Journal

3/28- Reinoculating fresh plate from 3/18 plate with 100 ug/mL amp.

3/18 Began plate with 100ug/ml amp, inoculate from purchased slant (CArolina) A- 24 hrs B-24 hrs C- 24 hrs D- 24 hrs

E- 24 hrs

3/1 Starting test plates- each with a 1-- ug/mL amp (15 ul) conc Control A- .5mL, 33.3mg/ml B- .25mL, 16.7 mg/ml C- 125 ul, 8.3 mg/ml D- 62.5 ul, 4.2 mg/ml E- 31.25 ul sol, 2.08 mg/ml

2/23 No glowing on A2, low growth

2/21 E+F still glowing, dim glowing on D. Testing to see if inhibited bac. will glow normally on fresh medium. Should have picked isolated colony? A2 inoculated from A onto normal agar.

2/18- Glowing observed on E + F, indicating too low of a concentration?

2/17 D- .125ul soln, 8.3mg/ml (pouring error?) E- 3.125ul soln, 2.08 mg/ml F 7.81 ul soln, 520 ug/ml

2/16 No luminescence observed on B 2/15, has "smoother" growth

2/15 Luminescence on C, none on A+B. Growth seems to have been affected, streak is a bit paler and more flat. Normal growth of B may be due to error- agar solidified around garlic.

2/14 It has been found that 1 dropper of garlic = 1 mL, concentration 1g/mL. Three plates with three concentrations: C (control) A- .5 mL sol'n, 33.3 mg/ml B- .25mL sol'n, 16.67 mg/ml

2/11 Growth on both plates- untreated plate is glowing, treated plate is not.

2/10 Started 2 agar plates- 1 with a dropper of garlic and one control. No luminescence on petri dish culture.

2/7 10 mL petri dish culture -> 1000mg amp = 1mL, .66g broth needed with 100 microl amp solution. The 1/28 culture is no longer glowing. 1/30 culture is glowing. Started a broth plate culture heavily inoculated from 1/30.

2/3: Glowing observed on 1/28 culture.

1/30: Two broth cultures evaporated- leave cover on next time. No growth on broth inoculated agar, some minimal growth on source inoculated agar. Started a 40 mL unfiltered broth culture with bac. from plate 1/28. First trials might have been done with old bac. No bubble stone- put tubing directly in broth, more bubbles.

From after break up until 1/30, I experimented with growing bacteria on broth cultures to no success.

1/14- Observed no luminescence in 1/13 culture. Started a clear broth culture. Possible causes: culture hasn't grown yet, ordered bac. is too old, inadequate aeration (larger flask?)

December 15th- 22nd:

Materials list has been finalized and ordered from Carolina, and orders placed for other materials (garlic extract, bubble stones) have been placed. While I still do not know how to sterilize the bubble stones, I found a pack of 100 stones, which should be sufficient.

December 5th- 13th:

The project (for now) will be narrowed down to only using garlic extract. If there is enough time, I will test some analogues. A substitute to water bath shakers has been found- bubble stone aerators commonly used in fish aquariums. As of right now, I'm not sure how I'll be able to sterilize them. The necessary concentration of garlic extract has been found in papers. More kinks and specifics concerning procedure have been worked out, and I am working on a step by step procedure.

Overview of project as of December 5th:

While looking for project ideas involving quorum sensing, I learned that the bacteria Pseudomonas aeruginosa, a deadly pathogen seen in immunocompromised individuals, utilizes quorum sensing to form biofilms, protecting the bacteria from harm. The quorum sensing mechanism, though a bit more complicated, works in a very similar fashion to the mechanism utilized by V. fischeri in bioluminescence. Therefore, I'd like to test various methods of inhibiting quorum sensing, using Vibrio fischeri as a model. Some methods I've found include:

Ultimately I'll probably test one or two natural methods, the pH method, and the acid method. For use medically, the AHL analogue or the blockage of AHL production method are probably the most useful; however, though I have identified some potential compounds, I still need to search for suppliers and determine how much they cost.
 * In vitro production of AHL blockage:
 * L/D-S-adenosylhomocysteine
 * sinefungin
 * butyryl-S-adenosylmethionine (butyryl-SAM)
 * Increase pH to above 7, causing lactonolysis (ring opening of AHL)
 * Temperature increase?
 * Lactonase enzymes?
 * Hypobromous and hypochlorous acids
 * Analogue of AHL molecule
 * Natural:
 * carrot
 * soybean
 * tomato
 * habanero
 * garlic

I plan to to first grow a control of V fischeri in both liquid and solid media and determine the time it takes to reach a high enough concentration to glow. I've found a piece of free software that can create timelapse movies from a simple webcam. Using this, I should be able to monitor the change in glow of the bacteria over time. Mr. Savage has also said that he has a Vernier light probe to determine the amount of light output the bacteria produce. By comparing the intensity of light, I can see how effective each method of inhibition is. I'll probably need to build some sort of lightproof box to keep out external light.

Also, the procedures I've read use a water bath shaker to grow V. fischeri in liquid media. I don't have one, so I don't know if the bacteria will grow correctly. It seems like many companies sell bioluminescence project kits using V. fischeri, and it doesn't look like they require one. Hopefully the bacteria will grow properly.

Week of Oct 10th-Oct 15th and Oct 17th-Oct 22nd
Researched and read papers on //Vibrio fischeri// (notes) and read procedures on culturing bacteria in liquid media. (http://dwb4.unl.edu/chemistry/biotech/A09.html) (http://www.biotopics.co.uk/microbes/tech1.html) Wrote first draft of summary to be submitted to colleges.

Located papers on the quorum sensing functions of //Vibrio fischeri//, located a photobacterium broth source, and continued researching the signaling cascade that leads to bioluminescence.

Week of Sept 27th-Oct 1st
Decided on first project- working with the quorum sensing capabilities of //Vibrio fischeri//. Things that need to be researched- photobacterium broth sources/recipe, procedures on growing //Vibrio fischeri//, and variables to be investigated.

Sunday, Sept. 26th 2010
Finished contract, beginning to research autoinducer project.

Saturday, Sept. 25th 2010
Watched first lecture on quorum sensing by Bonnie Bassler in HHMI's 2009 Holiday Lectures, searched for project possibilities.

Friday, Sept. 24th 2010
Searched for project possibilities, ( Project Ideas ) the most notable being the effects of commercial homeopathic antiseptics and antibiotics on bacterial growth. Began to outline syllabus.

Thursday, Sept. 23rd 2010
I created and organized the wiki I will use to keep track of progress. I also investigated project possibilities (Project Ideas) and watched the beginning of the first lecture of HHMI's Biodiversity Holiday Lectures on toxins. I decided that it was more urgent to look at other topics of interest first, since the topic would most likely not lead to a feasible project.