Superbroth for EVERYONE! (was NOT, but now IS)
Ian J. Mehr
ijmehr at merle.acns.nwu.edu
Wed Feb 28 10:53:06 EST 1996
In article <horowicz-2702961947360001 at mac056027.lif.icnet.uk>,
horowicz at icrf.icnet.uk says...
>I already got four requests to post the text version in 2 hours, so here
>it is. Acknowledgements to David Micklem and Gustilo for forwarding it to
>me, and to Dave Smith for posting it (or trying to) in the first place. No
>SUPER BROTH APPARATUS AND PROTOCOL
>To build the apparatus drawn in Figure 1, you will need the
> autoclaveable glass or rigid plastic tubing
> approximately 5 mm in outer diameter (a 1.0 mL glass
> pipet is suitable)
> autoclaveable glass container
> (Schott 1 Liter Duran flasks; 18 liter glass carboys)
> rubber stopper
> to fit the above container (The solid plastic inserts
> in Fig. 1 are custom made and appropriate holes are
> drilled in the caps of the Duran flasks. The wings are
> flexible and simply serve to hold the cap assembly
> air stones
> (presumably acquired from a pet store--aquarium-type
> aeration stones)
> flexible tubing
> (Tygon tubing from Nalgene is suitable.)
> Splitters, or "Y"s
> (only if aerating multiple flasks simultaneously)
> Hose clamps and/or Electrician's tie wraps
>The cap assembly needs 3 holes in the insert or stopper. Rigid
>tubing is inserted into only two of the holes and need only span
>the insert allowing enough distance on each side of the insert for
>attachment of flexible tubing. Once in the insert the rigid tubing
>should be tight enough to not leak air, but loose enough to allow
>adjustment for culture sampling (see below for explanation). A six
>inch length of flexible tubing is attached externally to the sample
>outlet tube of the cap assembly. This tube is closed with a thumb-
>type hose clamp. The air stone is attached to the rigid air inlet
>tube by a length of flexible tubing so that the stone is positioned
>just above the floor of the container. If any tubing joints are
>too loose, electrician's tie wraps or small hose clamps can be used
>to secure the connections. In the case of the rubber stopper, the
>cap assembly will need to be firmly attached to the flask before
>aerating the culture. A wire "cage" like the ones found on a
>champagne bottle corks will work.
>Set up a water bath of desired size and temperature adjacent to the
>air source. In our hands a 37 degree Celsius water bath placed in
>a fume hood supplied with "in house" air works well. This
>eliminates problems with culture blowout and laboratory
>contamination. No shaker is necessary; aeration from the air stone
>is sufficient for optimal growth. Depending on the quality of the
>air source an in line sterilization filter may be necessary to
>prevent contamination. A length of flexible tubing connects the
>air source and the apparatus. Multiple apparati are aerated
>simultaneously by branching a single air source with tubing
>splitters. This can result in uneven aeration from flask to flask.
>Air stones may also differ in their ability to diffuse air, thus
>adding to the difficulty of even aeration. Varying the lengths of
>tubing connecting multiple flasks helps reduce this problem.
>Testing the apparatus
>Check the apparatus for leaks and proper aeration using water.
>While aerating, a "sample" may be taken to familiarize oneself with
>the technique. Release the hose clamp on the sample outlet tube.
>Bend the opening of the flexible tube into a 1.5 mL Eppendorf or
>small glass culture tube, and while holding it in place with one
>hand, briefly place a finger from the other hand over the air
>outlet hole in the cap assembly. Beware! The sample will exit
>During growth, some culture may be trapped in the sample outlet
>tube and become anaerobic. This leads to incorrect OD readings.
>For this reason the sample outlet tube is designed to slide "up and
>down" in the cap assembly. It is pushed "down" into the culture
>only during sample collection. Vaccuum grease applied to the
>outside of the rigid sample outlet tube makes this process easier
>while maintining an air-tight seal.
>Preparing for Super Broth Growth
>Prepare the desired amount of Super Broth Media (SB).
> Super Broth Media Recipe:
> 32 g Tryptone
> 20 g Yeast Extract
> 5 g NaCl
> 5 mL 1.0 M NaOH
> Bring to 1 liter with house distilled water.
>330 mL works well in the 1 liter Duran flask apparatus. Aliquot
>the media to the flasks and cover with caps (without holes) or
>aluminum foil. Wrap the cap assembly (including the attached air
>stones) and any flexible tubing that requires sterilization in
>aluminum foil. The rigid tubing at the top of the assembly may
>protrude through the foil unless covered with a "dull" object such
>as an inverted Eppendorf tube. Autoclave the SB and other items.
>Prepare an overnight culture of the organism.
>Super Broth Growth
>Add antibiotic (100 ug/mL ampicillin) to and pre-warm the SB media.
>Pre-warming the SB media prevents cold-shocking the organisms and
>thus reduces the lag period prior to logarithmic growth. Measure
>the OD of the overnight culture to determine the volume required
>for inoculation. Inoculate the culture(s) and begin aeration.
>Correct air pressure and aeration will be different for any given
>system. It may best be judged by keeping the foam at or near 2/3
>or 3/4 capacity of the container. The two methods of regulating
>this are air pressure and anti-foam emulsion. Sigma anti foam
>emulsion A (cat# ) works well in our hands. It must be
>vigorously shaken prior to every addition. The bubbles/foam should
>NEVER be allowed to rise to the air outlet hole in the cap assembly
>as this will result in "blowout" of the culture.
>Under the conditions described the host E. coli BL21 (DE3)
>harboring the plasmid pET11aS105 inoculated at an initial OD550 of
>0.025 requires 13 hours to reach an OD of 100 (see Fig. 2).
>Imperial Cancer Research Fund, London, England
>horowicz at icrf.icnet.uk
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