Sealing Anaerobic Cultures with Cotton Plugs

Purpose: To make glass tubes anaerobic by plugging the top of the glass with cotton
Materials Needed:
1. Autoclaved cotton plugs (unbleached)
2. Borosilicate glass tubes
a. Small: 13 X 100 mm (Cat No 14-961-27, Fisher)
b. Large: 14 X 150 mm (Cat No 14-961-32, Fisher)
3. Rubber stoppers
a. Small: size 00 (Cat No 14-130A, Fisher)
b. Large: size 0 (Cat No 14-130-1, Fisher)
4. Bunsen burner
5. Saturated sodium bicarbonate
6. 35% Pyrogallol
7. Tweezers
Method
1. Prepare either a small or large glass tube with appropriate liquid culture
2. Plug the top of the glass tube with autoclaved cotton ball, leaving about 1/3 of the
cotton sticking out on top
3. Take Bunsen burner and char the cotton (this will make the cotton burn down to
about the level of the glass tube)
4. Add 200 ul of saturated sodium bicarbonate and 200 ul of 35% pyrogallol to the
top of the cotton (amount is the same for both small and large tubes0
5. Use tweezers to push the cotton plug approximately of the way down the tube
6. Plug the tube with the appropriate size rubber stopper
7. Place the tubes in an aerobic incubator at 37o C