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
>figures.
>
>David Ish-Horowicz
>
>
>
>SUPER BROTH APPARATUS AND PROTOCOL
>
>To build the apparatus drawn in Figure 1, you will need the
>following:
>
>     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
>           together.)
>
>     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
>quite rapidly.
>
>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.
>
>Typical Results
>
>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).
>
>David Ish-Horowicz
>Imperial Cancer Research Fund, London, England
>horowicz at icrf.icnet.uk




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