2.9. Differential Staining of Meat Exudate and E. coli

A dual staining procedure was conducted as described previously [51]. Ten microlitres of DAPI was added to the samples and spread across the surface using a sterile plastic spreader to detect the bacteria and then 10 µL of Rhodamine B was applied to the substrate in the same manner to detect the retained meat extract [51]. Following staining, the samples were dried in the dark at room temperature in a microbiological class 2 flow hood.

The samples were viewed, and images obtained using an epifluorescence microscope with black and white digital camera and a Cell F Image Analysis package to measure the percentage coverage of the area of the stained material and to determine the percentage surface coverage of the bacteria and organic material. A filter wavelength of 330–380 nm was used to detect the DAPI stained cells, and a 590–650 nm filter was used to detect the Rhodamine B stained organic material. The retained material on the surfaces was measured using percentage coverage of the field size for randomly selected areas across the test substratum. Each of the three samples had 15 areas independently selected and analysed for the percentage coverage of bacteria and meat extract (n = 45).