McFarland Standards

Understanding the McFarland Standard: A Close Look

• In the realm of microbiology, McFarland standards play a crucial role. They are used to calibrate the cloudiness, or turbidity, of bacterial suspensions. This ensures the bacterial count stays within a specific range, standardizing microbial testing techniques.
• Among the many applications of McFarland standards is antibiotic susceptibility testing. In this process, the minimum inhibitory concentration is gauged, a practice that is routine in both medical microbiology and sophisticated research circles.
• It's vital to have the bacterial suspension just right. If it's either too dense or too dilute, inconsistencies can arise in test results, which can manifest as either false resistance or susceptibility towards a particular antimicrobial agent.
• To trace McFarland standards to their roots, we would find mixtures with precise amounts of barium chloride and sulfuric acid. These were the elements of the original McFarland standard.
• On combining these two compounds, barium sulfate precipitates out. This infusion creates a cloudiness, or turbidity, in the mixture, synonymous with the McFarland standard.
• How do we prepare a 0.5 McFarland standard? Begin by blending 0.05 mL of 1.175% barium chloride dihydrate (BaCl2•2H2O) with 9.95 mL of 1% sulfuric acid (H2SO4). That's the recipe for this level of McFarland standard.
• Fast forward to today, we have McFarland standards based on suspensions of latex particles. This approach notably extends the shelf life and stability of these bacterial suspensions, offering a modern, robust standard.
• For easy comparison, the McFarland standard can be visually compared directly to a bacteria suspension in a sterile saline solution or a nutrient broth.
• Adjustments are straightforward with the McFarland standard. If the bacterial suspension appears excessively turbid, dilute it. Conversely, if it's not cloudy enough, it's relatively simple to add more bacteria to the mix.

What are Uses of McFarland standard?

1. It is used in the antimicrobial susceptibility testing process.
2. In the realm of microbiology, McFarland standards play a crucial role. They are used to calibrate the cloudiness, or turbidity, of bacterial suspensions. This ensures the bacterial count stays within a specific range, standardizing microbial testing techniques.

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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