CA2459320A1 - Rapid detection of replicating cells - Google Patents

Rapid detection of replicating cells Download PDF

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CA2459320A1
CA2459320A1 CA002459320A CA2459320A CA2459320A1 CA 2459320 A1 CA2459320 A1 CA 2459320A1 CA 002459320 A CA002459320 A CA 002459320A CA 2459320 A CA2459320 A CA 2459320A CA 2459320 A1 CA2459320 A1 CA 2459320A1
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target cells
detecting
microcolonies
cells
category
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CA2459320C (en
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Don Straus
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Rapid Micro Biosystems Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
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    • B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • B—PERFORMING OPERATIONS; TRANSPORTING
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Abstract

The invention provides efficient, rapid, and sensitive enumeration of living cells by detecting microscopic colonies derived from in situ cell division using large area imaging. Microbial enumeration tests based on the invention address an important problem in clinical and industrial microbiology - the long time needed for detection using traditional methods - while retaining k ey advantages of the traditional methods based om microbial culture. Embodiment s of the invention include non-destructive aseptic methods for detecting cellular microcolonies without labeling reagents. These methods allow for th e generation of pure cultures which can be used for microbial identification a nd determination of antimicrobial resistance.

Claims (120)

1. A method for detecting living target cells in a sample, said method comprising the steps of:
(a) providing living target cells present in said sample in a detection zone comprising a detection area at a density of less than 100 target cells per mm2 of the detection area, wherein within said detection zone said cells are randomly dispersed and immobilized;
(b) allowing the formation of one or more microcolonies of said target cells by in situ replication; and (c) detecting said one or more microcolonies;
wherein the longest linear dimension of said detection area is greater than 1 mm; said one or more microcolonies have a mean measurement of less than 50 microns in at least two orthogonal dimensions; and said cells in said one or more microcolonies remain competent to replicate following said detecting.
2. The method of claim 1, wherein said target cells are randomly dispersed in a detection zone at a density of less than 10 target cells per mm2 of the detection area.
3. The method of claim 1, wherein said target cells are randomly dispersed in a detection zone at a density of less than 1 target cells per mm2 of the detection area.
4. The method of claim 1, wherein said detecting detects a single microcolony in the detection area.
5. The method of claim 1, wherein said detecting detects overlapping or contiguous microcolonies.
6. The method of each of claim 1 wherein said detecting does not entail magnification of more than 5x.
7. The method of claim 1, wherein said detecting does not entail magnification of more than 2x.
8. The method of claim 1, wherein said detecting does not entail magnification of more than 1x.
9. The method of claim 1, wherein said detecting does not entail magnification of more than 0.2x.
10. The method of claim 1, wherein the mean number of cells in said one or more microcolonies is less than 50,000 cells.
11. The method of claim 10, wherein said one or more microcolonies comprise less than 10,000 cells.
12. The method of claim 10, wherein the mean number of cells in said one or more microcolonies is less than 1000.
13. The method of claim 10, wherein the mean number of cells in said one or more microcolonies is less than 100.
14. The method of claim 10, wherein the mean number of cells in said one or more microcolonies is less than 10.
15. The method of claim 10, wherein said one or more microcolonies have a mean measurement of less than 25 microns in the longest linear dimension.
16. The method of claim 10, wherein said one or more microcolonies have a mean measurement of less than 10 microns in the longest linear dimension.
17. The method of claim 1, wherein said target cells are bacteria.
18. The method of claim 1, wherein said target cells are eukaryotic cells.
19. The method of claim 18, wherein said target cells are mold or fungal cells.
20. The method of claim 18, wherein said target cells are human, animal, or plant cells.
21. The method of claim 1, wherein said target cells are parasites of humans, animals, or plants.
22. The method of claim 1, wherein said detecting detects and identifies microcolonies from more than one non-overlapping category of cell.
23. The method of claim 1, wherein said sample comprises fluids or tissues obtained from a multicellular organism.
24. The method of claim 1, wherein said sample comprises the bodily fluids or tissues of an animal.
25. The method of claim 1, wherein said sample is derived from a human.
26. The method of claim 1, wherein said sample is derived from a non-human vertebrate.
27. The method of claim 1, wherein said sample is selected from the group consisting of:
respiratory, urogenital, reproductive tract, central nervous system, urine, blood, dermal, plasma, serum, saliva, wound tissue, wound exudate, biopsy, feces, reproductive tract, and solid tissue samples, and derivatives thereof.
28. The method of claim 1, wherein said sample is a blood or urine sample.
29. The method of claim 1, wherein said sample is derived from a plant.
30. The method of claim 1, wherein said sample is obtained by sampling environmental air or water, or surfaces, objects, or organisms exposed to the environment.
31. The method of claim 1, wherein said sample is obtained from a material selected from the group consisting of raw, finished, or in-process material in the manufacture of pharmacological, cosmetic, blood, or other products for topical or internal use in humans or animals; raw, in-process, or finished material in the manufacture of foods or beverages; raw, in-process, or finished material in the manufacture of medical or in vitro diagnostic devices, chemical products; industrial surfaces; instrumentation;
and machinery.
32. The method of claim 1, wherein said detection zone is contacted with a liquid medium comprising one or more substances that facilitate replication of target cells.
33. The method of claim 1, wherein said cells are deposited directly on a solid or semi-solid growth medium.
34. The method of claim 1, wherein, prior to step (a), a selection method is used to deposit complexes of one or more of said target cells and a selection moiety in said detection zone, wherein said selection method is selected from the group consisting of magnetic selection, centrifugation, settling, and filtration.
35. The method of claim 34, wherein, prior to step (a), said target cells are contacted with target cell-specific magnetic selection moieties and complexes of one or more of said target cells and said selection moiety are subsequently deposited on said detection surface using magnetic force.
36. The method of claim 35, wherein said target cell-specific magnetic selection moieties comprise magnetic particles that are conjugated to category-binding molecules.
37. The method of claim 34, wherein said target cells are contacted in a liquid with said target-cell specific selection moieties that have an average density greater than the average density of said liquid and wherein complexes of one or more of said target cells and said selection moiety are subsequently deposited on said detection surface using gravitational, centrifugal, or centripetal force.
38. The method of claim 1, wherein said target cells are deposited in said detection zone using a selection method selected from the group consisting of magnetic selection, centrifugation, settling, and filtration, wherein a selection moiety is not employed.
39. The method of claim 1, wherein, prior to step (a), said sample is treated to liquefy and/or homogenize said sample.
40. The method of claim 1, wherein, prior to step (a), said sample is treated to remove substances or objects other than said target cells.
41. The method of claim 1, further comprising the step of determining the effect of one or more substances or treatments on one or more attributes of said target cells.
42. The method of claim 41, wherein said attribute is the ability to undergo cell replication.
43. The method of claim 41, wherein said one or more substances are present in a medium used to support the replication of said target cells.
44. The method of claim 41, wherein said attribute is the ability of said target cells to replicate following a sterilization treatment.
45. The method of claim 41, wherein said attribute is the ability of said target cells to replicate in the presence of one or more potential inhibitors of replication.
46. The method of claim 41, wherein said target cells are bacteria, fungi, parasites, or cultured cells, and said substances are antibacterial agents, agents, anti-fungal agents, or anti-parasitic agents.
47. The method of claim 45, wherein said one or more inhibitors are antimicrobial compounds.
48. The method of claim 45, wherein said one ore more inhibitors are anti-tumor compounds.
49. The method of claim 41, wherein said attribute is the viability, change in an optical property, metabolic or enzymatic activity, or biochemical constituency of said target cells.
50. The method of claim 35, further comprising the steps of,:
(d) contacting said target cells with one or more substances or treating said cells with one or more treatments; and (e) determining the effect of said one or more substances or said one or more treatments on one or more attributes of said target cells.
51. The method of claim 1, wherein said detection zone comprises a material selected from the group consisting of glass, plastic, the surface of wells of microtiter plates, bibulous membranes, plastic strips, the surfaces of capillary tubes, the surfaces of microfluidic chambers, and the surfaces or microfluidic channels.
52. The method of claim 1, wherein the replication of said cells in said microcolonies is continued after said detecting.
53. The method of claim 1, wherein step (c) comprises at least two cycles each of which comprises a period in which cells are allowed to replicate followed by a detection step.
54. The method of claim 1, further comprising the step of repeating steps (a) -(c) with one or more additional samples, wherein said repeating is automated.
55. The method of claim 54, wherein said samples are automatically loaded into an instrument that comprises a detector.
56. The method of claim 54, wherein said samples are automatically deposited in a series of detection zones that are physically associated and that are automatically and successively loaded into an instrument that comprises a detector.
57. The method of claim 1, wherein said detecting comprises illuminating one or more microcolonies to generate a detectable signal.
58. The method of claim 57, wherein said detecting detects light emitted, scattered, reflected, or absorbed as a result of illumination of said one or more microcolonies.
59. The method of claim 1, wherein said detecting detects fluorescence.
60. The method of claim 59, wherein said fluorescence is autofluorescence emitted by said microcolonies.
61. The method of claim 57, wherein said illuminating employs one or more lasers.
62. The method of claim 57, wherein said illuminating employs one or more light-emitting diodes.
63. The method of claim 57, wherein said illuminating employs a source of white-light.
64. The method of claim 57, wherein said illuminating is through one or more optical filters that only pass selected wavelengths of light.
65. A method for detecting microcolonies of target cells, said method comprising the steps of:
(a) providing target cells in a detection zone, wherein within said detection area said cells are randomly dispersed and immobilized;
(b) allowing the formation of one or more microcolonies of said target cells by in situ replication, wherein at least one of said microcolonies comprises fewer than 100 target cells; and (c) detecting one or more naturally occurring optical properties of said one or more microcolonies using less than 5 fold magnification.
66. The method of claim 65, wherein said optical property or properties comprises autofluorescence.
67. The method of claim 65, wherein said optical property or properties comprises thermal radiation.
68. The method of claim 65, wherein said optical property or properties comprises optical absorbance.
69. The method of claim 68, wherein said optical absorbance is in the infrared region.
70. The method of claim 65, wherein said optical property or properties comprises fluorescence polarization.
71. The method of claim 65, wherein said optical property or properties comprises optical reflectance.
72. The method of claim 65, wherein said optical property or properties comprises light scattering.
73. The method of claim 1, wherein said detecting detects a property of said one or more microcolonies that does not depend on the addition of a signaling moiety or category-binding molecule.
74. The method of claim 1, further comprising the step, prior to or during step (c), of labeling said one or more microcolonies with a signaling moiety, wherein said detecting in step (c) detects the signal generated by signaling moieties.
75. The method of claim 1, further comprising the step, prior to or during step (c), of contacting said sample with a signaling moiety that associates either directly or indirectly with said target cells.
76. The method of claim 75, wherein said signaling moiety is associated with a category-binding molecule.
77. The method of claim 1, further comprising the step, prior to or during step (c), of contacting said sample with a category-binding molecule under conditions that allow the formation of one or more complexes between said category-binding molecule and one or more category-specific binding sites on one or more of said target cells.
78. The method of claim 77, wherein said category-binding molecule comprises an antibody, aptamer, or ligand.
79. The method of claim 77, wherein said detecting employs optical filters capable of discriminating between the signal signatures of different families of labeled category-binding molecules.
80. The method of claim 77, wherein said category-binding molecule is labeled, either directly or indirectly, with one or more signaling moieties.
81. The method of claim 80, further comprising the step, prior to step (c), of removing any unbound category-binding molecules from said one or more complexes.
82. The method of claim 77, wherein said category binding molecule is a member of an ensemble of category-binding molecules, wherein said ensemble comprises one family of category-binding molecules specific for each non-overlapping category of target cells to be detected.
83. The method of claim 82, wherein each of said families of category-binding molecules is labeled with a signaling moietie that emits a signal of a distinct signal class or signal signature.
84. The method of claim 83, wherein in step (c) said detecting detects said non-overlapping categories of target cells by detection of and discrimination between the distinct signal signatures of said signaling moieties.
85. The method of claim 76, wherein said signaling moiety is a particle or is physically associated with a particle.
86. The method of claim 75, wherein said signaling moiety has fluorescent signaling character.
87. The method of claim 86, wherein said signaling moiety is selected from the group consisting of organic fluorophores, up-regulated phosphors, lanthanides, quantum dots, enzymes that generate fluorescent product from non-fluorescent substrates, and fluorescently dyed particles.
88. The method of claim 86, wherein said signaling moiety is a fluorescent stain for cells.
89. The method of claim 75, wherein said signaling moiety has chromogenic signaling character.
90. The method of claim 86, wherein said signaling moiety has chemiluminescent signaling character.
91. The method of claim 75, wherein said signaling moiety has light-scattering signaling character.
92. The method of claim 91, wherein said signaling moiety is a resonance light scattering particle or plasmon resonance particle.
93. The method of claim 75, wherein said signaling moiety is a viability stain for staining living cells.
94. The method of claim 82, wherein said ensemble of category-binding molecules has a family complexity of 1.
95. The method of claim 82, wherein said ensemble of category-binding molecules has a family complexity that is greater than 1.
96. The method of claim 95, wherein said ensemble has a family complexity >= 5.
97. The method of claim 75, wherein said signaling moiety comprises one or more compounds that are not detectable until upon association with said target cells, said signaling moiety isacted on by a constituent of said target cells or by a physiological, physical, or micro-environmental state of said target cells.
98. The method of claim 1, wherein said replication and said detecting occur in a vessel constructed so as not to allow additional cells to enter or cells in the sample to exit.
99. The method of claim 1, wherein said replication and said detecting occur in a vessel that has a bar code or equivalent label for tracking the sample automatically.
100. The method of claim 1, wherein said replication and said detecting occur on a surface with registration marks to facilitate alignment of multiple images of the same surface.
101. The method of claim 1, wherein said detecting detects control marks or control cells in a specified region of the detection zone.
102. The method of claim 1, wherein said detecting employs optical filters adapted to detect a signal derived from the illumination of said target cells.
103. The method of claim 1, wherein said detecting employs a photoelectric detector.
104. The method of claim 1, wherein said detecting employs a photoelectric array detector.
105. The method of claim 104, wherein said photoelectric detector comprises a CCD
detector.
106. The method of claim 1, wherein said detecting does not employ an image intensifier.
107. The method of claim 1, wherein said detecting employs a photomultiplier tube detector.
108. The method of claim 1, wherein said detecting employs a photodiode detector.
109. The method of claim 1, wherein said detecting employs a photosensitive film.
110. An instrument for detecting microcolonies of target cells, said instrument comprising:
(a) a photoelectric array detector having an optical resolution of less than microns and encircled or ensquared energy values of greater than 50%
per pixel; and (b) an illumination source, wherein said instrument is capable of illuminating and simultaneously imaging a detection area having at least one dimension that is >= 1 cm, and wherein said instrument does not optically magnify more than 5 fold.
111. The instrument of claim 110, wherein said instrument does not comprise an image intensifier.
112. The instrument of claim 110, further comprising an automatic focus for focusing on said detection zone.
113. The instrument of claim 110, further comprising a computer to which data collected by said photodetector is transmitted for image analysis.
114. The method of claim 1, further comprising the step, during or after step (c), of quantifying the number of microcolonies.
115. The method of claim 1, further comprising the step, during or after step (c), of determining the category of said target cells by analyzing an image of said detection area using image analysis software.
116. The method of claim 1, further comprising the step, during or after step (c), of determining the locations in the detection zone of said one or more microcolonies by analyzing an image of said detection area using image analysis software.
117. The method of claim 116, further comprising the step, during or after step (c), of comparing said locations in the detection zone of individual microcolonies to previously determined locations of the same microcolonies.
118. The method of claim 117, wherein said image analysis software comprises algorithms for discerning objects that change size over time from objects that do not change size over time.
119. The method of claim 114, wherein said determining comprises analyzing an image of said detection area.
120. The method of claim 44, wherein said sterilization treatment is selected from the group consisting of heat sterilization, irradiation, toxic gas exposure, and disinfectant treatment.
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