Skip to content

Latest commit

 

History

History
60 lines (46 loc) · 2.54 KB

FecalMIC.md

File metadata and controls

60 lines (46 loc) · 2.54 KB
Raw

Fecal Minimal Inhibitory Concentration (MIC) assay protocol

Goal:

To determine the effects of drugs on gut microbiota composition

Materials

  • Sterile H2O or DMF (Sigma 319937)
  • Syringe Filters (Nylon-VWR 28145; SFCA-Corning 431219)
  • 96 well culture plates (Falcon 351177)
  • Deep well plates (VWR 75870-796)
  • 50 mL reservoirs (USA Scientific 1930-2530)

Preparation of drug stocks:

  • Dissolve drug in water or DMF at a concentration of 25.6 mg/mL
  • Filter-sterilize and store at -20˚C or as appropriate.

Preparation of plate:

Note: Can be done a day in advance on the bench. Place the prepared plate in the anaerobic chamber 24 hours before the experiment. If preparing plate on the day of the experiment, prepare plate in the anaerobic chamber.

  1. In a deep well 96-well plate, add 1mL of media (BHI CHV or mGAM) to each well of first column. If using DMF, add a second column to act as vehicle control
  2. Add additional 980 uL of media to the first row only
  3. Add 20uL of drug stock (or vehicle) to the first row
  4. Serially dilute the media containing the drug by pipetting 1 mL and dispensing onto the next row skiping the last row.

The well plate should contain the following drug concentrations:

Row Drug
A 256 ug/mL
B 128 ug/mL
C 64 ug/mL
D 32 ug/mL
E 16 ug/mL
F 8 ug/mL
G 4 ug/mL
H 0 ug/mL
  1. Using a multi-channel pipette, transfer 200 uL of drug containing media onto 250uL 96-well plate
  2. Place the plate in anaerobic chamber 24 hours before the experiment if prepared on the bench to remove oxygen

Preparation of fecal samples:

  1. Take 100mg aliquot of fecal sample out of -80˚C freezer and into the anaerobic chamber
  2. Place the aliquot in a tube with 1mL media (BHI CHV or mGAM)
  3. Vortex for 5-10 sec to homogenize the fecal sample
  4. Let the homogenized mixture sit for a few minutes to let the solids settle

Experimental Protocol

Note: Must be done in the anaerobic chamber

  1. Using a multi-channel pipette, transfer 1 uL of fecal sample to the prepared plate (0.5% loading inoculum)
  2. Carefully place the cover on the plate and tape around the edges to seal the plate
  3. Place the plate into the plate reader
  4. Set up the plate with the following parameters:
    1. Kinetic loop set for 24 hours
    2. Read every 15 minutes
    3. Shake for 30 sec before reading
  5. When finished, save and export the data