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.
- 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.
- 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.
- 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.
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