Sample preparation for flow cytometry
Described herein are methods and reagents for identifying and analyzing at least one microorganism (e.g. bacteria) in a sample and reducing the background signal intensity obtained when analyzing the sample by flow cytometry. The sample is prepared by combining the sample with a background signal-reducing molecule or with a nucleic acid stain covalently linked to a quencher. A portion of the particulate matter in the sample can optionally be removed with a resin prior to staining with a nucleic acid stain.
1. A method of analyzing a sample for the total amount of viable microorganisms, comprising:
obtaining a sample to be tested for the total viable amount of an at least one target microorganism;
preparing the sample by:
i) adding an excess amount of background signal-reducing molecules wherein the background signal-reducing molecule is selected from the group consisting of a molecule with the structure Y—(CH═CH) n —CH═Z or salts thereof, wherein,
Y is:
and is either substituted or unsubstituted;
n is 0 or an integer up to about 5;
X is either carbon or sulfur;
R 1 is optionally hydrogen, an alkyl group having from 1-6 carbon atoms, a sulfite moiety or an alkyl amide, and may be selected in order to decrease cell permeability of the background-signal reducing molecule into the viable target microorganism; and,
Z is either the same or different from Y; and
ii) adding a nucleic acid stain that permeates and labels target viable cells of the microorganism;
and
analyzing the prepared sample for total viable amount of the at least one microorganism using flow cytometry.
2. The method of claim 1 , wherein the sample is selected from the group consisting of a food sample, an environmental sample, a cosmetic sample, and a biological sample.
3. The method of claim 1 , wherein the concentration of the background signal-reducing molecules is about 0.1 μM to about 50 μM when combined with the sample.
4. The method of claim 1 , wherein the background signal-reducing molecules are incubated with the sample for about 2 minutes to about 1 hour prior to the addition of the nucleic acid stain.
5. The method of claim 1 , wherein a quencher is attached to the background signal-reducing molecule.
6. The method of claim 1 , wherein the background signal-reducing molecule is a hemicyanine or closed chain cyanine.
7. The method of claim 1 , wherein n is 0 or an integer up to about 3.
8. The method of claim 1 , wherein Y is selected from the group consisting of a benzene moiety fused thereto; a benzene moiety derivative fused thereto; and a quinolone substituent,
wherein the benzene moiety, benzene moiety derivative, and quinolone can be substituted or unsubstituted.
9. The method of claim 8 , wherein n is 0 or an integer up to about 3; X is sulfur, R 1 is an alkyl amide; and Y has a benzene or benzene derivative fused thereto.
10. The method of claim 1 , wherein the background signal-reducing molecule is selected from the group consisting of
11. The method of claim 1 , wherein the prepared sample is analyzed by flow cytometry.
12. The method of claim 11 , wherein the background signal intensity is reduced by at least 50% to at least 90%.
13. The method of claim 1 , wherein the sample is prepared by adding resin to the sample and removing the resin from the sample thereby removing at least a portion of particulate matter in the sample.