Jan 30, 2023
Minimum Inhibitory Concentration
This protocol is a draft, published without a DOI.
- 1San Diego State University
Protocol Citation: Amanda T Alker, Nicholas J Shikuma 2023. Minimum Inhibitory Concentration. protocols.io https://protocols.io/view/minimum-inhibitory-concentration-b5iwq4fe
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 23, 2022
Last Modified: January 30, 2023
Protocol Integer ID: 58678
Keywords: antibiotic selection, minimum inhibitory concentration
Funders Acknowledgement:
Gordon and Betty Moore Foundation
Grant ID: GBMF9344
National Science Foundation
Grant ID: 1942251
Office of Naval Research
Grant ID: N00014-20-1-2120
Abstract
Protocol for determining the minimum inhibitory concentration of antibiotics for marine bacteria.
Day 1
Day 1
Streak out marine microbes onto Marine Agar plates and incubate overnight at 25-28ºC.
Note
Some strains take longer to grow to single colonies. Incubation times longer than 24 hours may be required for some slower growing strains. Monitor growth in future steps accordingly.
Day 2
Day 2
Inoculate a single colony into 5mL Marine Broth (2216) media in the late afternoon/ early evening. Incubate overnight at 25ºC, shaking at 200 rpm.
Day 3
Day 3
Measure optical density (OD600) of overnight culture growth and document
Pipette 100µL of overnight culture from each strain onto Marine Agar plates containing either Strep (for background resistance), Kan or Gent (MIC) at each concentration (25µg/mL, 50µg/mL, 100 µg/mL and 200µg/mL).
Use beads or plate spreader to spread the culture evenly on the plate. Remove the beads and incubate overnight at 25ºC overnight.
Day 4
Day 4
Observe and document growth on all concentration plates.
For the MICs (Kan/Gent) identify the lowest concentration of media in which no colonies are observed.
Note
If there are no colonies at any concentration, screen lower concentrations of Kanamycin/Gentamicin in the media (i.e. 5, 10, 15, 20µg/mL)
For the Streptomycin Resistant strain, select a single colony from the highest dose of Streptomycin that has growth and streak it out onto a plate containing the same concentration AND a plate containing the next highest concentration of antibiotics. Incubate overnight at 25C.
Day 5
Day 5
Observe and document growth on both the MIC plates (2 days old) and the Streptomycin resistance plates. If Streptomycin resistance is sustained at less than 200µg/mL, then continue step 7, passaging the strains onto higher concentrations of antibiotics. Once robust growth occurs at 200µg/mL, inoculate a single colony into 5mL Marine Broth containing Strep 200µg/mL and incubate 25ºC overnight, shaking at 200rpm.
Day 6 or later
Day 6 or later
Store culture in cryovials for long term storage. Add 500µL overnight culture to 500µL 50% glycerol, pipette mix and store in the -80C for future use.