McFarland Standards – Requirement, Procedure, and Result | Biology Ideas

What is Mcfarland Standard?

• McFarland standards are used as a reference to adjust the turbidity of bacterial suspensions so that the number of bacteria will be within a given range to standardize microbial testing.
• An example of such testing is antibiotic susceptibility testing by measurement of minimum inhibitory concentration which is routinely used in medical microbiology and research.
• If a suspension used is too heavy or too dilute, an erroneous result (either falsely resistant or falsely susceptible) for any given anti-microbial agent could occur.
• Original McFarland standards were mixing specified amounts of barium chloride and sulfuric acid together.
• Mixing the two compounds forms a barium sulfate precipitate, which causes turbidity in the solution.
• A 0.5 McFarland standard is prepared by mixing 0.05 mL of 1.175% barium chloride dihydrate (BaCl2•2H2O), with 9.95 mL of 1% sulfuric acid (H2SO4).
• Now there are McFarland standards prepared from suspensions of latex particles, which lengthens the shelf life and stability of the suspensions.
• The standard can be compared visually to a suspension of bacteria in sterile saline or nutrient broth.
• If the bacterial suspension is too turbid, it can be diluted with more diluent. If the suspension is not turbid enough, more bacteria can be added.

What are Uses of McFarland standard?

1. It is used in the antimicrobial susceptibility testing process.
2. McFarland standards are used as a reference to adjust the turbidity of bacterial suspensions so that the number of bacteria will be within a given range to standardize microbial testing.

Measurement Of Cell Mass by Turbidimetry

• This is a most convenient is the measurement of cell mass by turbidimetry.
• Increase the total cell mass, as well as in cell numbers, accompany population growth.
• Turbidimetric methods are rapid and sensitive. They depend on the fact that microbial cells scatter light striking them.
• Because microbial cells in the population are of roughly constant size, the amount of scattering is proportional to the concentration of cells present.
• When the concentration of bacteria reaches about one million cells per mL, the medium appears slightly cloudy or turbid.
• Further increase in the concentration of bacteria cells results in greater turbidity and less light is transmitted through the medium.
• The extent of the light scattering can be measured by a spectrophotometer and is almost linearly related to bacterial concentrations at low absorbance levels.
• The population growth can be easily measured spectrophotometrically as long as the population is high enough to give detectable turbidity.

McFarland Nephelometer Standards

McFarland Standard No.	0.5	1	2	3	4
1.0% Barium chloride (ml)	0.05	0.1	0.2	0.3	0.4
1.0% Sulfuric acid (ml)	9.95	9.9	9.8	9.7	9.6
Approx. cell density (1X10^8 CFU/mL)	1.5	3.0	6.0	9.0	12.0
% Transmittance*	74.3	55.6	35.6	26.4	21.5
Absorbance*	0.08 to 0.1	0.257	0.451	0.582	0.669

• * (at a wavelength of 600 nm)
• McFarland Latex Standards from Hardy Diagnostics (2014-12-10), measured at the UCSF DeRisi Lab

McFarland Standard No.	0.5	1	2	4	6	8
Absorbance @600nm	0.063	0.123	0.242	0.431	0.653	0.867

Requirements of McFarland standard

1. Test culture (freshly grown and young) on nutrient agar slant.
2. 5.0 mL sterile saline in tubes to suspend the culture (if inoculum is not in nutrient broth).
3. Two side tube Erlenmeyer flasks containing sterile nutrient liquid medium.
4. Spectrophotometer.
5. 200-250 rpm rotary shaker.
6. 1.0 ml sterile pipette.

Preparation of McFarland standard

1. Suspend a small portion of the culture in sterile saline.
2. Inoculate the cell suspension into one of the nutrient medium flasks using the sterile 1mL pipette
3. Read the absorbance of the medium at 530nm [use the uninoculated flask as a control to adjust 100% transmittance].
4. Put both the flasks on the rotary shaker.
5. Periodically [30 minutes interval] remove the flasks from the shaker and read the absorbance at 530nm.
6. When the culture enters its stationary phase (no more increase in absorbance), terminate the experiment.
7. Plot a graph of absorbance versus time.
8. Determine the generation time using the plot.

Reference And Sources

• https://en.wikipedia.org/wiki/McFarland_standards
• https://microbenotes.com/mcfarland-standards/
• https://paramedicsworld.com/microbiology-practicals/preparation-of-mcfarland-standard-for-antibiotic-susceptibility-test-ast-in-laboratory/medical-paramedical-studynotes
• https://microbeonline.com/preparation-mcfarland-turbidity-standards

Further Readings

1. Instruments used in Microbiology Laboratory
2. Fimbriae vs Pili
3. Fimbriae vs Flagella
4. Aseptic Transfer Technique
5. Growth Curve of Bacteria
6. Spread Plate Technique
7. MacConkey agar
8. Preparation and Sterilization of culture media
9. Monochrome Staining
10. Acid fast staining of bacteria
11. Negative Staining
12. Instruments used in Microbiology Laboratory
13. Streak Plate Technique
14. Algae
15. Bacterial Growth and Nutrition
16. Bacterial Flagella, Fimbriae and Pili, Capsule
17. Serial Dilution in Microbiology
18. Negative Staining

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