wherein, The filter membrane has a refractive index of less than 1.5 at a wavelength of 589.29 nm.

6. The method according to any one of claims 1-3, wherein, The filter membrane is made of a material having a filter membrane refractive index, and the culture medium has a culture medium refractive index, wherein the refractive index difference RID between the filter membrane refractive index and the culture medium refractive index is less than 0.35 at the selected scanning wavelength.

7. The method according to any one of claims 1-3, wherein, The filter membrane has transparency and refractive index, which are selected such that when the filter membrane is immersed in the culture medium, a scanning and image analysis procedure will reveal whether microorganisms with a cell size of 0.6-0.7 µm are present on the filter membrane. The scanning and image analysis procedure includes wave characteristic scans selected from at least one wavelength of phase velocity, group velocity dispersion, wave dispersion, wavefront, wave phase, group delay dispersion, or time of flight.

8. The method according to any one of claims 1-3, wherein, The at least one selected scanning wavelength includes multiple scanning wavelengths.

9. The method according to any one of claims 1-3, wherein, The scan is performed at a scan depth of at least 0.1 μm.

10. The method according to any one of claims 1-3, wherein, The filter membrane has a thickness, and the 3D scan includes scanning the entire thickness of the filter membrane.

11. The method according to claim 10, wherein, The 3D scan includes scanning the entire thickness of the filter membrane and includes at least a certain volume of the culture medium located adjacent to the front side of the filter membrane.

12. The method according to any one of claims 1-3, wherein, The scan is performed using a scanner that includes a light emitter and an image acquisition device, wherein the image acquisition device has an optical axis.

13. The method according to claim 12, wherein, The scanning path coincides with the optical axis.

14. The method according to claim 12, wherein, The scanning path is substantially orthogonal to the optical axis.

15. The method according to any one of claims 1-3, wherein, The scanning and image analysis procedure includes continuously performing scans along the scanning path and generating a set of acquired images for each scan.

16. The method according to claim 15, wherein, The scanning and image analysis procedure includes performing a background scan along the scanning path and generating a set of background images.

17. The method according to claim 16, wherein, The scanning and image analysis procedure includes processing each set of acquired images by subtracting the corresponding pixel value of the corresponding background image from the corresponding pixel value of the corresponding acquired image in the set of acquired images.

18. The method according to claim 17, wherein, The scanning and image analysis procedure includes synthesizing one or more resulting images from images acquired from a corresponding group, wherein each resulting image is synthesized from a group of acquired images.

19. The method according to claim 18, wherein, Each group of acquired images is associated with a time attribute, which indicates the time at which a selected image was acquired, to provide a group of acquired images generated from continuous scanning as a function of time.

20. The method according to claim 19, wherein, Each of the resulting images is associated with a temporal attribute of the acquired image set used for image synthesis.

21. The method according to any one of claims 1-3, wherein, The scanning and image analysis procedures include bright-field microscopy, dark-field microscopy, and / or fluorescence microscopy.

22. The method according to any one of claims 1-3, wherein, The scanning and image analysis procedures include tilted illumination, off-axis illumination, multi-axis illumination, and / or inline illumination.

23. The method of claim 22, wherein the illumination varies from one scan to the next.

24. The method according to any one of claims 1-3, wherein, The scanning and image analysis procedures include light transmission scanning and / or light reflection scanning.

25. The method according to any one of claims 1-3, wherein, The scanning and image analysis procedures include hyperspectral scanning and imaging.

26. The method according to any one of claims 1-3, wherein, The scanning and image analysis procedure includes a wave characteristic scan of at least one wavelength, wherein the wave characteristic includes the characteristics of the at least one wavelength, the characteristics depending on the material through which it passes.

27. The method according to claim 26, wherein, The baud rate scan includes acquiring multiple baud rate images along a scan path such as the at least one scan path.

28. The method according to claim 26, wherein, The scanning and image analysis procedure includes performing a wave pattern background scan along the scanning path, generating a set of background wave pattern images, and comparing at least one of the acquired wave pattern images with a corresponding image of the background wave pattern image.

29. The method according to any one of claims 1-3, wherein, The at least one selected scanning wavelength includes one or more wavelengths in the range of 200 nm to 1200 nm.

30. The method according to any one of claims 1-3, wherein, The at least one selected scanning wavelength includes one or more excitation wavelengths for exciting fluorophores, wherein the fluorophores are fluorophores present in the culture medium, fluorophores expected to be generated in the culture medium upon microbial activation, and / or fluorophores of expected biofluorescently active microorganisms in the sample.

31. The method according to any one of claims 1-3, wherein, The scanning and image analysis procedures include hyperspectral microscopy and / or Raman spectroscopy.

32. The method according to claim 31, wherein, The scanning and image analysis procedure includes determining at least one morphological parameter of one or more microorganisms and / or one or more colonies.

33. The method according to claim 31, wherein, The scanning and image analysis procedure includes determining at least one time parameter for one or more microorganisms and / or one or more colonies.

34. The method according to any one of claims 1-3, wherein, The 3D scan is performed using two or more selected scanning wavelengths, and the image analysis procedure includes spectral analysis.

35. The method according to any one of claims 1-3, wherein, At least a portion of the culture medium is added before the filter unit is applied to the container.

36. The method according to any one of claims 1-3, wherein, At least a portion of the culture medium is added after the filter unit is applied to the container.

37. The method according to any one of claims 1-3, wherein, The container includes a bottom and walls that define the top of the opening.

38. The method according to any one of claims 1-3, wherein, Applying the filter unit to the container includes applying the filter unit to the container such that the front side of the filter membrane faces the bottom of the container.

39. The method according to any one of claims 1-3, wherein, Applying the filter unit to the container includes applying the filter unit to the container such that the back side of the filter membrane faces the bottom of the container.

40. The method according to any one of claims 1-3, wherein, The bottom of the container is at least 50% transparent to the selected scanning wavelength.

41. The method according to any one of claims 1-3, wherein, The 3D scan is performed from the front of the filter membrane.

42. The method according to any one of claims 1-3, wherein, The culture medium is added in liquid form.

43. The method according to any one of claims 1-3, wherein, The method further includes adding an additive to the container, the additive being a biocide and / or an antibiotic.

44. The method according to any one of claims 1-3, wherein, The method includes determining the number of live microorganisms and / or colonies on and in the filter membrane, and correlating that number with the volume of the liquid sample to determine the number of microorganisms per volume unit.

45. The method according to any one of claims 1-3, wherein, The method includes determining the characteristics of the filtrate on the front side of the filter membrane as a function of time.

46. ​​The method according to claim 19, wherein, The scanning and image analysis procedure includes performing the continuous scanning and generating the acquired image set, wherein the first scan is a reference scan, and after each subsequent scan, the subsequent set of acquired images is processed by subtracting the corresponding pixel value of the corresponding background image from the corresponding pixel value of the corresponding image of the subsequent set of acquired images, synthesizing at least one result image, and analyzing the result image to indicate the characteristics of live microorganisms and / or detected live microorganisms.

47. The method according to any one of claims 1-3, wherein, The method includes analyzing multiple fluid samples obtained from the same parent sample, wherein at least two of the liquid samples are subjected to different culture media and / or additives, the additives being biocides and / or antibiotics, and the method includes comparing the resulting images of the analyses from the individual fluid samples.

48. The method according to any one of claims 1-3, wherein, The method includes a two-step filtration process, comprising providing a pre-filter unit having a pre-filter membrane having a front and a back side, wherein the fluid sample is squeezed through the pre-filter membrane from the front side before the sample is squeezed through the filter membrane, wherein the pre-filter membrane has a larger cutoff size than the filter membrane.

49. The method according to claim 48, wherein, The method includes releasing the pre-filter unit after the filtration process.

50. The method according to claim 48, wherein, The method includes analyzing the pre-filter unit after the filtration process, wherein the analysis includes... • Apply the pre-filter unit to the container. • Add the culture medium to the container, and • Perform a scanning and image analysis procedure on the filter membrane using at least one of the selected scans. Furthermore, the scan described is an optical 3D scan and includes acquiring multiple images along the scan path.

51. A system for analyzing the bioactivity of fluid samples, the system comprising: • A filter unit comprising a filter membrane having a front and a back side and a filter ring assembly surrounding the filter membrane. • A filter housing adapted to hold the filter unit in a temporary fixed position, and • A container suitable for the filter unit, • At least one culture medium added to the container, and • A scanning and image analysis system suitable for performing scanning and image analysis procedures on a filter membrane using at least one selected scanning wavelength. The filter housing includes an inlet for feeding a fluid sample into the housing and through the filter membrane when the filter unit is in its temporary fixed position. The filter unit is adapted to be released from its temporary fixed position within the housing and transferred to the container; The scan is an optical 3D scan and includes acquiring multiple images along at least one scan path, and the optical 3D scan includes scanning at least a portion of the thickness of the filter membrane.

52. The system according to claim 51, wherein, The filter membrane is at least 2% transparent to the selected scanning wavelength.

53. The system according to any one of claims 51 or 52, wherein, The filter membrane is made of a material having a refractive index of less than 1.5 at the selected scanning wavelength.

54. The system according to any one of claims 51-52, wherein, The filter membrane is made of a material having a refractive index of less than 1.5 at a wavelength of 589.29 nm.

55. The system according to any one of claims 51-52, wherein, The filter membrane is made of a single material.

56. The system according to any one of claims 51-52, wherein, The filter membrane has transparency and refractive index, which are selected such that when the filter membrane is immersed in the culture medium, a scanning and image analysis procedure, including a wave characteristic scan of phase velocity, group velocity dispersion, wave dispersion, wavefront, wave phase, group delay dispersion, or time of flight at least one wavelength, will reveal the presence of microorganisms with a cell size of 0.6-0.7 µm on the filter membrane.

57. The system according to any one of claims 51-52, wherein, The filter membrane comprises one or more polymer materials, one or more glasses, one or more ceramics, one or more semiconductor materials, one or more metals, or any combination thereof.

58. The system according to any one of claims 51-52, wherein, The thickness of the filter membrane is from 0.05 μm to 0.5 cm.

59. The system according to any one of claims 51-52, wherein, The cutoff particle size of the filter membrane is from 0.01 μm to 500 μm.

60. The system according to any one of claims 51-52, wherein, The filter membrane has a substantially flat front side.

61. The system according to any one of claims 51-52, wherein, The filter ring device is rigid at 20°C.

62. The system according to any one of claims 51-52, wherein, The filter housing includes a front portion and a rear portion, wherein the rear portion is adapted to hold the filter unit in the temporary fixed position.

63. The system according to claim 62, wherein, The rear portion includes a release device for releasing the filter unit from its temporary fixed position, wherein the rear portion is adapted to mechanically retain the filter unit in the temporary fixed position.

64. The system according to claim 63, wherein, The release device is a spring-loaded release device including at least one spring, wherein when the filter unit is held in its temporary fixed position, the at least one spring is in an unloaded position.

65. The system according to any one of claims 51-52, wherein, The container has a substantially flat bottom or a convex bottom, and the container advantageously has an inner wall dimension adapted to the outer periphery of the ring of the filter unit.

66. The system according to claim 65, wherein, When the filter is arranged in the container, the bottom of the container has an inner surface that aligns with the front side of the filter membrane.

67. The system according to claim 65, wherein, The bottom of the container is at least 2% transparent to the selected scanning wavelength.

68. The system according to any one of claims 51-52, wherein, The containers form a multi-container plate.

69. The system according to any one of claims 51-52, wherein, The filter unit includes two or more laterally positioned filter membranes, each of which is surrounded by the ring device.

70. The system according to claim 69, wherein, The system also includes a container separator adapted to divide the container into lateral portions associated with the respective filter membranes of the laterally positioned filter membranes.

71. The system according to claim 69, wherein, The laterally positioned filter membranes differ in at least one property.

72. The system according to any one of claims 51-52, wherein, The filter membrane includes optically detectable markers located at predefined positions.

73. The system according to any one of claims 51-52, wherein, The system further includes a pre-filtration unit, which comprises a pre-filtration membrane having a cutoff size larger than that of the filtration unit.

74. The system according to any one of claims 51-52, wherein, The filter membrane is made of a material having a filter refractive index and the at least one culture medium has a culture medium refractive index, wherein the refractive index difference RID between the filter refractive index and the culture medium refractive index is less than 0.35 at the selected scanning wavelength.

75. The system according to any one of claims 51-52, wherein, The scanning and image analysis system is adapted to perform a 3D reflective scanning and image analysis procedure of the filter membrane from the front side using at least one selected scanning wavelength in the visible light range, including acquiring multiple images along the scanning path.

76. The system of claim 74, wherein the selected scanning wavelength is 589.29 nm.

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