Rapid detection of replicating cells

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.

1 - 28 . (canceled) 
     
     
         29 . A method for detecting microcolonies of living target cells in a sample, said method comprising:
 (a) depositing 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;   (b) allowing the formation of one or more microcolonies of said target cells by in situ replication;   (c) detecting the one or more microcolonies without using a labeling reagent; and   (d) determining the effect of one or more substances or treatments on one or more attributes of the target cells; wherein   the cells in the one or more microcolonies remain competent to replicate following the detecting.   
     
     
         30 . The method of  claim 29 , wherein the cells are randomly dispersed and immobilized within the detection zone. 
     
     
         31 . The method of  claim 29 , wherein a longest linear dimension of the detection area is greater than 1 mm. 
     
     
         32 . The method of  claim 29 , wherein the one or more microcolonies have a measurement of less than 50 microns in at least two orthogonal dimensions. 
     
     
         33 . The method of  claim 29 , wherein the detecting does not entail magnification of more than 5 times. 
     
     
         34 . The method of  claim 29 , wherein the detecting detects a property of the one or more microcolonies without an addition of a signaling moiety or category binding molecule. 
     
     
         35 . The method of  claim 29 , wherein one of the one or more attributes is an ability to undergo cell replication. 
     
     
         36 . The method of  claim 29 , wherein the one or more substances are present in a medium used to support replication of the target cells. 
     
     
         37 . The method of  claim 29 , wherein the attribute is an ability of the target cells to replicate following a sterilization treatment. 
     
     
         38 . The method of  claim 37 , wherein the sterilization treatment is selected from the group consisting of heat sterilization, irradiation, toxic gas exposure, and disinfectant treatment. 
     
     
         39 . The method of  claim 29 , wherein the attribute is an ability of the target cells to replicate in the presence of one or more inhibitors of replication. 
     
     
         40 . The method of  claim 39 , wherein the one or more inhibitors are antimicrobial compounds. 
     
     
         41 . The method of  claim 39 , wherein the one or more inhibitors are anti-tumor compounds. 
     
     
         42 . The method of  claim 29 , wherein the attribute is a viability, a change in an optical property, metabolic or enzymatic activity, or biochemical constituency or the target cells. 
     
     
         43 . The method of  claim 29 , wherein the target cells are bacteria, fungi, parasites, or cultured cells, and the substances are antibacterial agents, anti-fungal agents, or anti-parasitic agents. 
     
     
         44 . The method of  claim 29 , wherein the target cells are randomly dispersed in the detection zone at a density of less than 10 target cells per mm 2  of the detection area. 
     
     
         45 . The method of  claim 29 , wherein the target cells are randomly dispersed in the detection zone at a density of less than 1 target cells per mm 2  of the detection area. 
     
     
         46 . The method of  claim 29 , wherein the mean number of cells in the one or more microcolonies is less than 50,000 cells. 
     
     
         47 . The method of  claim 29 , wherein the detecting detects and identifies microcolonies from more than one non-overlapping category of cell. 
     
     
         48 . The method of  claim 29 , wherein the detection zone is contacted with a liquid medium comprising one or more substances that facilitate replication of target cells.