Hybridizing Bacterial Cells with an Oligonucleotide Probe Labeled with a Fluorophore Using Fluorescence In Situ Hybridization (FISH)

To begin, take bacterial cells fixed in paraformaldehyde. Centrifuge the cells at 14, 000 x g for 10 minutes. Then remove the supernatant.

Resuspend the pellet in 100 microliters of 96%analytical grade ethanol and incubate for one minute at room temperature. Repeat the centrifugation for five minutes, then remove the ethanol and air dry the cell pellet. Now hybridize the cells in a solution containing fluorescently labeled oligonucleotide for three hours at 46 to degrees Celsius.

Immediately after hybridization, centrifuge the samples in a rotor preheated at 46 degrees Celsius for 15 minutes at the maximum allowed temperature. Then wash the samples in a buffer with suitable stringency for 15 minutes at 48 degrees Celsius. After centrifuging the sample, once again, wash the cells with 500 microliters of ice cold PBS.

Resuspend them in 20 microliters of PBS and store them at four degrees Celsius until further use.