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    K. rhaeticus HS media and culturing
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    K. rhaeticus HS media and culturing

    Introduction

    As copied from Imperial's 2014 protocol, added equipment section through inference from their instructions. Hestrin-Schramm Media has been found to be the most preferrable media for BC production. Contains carbon source, enriched nitrogen source, and a small amount of citric acid. There are two options for culturing: in plates or in a liquid medium. The steps for both are quite similar and so both are outlined below.

    Materials

    • Equipment
      • Autoclave
      • Incubator
      • 2 x 500 mL bottles
      • Optional: Motion shaker (Shaky) for fast growth
      • Smear loop (for plate streaking)
    • For 500mL Media:
      • 10g glucose (2%w/v) (autoclaved separately)
      • 2.5g yeast extract (0.5% w/v)
      • 2.5g peptone (0.5% w/v)
      • 1.35g Na2HPO4 (0.27% w/v)
      • 0.75g citric acid (0.15% w/v)
      • 500ml distilled H20 (dH2O)
      • (antibiotics as necessary)
    • if HS+cellulase media required (added for fast growth liquid medium)
      • 0.5mL cellulase
    • if making HS-agar plates
      • 7.5g of agar (agar-agar)

    Procedure

    • Making the media
    1. In one bottle, add 250 mL of dH20 and 10g of glucose. Stir vigorously to dissolve glucose.
    1. In another bottle, add 250 mL of dH20 and the rest of the ingredients. (see materials list for specific ingredients)
    • Important! This is the step where you should add ingredients to "customize" the solution.
    • If making agar plates, add 7.5 g agar into the solution.
    • If HS + cellulase media required, add 0.5 mL cellulase. Add cellulase for fast growth liquid medium
    1. 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)
    1. Pour the contents of the first bottle (contains glucose) into the second bottle. This is your 500 mL media.
    • Culturing: plates
    1. Pour media into plates, and let set
    1. Streak K. rhaeticus onto plates, and incubate at 30o C. Colonies should appear in 48-72 hours
    • Culturing: liquid medium
    1. Inoculate K. rhaeticus into media (1:10 ratio)
    1. Incubate at 30o C
    1. For slow but quiality growth, use standing solution.
    1. For fast growth, grow with shaking at 180 rpm

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