Dandekar & Chandler:MIC Protocol

MIC Protocol:

Materials Needed

• 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

Day 1:

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

Day 2:

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.

Day 3:

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.