Silver stain for flagella

1. Silver stain for flagella • Bactria: • Escherichia coli • Pseudomonas aeruginosa Chapter 15, p 89

2. objective • Stain bacterial flagella which are responsible for bacterial motility

3. principle • Use mordant (stain solution A) to enclose flagella • Use sliver stain (stain solution B) to dye the protein of flagella

4. Stain agents • Solution one (sliver stain A): • 25 mL of saturated aluminum potassium sulfate (KAl(SO4)2 ) • 50 mL of 10% tannic acid • 5 mL of 5% ferric chloride (FeCl3) • Solution two (sliver stain B): • Ammonium hydroxide (AlOH3) • 5% sliver nitrate

5. procedures • Clean slide • Drop water on the slide • Pick up bacterial colony and spread into the drop of water • Spread bacterial suspension • Air dry (DO NOT use flame) • Add solution ONE • Stain for 4 min • Wash slightly

6. Add solution TWO • Stain for 2 min • Place slide on an alcohol burner • Do not overheat • Heat the slide until steam (about 4 min) • Wash • Dry • Observe under microscope

8. Pseudomonas aeruginosa

9. Escherichia coli

10. Bacillus subtilis

11. Motility test • Bactria: • Escherichia coli • Staphylococcus aureus • Medium • TSB + 0.4% agar (4 g/L)(2)

12. Procedure • Stab (about 1 cm) into medium • Incubate at 37℃ for 24 hours