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Author Topic: Bioluminescent Bacteria Culturing  (Read 543 times)

da3v

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Bioluminescent Bacteria Culturing
« on: September 22, 2015, 02:31:48 PM »

Before jumping into doing a bioluminescent bacterial transformation (adding glowing genes to normally non-glowing bacteria) we thought it would be a good idea to get some experience working with naturally bioluminescent bacteria.  We got a http://www.carolina.com/bacteria/bioluminescent-bacterium-kit/154750.pr kit, which included a tube of Vibrio Fischeri culture, and 8 tubes of agar to grow it on.  Vibrio Fischeri is a marine bacteria, so the agar is a special "photobacterium agar".


Transferring culture to new tubes is done via an innoculation loop.  In the past, I have used sterile disposable loops, but this gets expensive fairly quickly, so I wanted to move on to a reusable metal loop.  cosmicaug and I looked at a few loop sterilization technologies, and I tried the following:


Bacloop electric loop sterilizer (http://shellab.com/product/bacloop-metal-inuculation-loop-sterilizer/ ). It appears to be an isolation transformer (I didn't do too much reverse engineering...yet) and whether or not there is switching/sensing/regulating or whether it is just two terminals handing out voltage to whatever gets laid across them, I am not sure. It does a good job of quickly and fairly uniformly heating the loop and wire leading up to it to a nice red-orange glow. What I didn't like was the fact that the wire heats whatever is on it, and that pops off as often as it burns.  If it happens to be covered with agar containing cooties, and that agar goes flying into tiny pieces in the air, that doesn't seem like a good thing. The bacloop is intended for use inside a glovebox, and this might not be as much of a concern in that case, but for our purposes, I'll save it for later exploration.


I then moved on to using an alcohol lamp. Bringing the loop in slowly from above heats whatever is on the loop, burns it, then heats the loop. Slower, but no popping flying bits of stuff. I loosely followed the procedure outlined on page 5 of http://www.carolina.com/pdf/care-sheets/Techniques-for-Studying-Bacteria-and-Fungi.pdf (I did not do as pretty of a job of juggling the tubes and caps between my fingers.)
I inoculated 2 tubes and labeled them vib 9/16/2015.


I checked in on 9/18/2015. The two subculture tubes were visibly glowing, the original culture was also glowing, but it was very faint. (Eyes needed to adjust to darkness for approx 5 min before the glow could be easily seen.) I then sub-sub-cultured from the 9/16 tubes and marked the new tubes 9/18 vib.


On 9/19/2015, at roughly 4PM, four of us looked in on the results, and tried culturing in more of a "workshop-like" setting. All 4 subculture tubes were relatively bright, the original was barely visible.  We selected the brightest tube, and each did a subculture from it to a new tube.  When we checked back at midnight, 3 of the 4 tubes had a small glowing area visible in them. Mine did not. :-( My current theory is that I did a really thorough job of spreading the sample in the new tube, and that it would take longer for visible colonies to form. It is also possible that I made a mistake while talking and demo-ing.  I am hoping to check back in tonight and find out.




Things we learned:
The glow is really, really faint, and we need to do a better job of lightproofing the door.
In a workshop setting, we need to get everything laid out in advance. There was a lot of re-gloving and re-spraying after we went rummaging for markers, lighters, the instruction booklet, etc.
The person doing the demo needs to be extra-careful, as attention is divided between explaining and doing.




Todo:
Have Mike.Lu do a subculture while we still have some left
Check in on Saturday's subculture and see how it turned out
Get more kits
Find way to light-proof the door
Experiment with dim red safe-lights
Look into culturing Vibrio fischeri in liquid media
Look into making our own agar by adding salt and ? to regular media.
Look into pro's and con's of culturing Vibrio phosphoreum (colder incubation, brighter glow, but what pathogenic strains are likely to be co-cultured? )
Build a laminar hood
« Last Edit: September 22, 2015, 02:43:20 PM by da3v »