Safranin O and TB staining procedure
| Safranin O staining | |
| Reagents: 1% HCl in 70% ethanol 1% acetic acid in water Weigert’s iron haematoxylin working solution 0.1% fast green in distilled water 0.1% safranin in distilled water | |
| Staining procedure (at room temperature): | Time |
| Incubate with Weigert‘s haematoxylin working solution | 5 min |
| Differentiate in 1%HCl/70%ethanol (under gentle shaking) | 5 s |
| Rinse in running tap water | 15 s |
| Incubate in 0.1% safranin O | 30 s to 1 min |
| Rinse in 1% acetic acid | 15 s |
| Rinse in running tap water | 15 s |
| Incubate with fast green 0,1% | 5–7 min |
| Rinse in 96% ethanol | 15 s |
| Rinse in 100% ethanol until no colour can be removed | up to 1 min |
| Incubate in xylene | 5 min |
| Seal the slides in permanent mounting medium (eg, Eukitt) | |
| TB staining | |
| Reagents: TB stock solution 10×: 1% TB O, 1% natriumtetraborate in water. Stock solution should be filtrated two times before use. 1× TB working solution is prepared in water. | |
| Staining procedure (at room temperature): | Time |
| Incubate in TB working solution (1×) | 8–30 s |
| Rinse in distilled water | 15 s |
| Rinse in 96% ethanol (metachromatic dye becomes visible) | 10–20 s |
| Dehydrate in absolute ethanol | 5 min |
| Incubate in xylene | 5 min |
| Mount with permanent mounting medium (eg, Eukitt) | |
TB, toluidine blue.