A METHOD AND A SYSTEM FOR ANALYSING A FLUID SAMPLE FOR A BIOLOGICAL ACTIVITY

Abstract

A method and a system for analyzing a fluid sample for a biological activity. The method includes, providing a filter unit including a membrane having a front face and a rear face, passing the fluid sample through the filter membrane from its front face, applying the filter unit in a container, adding a medium into the container, and performing a scanning and image analyzing procedure of the filter membrane using at least one selected scanning wavelength. The scanning is an optical 3D scanning and includes acquiring a plurality of images along at least one scanning path.

Claims

1. A method for analyzing a fluid sample for a biological activity, the method comprising providing a filter unit comprising a membrane having a front face and a rear face, passing the fluid sample through the filter membrane from its front face, applying the filter unit in a container, adding a medium into the container, and performing a scanning and image analyzing procedure of the filter membrane using at least one selected scanning wavelength, and wherein the scanning is an optical 3D scanning and comprises acquiring a plurality of images along at least one scanning path.

2. The method of claim 1, wherein the scanning comprises a time-lapse series scanning, the time-lapse series scanning comprises comparing each of a number of subsequent scans with one or more scans that is/are previous scan(s) to said respective subsequent scan.

3. (canceled)

4. The method of claim 1, wherein said filter membrane is of a material having a refractive index of less than about 1.5 at a wavelength selected from the selected scanning wavelength and 589.29 nm.

5. (canceled)

6. The method of claim 1, wherein said filter membrane is of a material having a filter membrane refractive index and said medium has a medium refractive index where the refractive index difference (RID) between said filter membrane reference index and said RID is less than about 0.35 at a wavelength selected from the selected scanning wavelength and 589.29 nm.

7.-9. (canceled)

10. The method of claim 1, wherein the filter membrane has a thickness and the 3D scanning comprises scanning through at least a part of the filter membrane thickness.

11. The method of claim 10, wherein the 3D scanning comprises scanning through the entire filter membrane thickness and including at least a volume of the medium located adjacent the front face of the membrane.

12.-14. (canceled)

15. The method of claim 1, wherein the scanning and image analyzing procedure comprises performing consecutively scans along the scanning path and for each scan generating a set of acquired images, wherein the scanning and image analyzing procedure comprises performing a background scan long the scanning path and generating a set of background images and wherein the scanning and image analyzing procedure comprises processing each set of acquired images by a method comprising subtracting pixel values of said respective background images from corresponding pixel values of the respective acquired images of said set of acquired images.

16.-18. (canceled)

19. The method of claim 15, wherein each set of acquired images is associated with a time attribute representing the time of acquiring a selected one of the images, to provide that the sets of image generated from the consecutive scans represent sets of acquired images as a function of time.

20. (canceled)

21. The method of claim 1, wherein the scanning and image analyzing procedure comprises at least one of bright field microscopy, dark field microscopy, fluorescence microscopy, hyperspectral scanning and imaging, hyperspectral microscopy, Raman spectroscopy, light transmission scanning and/or light reflection scanning.

22.-28. (canceled)

29. The method of claim 1, wherein the at least one selected wavelength comprises one or more excitation wavelength for excitation of a fluorophore selected from a fluorophore present in the medium, a fluorophore expected to be generated in the medium upon microbial activation or a fluorophore of a biofluorescence active microorganism expected in the sample.

30. (canceled)

31. The method of claim 1, wherein the scanning and image analyzing procedure comprises determining at least one parameter selected from a morphological parameter a temporal parameter of one or more microorganism and/or colonies.

32. (canceled)

33. The method of claim 1, wherein the 3D scanning is performed using two or more selected scanning wavelength and wherein the image analyzing procedure comprises a spectral analysis, such as a multiplexing between said two or more wavelength.

34. The method of claim 1, wherein the container comprises a bottom and a wall defining an open top, wherein at least a part of the medium is added prior to applying the filter unit in the container and wherein the application of the filter unit in the container comprises applying the filter unit in the container with the front face of the filter membrane facing a bottom of the container, wherein the container bottom is at least 50% transparent for said selected scanning wavelength and wherein the 3D scanning is performed from the front face of the filter membrane.

35. The method of claim 1, wherein the container comprises a bottom and a wall defining an open top, wherein at least a part of the medium is added after application of the filter unit in the container, wherein the application of the filter unit in the container comprises applying the filter unit in the container with the rear face of the filter membrane facing the bottom of the container a and wherein the 3D scanning is performed from the open top.

36.-40. (canceled)

41. The method of claim 1, wherein the medium is added in liquid form.

42. The method of claim 1, wherein the method further comprises adding an additional substance to the container, the additional substance is preferably a biocide and/or an antibiotic.

43. (canceled)

44. The method of claim 1, wherein the method comprises determining a characteristic as a function of time of the filtrate collected by the filter membrane.

45. The method of claim 1, wherein the scanning and image analyzing procedure comprises performing said consecutive scans and generation of sets of acquired images, wherein a first scan is a reference scan, after each subsequent scan processing the subsequent set of acquired images by a method comprising subtracting pixel values of respective background images from corresponding pixel values of the respective images of said set of subsequent acquired images, synthesizing at least one resulting image and analyzing said resulting image for indication of living microorganism and/or a characteristics of a detected living microorganism.

46. The method of claim 1, wherein the method comprises analyzing a plurality of fluid samples obtained from the same mother sample, wherein at least two of the liquid samples are subjected to different medium and/or additional substance and the method comprises comparing resulting images from the analysis of the respective fluid samples, preferably the method comprises comparing timely corresponding resulting images from the analysis of the respective fluid samples.

47.-50. (canceled)

51. A system for analyzing a fluid sample for a biological activity, the system comprises a filter unit, comprising a membrane having a front face and a rear face and a filter collar arrangement surrounding said membrane, a filter housing adapted for holding the filter unit in a temporarily fixed position, and a container adapted for the filter unit, wherein said filter membrane is of a material having a refractive index of less than about 1.5 at the wavelength 589.29 nm, wherein the filter housing comprises an inlet for feeding a fluid sample into the housing and through said filter membrane when said filter unit is in its temporarily fixed position, and wherein the filter unit is adapted for being released from its temporarily fixed position within the housing and transferred to the container.

52.-75. (canceled)

Description

BRIEF DESCRIPTION OF THE EXAMPLES AND DRAWING

[0190] The invention is being illustrated further below in connection with preferred embodiment and with reference to the figures. The figures are schematic and may not be drawn to scale.

[0191] FIG. 1a is a top view of a filter unit suitable for the method of the invention.

[0192] FIGS. 1b and 1c illustrated filter units comprising laterally located filter membranes.

[0193] FIG. 2 is a perspective view of a system of the invention comprising a filter housing holding a not shown filter unit, a syringe and a container adapted for the filter unit.

[0194] FIG. 3 is a cross sectional view of a filter housing and a filter unit.

[0195] FIG. 4 is a perspective view of a filter housing and a filter unit.

[0196] FIGS. 5-8 illustrates the step of applying the filter in the container.

[0197] FIG. 9 illustrates images of a time lapse series.

[0198] FIGS. 10a-10c show an example of scanning results of water bacteria collected and cultivated on a PTFE filter.

[0199] FIGS. 11a-11b show an example of a scanned image of water bacteria collected and cultivated on a PTFE filter.

[0200] FIGS. 12a-12d illustrate a sequence of steps of an example of a method of the invention.

[0201] The filter unit illustrated in FIG. 1 comprises a filter membrane 1 and a collar arrangement 2 surrounding the filter membrane. As described above the filter membrane advantageously is substantially flat and comprises a front face and a rear face.

[0202] The filter units shown in FIGS. 1b and 1c comprise laterally located filter membranes 1a-1d. The filter unit of FIG. 1b comprises two laterally located filter membranes 1a and 1b. The laterally located filter membranes 1a and 1b are both surrounded by a collar arrangement 2a, 2b, wherein a first part of the collar arrangement 2a defines the periphery of the filter unit and a second part of the collar arrangement 2b defines a cross separation between the two laterally located filter membranes 1a, 1b.

[0203] The filter unit of FIG. 1c comprises four laterally located filter membranes 1a, 1b, 1c, 1d. The laterally located filter membranes 1a, 1b, 1c, 1d are all surrounded by a collar arrangement 2a, 2b, wherein a first part of the collar arrangement 2a defines the periphery of the filter unit and a second part of the collar arrangement 2b defines two cross separations between the four laterally located filter membranes 1a, 1b, 1c, 1d.

[0204] The laterally located filter membranes may be arranged in any desired configuration.

[0205] To ensure an evenly filtration over the various laterally located filter membranes, it may be desired that the flow resistance over the respective laterally located filter membranes are substantially equal. The filter unit with laterally located filter membranes ensures that several determinations may be performed using one single filter unit e.g. as described above.

[0206] The system shown in FIG. 2 discloses an example of a filter housing 5 holding a not shown filter unit, a syringe 3 and a container 4 adapted for the filter unit.

[0207] The filter housing 5 comprises a front part 5a and a rear part 5b. The not shown filter unit is held in a temporarily fixed position between the front part 5a and the rear part 5b of the filter housing.

[0208] The syringe 3 is arranged to feed a fluid sample into an inlet 5a1 of the front part 5a of the filter housing 5. As explained above the sample may be driven through the filter membrane by any means and hence the syringe is an optional part of the system. The container 4 is a part of a container plate 4a comprising a plurality of containers, such as wells.

[0209] In FIG. 3, an example of a filter housing 15a, 15b and an example of a filter unit 16 are shown in more details.

[0210] The filter unit comprises a filter membrane 17 with a front face F and a rear face R and a collar arrangement 18 surrounding the filter membrane 17. The collar arrangement 18 extends beyond the rear face R of the filter membrane 17 to form a mounting arrange 19, for being mechanically held in a temporarily fixed position by the rear part 15b of the filter housing.

[0211] The filter housing comprises a front part 15a and a rear part 15b. The front part 15a comprises an inlet 11, for example shaped with a LUER coupling suitable for a syringe or other means for injecting a fluid sample. Inside the front part 15a of the filter housing, a gasket 12 is arranged, such that when the filter housing is assembled with the filter unit 16, the collar arrangement 18 of the filter unit seal against the gasket 12 of the front part 15a. The front part further comprises a number of sliding frames 13 (only two are shown) of a locking arrangement for locking the front part 15a to the rear part 15b.

[0212] The rear part 15b of the filter housing comprises a first and a second displaceable portions 15c and 15d and a spring arrangement 21 arranged to hold the first and the second displaceable portions 15c and 15d and thereby to provide a release arrangement is adapted for mechanically releasing the filter unit from its temporarily fixed position at the rear part.

[0213] The rear part 15b of the filter housing comprises a gasket 19 located at the first portion 15c, which will seal against the inner side of the collar arrangement 18 when the filter housing is assembled with the filter unit 16. A support structure 22 is arranged to support the filter membrane and to ensure that the filter membrane do not deform or burst when the fluid sample is pressed through the filter membrane 17. At the second portion 15d is located a click on and/or friction structure 20, adapted to be mated with the mounting arrangement 19, for mechanically holding the filter unit in a temporarily fixed position.

[0214] The first portion 15c comprises a rear most flange 15c′ and when pulling the second portion 15d towards the rear most flange 15c′ the filter unit when held at its temporarily fixed position by the rear part 15b, will be released from this position. At the same time, the spring arrangement 21 is depressed. The filter unit 16 is not released until the spring arrangement 21 is depressed. Thereby the spring arrangement 21 ensures that the filter unit 16 is safely held in its temporarily fixed position until actively released therefrom.

[0215] The second portion 15d of the rear portion 15b of the filter housing further comprises a number projecting locking elements 14 of the locking arrangement, adapted to be mated with the same number of sliding frames 13 of the front part 15a for locking the front part 15a to the rear part 15b. The locking elements 14 may be inserted to the sliding frames 13 by a twist, and held in that position for as long as desired. When the locking arrangement is in its locked position the first and the second displaceable portions 15c and 15d are held in a fixed relation to each other.

[0216] The rear part 15b further comprises an outlet 23, through which the filtered sample may escape. As mentioned, the rear part 15b could instead comprise a collecting chamber for collecting the filtered sample or an additional disposable collection chamber could be temporarily mounted to the rear portion 15b for collecting the filtered sample.

[0217] In FIG. 4, the filter housing and the filter unit as seen in perspective. The reference numbers are as for FIG. 3.

[0218] FIGS. 5-8 illustrates the step of applying the filter in the container. In FIG. 5 the rear part 15b of the filter housing has been demounted from the not shown front part of the filter housing. The filter 16 is held in its temporarily fixed position to the rear part 15b and the user is about to applying the filter unit in the container 4.

[0219] In FIG. 6. The user has inserted the filter unit together with a part of the rear part 15b into the container 4 and depressed the spring arrangement to release the filter unit 16 from the rear part 15b.

[0220] In FIG. 7, it can be seen that the filter unit 16 have been released and the user is removing the rear part 15b.

[0221] In FIG. 8, the filter unit 16 is now ready for adding the medium.

[0222] FIG. 9 shows an example of a time lapse image series of a single bacterium forming a colony during 15 hours on a membrane filter.

[0223] The depicted bacterium is one among many bacteria collected from a fluid sample on a PTFE filter (pore cut off size: 0.2 μm) and embedded in PCA medium (Principal Components Analysis medium).

[0224] The image series clearly illustrates the transition from a single bacterium to a micro-colony forming unit by multiplications.

[0225] FIG. 10a shows an example of a scanned image of water bacteria collected and cultivated on a PTFE filter after 5 hours of growth. The bacteria were collected on the PFTE filter and embedded in WPCA medium (Water Plate Count Agar). The filter parameters are: Pore cut off size of 0.2 μm and diameter of 13 mm.

[0226] FIG. 10a shows a background scan subtracted (or corrected) image of the scanned filter image in 10a. The corrected image enhances the temporal changes developed since the time lapse image 1 was recorded (first time-lapse scanned image). The first time-lapse image was applied as background subtraction image.

[0227] FIG. 10c shows a plot of the CFU concentration as function of cultivation time detected by bacterial growth on filter. Note the small drop in CFU after 8 hours. This drop is due to fission of some colonies.

[0228] FIG. 11a shows an example of a scanned image of water bacteria collected and cultivated on a PTFE filter after 6 hours of growth. The bacteria were collected on the PFTE filter and embedded in WPCA medium. The filter parameters are: Pore cut off size of 0.2 μm and diameter of 13 mm.

[0229] FIG. 11a shows a detail of the scanned image, illustrating the heterogeneity of cell forming units on the filter.

[0230] FIGS. 12a-12d show a sequence of steps of an example of a method of the invention. In FIG. 12, the filter unit is mounted in the filter housing 15 and a fluid sample is passed into the housing 12 via the inlet 11 and out of the housing 15 via the outlet 23.

[0231] In FIG. 12b, the filter unit 16 has been withdrawn from the filter housing. In a container 4 a layer of medium M has been applied and the filter unit 16 has been placed in the container 4 with the front face facing the bottom of the container 4. A scanning and image analyzing systems comprising an illumination device (light emitter) 24 and an image acquisition device 25 has been arranged to perform a scanning and image analyzing procedure of the filter membrane using at least one selected scanning wavelength.

[0232] FIG. 12c illustrates two series if images from two subsequent scans.

[0233] FIG. 12d illustrates a result of determined biological activity showing colony-forming unit (CFU) per mL sample as a function of time in hours.