Rapid detection of replicating cells
View Patent ↗The invention enables 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 in traditional tests—while retaining key advantages of the traditional methods based on microbial culture. Embodiments of the invention include non-destructive aseptic methods for detecting cellular microcolonies without labeling reagents. These methods allow for the generation of pure cultures which can be used for microbial identification and determination of antimicrobial resistance.
1. An instrument for detecting microcolonies of target cells in a sample, said instrument comprising:
(a) a photoelectric array detector having associated optics to detect a detection area having at least one dimension that is ≧1 cm without magnifying said detection area by more than 5-fold and an optical resolution of less than 50 microns;
(b) an illumination source that illuminates said detection area; and
(c) a computer programmed to receive data collected by said photoelectric array detector,
wherein said data is a digital representation of said detection area, and programmed for image analysis comprising analyzing said data to detect one or more microcolonies having a measurement of less than 50 microns in at least two orthogonal dimensions, and programmed to quantify the number of microcolonies detected in said detection area,
wherein said instrument detects a property of said one or more microcolonies that does not depend on the addition of a signaling moiety or category binding molecule, and wherein said cells in said one or more microcolonies remain competent to replicate following detection.
2. The instrument of claim 1 , further comprising an incubator for microbial replication.
3. The instrument of claim 2 , wherein said computer is programmed to detect said detection area at multiple time points during incubation, wherein said sample is stored in said incubator between said time points, and movement of said sample between said incubator and said detector is automated.
4. The instrument of claim 1 , further comprising an automatic focus for focusing on said detection area.
5. The instrument of claim 1 , wherein said instrument does not comprise an image intensifier.
6. The instrument of claim 1 , wherein said instrument detects living cells at a density of less than 100 target cells per mm 2 of the detection area, wherein within said detection zone said cells are randomly dispersed and immobilized.
7. The instrument of claim 1 , wherein said associated optics do not magnify.
8. The instrument of claim 1 , wherein said one or more microcolonies have a measurement of less than 10 microns in the longest linear dimension.
9. The instrument of claim 1 , wherein said target cells are bacteria.
10. The instrument of claim 1 , wherein said sample comprises an essentially planar solid or semi-solid growth medium.
11. The instrument of claim 1 , wherein said image analysis further comprises detecting growing microcolonies.
12. The instrument of claim 1 , wherein said instrument automatically loads said sample.
13. The instrument of claim 1 , wherein said detecting detects light emitted, scattered, reflected, or absorbed as a result of illumination of said one or more microcolonies.
14. The instrument of claim 1 , wherein said detecting detects autofluorescence emitted by said microcolonies.
15. The instrument of claim 1 , wherein said illumination source employs one or more lasers.
16. The instrument of claim 1 , wherein said illumination source employs one or more light-emitting diodes.
17. The instrument of claim 1 , further comprising one or more optical filters that only pass selected wavelengths of light.
18. The instrument of claim 1 , wherein said instrument automatically tracks said sample by a bar code or equivalent label.
19. The instrument of claim 1 , wherein said instrument is programmed to align multiple images of the detection area via registration marks.
20. The instrument of claim 1 , wherein said photoelectric array detector comprises a CCD detector, photomultiplier tube detector, or a photodiode detector.
21. The instrument of claim 20 , wherein said CCD detector is cooled.
22. The instrument of claim 1 , wherein said computer is programmed to determine the locations in the detection area of said one or more microcolonies.
23. The instrument of claim 22 , wherein said computer is programmed to compare said locations in the detection area of individual microcolonies to previously determined locations of the same microcolonies.
24. The instrument of claim 1 , wherein said image analysis discerns objects that change size over time from objects that do not change size over time.
25. The instrument of claim 1 , further comprising an automated X-Y stage for positioning said sample relative to said illumination source and detector.
26. The instrument of claim 1 , wherein said computer automatically saves output data for querying.
27. An instrument for detecting microcolonies of target cells, said instrument comprising:
(a) a photoelectric array detector having associated optics to detect a detection area having at least one dimension that is ≧1 cm without magnifying the detection area by more than 5-fold and an optical resolution of less than 50 microns and encircle or ensquared energy values of greater than 50% per pixel;
(b) an illumination source configured to illuminate said detection area having at least one dimension that is ≧1 cm;
(c) a computer programmed to receive data collected by said photoelectric array detector, wherein said data is a digital representation of said detection area, programmed for image analysis comprising analyzing said data to detect one or more microcolonies having a measurement of less than 50 microns in at least two orthogonal dimensions, and programmed to quantify the number of microcolonies detected in said detection area;
(d) an incubator for microbial replication;
(e) an automated X-Y stage for positioning a sample relative to said illumination source and detector; and
(f) an automatic focus for focusing on a detection area in said sample;
wherein said instrument detects a property of said one or more microcolonies that does not depend on the addition of a signaling moiety or category binding molecule, and wherein said cells in said one or more microcolonies remain competent to replicate following detection.
28. The instrument of claim 1 , wherein the computer is programmed to detect said microcolonies by an object-finding utility that joins contiguous pixels that have a value above an automatic or user-defined threshold to establish a contour line around the perimeter of the object, wherein the perimeter pixels and those inside are defined as the object.
29. The instrument of claim 27 , wherein the computer is programmed to detect said microcolonies by an object-finding utility that joins contiguous pixels that have a value above an automatic or user-defined threshold to establish a contour line around the perimeter of the object, wherein the perimeter pixels and those inside are defined as the object.
30. The instrument of claim 1 , wherein the associated optics comprise a collection lens and a focusing lens.
31. The instrument of claim 27 , wherein the associated optics comprise a collection lens and a focusing lens.