How to perform serial dilutions and make microbial suspensions using McFarland Standards

Summary

This Hardy Diagnostics tutorial explains how to prepare bacterial suspensions using McFarland standards, a technique essential in microbiology labs. The video demonstrates how to adjust the turbidity of a bacterial culture to a 0.5 McFarland standard using aseptic technique and a wicker room card for visual comparison. It also covers the process of serial dilution to reduce bacterial concentration, explaining the steps for achieving various dilution factors. The tutorial emphasizes the importance of accurate turbidity adjustment for reliable antimicrobial susceptibility testing results, and it highlights the role of Hardy Diagnostics in supporting microbiologists.

Takeaways

• 😀 McFarland standards are used by microbiologists to adjust the density of bacterial suspensions.
• 😀 McFarland standards help create semi-quantitative bacterial suspensions and serial dilutions.
• 😀 Originally, McFarland standards were made with barium chloride and sulfuric acid; now, latex particles are commonly used for more stability.
• 😀 McFarland standards are commercially available in various concentrations from 0.5 to 8.0.
• 😀 A 0.5 McFarland standard has an approximate bacterial cell density of 1.5 * 10^8 CFUs per milliliter.
• 😀 Use a fresh culture (less than 24 hours old) for preparing bacterial suspensions to avoid dead bacteria.
• 😀 A sterile cotton swab is used to collect bacterial colonies for inoculation into a saline tube.
• 😀 The bacterial suspension is homogenized by vortexing or inverting to ensure an even suspension.
• 😀 The turbidity of the bacterial suspension is compared against the 0.5 McFarland standard using a white card with black lines.
• 😀 Adjust the turbidity of the suspension to match the 0.5 McFarland standard to avoid false resistance or susceptibility results in antimicrobial testing.
• 😀 Bacterial suspensions can be serially diluted to decrease the concentration by factors like 100-fold or 10-fold, allowing for the desired bacterial concentration for testing.

Q & A

What are McFarland standards used for?

McFarland standards are used by microbiologists to adjust the density of bacterial suspensions, a common and essential technique in microbiology labs.

What were McFarland standards originally made from?

McFarland standards were originally prepared by adding barium chloride to sulfuric acid, resulting in a turbid barium sulfate precipitation.

Why are latex particle suspensions preferred today over the original method for McFarland standards?

Latex particle suspensions are preferred because they are much more stable compared to the original barium sulfate method.

What is the bacterial cell density of a 0.5 McFarland standard?

The bacterial cell density of a 0.5 McFarland standard is approximately 1.5 * 10^8 CFUs per milliliter of sample.

Why should cultures used for preparing McFarland standards be no older than 24 hours?

Cultures older than 24 hours may contain a higher percentage of dead bacteria, which can affect the accuracy of the suspension's turbidity.

How should a bacterial suspension be prepared for a 0.5 McFarland standard?

Using aseptic technique, pick 3 to 5 well-isolated colonies and inoculate them into a 5 ml tube of saline. Vortex or invert the tube to ensure even suspension of the bacteria.

What is the purpose of using a Wickerham card when adjusting the bacterial suspension?

The Wickerham card, with contrasting black lines on a white background, is used to compare the turbidity of the bacterial suspension to that of the 0.5 McFarland standard. When the suspension matches, the black lines should be obscured.

What could happen if the turbidity of the bacterial suspension is too high or too low?

If the turbidity is too high or too low, it may lead to false resistant or false susceptible results when performing antimicrobial susceptibility testing.

How is the bacterial concentration adjusted during serial dilutions?

To increase turbidity, more bacteria are added; to decrease turbidity, more saline or broth is added. For serial dilutions, aliquots of bacterial suspension are transferred into new tubes containing saline to reduce the concentration step by step.

What is a typical dilution factor used in McFarland standard dilutions?

In McFarland standard dilutions, a 1 to 100 dilution is commonly used. This involves transferring an aliquot of bacterial suspension into another tube containing saline and vortexing to homogenize.