Dandekar & Chandler:MIC protocol
Materials needed (all must be sterile):
- • Mueller Hinton Broth
- - From Parsek lab: MHB broth BBL II, Cation Adjusted (11g/500mL)
- • Tryptic Soy Broth
- • Phosphate Buffered Saline
- • Costar 3370: 96 well assay plates
- • Breathe-Easy 9123-6100 gas permeable sealing membrane
1. Streak relevant strains onto LB plate and incubate at 37C
- a. TT Box 1:
- i. WT 3610 - H4
- ii. 1.19 (E236A) - B1
- iii. 3.3 (235G) - B7
- iv. 3.4 (E236Q) - B8
1. In the morning, inoculate a small clump of cells directly into 3mL TSB in glass tubes (one for each strain). Shake at 37C.
2. When all tubes are visibly turbid (after about 3-4 hours), dilute in PBS to McFarland Standard 1.
- a. 50 μL BaCl2 + 4.45 mL H2SO4
3. Setup MIC plate:
- a. Fill each well with 100 μL MHB.
- b. Fill first column of wells with 100 μL additional MHB (200 μL total volume).
- c. Add highest desired concentration of antibiotic to first column (calculated for 200 μL total volume).
- d. Dilute 2-fold across columns by transfering 100 μL at a time. Change tips between transfers.
- e. Remove 100 μL from last column so all wells are at a total volume of 100 μL.
4. Seed each well with 2.5 μL diluted cells.
- a. Set up 1-2 negative control wells: MHB without antibiotic or cells
- b. Set up 1-2 positive control wells: MHB without antibiotic but with cells
5. Remove plate lid and seal with breathe-easy membrane. 6. Place plate in 37C incubator, non shaking.
1. After 20-24 hours of incubation, remove the membrane.
2. Observe and record MIC as lowest concentration antibiotic where well is clear of growth.
It can be helpful to have a template to record your wells, especially if you are making MIC calls by eye;
Thao and Nicole like to scale this image to record runs in their lab notebooks