Mueller-Hinton broth

Dilute mid-log phase bacteria to 1 x 10s cells/mL in Mueller-Hinton broth. 

Mueller-Hinton broth (Oxoid). This is prepared by dissolving 21 g of powder in one litre of distilled water and autoclaving at 121°C for 15 min. Alternatively, smaller amounts can be prepared and filter sterilized. 

The bacterium is grown on blood agar plates and stored in a mixture of Mueller-Hinton broth containing 20% (v/v) glycerol. In this mixture, bacteria can be stored in small aliquots for several years at -70°C. 

If larger culture volumes are required then 2.5-L Ehrlenmeyer flasks containing a maximum of one liter of Mueller-Hinton broth are prepared and autoclaved. Once cooled, this broth may be seeded directly with colonies from the agar plate however, this often results in poor yields and slow growth. It is better to seed this volume of culture with a small amount of overnight broth culture. 

To prepare a seeding culture, fill a 25 pL universal with approximately 10 pL of sterile Mueller-Hinton broth and seed these with 1-2 colonies from the blood agar plate. This is then incubated at 37°C overnight with vigorous shaking at approximately 200 rpm. 

These included peptone water (bacteriological peptone, Oxoid 1.0%, Analar NaCl 0.5%, pH 12-1 A), nutrient broth (Oxoid), and Mueller-Hinton broth (Oxoid). 

From the beginning, laboratory studies on the antibacterial properties of cefaclor pointed to an inherent chemical instability in solution and in neutral to alkaline bacteriological media. Test medium composition, pH, and inoculum density can significantly affect in vitro susceptibility assay results. For example, in both Mueller-Hinton broth and agar, chemical degradation of cefaclor is observed as a rapid loss of antimicrobial activity (50%) after 6 hr at 37°C (Preston, 1979). Special care is, therefore, recommended in the interpretation of in vitro test results. 

Antibacterial assay. The antibacterial activity of all test compounds was evaluated by the same procedure as in the antifungal assay except that Mueller-Hinton broth was used as the medium and the test organism inoculum was 10 CPU each. The inoculated tubes containing test compounds were incubated at 37 C for 24 h and scored for growth of each test organism. The MIC for each species represents the lowest concentration of the test compound at which complete inhibition of growth occurred. 

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