Detection of cells in a liquid sample

A method of counting cells in a liquid sample, said method comprising the steps of:

• a) mixing a staining composition comprising a dimeric nucleic acid dye and a buffering agent with said sample;
• b) optionally sonicating the mixture of step a);
• c) incubating the mixture at a temperature from about 50° C. to about 95° C. for less than 10 minutes, wherein the mixture is incubated without an ion-chelating agent or detergent;
• d) optionally sonicating the incubated mixture of step c);
• and
• e) counting the cells that are stained with said dye within said mixture, or a part thereof, wherein
• the dimeric nucleic acid dye is cell-impermeant to HeLa cells after 30 minutes of incubation at 37° C. and neutral pH and has the formula Q1-BRIDGE-Q2, wherein Q1 and Q2 are nucleic acid dye moieties and BRIDGE is connecting Q1 and Q2 and has the formula:
• —(CH2)x—C(═O)NH—(CH2)α—[O—(CH2)β]b—[O—(CH2)γ]c-NH(O═C)—(CH2)x—,
• wherein:
• each x, independently, is an integer selected from 1 to 11,
• α may be an integer selected from 2 to about 20,
• β and γ, are independently zero, 2 or 3,
• b is zero or an integer selected from 1 to about 20, and
• c is zero, 1 or 2, and wherein
• the buffering agent is an organic buffering agent comprising an amine which is effective in rendering the dimeric nucleic acid dye cell-permeant under the incubating conditions of step c) to achieve a reliable counting of the cells.