Table 2 Summary of steps for live-FISH procedure.
From: Fluorescence in situ hybridization (FISH) and cell sorting of living bacteria
Stage | Step no. | Description |
|---|---|---|
Pre-hybridization | 1 | Mix 1 ml of overnight culture with 500 µl CMFASWa. |
2 | Centrifuge at 13,000 × g for 5 min at RTb. | |
3 | Wash twice in 500 µl CMFASWa at 13,000 × g for 5 min at RTb. | |
Heat shock | 4 | Resuspend pellets in 1 ml ice cold 160 mM MgCl2/40 mM CaCl2. |
5 | Centrifuge at 13,000 × g for 5 min at 4 °C. | |
6 | Resuspend pellets in 50 µl ice cold 100 mM CaCl2 + 4 µl probe (500 ng/µl). | |
7 | Incubate 15 min on ice in the dark. | |
8 | Incubate for 60 sec at 42 °C. | |
Hybridization | 9 | Add immediately 500 µl of pre-warmed hybridization buffer. |
10 | Incubate at 46 °C in the dark for 2 hrs. | |
Washing | 11 | Centrifuge at 13,000 × g for 5 min at RTb. |
12 | Wash twice in 1 ml pre-warmed wash buffer at 13,000 × g for 15 min at 48 °C. | |
13 | Re-suspend in 500 µl CMFASWa and keep on ice in the dark until sorting. |
