Question

A plaque assay is performed beginning with \(1 \mathrm{mL}\) of a solution containing bacteriophages. This solution is serially diluted three times by combining 0.1 mL of each sequential dilution with \(9.9 \mathrm{mL}\) of liquid medium. Then 0.1 mL of the final dilution is plated in the plaque assay and yields 17 plaques. What is the initial density of bacteriophages in the original 1 mL?

Short answer

Answer: The initial density of bacteriophages in the original 1 mL solution is 170,000 plaques/mL.

Step-by-step solution

Calculate the dilution factor for each step

In each dilution step, we are combining 0.1 mL of the previous solution with 9.9 mL of liquid medium. The dilution factor can be calculated by dividing the final volume of the mixture by the volume of the starting solution: Dilution factor = \(\frac{9.9 \,\mathrm{mL} + 0.1\, \mathrm{mL}}{0.1\, \mathrm{mL}} = 10\) Since the solution is diluted three times serially, we must take the cube of the dilution factor: Total dilution factor = \(10^3 = 1000\)

Calculate the final concentration of bacteriophages from the plaques

We are given that 0.1 mL of the final dilution yields 17 plaques. To calculate the concentration of bacteriophages in the final dilution, we need to divide the number of plaques by the volume plated: Final concentration of bacteriophages = \(\frac{17 \,\mathrm{plaques}}{0.1\, \mathrm{mL}} = 170 \,\mathrm{plaques/mL}\)

Calculate the initial concentration of bacteriophages

Now, we can multiply the final concentration by the total dilution factor to get the initial concentration of bacteriophages in the original 1 mL solution: Initial concentration of bacteriophages = \(170 \,\mathrm{plaques/mL} \times 1000 = 170000 \,\mathrm{plaques/mL}\) Thus, the initial density of bacteriophages in the original 1 mL solution is \(170,000 \,\mathrm{plaques/mL}\).

Key concepts

Serial Dilution

Serial dilution is a critical technique used in microbiology to estimate the concentration of organisms, such as bacteriophages, in a sample. It involves the step-by-step dilution of a substance into a solvent, usually in a tenfold ratio, which makes it easier to quantify organisms that are too concentrated to measure accurately.

When performing a serial dilution, one takes a small portion of the original solution and adds it to a new solvent to create a diluted mixture. This process is repeated multiple times, each time using a new portion from the previously diluted mixture. This stepwise approach exponentially decreases the concentration of the substance. Typically, each step involves diluting the sample by the same factor, commonly a factor of 10.

Application in Plaque Assays

In plaque assays, serial dilutions are used to reach a concentration that will result in a countable number of plaques. This is essential because if there are too many plaques, they will merge, making an accurate count impossible.

Bacteriophage Density

Bacteriophage density refers to the number of bacteriophage particles present in a given volume of medium. It is a critical factor in virology studies and various microbiological assays, like plaque assays.

A plaque assay is a method used to measure virus concentration in a sample by counting the number of plaques formed on a host bacterial culture. These plaques are essentially areas where virus particles have infected and lysed host cells. The key to a successful plaque assay is to achieve a dilution that offers well-separated and countable plaques.

Importance of Accurate Density Measurement

Accurately determining bacteriophage density allows researchers to study viral infectivity, replication, and other characteristics. Uncovering these properties is essential for developing treatments and understanding bacteriophage behavior.

Concentration Calculation

Concentration calculation is a mathematical operation used to determine the amount of a substance within a specified volume of solution. In microbiology, it allows researchers to estimate the number of microbial entities, such as bacteriophages, in a sample.

The concentration is usually expressed in units of entities per milliliter (e.g., plaques per mL). To calculate the initial concentration from a plaque assay, one can use the number of plaques formed, the volume in which the bacteriophages were plated, and the dilution factors applied during the serial dilution process.

Utilizing the Dilution Factors

In the exercise provided, the key to finding the concentration was determining the total dilution factor and then using it alongside the count of plaques obtained from the assay. By combining both, the initial bacteriophage density could be calculated, which provides vital information for understanding and manipulating phage concentrations in various applications.