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. 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 mm 2 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,
(c) labeling said one or more microcolonies with a labeling reagent; and
(d) detecting said one or more microcolonies by detecting the signal generated by said labeling reagent;
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; wherein said detecting does not entail magnification of more than 5×; and wherein 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 1 target cells per mm 2 of the detection area.
3. The method of claim 1 , wherein said detecting detects a single microcolony in the detection area.
4. The method of claim 1 , wherein said target cells are bacteria or eukaryotic cells.
5. The method of claim 4 , wherein said target cells are fungal, animal, or plant cells.
6. 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.
7. 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.
8. The method of claim 1 , wherein said detection zone comprises a material selected from the group consisting of glass, plastic, bibulous membranes, and plastic strips.
9. The method of claim 1 , wherein the replication of said cells in said microcolonies is continued after said detecting.
10. The method of claim 1 , wherein said detecting comprises illuminating one or more microcolonies to generate a detectable signal.
11. The method of claim 10 , wherein said illuminating employs one or more lasers or light-emitting diodes.
12. The method of claim 10 , wherein said illuminating employs a source of white-light.
13. The method of claim 10 , wherein said illuminating is through one or more optical filters that only pass selected wavelengths of light.
14. The method of claim 1 , wherein said detecting detects fluorescence.
15. The method of claim 14 , wherein said detecting employs optical filters adapted to detect the signal derived from the illumination of said target cells.
16. The method of claim 1 , wherein said labeling reagent is a fluorescent stain for cells.
17. The method of claim 1 , wherein said labeling reagent signaling moiety is a viability stain for staining living cells.
18. The method of claim 1 , wherein said labeling reagent comprises one or more compounds that are not detectable, until upon association with said target cells, said labeling reagent is acted on by a constituent of said target cells.
19. The method of claim 18 , wherein said labeling reagent is an esterase substrate.
20. The method of claim 1 , wherein said detecting employs a photoelectric detector.
21. The method of claim 1 , wherein said detecting employs a photoelectric array detector.
22. The method of claim 1 , further comprising the step, during or after step (d), of quantifying the number of microcolonies.
23. The method of claim 1 , further comprising the step, during or after step (d), of detecting microcolonies that grow over time.