User:Anand Gupta/Notebook/Microbial Fuel Cell/2009/04/22

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Bacteria Preparation Protocol

When bacteria arrive, they will be plated out so that quantities bacteria are ready for the series of protocols that follow. For the fuel cell, we'll be using overnight cultures (in broth as opposed to on plates) so as to maximize reaction surface area and bacterial concentration. We'll be using LB nutrient agar for the first cell to ensure maximal growth.

Preflight Checklist

Protocol #1 Materials

  • Plates
  • LB agar
  • dH2O
  • Autoclave-safe bottle
  • E Coli K-12 Stock plate
  • Inoculating loops
  • Alcohol [surface cleaning], Chlorox [disinfectant]
  • [Optional] Bacti-incinerator / Bunsen Burner

Plate Prep

1) Dissolve [X g]/pellets LB agar in [Y ml] distilled H2O [as per material specification].

2) Autoclave and allow to cool.

3) Reheat to liquid (~4 mins in microwave).

Pour plates

4) Open agar bottle, flame mouth briefly to sterilize--keep bottle tilted when open, with cap adjacent.

5) Clamshell plate an tilt agar bottle to pour LB into plate. Add agar to fill line and close lids promptly.

6) Store inverted at 4C until use.

We'll prepare [X plates], on to which our bacteria will be plated from the starter plates.

Plate Inoculation

1) Heat inoculating loop to sterilize(if not sterile), and touch to agar to cool.

2) From stock plate, touch single bacterial colony with inoculating loop.

3) On fresh plate, streak loop in zig-zag pattern across plate in three directions and densities--first third is dense, second is less dense, and third is wide enough to isolate individual colonies. Keep plate clamshelled throughout.

4) Close and mark plate. Store at 37C in incubator.

Note on marking; all plates should be marked in following way: [Your initials], [Date of prep], MFC [Protocol #], [Media]