Preparation of McFarland Turbidity Standards

McFarland turbidity standards are prepared by mixing various volumes of 1% sulfuric acid and 1% barium chloride to obtain solutions with specific optical densities. 0.5 McFarland turbidity standard provides an optical density comparable to the density of a bacterial suspension 1.5x 10^8 colony forming units (CFU/ml). 0.5 McFarland standard is commercially available.

For performing antimicrobial susceptibility testing using Kirby Bauer disc diffusion method, a cell suspension of organisms equivalent to a 0.5 McFarland standard is used.

Procedure

1. Prepare a 1% solution of anhydrous barium chloride (BaCl₂).
2. Prepare a 1% solution of sulfuric acid (H₂SO₄)
3. Combine and completely mix the barium chloride and sulfuric acid solutions to form a turbid suspension and BaSO₄ in a specific proportion for each McFarland turbidity standard as shown in Table 1.
4. Place the resulting mixture in a foil-covered screw-cap tube.
5. Store the it at room temperature (25 °C) when not in use. McFarland standard density solution will precipitate and clump over time, and it needs vigorous vortexing before each use. Mark the tube to indicate the level of liquid, and check before use to be sure that evaporation has not occurred.
6. Prepare a fresh standard solution every 6 months.

An alternative way is to use a McFarland densitometer for preparing bacterial suspension as per the required McFarland standard. Here the preparation of BaCl ₂ and H₂SO₄ can be avoided. 

Table 1: McFarland turbidity standards

Matching with turbidity standards

Density of the suspension of bacterial cells is compared to the McFarland standard (0.5  McFarland turbidity standard for antimicrobial susceptibility testing purposes) by holding the suspension and McFarland standard in front of light against a white background with contrasting black lines.

If the density is too heavy, the suspension should be diluted with saline or broth (whichever was used to make the suspension). If the density is not sufficient, additional bacteria should be added to the suspension. The adjusted suspensions should be used as inocula within 15 minutes.

Uses

• Standardization of inoculum for
  • Antimicrobial susceptibility testing 
    If the inoculum does not contain a concentration of bacteria that approximates the 0.5 McFarland turbidity standard, antimicrobial susceptibility test results will be affected. For instance, a resistant organism could appear susceptible if too few bacteria are used in the inoculum.
  • Various other purposes (such as DNA extraction, tests, etc. ).

References

1. Zapata, A., & Ramirez-Arcos, S. (2015). A comparative study of McFarland turbidity standards and the Densimat photometer to determine bacterial cell density. Current microbiology, 70(6), 907–909. https://doi.org/10.1007/s00284-015-0801-2 
2. Swenson, J. M., & Thornsberry, C. (1984). Preparing inoculum for susceptibility testing of anaerobes. Journal of clinical microbiology, 19(3), 321–325. https://doi.org/10.1128/jcm.19.3.321-325.1984