Digital microbiology
Methods, compositions, and kits are provided for rapidly analyzing microbial growth and/or number in a plurality of water-in-oil emulsion droplets.
1. A method for rapidly assaying a food matrix for a number of target microorganisms per unit mass or volume, the method comprising:
i) homogenizing a portion of the food matrix, wherein the portion of the matrix has a known mass or volume;
ii) encapsulating the homogenized matrix in a plurality of water-in-oil emulsion droplets, wherein the water-in-oil emulsion droplets further encapsulate a microbiological growth medium; and
iii) incubating the plurality of water-in-oil emulsion droplets at a temperature permissive of microbiological growth, and for a period of time sufficient to allow the target microorganisms to double from 5 to 45 times; and
iv) detecting autofluorescence in each of the plurality of water-in-oil droplets and determining from the incubated water-in-oil emulsion droplets:
a number of water-in-oil emulsion droplets that contain microorganisms, wherein the number of droplets that contain microorganisms is the number of droplets exhibiting autofluorescence, thereby determining a number of positive droplets; and
a number of water-in-oil emulsion droplets that do not contain microorganisms, wherein the number of droplets that do not contain microorganisms is the number of droplets exhibiting the absence of autofluorescence, thereby determining a number of negative droplets; and
v) determining from the number of positive droplets and negative droplets a total number of target microorganisms, thereby determining the number of target microorganisms per unit mass or volume of the food matrix.
2. The method of claim 1 , wherein the target microorganisms are selected from the group consisting of bacteria, yeasts, and molds and wherein the incubation is performed for a period of time corresponding to:
about 4 hours and about 16 hours when the target microorganisms are bacteria;
about 6 hours and about 12 hours when the target microorganisms are yeasts; and
about 8 hours and about 36 hours when the target microorganisms are molds.
3. The method of claim 1 , wherein the microbiological growth medium comprises partially digested protein.
4. The method of claim 1 , wherein the microbiological growth medium comprises: a) buffered peptone water; or b) a surfactant.
5. The method of claim 4 , wherein the surfactant is a non-ionic surfactant.
6. The method of claim 5 , wherein the non-ionic surfactant is a poloxamer.
7. The method of claim 6 , wherein the poloxamer has a molecular weight of about 1,800 g/mol.
8. The method of claim 7 , wherein the poloxamer comprises about 80% polyoxyethylene.
9. The method of claim 4 , wherein the microbiological growth medium comprises surfactant at a concentration of about 0.01% and no more than about 5%.
10. The method of claim 1 , wherein, prior to the incubation, the encapsulated microbiological growth medium has a pH of about 7.2.
11. The method of claim 1 , wherein the food matrix comprises animal protein, a dairy product, cheese, ground beef, ham, unpasteurized milk, plant matter, or 20 to 45% fat.
12. The method of claim 1 , wherein the determining the number of positive and negative droplets comprises collecting data from the droplets travelling serially through a detection region of a detecting device.
13. The method of claim 1 , wherein the determining the number of positive and negative droplets comprises imaging the plurality of water-in-oil droplets in parallel.
14. The method of claim 1 , wherein the determining the number of positive droplets further comprises correcting the number by applying a Poisson distribution correction to account for incorporation of multiple target microorganisms in a single droplet.
15. The method of claim 12 , wherein the target microorganism is mold and the oil phase comprises at least one antifungal agent.
16. The method of claim 15 , wherein the at least one antifungal agent is selected from the group consisting of 2,6-dichloro-4-nitroaniline, 4,5,6,7-tetrachloro-2′,4′,5′, 7′-tetraiodofluorescein, (RS)-1-[2-(allyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole, and chitosan.
17. The method of claim 16 , wherein:
a) the concentration of 2,6-dichloro-4-nitroaniline ranges from about 5 mg/L to about 200 mg/L;
b) the concentration of 4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein ranges from about 50 mg/L to about 375 mg/L; or
c) the concentration of (RS)-1-[2-(allyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole ranges from about 0.1 to about 2.5 g/L.