dont even know where to start You prepared a 10-fold dilution of a culture out to 10-9. You initially estimated there were between 3 x 108 cells/ml and 5 x 109 cells/ml in the original cell suspension. Which tube dilutions would you plate to ensure you captured significant counts on plates? What volumes would you use to inoculate the plates? How do the tube and plate dilutions compare in your procedure?
To determine the appropriate tube dilutions and plate volumes, we need to consider the dilution factor and the cell concentrations mentioned in the question.
First, let's calculate the dilution factor for the 10-fold dilution. A 10-fold dilution means that for each step, the concentration is reduced by a factor of 10. Therefore, the dilution factor for a 10-fold dilution is 10. For a 10-fold dilution repeated 9 times, we multiply the individual dilution factors together: 10 x 10 x 10 x 10 x 10 x 10 x 10 x 10 x 10 = 10^9 (10 to the power of 9).
Next, we need to determine which tube dilutions to plate in order to capture significant counts on the plates. Since the initial estimated cell concentration ranges from 3 x 10^8 cells/ml to 5 x 10^9 cells/ml, we want to find tube dilutions that would give us a cell concentration range suitable for counting on the plates.
To ensure significant counts on the plates, we generally aim for a range of 30-300 colonies per plate. Let's assume we are using standard Petri dishes with a volume of 20 ml.
1. Lowest tube dilution: We want at least 30 cells to be plated, so we need a volume that will result in 30 colonies in the case of the smallest estimated cell concentration (3 x 10^8 cells/ml). We can calculate the volume required using the formula:
(30 colonies) / (3 x 10^8 cells/ml) = volume in ml
For simplicity, we will round the cell concentration to 3 x 10^8 cells/ml.
(30 colonies) / (3 x 10^8 cells/ml) = 0.1 ml
Therefore, for the lowest tube dilution, we should use 0.1 ml.
2. Highest tube dilution: Similarly, we want to ensure not more than 300 colonies on the plate, so we should calculate the volume required for the highest estimated cell concentration (5 x 10^9 cells/ml). Again, we will round the cell concentration to 5 x 10^9 cells/ml.
(300 colonies) / (5 x 10^9 cells/ml) = 0.06 ml
Therefore, for the highest tube dilution, we should use 0.06 ml.
Comparing the tube and plate dilutions:
The tube dilutions are carried out to obtain a suitable concentration range that can be plated on the plates. These dilutions help in reducing the concentration of cells to a manageable range for counting. In this case, the 10-fold dilution is carried out nine times, resulting in a final dilution of 10^-9.
The plate dilutions, on the other hand, involve inoculating the diluted samples from the tubes onto agar plates to allow the growth of colonies. The volume used for inoculation determines the number of cells placed on the plate and subsequently the number of colonies that will grow.
Therefore, the tube dilutions help in obtaining a suitable concentration range, while the plate dilutions involve inoculating the appropriate volume to achieve the desired colony count on the plates.