Rl4. Clark's Flagellar Stain Solution A Basic fuchsin, special 1.2 g 95% ethanol 100 ml
Mix and let stand overnight at room temperature.
Solution B Tannic acid 3.0 g NaCl 1.5 g Distilled water 200 ml
Mix solutions A and B. Adjust pH to 5.0 with 1 N NaOH or 1 N HCl, if necessary. Refrigerate 2-3 days before use. Stain is stable l month at 4°C or may be stored frozen indefinitely (50 ml portions). To use, thaw stain, remix, and store at 4°C. Optimum staining time for each batch varies 5-15 min. To determine staining time (after 2-3 days refrigeration at 4°C), stain a known flagellated organism on 3 or more cleaned slides for various times (e.g., 5, 10, 15 min). Mark best staining time on all containers.
IMPORTANT: Stain will not work unless slides are clean. To clean slides, soak them 4 days at room temperature in cleaning solution (either acid dichromate or 3% concentrated HCl in 95% ethanol). Rinse 10 times in fresh tap water and twice in distilled water. Air-dry at room temperature. Store in covered container.