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    9/22: Bioreactor Prep Work
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    9/22: Bioreactor Prep Work

    Saturday, 7/27/2019Introduction/Background ObjectivesMaterialsK. rhaeticus Plates & Liquid Culture MaterialsYamanaka MaterialsWhat we did today:For Next timeProcedure K. rhaeticus Plates & Liquid Culture More Yamanka Media & More Glucose Solution
    Saturday, 7/27/2019
    Introduction/Background
    These are the steps and procedures that we need to follow to ensure we have prepared everything we need for running our first test in our bioreactor.
    Objectives
    ●
    Prepare the media and the culture needed to seed our bioreactor
    Materials
    K. rhaeticus Plates & Liquid Culture Materials
    ●
    Equipment
    ○
    Autoclave
    ○
    Incubator
    ○
    2 x 250 mL bottles
    ○
    Optional: Motion shaker (Shaky) for fast growth
    ○
    Smear loop (for plate streaking)
    ●
    For 250mL Media:
    ○
    5g glucose (2%w/v) (autoclaved separately)
    ○
    1.25g yeast extract (0.5% w/v)
    ○
    1.25g peptone (0.5% w/v)
    ○
    0.675g Na2HPO4 (0.27% w/v)
    ○
    0.375g citric acid (0.15% w/v)
    ○
    250ml distilled H20 (dH2O)
    ○
    (antibiotics as necessary)
    ●
    if making HS-agar plates
    ○
    3.25g of agar (agar-agar)
    Yamanaka Materials
    Materials found on this recipe: https://benchling.com/protocols/20LDBwi1/preparing-yamanaka-media BEFORE STARTING TO MEASURE/MIX ANYTHING MAKE SURE YOU HAVE ENOUGH INGREDIENTS
    What we did today:
    ●
    Trouble shooted bioreactor leaking issues
    ○
    the DO port is leaking so we need a 12mm O ring for that
    ●
    Checked and added new sparge system into the bioreactor
    ○
    we installed new sparge stones so that there would be an even oxygen flow throughout all the spargers
    ●
    Threw out contaminated BC from the vessel experiment into the biohazard bin
    ●
    Made new liquid cultures
    ●
    Made 10 yamanaka +agar + MANNITOL plates
    ○
    We're using mannitol because we ran out of glucose
    ●
    Made more Yamanaka media(4000ml) using 3600 ml for bioreactor
    For Next time
    ●
    Streak the agar plates t86i
    Procedure
    K. rhaeticus Plates & Liquid Culture
    Creating 250 mL of HS+ Glucose + Agar Media for 10 HS + Glucose + Agar Plates
    1.
    In one bottle, add 125 mL of dH20 and 5g of glucose. Stir vigorously to dissolve glucose.
    2.
    In another bottle, add 125 mL of dH20 and the rest of the ingredients. (see materials list for specific ingredients)
    a.
    If making agar plates, add 3.25 g agar into the solution.
    3. Autoclave both bottles. (The bottles are separate because the yeast excract and peptone cannot be autoclaved with sugar. If autoclaved together, there's the possibility for a Maillard reaction to occur, which can result in toxic byproducts)
    4. Pour the contents of the first bottle (contains glucose) into the second bottle. This is your 250 mL media.
    Culturing: Plates
    1.
    Pour media into 10 plates, and let set
    2.
    Streak K. rhaeticus onto plates, and incubate at 30o C. Colonies should appear in 48-72 hours
    More Yamanka Media & More Glucose Solution
    1.
    Make 3,200 mL of Yamanaka + Glucose Media
    a.
    This breaks down into: 1,600 mL of Yamanaka w/o Glucose and 1,600 mL of Glucose solutions
    2.
    Make sure ahead of time that you have enough bottles and volume in bottles present to hold this much media. At the end of the media making process, there should be an equal amount of Yamanka w/o Glucose bottles when compared with Glucose solution bottles
    3.
    Following this recipe: https://benchling.com/protocols/20LDBwi1/preparing-yamanaka-media, make sure to scale up all of your measurements to make the amount of media necessary--DO NOT START MIXING/MEASURING IF YOU DON'T HAVE ENOUGH OF A COMPONENT
    4.
    Use proper labeling techniques! Include the ratio/concentration on the labels.

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