Rapid detection of replicating cells

• US 9,090,462 B2
• Filed: 03/19/2010
• Issued: 07/28/2015
• Est. Priority Date: 09/06/2001
• Status: Expired due to Fees

• Current Assignee: Rapid Micro Biosystems, Inc.
• Original Assignee: Rapid Micro Biosystems, Inc.
• Inventors: Straus, Don
• Primary Examiner(s): Ware, Debbie K
• Assistant Examiner(s): Srivastava, Kailash C
• Application Number:
  US12/727,598
• Publication Number:
  US 20100248281A1
• Time in Patent Office: 1,957 Days
• US Class Current: 1/1
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 20/00 Nanooptics, e.g. quantum optics or photonic crystals
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/06 Quantitative determination
• CPC Class Codes: C12Q 1/18 Testing for antimicrobial activity of a material
• CPC Class Codes: G01N 15/1429 Signal processing
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 2015/1486 Counting the particles
• CPC Class Codes: G01N 2015/1488 Methods for deciding
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 2021/6471 Special filters, filter wheel
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 2333/195 from bacteria
• CPC Class Codes: G01N 2333/245 Escherichia (G)
• CPC Class Codes: G01N 2333/31 from Staphylococcus (G)
• CPC Class Codes: G01N 2333/32 from Bacillus (G)
• CPC Class Codes: G01N 2333/33 from Clostridium (G)
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/56916 : Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
• CPC Class Codes: G01N 33/56938 : Staphylococcus
• CPC Class Codes: G01N 33/56966 : Animal cells
• CPC Class Codes: G01N 33/56983 : Viruses
• CPC Class Codes: G01N 33/58 : involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/582 : with fluorescent label

Abstract

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.

Claims

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,

(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 mm² 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.