Mueller Hinton II (Cation Adjusted)

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Purpose

Mueller Hinton II broth is used for quantitative susceptibility testing for gram-negative and gram-positive facultative and aerobic bacteria. The cation adjustment is necessary for the in vitro testing of Pseudomonas aeruginosa.

Materials

• 3.0g/L Beef Extract
• 17.5g/L Acid Hydrolysate of Casein
• 1.5g/L Starch
• 20-25mg/L Ca²⁺
• 10-12.5mg/L Mg²⁺
• dH₂O

Procedure

1. Create a stock solution of Ca²⁺ by adding 8.36g of MgCl₂·6H₂O into 100mL dH₂O and sterile filter solution resulting a final concentration of 10mg/mL.
2. Create a stock solution of Mg²⁺ by adding 3.68g of CaCl₂·2H₂O into 100mL dH₂O and sterile filter solution resulting a final concentration of 10mg/mL.
3. In a 1L bottle add 3.0g Beef Extract, 17.5g Acid Hydrolysate of Casein, .5g Starch to 997mL of dH₂O
4. Autoclave media at 121°C for one hour
5. After autoclave cycle is complete, allow media to cool down to 50°C before adding 1mL of Mg²⁺ stock and 2mL Ca²⁺

• for procedure see MIC P. aeruginosa