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Institute for Systems Biology
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Seattle, WA 98103-8904


Halo Growth without Incubator

Purpose:
We decided that since some schools might not have an evironmental shaker-incubator that we should create a way for them to conduct the halo growth experiment in light and dark conditions without it.  Throughout the entire summer we devoted a lot of our time to trying different variations of this experiment in order to optimize it so it could be easy to be done in the classroom.  We tried using glass flasks, plastic tubes, stoppers, drilling holes, a variety of different sized tubing, and different manifolds.  The protocol, results, and conclusions below is one of our more successful experiments.

Protocol:
Used 18 50ml conical tubes.  Wrapped 9 of the tubes in black electrical tape to block out light.  There were 6 light and 6 dark aerobic tubes and 3 light and 3 dark anaerobic tubes.  Added 20ml CM and 4ml Halobacterium to the aerobic tubes.  In the anaerobic tubes we added 40ml CM and 8ml Halo.  We setup the tubes as per the diagram below.  For the aerobic samples we used a #54 drill bit to drill a hole in the cap of the tubes.  We had an AZOO Air Pump 5500 with two outlets that we used.  We connected Manifold B to the air pump though a 3/16th inch ID tube.  The tube fed into a #2 rubber stopper in manifold with pipette tip(we cut off the pointed tip) to increase air flow.  From each outlet in the manifold we connected 3/16th inch ID tubing.  At the other end of the 3/16th inch ID tube we added a piece of 1 inch long 1/16th inch ID tubing.  This was to make a better seal between the tubes and make the bubbling more consistent.  The final piece of tubing that went into the conical tubes was a 1/16 x .010 blue and stiff tubing.  Each piece was 140mm long and the ends were cut at an angle.  Fill the top of each tube with the same amount of hydrophobic glass wool.  After 48 hours take the OD600 of the aerobic samples.  Also take 1.6ml of the solution and spin it down in eppendorf tubes in a microcentrifuge.  Collect the same data for the anaerobic samples after 72 hours.


Results:



Observations/Conclusions:
The OD600 was consistent and good even though there was evaporation and accumulation of salt at the bottom of the tubes.  There was a color difference between the light and dark pellets, but it wasn't drastic enough so we need to find a way to reduce evaporation in order to hopefully improve the pellets.  In the past using halo that is in its plateau stage (has a high OD600, like above 0.9) has yielded better pellets that mor obviously show the difference between light and dark.

Next Step:
Repeat the experiment except :
-use tin foil instead of electrical tape (attempt to reduce temperature in dark tubes)
-use halo with a high OD600
-use more glass wool at top of 50ml conical tube