Quorum Sensing Lab Notes

Quorum Sensing - change in bacterial gene expression at high population densities

Vibrio Fischeri- quorum sensing – phenotype: glow
- every 2 groups gets 1 test tube of seawater agar
- heavily streak v. fischeri (photobacterium)onto a slant cap loosely, wrap in aluminum foil, then incubate at 25 degrees Celsius for 18-24 hrs
Psuedomonas Aeruginosa
-gram (-) quorum sensing
- HSL in autoinduser
- Quorum sensing – phenotype = biofilm production snot-looking floating goobers that have purple flecks
- Rhamnolipids = goobers
- Pyocianin = purple (sorta smells like grapes)

Lab 2: strains of P. aeruginosa
- PA01 = wild type
- JP2 = lacks the gene for HSL production

- centrifuge PA01 broth thru filter w/ 0.25 micro liters holes to remove bacteria, leave the AI in broth
- Make tubes of AI broth:
100% AI/ 0% PTSB
50% AI/ 50% PTSB
25% AI/ 75% PTSB
10% AI/ 90% PTSB
inoculate each tube w/ 50mL of broth cultured JP2
make positive and negative control tubes
10mL PTSB plus 50 micro liters of bacterial broth
(+) control = PA01
(-) control = JP2

The recipe for PTSB (peptone tryptic soy broth): 50 grams of peptone, 2.5 grams tryptic soy broth powder, and one liter of distilled water.

The recipe for Seawater Agar: 5g tryptone, 2.5g yeast, 0.3g Ammonium chloride, 0.3g Magnesium sulfate, 15g agar, 1g calcium carbonate, 3g monopotassium phosphate, 30g sodium chloride, 23.5g sodium glycerol phosphate in 1L of distilled water.
-Null Hypothesis = addition of HSL in a sterile – filtered PA01 broth will NOT make JP2 express quorum sensing phenotype
-Hypothesis = at X% AI broth, JP2 will express the quorum sensing phenotype

- PTSB = Peptone tryptic soy broth
- AI = Autoinducer
- HSL = homoserine lactone

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