Final answer:
To calculate an end point dilution assay, perform serial dilutions of the original sample, plate samples to count colonies, and use the dilution factor for the countable range for calculations. The C1V1 = C2V2 formula, along with spectrophotometers, are used for direct concentration measurements.
Step-by-step explanation:
To calculate end point dilution assay, you start by understanding the principle of serial dilutions. Typically, a fixed volume from the original culture is mixed with a solution to create a dilution factor. A 1.0 mL sample added to 9.0 mL of sterile broth creates a 1:10 dilution. Subsequent dilutions are made by transferring 1.0 mL from the previous dilution into 9.0 mL of new solution. This process is repeated to achieve a range of dilution factors. After mixing thoroughly to ensure a random distribution, samples from each dilution are plated on a solid medium and incubated. The average number of colonies from 2-3 plates per dilution is counted. The dilution factor at which colonies are countable, typically between 30 and 300, is used as the end point for calculating the original cell concentration.
Using the formula C1V1 = C2V2, where C1 is the concentration of the original solution, V1 is the volume being transferred, C2 is the concentration of the dilution, and V2 is the final volume, concentrations at each dilution step can be calculated. With nucleic acid assays, spectrophotometers can be employed to determine concentrations directly. For example, after measuring the absorbance of a DNA sample, concentration in g/l or mg/ml can be recorded.
Understand the principle of serial dilution, and use of the dilution equation and precise measurement tools like pipettes and spectrophotometers are critical to calculations in an end point dilution assay.