Procedures

Bacteria

Pouring an Agar Plate:

• Melt bottled agar at 95o in a hot water bath.
• Pour the hot, liquid agar into a sterile petri dish cracked open slightly.
• Let the agar cool and set, then flip and store upside down to avoid contamination.

Plating the P. putida:

• Retrieve a sample from either a plate or test tube of bacteria with a wire loop.
• Streak the loop onto a third of the plate and rotated and repeated to spread out colony growth.
• Replace the lid and store upside down in an incubator.

Transferring P. putida from Plate to Broth:

• Retrieve a sample from a plate with a wire loop.
• Immerse the loop into a sterile test tube of nutrient broth and move the loop back and forth to dislodge the bacteria into the broth.
• Seal tightly and place into an incubator for growth.

Introducing Toluene to P. putida:

• Grow bacteria until enough sustained growth and obtain a sample.
• Immerse the bacteria in a test tube of nutrient broth and toluene.
• Incubate and grow the bacteria.
• Plate, incubate, and grow the bacteria.
• Repeat with a gradually increasing percentage of toluene.

Measuring Optical Density:

• Find the optimum wavelength for measuring nutrient broth in the spectrophotometer .
• Create a "blank" sample by pouring nutrient broth (without any bacteria) into a cuvette and zeroing the spectrophotometer to this absorbance reading.
• Put the sample of nutrient broth with bacteria into a cuvette and put it inside the spectrophotometer to read its absorbance.

Gram Staining Bacteria Growth:

• Place a sample of bacteria onto a clean slide with a sterilized wire loop.
• Allow the slide to dry and pass it through a Bunsen flame breifly to set the bacteria.
• Flood the slide with crystal violet solution, Gram's Iodine solution, alcohol, and safranin solution in rounds with washing in between.
• Blot the slide dry, place a drop of oil over the bacteria, and examine underneath a microscope.

Carrying Out a Folin's Reagant Test:

• Combine nutrient broth, water, Folin-Ciocalteau reagent.
• Wait for at least 30 seconds, then add sodium carbonate solution, and shake to mix.
• Let the mixture sit for 2 hours and then measure the absorbance in the spectrophotometer.

Bioreactor

Constructing the Bioreactor:

• Pick a container and lay out with a stir plate and clamp.
• Locate places for the probe and air holes and drill.
• Clean the bioreactor, set up, add stir bar.
• Fill with reactants and record results.

Measuring the Results of Bioreactor:

• Set up the probes inside the bioreactor.
• Attach to a computer with Vernier Logger Pro software.
• Choose an interval to record data and a length to run.
• Start the software immediately after the reactants are added.

Setting up Warm and Cold Environments:

• Warm: setup the bioreactor inside of an incubator.
• Cold: setup the bioreactor inside of a refrigerator.
• Record results as before.