Standard plate count

Summary

This video explains the technique of serial dilution to estimate the number of viable bacteria in a sample, such as alfalfa sprouts. It covers the process of preparing dilutions by mixing samples with sterile water, calculating dilution factors, and transferring the diluted sample through successive tubes. The video demonstrates proper aseptic techniques, the preparation of agar plates, and incubation. After counting bacterial colonies on the plates, the number of bacteria in the original sample is estimated. The final count is reported as colony-forming units (CFU) per gram or milliliter, with a focus on accuracy and proper data recording.

Takeaways

• 😀 Serial dilution is a method used to reduce the concentration of a sample in steps, making it easier to quantify bacterial counts.
• 😀 A 1:10 dilution (10^-1) is created by adding 1 mL of a sample to 9 mL of sterile water, resulting in a total volume of 10 mL.
• 😀 The dilution factor multiplies with each subsequent transfer, e.g., 10^-1 × 10^-1 = 10^-2 for the second dilution tube.
• 😀 For solid samples, 1 gram of the sample is treated as equivalent to 1 mL of liquid for dilution purposes.
• 😀 Alternate dilution methods, like using 99 mL dilution bottles, can also be applied for serial dilution.
• 😀 To calculate total dilution in any tube, multiply the dilution of the transfer with the dilution factor of the previous tube.
• 😀 Aseptic technique is critical throughout the process to avoid contamination, including flaming the mouths of tubes and using sterile pipettes.
• 😀 After creating the dilutions, the samples are plated on agar, which is incubated to allow bacterial colonies to grow.
• 😀 Plates with 30-300 colonies are ideal for counting, as they provide a statistically reliable estimate of bacterial concentration.
• 😀 Results are reported as Colony Forming Units (CFUs) per milliliter or gram, not as individual bacterial cells.
• 😀 To calculate the bacterial concentration, the number of colonies is multiplied by the reciprocal of the dilution factor (e.g., 89 colonies at 10^-5 dilution = 8.9 × 10^6 CFUs per gram).

Q & A

What is the purpose of using the serial dilution technique in microbiology?

The serial dilution technique is used to quantify the number of bacteria in a sample. By progressively diluting the sample, it ensures that the bacteria can be counted and used to estimate the concentration of viable bacteria in the original sample.

How is a dilution series typically made in the procedure described?

A dilution series is made by adding a measured volume or weight of the sample to a volume of sterile water, progressively making the solution less concentrated with each step. For example, a one-to-nine dilution results in a tenfold decrease in concentration for each step.

What does the notation '10 to the negative 1' represent in dilution calculations?

'10 to the negative 1' represents a 1:10 dilution, meaning one part sample is added to nine parts of sterile water, resulting in a solution that is one-tenth as concentrated as the original.

How is the total dilution calculated when transferring from one tube to another?

To calculate the total dilution, multiply the dilution of the transfer by the dilution in the previous tube. For example, if you transfer from a 10^-1 dilution to a 10^-2 dilution, the total dilution in the second tube is 10^-2.

Why is it important to calculate the total dilution in each dilution tube?

It is important to calculate the total dilution in each tube to accurately determine the concentration of bacteria in the original sample and ensure the results from counting colonies are correct.

What technique is used to mix the contents of the dilution tubes?

The contents of the dilution tubes are mixed either with a vortex mixer or by flicking the tube vigorously with the index finger, ensuring proper mixing without contamination.

Why is it necessary to use aseptic technique when making serial dilutions?

Aseptic technique is necessary to prevent contamination of the samples, ensuring that the bacteria being counted are only those from the original sample and not from external sources.

How are bacterial colonies counted after incubation?

Bacterial colonies are counted on plates where the number of colonies is between 30 and 300. Plates with too many colonies are marked as 'TNTC' (too numerous to count), while those with fewer than 30 are marked as 'TFTC' (too few to count).

What is the significance of counting 'colony forming units' (CFUs)?

Colony forming units (CFUs) are used to report the number of viable bacteria in a sample, as some bacteria may remain clumped together and form a single colony, making it impossible to count individual cells.

How do you calculate the number of bacteria per gram or milliliter in the original sample?

To calculate the number of bacteria, multiply the number of colonies counted on the plate by the reciprocal of the dilution factor for that plate. For example, if there are 89 colonies at a 10^-5 dilution, the result is 89 × 10^5 CFU per gram.