Question

1:10 dilution steps (0.1 mL added to 0.9 mL at each step) filtrate 0.1 ml 0.1 mL 0.1 mL + host + STA P-20 plate X plate Y plate z volume transfer to experiment

A microbiology student was carrying out an independent research project in which he needed to know the number of infectious phage particles added during a particular step in an experimental protocol. In preparation for the experiment, he passed a crude mixture of phage plus infected bacteria through a 0.45 micrometer filter and collected the filtrate (portrayed below in the green-capped Eppendorf tube):

Once the filtrate was prepared, the student used the P-20 shown in the photograph to transfer a volume from the green-capped tube to begin his experiment.

Next, he removed a volume of 0.1 mL from the green-capped tube and serially diluted the filtrate as shown in the diagram. Three plaque assay plates were prepared by mixing 0.1-mL volumes from three dilution tubes with host bacteria plus molten soft top agar (STA), then pouring a thin layer onto bottom agar for plates designated "X", "Y", and "Z".

After incubation, the countable plate in his plaque assay was plate "Z" which had 72 plaques.

Based on the results from his plaque assay, the student now knows how many infectious particles were transferred from the filtrate when he used the P-20 set at "120" to initiate his experiment. Express this answer in the two blanks below using good scientific notation:

Answer 1

From the picture of P20, which is a 20 uL micropippette, the reading set is 120, which means that the student used 12 uL for the experiment.

From the original tube, the student has performed 4 serial dilutions of 1 / 10 (by taking 0.1 mL in a total volume of 1 mL: 0.1 / 1 = 1 / 10).

Now let us write down the dilution of each tube:

Green capped tube: Original culture

Tube 1: 1 / 10

Tube 2 (plate X) : 1 / 10 * 1 / 10 = 1 / 100 or 10^-2 dilution.

Tube 3 (plate Y) : 1 / 10 * 1 / 10 * 1 / 10 = 1 / 1000 or 10^-3 dilution.

Tube 4 (plate Z) : 1 /10 * 1 / 10 * 1/10 * 1/10 = 1 / 10000 or 10^-4 dilution.

Number of plaques obtained in plate Z = 72.

volume plated = 0.1 mL.

From the data obtained for plate Z, the concentration of infectious particles in the original culture can be calculated using the formula:

PFU / mL = No. of plaques * DF / volume plated

where DF is the dilution factor. DF is the inverse of the dilution used for plating. Since the dilution used for plating is 10^-4, which means that the culture is diluted 10⁴ times, the DF here would be 10⁴.

PFU / mL = 72 * 10⁴ / 0.1 = 720 * 10⁴ = 7.20 * 10⁶.

In order to represent the values in scientific notation, the representation should have a single digit whole number and the decimals are counted and added up as exponents of 10. In this case, 720 is represented as 7.20, the number of decimals is 2, which is added up as exponents to the existing 10⁴ as 10⁴⁺² = 10⁶.  

Therefore the concentration of phage particles in the green cap tube is 7.2 * 10⁶ PFU / mL.

Now, the student has used 12 uL or 12 / 1000 = 0.012 mL of the above culture for his experiment. The number of infectious particles used for the experiment is :

7.2 * 10⁶ * 0.012 = 0.0864 * 10⁶ = 8.64 * 10⁴ PFU / 12 uL.

In the above scientific notation, the decimals are converted in to one whole number (8) and remaining digits as decimals, for which we needed to shift two decimal places from the left, therefore we need to minus the value in this case: 0.0864 is represented as 8.64, the number of decimals "deducted" here is 2 and so this would be subtracted from the existing exponent of 10: 8.64 * 10^6-2 = 8.64 * 10⁴.