What are McFarland Standards – Its Principle, preparation, and uses

Introduction

McFarland Standards are a series of standardized suspensions of bacteria that are used to determine the concentration of microorganisms in a sample. They are widely used in microbiology for various applications, including microbial susceptibility testing, quality control, and bacterial density measurements. McFarland Standards are essential in ensuring the reproducibility and accuracy of results in microbiology, making them a crucial tool in the field.

What are McFarland Standards?

McFarland Standards are suspensions of microorganisms that have been standardized to a specific optical density. The standards are used to determine the concentration of bacteria in a sample and are expressed as colony-forming units (CFU) per milliliter of sample. McFarland Standards are prepared by mixing a specific amount of bacterial culture with a specific amount of a chemical reagent, such as barium chloride or sulfuric acid, to achieve a specific turbidity level. The turbidity level is measured using a spectrophotometer, which determines the optical density of the suspension.

A brief history of McFarland Standards

The McFarland Standards were first introduced in 1907 by John McFarland, a Scottish bacteriologist. McFarland developed the standards to standardize bacterial density measurements and improve the accuracy and reproducibility of microbiology tests. Originally, the standards were based on the use of a photometer and a suspension of E. coli, but over time, the method was refined to use different chemicals and microorganisms to achieve a standardized turbidity level. Today, McFarland Standards are widely used in microbiology laboratories around the world and have become an essential tool for accurate and reliable microbiology testing and research.

Principle of McFarland Standard

A McFarland Standard is a chemical solution that is used to determine the concentration of microorganisms in a sample. The solution is made by combining barium chloride and sulfuric acid, which react to produce a fine precipitate of barium sulfate. This solution is then shaken well to produce turbidity that is visually comparable to a bacterial suspension of known concentration.

McFarland turbidity standards are prepared by mixing different volumes of 1% sulfuric acid and 1% barium chloride to obtain solutions with specific optical densities. By adjusting the volume of these two chemical reagents, McFarland standards of varying degrees of turbidity can be prepared which represent different bacterial densities or cell counts. For instance, the 0.5 McFarland turbidity standard provides an optical density comparable to the density of a bacterial suspension with a 1.5 x 10^8 colony forming units (CFU/ml).

McFarland Standard	1% BaCl2(ml)	1% H2SO4 (ml)	Cell count in CFU/ml
0.5	0.05	9.95	1.5 x 108
1.0	0.1	9.9	3.0 x 108
2.0	0.2	9.8	6.0 x 108
3.0	0.3	9.7	9.0 x 108
4.0	0.4	9.6	12.0 x 108

How to prepare McFarland Standard?

Preparing McFarland Standards involves combining specific amounts of barium chloride and sulfuric acid to create a turbid solution with a known concentration of microorganisms. The following steps are commonly used to prepare McFarland Standards:

1. Gather the necessary materials: Barium chloride solution (1% w/v), sulfuric acid solution (1% v/v), volumetric flask, distilled water, and a spectrophotometer.
2. Clean and dry the volumetric flask to avoid contamination.
3. Calculate the amount of barium chloride and sulfuric acid needed to prepare the desired McFarland Standard using a formula or table. For example, to prepare a 0.5 McFarland Standard, you would need to mix 0.05 ml of 1% barium chloride solution with 9.95 ml of 1% sulfuric acid solution.
4. Carefully measure the required amount of barium chloride solution and add it to the volumetric flask.
5. Add the required amount of sulfuric acid solution to the volumetric flask.
6. Swirl the flask gently to mix the contents. Avoid vigorous shaking, as this can create air bubbles that can affect the turbidity of the solution.
7. Check the turbidity of the solution using a spectrophotometer. Place the McFarland Standard in the sample holder of the spectrophotometer and measure the absorbance at a wavelength of 625 nm.
8. If the absorbance value is not within the expected range, adjust the volume of the barium chloride or sulfuric acid solution accordingly and repeat the measurement until the desired turbidity is achieved.
9. Label the McFarland Standard with the appropriate McFarland turbidity value and store it in a dark, cool place to prevent deterioration.

Uses of McFarland Standards

McFarland Standards are widely used in microbiology for various purposes. Here are some common uses of McFarland Standards:

1. Antibiotic susceptibility testing: McFarland Standards are used to standardize the concentration of bacterial suspensions used in antibiotic susceptibility testing. This ensures that the bacterial concentration is consistent across all tests, allowing for an accurate interpretation of antibiotic sensitivity.
2. Quality control of media: McFarland Standards can be used to check the quality of growth media by inoculating it with a known concentration of bacteria. This helps to ensure that the media is suitable for growing bacteria and that the bacterial concentration is within the desired range.
3. Microbial quantification: McFarland Standards can be used to determine the concentration of bacteria in a sample. By comparing the turbidity of a bacterial suspension with a McFarland Standard of known concentration, the bacterial density can be estimated.
4. Hematology: McFarland Standards can be used in hematology to standardize the concentration of blood cells for cell counting. This helps to ensure accurate cell counts and to standardize the results across different laboratories.
5. Environmental monitoring: McFarland Standards can be used to monitor the bacterial content of environmental samples such as water, soil, and air. By comparing the turbidity of the sample with a McFarland Standard, the bacterial concentration can be estimated.
6. Research and development: McFarland Standards are commonly used in the research and development of new diagnostic tools, vaccines, and antibiotics. They provide a reliable and standardized way to measure bacterial concentration, which is essential in developing accurate and effective diagnostic and therapeutic tools.

Limitations of McFarland Standards

While McFarland Standards are a widely used tool in microbiology, there are some limitations to their use that should be considered:

1. Incompatibility with Colored Media: The use of colored media can affect the proper contrast of McFarland Equivalence Standards, leading to incorrect results or incorrect densities.
2. Limitations with Older Cultures: McFarland Standards may not compare well with expected bacterial counts when used with bacterial suspensions older than 24 hours.
3. Instrumentation Dependency: McFarland Standards are adjusted based on spectrophotometer analysis. The use of any other instrumentation may not give reliable results during the testing procedure.
4. Specific Requirements for Latex Standards: While using the latex standard, the tubes for the suspension should be the same diameter as the McFarland Latex Standard tube.
5. Storage Time Affects Turbidity: Exposure of the McFarland standard to light during the storage period can affect the turbidity measurement.
6. Interference from impurities: Impurities in the sample can interfere with the turbidity measurement, leading to inaccurate results. For example, blood, pus, or other particulate matter in the sample can affect the turbidity of the solution and lead to errors in the estimation of bacterial concentration.
7. Limited applicability: McFarland Standards are primarily used for bacteria that grow in liquid media. For bacteria that grow on solid media or require special growth conditions, McFarland Standards may not be applicable.

Conclusion

In conclusion, McFarland Standards play a critical role in standardizing the concentration of microorganisms in bacterial suspensions for accurate and reproducible results in microbiology experiments and tests. Despite its limitations, such as variations in the preparation of colored media, the use of spectrophotometers for analysis, and storage conditions affecting turbidity measurement, the McFarland Standard remains an essential tool in microbial testing. The future of McFarland Standardization could involve incorporating new technologies for precise and automated preparation of the standard, including the use of robotics and machine learning algorithms. As microbiology continues to evolve, McFarland Standards will continue to be an indispensable tool for ensuring accurate and reliable results in microbial testing.

References

• http://www.dalynn.com/dyn/ck_assets/files/tech/TM53.pdf
• Cappuccino J.G. and Sherman N. 2008. Microbiology: A Laboratory Manual, 8th ed. Pearson Benjamin Cummings, San Francisco, CA, USA.
• Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). In Clinical Microbiology Procedures Handbook, Fourth Edition. American Society of Microbiology. https://doi.org/10.1128/9781555818814
• Procop, G. W., Church, D. L., & Koneman, E. W. (2020). Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. Jones & Bartlett Learning.