How to prepare 0.5 McFarland turbidity standard ?

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0.5 McFarland turbidity standard is used to standardize the inoculum for performing antimicrobial susceptibility test. Before antibiotic disks are placed on Muller Hinton agar plate, the bacterial suspension should be matched with the turbidity of 0.5 McFarland turbidity standard. It contains 1.5 X 108  CFU/ml of bacteria.

Note: If we skip matching the turbidity of bacterial suspension with 0.5 McFarland turbidity standard, then we may get heavy growth of organism on the media plate that may affect the zone size of the antibiotics.  

Procedure to prepare 0.5 McFarland turbidity standard consists of the following steps:

  • Add 0.5 ml of 0.048 mol/L BaCl2 (1.175% w/v BaCl2 × 2H2O) to 99.5 mL of 0.18 mol/L H2SO4 (1% v/v).
  • Verify the correct density of the turbidity standard by using a spectrophotometer with a 1-cm light path and matched cuvette to determine the absorbance. The absorbance at 625 nm should be 0.08 to 0.10 for the 0.5 McFarland standard.
  • Distribute 4 to 6 mL into screw-cap tubes of the same size as those used in growing or diluting the broth culture inoculum.
  • Tightly seal the tubes and store them in the dark at room temperature.
  • Vigorously agitate this turbidity standard on a mechanical vortex mixer just before use.
  • Replace standards or recheck their densities three months after preparation

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