Preparation of Iron-Depleted Cation-Adjusted Mueller Hinton Broth Instructional Video

Preparation of iron-depleted cation-adjusted Mueller–Hinton broth for cefiderocol susceptibility testing.

Methodology according to CLSI M100 document.

To start preparing approximately 1 L of iron-depleted cation-adjusted Mueller–Hinton broth, autoclave 1 L of Mueller–Hinton II broth or Mueller–Hinton broth according to the manufacturer’s specifications.

Becton-Dickinson (BD) BBL and BD Difco were found to produce MIC values most predictive of efficacy in vivo.*

Allow the broth to cool down to room temperature between 20 and 25°C, then place it on a stirring plate.

To avoid contamination with iron during the preparation of the iron-depleted cation-adjusted Mueller–Hinton broth, use distilled or deionized water, plastic instead of metal spatulas, Teflon^TM-coated magnetic bars, and high-purity reagents.

Put a sterile, Teflon^TM-coated magnetic bar into the flask.

Then add 100 g Chelex^® 100 chelating resin, analytical grade [100–200 mesh, Na⁺ form].

Cover the flask and stir for 6 hours.

After completing the 6-hour chelation, pour the broth into a bottle-top filter device.

Filter the broth through a 0.2 µm pore size filter into the storage bottle using a vacuum-generating suction tube.

Place the filtered broth on a stirring plate and use a new Teflon^TM-coated magnetic bar to stir it.

Insert the pH meter probe into the broth and check the pH.

Expect the broth to be alkaline (pH is approximately 8).

Add hydrochloric acid from a 6 molar stock solution drop-by-drop until the pH is adjusted to between 7.2 and 7.4.

If necessary, adjust the pH with sodium hydroxide.

Both hydrochloric acid and sodium hydroxide should be made with deionized or distilled water.

Chelation removes other cations to negligible levels; therefore, calcium, magnesium, and zinc need to be added.

Prepare stock solutions of ultrapure ZnSO₄, CaCl₂ and MgCl₂ with deionized or distilled water.

To adjust the concentration of calcium, magnesium, and zinc, calculate the small amount you need to add from these stock solutions.

Add calcium chloride to reach a final calcium concentration of 22.5 µg/mL.

Add magnesium chloride to reach a final magnesium concentration of 11.25 µg/mL.

Finally, add zinc sulfate to reach a final zinc concentration of 0.75 µg/mL.

Insert a pH meter probe into the storage bottle to check that the pH has not changed.

If the pH has changed, adjust with hydrochloric acid or sodium hydroxide, as necessary.

Filter the broth as before.

Check that the total concentration of iron in the broth is less than 0.03 µg/mL by using the Visocolor^® assay (Macherey-Nagel, Düren, Germany).

Transfer a sample of the broth, according to the manufacturer’s instructions, into the two sample tubes included in the kit.

Add reagent 1 according to the manufacturer’s instructions into the tube on the right-hand side.

Close the tube and mix it.

Then add reagent 2 to the same tube.

Close the tube, mix it, and wait for 3 minutes.

Remove the caps from the tubes.

And from above, visually inspect the colors in the two tubes and turn the disk until the colors match.

Verify that the iron concentration in the sample is ≤0.03 µg/mL.

If the iron concentration is >0.03 µg/mL, discard the media and start from the beginning to prepare a new batch of iron-depleted broth.

Before restarting the preparation of the iron-depleted broth, troubleshoot which step of the broth preparation (chelation or inadvertent introduction of iron in subsequent steps) could have caused the elevated levels of iron in the final product.

If the iron concentration is ≤0.03 µg/mL, the sterile, filtered, iron-depleted cation-adjusted Mueller–Hinton broth can be stored at 4°C until use, for a maximum of 2 months.

*This information is not included in the CLSI M100.