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 McFarland standard 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.

Table 1: McFarland turbidity standards

McFarland turbidity standard no. 0.5 1 2 3 4
1% barium chloride (ml) 0.05 0.1 0.2 0.3 0.4
1% sulfuric acid (ml) 9.95 9.9 9.8 9.7 9.6
Approx. cell density (1×1^8 CFU/ml) 1.5 3 6 9 12

Matching with turbidity standards

Density of the suspension of bacterial cells is compared to the McFarland turbidity standard (0.5  McFarland turbidity standard for antimicrobial susceptibility testing purpose) by holding the suspension and McFarland turbidity standard in front of a 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.

Bacterial suspension prepared to match the turbidity of the 0.5 McFarland turbidity standard
Bacterial suspension prepared to match the turbidity of the 0.5 McFarland turbidity standard

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 ).

8 thoughts on “Preparation of McFarland Turbidity Standards

    1. Tankeshwar Acharya

      - Edit

      Reply

      Sorry i did not understand your question, in which portion you are specifying. Just drawing your attention to basic chemical reaction:
      Bacl2 reacts with H2So4 and BaSo4 is formed.

  1. auramine stait for the ditection of fast acid bacilius on myside the principle ot i can”t understood i need youre helper Thanks.

  2. Please write on asssesment of lotic water quality.procedure. and the methodology because its difficult to asses water quality due to transportation means. Thanks

  3. Hello. I would like to us on how we can prepare a 6×10^8 cfu/ml using mcfarland standards?? We will immerse our bacterial concentration to a water.. how we can calculate it, for example. We have 10 L of water and we want to inoculate a 6×10^8??

  4. Hello..
    Thank you for this post..
    I would like to ask a question, I’m working on the determination of D value for several bacteria, and I need to prepare an inoculum of know microbial load in order to contaminate a food material and then determine the D value…
    Would it possible in this case to use a McFarland standard? Or should I prepare a standard curve of O.D 600 values against several dilutions of the bacterial suspension in order to specify the bacteria load later before the contamination takes place?

    If the answer is yes, and I used a 0.5 McFarland for example, would the result be affected by the difference in bacterial shapes and sizes? Are this numbers more consistent with an E.coli suspension? Or would it work no matter what bacterial species I had?

    Thanks a lot.. 🙂

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