METHOD AND DEVICE FOR DETECTING BACTERIA AND DETERMINING THE CONCENTRATION THEREOF IN A LIQUID SAMPLE

• US 20150118708A1
• Filed: 10/28/2014
• Published: 04/30/2015
• Est. Priority Date: 10/29/2013
• Status: Active Grant

• Current Assignee: IDEXX Laboratories Incorporated
• Original Assignee: IDEXX Laboratories Incorporated
• Inventors: Hammond, Jeremy, Russell, James
• Granted Patent: US 9,644,230 B2
• Time in Patent Office: Days
• US Class Current: 435/39
• CPC Class Codes: B01L 2200/0652 Sorting or classification of particles or molecules
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0851 Bottom walls
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/502 with fluid transport, e.g. in multi-compartment structures centrifugal-type cuvettes G01N21/07; analysis by separation into components G01N30/00; automatic analysers G01N35/00
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: C12Q 1/06 Quantitative determination
• CPC Class Codes: G01N 15/01 specially adapted for biological cells, e.g. blood cells investigating sedimentation of particle suspensions in blood G01N15/05
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1447 Spatial selection
• CPC Class Codes: G01N 2015/1452 Adjustment of focus; Alignment
• CPC Class Codes: G01N 2015/1486 Counting the particles
• CPC Class Codes: G01N 2333/195 from bacteria
• CPC Class Codes: G01N 33/493 urine

Abstract

A method for detecting bacteria and determining the concentration thereof in a liquid sample includes the steps of taking an optical section through a container holding a volume of the liquid sample at a predetermined field of view and at a predetermined focal plane depth or angle and after a period of time has elapsed to allow non-bacteria in the sample to settle to the bottom of the container. Since bacteria auto arranges in the liquid sample, forming a lattice-like grid pattern, an optical section through the volume of auto-arranged bacteria may be used to measure the quantity of bacteria residing in that section. A container for holding the liquid sample has particular structure which aids in separating the non-bacteria from the bacteria.

Claims

1. A method for detecting bacteria and determining the concentration thereof in a liquid sample, which comprises the steps of:

taking at least one optical section through a volume of the liquid sample at a predetermined field of view and at a predetermined focal plane depth or angle in the volume and after a predetermined time has elapsed to allow bacteria in the liquid sample to auto arrange;

counting the number of bacteria present within the at least one optical section;

calculating the number of optical sections into which the volume of the liquid sample may be divided thereby determining a total number of possible optical sections;

multiplying the number of bacteria present in the at least one optical section by the total number of possible optical sections thereby determining at least an approximation of the total number of bacteria within the volume of the liquid sample; and

determining the concentration of bacteria within the liquid sample based on the at least approximation of the total number of bacteria within the volume of the liquid sample.

2. A method for detecting bacteria and determining the concentration thereof in a liquid sample as defined by claim 1, wherein the predetermined time allowed for bacteria in the liquid sample to auto arrange is between about three minutes and about ninety minutes.

3. A method for detecting bacteria and determining the concentration thereof in a liquid sample as defined by claim 1, wherein the predetermined time allowed for bacteria in the liquid sample to auto arrange is between about three minutes and about 10 minutes.

4. A method for detecting bacteria and determining the concentration thereof in a liquid sample as defined by claim 1, wherein the at least one optical section taken through the volume of the liquid sample has a focal plane angle of about zero (0) degrees to about ninety (90) degrees relative to a vertical plane through the volume.

5. A method for detecting bacteria and determining the concentration thereof in a liquid sample as defined by claim 1, wherein the at least one optical section taken through the volume of the liquid sample has a focal plane angle of about zero degrees relative to a vertical plane through the volume.

6. A method for detecting bacteria and determining the concentration thereof in a liquid sample as defined by claim 1, wherein the at least one optical section taken through the volume of the liquid sample has a focal plane angle of about ninety degrees relative to a vertical plane through the volume.

7. A method for detecting bacteria and determining the concentration thereof in a liquid sample as defined by claim 6, wherein the optical section has a focal plane depth of at least one of about 100 microns, about 200 microns, about 400 microns, about 600 microns, about 800 microns, about 1,000 microns and about 1,200 microns above the bottom of the volume of the liquid sample.

8. A method for detecting bacteria and determining the concentration thereof in a liquid sample, which comprises the steps of:

taking a plurality of optical sections through a volume of the liquid sample at a predetermined field of view and at one or more predetermined focal plane depths or angles in the volume and after a predetermined time has elapsed to allow bacteria in the liquid sample to auto arrange;

counting the number of bacteria present within each optical section of the plurality of optical sections;

calculating an average of the number of bacteria present by dividing the total number of bacteria present in the plurality of optical sections by the number of optical sections taken through the volume of the liquid sample thereby determining an average number of bacteria present within the optical sections of the plurality of optical sections;

calculating the number of optical sections into which the volume of liquid sample may be divided thereby determining a total number of possible optical sections;

multiplying the average number of bacteria present in the optical sections of the plurality of optical sections by the total number of possible optical sections thereby determining at least an approximation of the total number of bacteria within the volume of the liquid sample; and

determining the concentration of bacteria within the liquid sample based on the at least approximation of the total number of bacteria within the volume of the liquid sample.

9. A method for detecting bacteria and determining the concentration thereof in a liquid sample, which comprises the steps of:

taking at least one optical section through a volume of the liquid sample at a predetermined field of view and at a predetermined focal plane depth or angle in the volume and after a predetermined time has elapsed to allow bacteria in the liquid sample to auto arrange;

determining the average spacing between bacteria present within the at least one optical section thereby determining the average bacteria spacing;

calculating the three dimensional area occupied by the volume of the liquid sample thereby determining a three dimensional volumetric area;

dividing the three dimensional volumetric area by the average spacing between bacteria thereby determining at least an approximation of the total number of bacteria within the volume of the liquid sample; and

determining the concentration of bacteria within the liquid sample based on the at least approximation of the total number of bacteria within the volume of the liquid sample.

10. A method for detecting particles in a liquid sample and distinguishing a first type of particles from at least a second type of particles in the liquid sample, which comprises the steps of:

at least partially filling a container with a volume of the liquid sample containing the first type of particles and the at least second type of particles, the container having at least one surface made from a predetermined material which causes the at least second type of particles in the liquid sample to exhibit an aversion thereto and the first type of particles in the liquid sample to exhibit no aversion thereto, the particles of the at least second type of particles in the liquid sample primarily not occupying an aversion region of the volume of the liquid sample in proximity to the surface of the container, and the particles of the first type of particles in the liquid sample occupying the aversion region of the volume of the liquid sample in proximity to the surface of the container; and

taking at least one optical section through the aversion region of the volume of the liquid sample at a predetermined field of view and at a predetermined focal plane depth or angle, the optical section optically detecting the particles of the first type of particles occupying the aversion region of the volume of the liquid sample in proximity to the surface of the container, as distinguished from the particles of the at least second type of particles which primarily do not occupy the aversion region.

11. A method for detecting particles in a liquid sample as defined by claim 10, wherein the surface of the container which causes the aversion thereto by the particles of the at least second type of particles is made from an acrylic material.

12. A method for detecting particles in a liquid sample as defined by claim 10, wherein the surface of the container which causes the aversion thereto by the particles of the at least second type of particles is made from poly(methyl methacrylate) (PMMA).

13. A container for holding a volume of a liquid sample and used for separating different types of particles within the volume of the liquid sample, the different types of particles within the volume of the liquid sample held by the container including a first type of particles which auto arrange within the volume of the liquid sample held by the container and a second type of particles which do not auto arrange within the volume of the liquid sample, the container comprising:

a bottom wall, the bottom wall having a recessed portion and a non-recessed portion adjacent the recessed portion, the container thereby defining a first zone situated at a first depth in the volume of the liquid sample and in vertical alignment with the non-recessed portion of the container bottom wall, and a second zone situated at a second depth in the volume of the liquid sample and in vertical alignment with the recessed portion of the container bottom wall, the first type of particles which auto arrange tending to occupy the second zone within the container, and the second type of particles which do not auto arrange tending to occupy the first zone within the container.

14. A container for holding a volume of a liquid sample as defined by claim 13, which further comprises:

at least one projection, the at least one projection extending upwardly from the non-recessed portion of the container bottom wall and at least partially into the volume of the liquid sample held by the container, the at least one projection being situated on the non-recessed portion of the bottom wall in proximity to the recessed portion of the bottom wall, the at least one projection further acting to separate the first type of particles which auto arrange and which tend to occupy the second zone within the container from the second type of particles which do not auto arrange and tend to occupy the first zone within the container.

15. A container for holding a volume of a liquid sample and used for separating different types of particles within the volume of the liquid sample, the different types of particles within the volume of the liquid sample held by the container including a first type of particles which auto arrange within the volume of the liquid sample held by the container and a second type of particles which do not auto arrange within the volume of the liquid sample, the container comprising:

a bottom wall; and

a plurality of projections spaced apart from each other over at least a portion of the axial length of the container, the projections extending upwardly from the bottom wall of the container and at least partially into the volume of the liquid sample held thereby, the projections defining a first zone and at least a second zone adjacent the first zone, the particles of the first type of particles which auto arrange tending to occupy the first zone within the container, and the particles of the second type of particles which do not auto arrange tending to occupy the at least second zone within the container.

16. A container for holding a volume of a liquid sample and used for separating different types of particles in the volume of the liquid sample, the different types of particles within the volume of the liquid sample held by the container including a first type of particles which exhibit a first dimension, and a second type of particles which exhibit a second dimension which is different from the first dimension exhibited by the particles of the first type of particles, the container comprising:

a bottom wall; and

at least one filter extending upwardly from the bottom wall and at least partially into the volume of the liquid sample held by the container, the at least one filter having a first axial side and a second axial side situated opposite the first axial side, the container defining a first zone within the volume of the liquid sample situated adjacent the first axial side of the at least one filter, and a second zone within the volume of the liquid sample situated adjacent the second axial side of the at least one filter, the at least one filter having a predetermined pore size which allows the particles of the second type of particles of the liquid sample to pass through the at least one filter and into the second zone, whereby the particles of the first type of particles tend to occupy the first zone within the container, and the particles of the second type of particles tend to occupy the second zone within the container.

17. A container for holding a volume of a liquid sample and used for separating different types of particles in the volume of the liquid sample, the different types of particles within the volume of the liquid sample held by the container including a first type of particles which exhibit a first dimension, and a second type of particles which exhibit a second dimension which is different from the first dimension exhibited by the particles of the first type of particles, the container comprising:

a bottom wall; and

a plurality of filters spaced apart from each other over at least a portion of the axial length of the container, the filters extending upwardly from the bottom wall and at least partially into the volume of the liquid sample held by the container, the plurality of filters defining at least a first zone within the volume of the liquid sample and a second zone within the volume of the liquid sample, each filter of the plurality of filters having a pore size which differs from the pore size of the next adjacent filter, at least one of the filters having a pore size which allows particles of the first type of particles to pass therethrough and which does not allow particles of the second type of particles to pass therethrough, whereby the particles of the first type of particles tend to occupy the first zone within the container, and the particles of the second type of particles tend to occupy the second zone within the container.

18. A container for holding a volume of a liquid sample and used for detecting different types of particles within the volume of the liquid sample, the different types of particles within the volume of the liquid sample held by the container including a first type of particles which auto arrange within the volume of the liquid sample held by the container and a second type of particles which do not auto arrange within the volume of the liquid sample, the container comprising:

a bottom wall, a first axial end and a second axial end situated opposite the first axial end, the bottom wall having a sloping surface thereby defining the container with a shallower section relatively closer to the first axial end thereof and a deeper section relatively closer to the second axial end thereof, such that a horizontally disposed optical section of the volume of the liquid sample held by the container taken by an optical imaging instrument and having a constant focal plane depth in the volume of the liquid sample, which focal plane depth is selected to be in close proximity to the bottom wall of the container over the shallower section thereof, will detect in a portion of the optical section in alignment with the shallower section of the container particles of the second type of particles which do not auto arrange, and will detect in a portion of the optical section in alignment with the deeper section of the container particles of the first type of particles which auto arrange.

19. A container for holding a volume of a liquid sample and used for detecting different types of particles within the liquid sample, the different types of particles within the volume of the liquid sample held by the container including a first type of particles and a second type of particles, the particles of the first type of particles either auto arrange within the volume of the liquid sample held by the container or have a first dimension, and the particles of the second type of particles either do not auto arrange within the volume of the liquid sample held by the container or have a second dimension which is different from the first dimension of the particles of the first type of particles, the container comprising:

a bottom wall; and

a plurality of spaced apart recesses, projections or particles formed in the bottom wall or situated in proximity to the bottom wall of the container for quality assurance purposes.

20. A container for holding a volume of a liquid sample and used for detecting different types of particles within the liquid sample, the different types of particles within the volume of the liquid sample held by the container including a first type of particles and a second type of particles, the particles of the first type of particles either auto arrange within the volume of the liquid sample held by the container or have a first dimension, and the particles of the second type of particles either do not auto arrange within the volume of the liquid sample held by the container or have a second dimension which is different from the first dimension of the particles of the first type of particles, the container comprising:

a bottom wall; and

a plurality of parallelly disposed and spaced apart plates, the plates extending vertically upwardly from the bottom wall of the container and at least partially into the volume of the liquid sample held by the container, adjacent plates of the plurality of parallelly disposed plates defining fluid flow channels therebetween.

21. A container for holding a volume of a liquid sample as defined by claim 20, wherein the container includes opposite lateral walls, the opposite lateral walls being joined to the bottom wall and extending upwardly therefrom, and a first axial end and a second axial end situated opposite the first axial end;

and wherein at least some of the plates of the plurality of parallelly disposed plates have differing axial lengths which increase from the longitudinal center of the container in symmetrical directions outwardly toward the opposite lateral sides thereof.