Fong:Serum bottle anaerobic culture

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Overview

Stoppered serum bottles are a relatively convenient and inexpensive way to culture strictly anaerobic bacteria. Media can be prepped in large batches filling 10 – 20 serum bottles which can then be stored at room temperature for several months ready for inoculation. Media is autoclaved in the sealed, purged bottles, so it is very important to follow careful sterile technique thereafter.

Materials

• 100 or 200ml serum bottles with 20mm openings
• 20mm blue or black butyl rubber stoppers
• Aluminum crimp seals and crimper

Procedure

Purge valve schematic
1. Turn on the vacuum and open the main valve of the ultra high purity N2 tank. The small choke valve on the regulator should also be open. Slowly adjust large regulator dial so that the tank is supplying ~5-10psi. Turn valve to the purge position as shown above. Nitrogen should be coming out of the needles on the apparatus.
2. Attach bottles (up to 5) to purging apparatus by inserting a needle into each bottle through the septum.
3. Turn valve to the vacuum position and watch for the pressure to equilibrate to around -20 in Hg (about 3-5 min).
4. Turn valve to purge position and watch for pressure to equilibrate to around 5-10psi (about 2-3 min). Note that you should be able to see air entering each of the bottles and blowing the surface of the media. If you do not see this, press needle further through the septum or replace the needle.
5. Repeat steps 3 and 4 four times (five cycles, total).
6. Remove needles from bottles during purge cycle so that a positive pressure of about 5-10psi is maintained inside the bottle. This helps prevent contamination and entrance of oxygen when piercing septum later.

Liquid transfer to/from serum bottles

1. Light ethanol burner and hold all open needles and syringes near the flame. Squirt a few drops of ethanol on the serum bottle septum, and carefully flame the ethanol to sterilize the opening. Allow all ethanol to burn off.
2. Make sure the plunger is pushed all the way into the syringe, flame the needle and then pierce the septum.
3. After filling the syringe, slowly remove the needle, allowing the pressure to release some when the needle tip is inside the septum (this prevents squirting when removing a full syringe). Keep the syringe near the flame, and flame needle before entering another bottle.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!