US 11,952,610 B2
Digital microbiology
Christophe Quiring, Marnes la Coquette (FR); Christine Favier, Marnes la Coquette (FR); Patrice Sarfati, Marnes la Coquette (FR); Jean Francois Mouscadet, Marnes la Coquette (FR); Ronald Lebofsky, Marnes la Coquette (FR); and Rebecca Dievart, Marnes la Coquette (FR)
Assigned to BIO-RAD EUROPE GMBH, Basel (CH)
Filed by BIO-RAD EUROPE GMBH, Basel (CH); and BIO-RAD LABORATORIES, INC., Hercules, CA (US)
Filed on Mar. 4, 2022, as Appl. No. 17/686,466.
Application 17/686,466 is a division of application No. 16/072,712, granted, now 11,268,124, previously published as PCT/IB2017/000086, filed on Jan. 25, 2017.
Claims priority of provisional application 62/286,897, filed on Jan. 25, 2016.
Prior Publication US 2022/0195484 A1, Jun. 23, 2022
Int. Cl. C12Q 1/06 (2006.01); C12N 9/26 (2006.01); C12N 9/34 (2006.01); C12N 9/38 (2006.01); C12N 9/40 (2006.01); C12N 9/42 (2006.01); C12N 9/48 (2006.01); C12Q 1/04 (2006.01)
CPC C12Q 1/06 (2013.01) [C12N 9/2428 (2013.01); C12N 9/2445 (2013.01); C12N 9/2465 (2013.01); C12N 9/2471 (2013.01); C12N 9/2474 (2013.01); C12N 9/485 (2013.01); C12Q 1/04 (2013.01); C12Q 1/045 (2013.01); C12Y 302/01003 (2013.01); C12Y 302/01022 (2013.01); C12Y 302/01035 (2013.01); C12Y 304/11 (2013.01)] 9 Claims
 
1. A method for rapidly assaying a target microorganism for a minimum inhibitory concentration of a test antimicrobial, the method comprising:
i) encapsulating a plurality of the target microorganisms in a plurality of water-in-oil emulsion droplets,
wherein the water-in-oil emulsion droplets further encapsulate a microbiological growth medium;
wherein:
a) a first portion of the water-in-oil droplets encapsulate the test antimicrobial at a first concentration, or do not encapsulate the test antimicrobial;
b) a second portion of the water-in-oil droplets encapsulate the test antimicrobial at a second concentration different than the first concentration;
wherein the target microorganism is a bacterium or a yeast; and
ii) incubating the plurality of water-in-oil emulsion droplets at a temperature permissive of microbiological growth in an absence of the test antimicrobial, and for a period of time sufficient to allow the target microorganisms, if not inhibited by the test antimicrobial, to divide from 5 to 20 times, or more; and
iii) determining from the incubated water-in-oil emulsion droplets by measuring autofluorescence:
a number of water-in-oil emulsion droplets that have an increase in microorganisms that results in increased autofluorescence, thereby determining a number of positive droplets; and
a number of water-in-oil emulsion droplets that have no increase in microorganisms and no increase in autofluorescence, thereby determining a number of negative droplets;
iv) determining the number of positive droplets and negative droplets at the tested antimicrobial concentrations; thereby assaying the minimum inhibitory concentration of the test antimicrobial.