How to Perform a Four-Fold Serial Dilution and Calculate Bacterial Counts
A serial dilution is a technique used to reduce the concentration of a solution by adding more solvent. It is often used in microbiology to estimate the number of bacteria in a sample by plating diluted samples on agar plates and counting the colonies that grow. A four-fold serial dilution means that each dilution step reduces the concentration by a factor of four.
In this article, we will explain how to perform a four-fold serial dilution and how to calculate the bacterial counts from the agar plates using the standard formula.
Steps to Perform a Four-Fold Serial Dilution
Prepare a stock solution of bacteria in a suitable liquid medium, such as nutrient broth. Label it as 1/1.
Prepare four empty tubes or flasks and label them as 1/4, 1/16, 1/64, and 1/256.
Add 3 mL of sterile water or saline to each of the four tubes or flasks.
Using a sterile pipette, transfer 1 mL of the stock solution (1/1) to the first tube or flask (1/4) and mix well. This is the first dilution step.
Using a new sterile pipette, transfer 1 mL of the first dilution (1/4) to the second tube or flask (1/16) and mix well. This is the second dilution step.
Repeat this process for the third and fourth dilution steps, transferring 1 mL of the previous dilution to the next tube or flask and mixing well.
You have now completed a four-fold serial dilution with five solutions: 1/1, 1/4, 1/16, 1/64, and 1/256.
Steps to Calculate Bacterial Counts from Agar Plates
Prepare agar plates with a suitable medium for bacterial growth, such as nutrient agar.
Using a sterile loop or spreader, inoculate each agar plate with 0.1 mL of one of the diluted solutions. Label each plate with the corresponding dilution factor.
Incubate the plates at an appropriate temperature and time for bacterial growth.
After incubation, count the number of colonies on each plate. Choose a plate that has between 30 and 300 colonies for accuracy. If none of the plates has this range, use the plate with the closest number of colonies.
Use the standard formula to calculate the bacterial count per mL of the original stock solution:Bacterial count per mL = (number of colonies x total dilution factor) / amount platedThe total dilution factor is the product of all the individual dilution factors up to the plate used for counting. The amount plated is usually 0.1 mL.
For example, if you counted 120 colonies on the plate inoculated with 0.1 mL of the 1/64 dilution, then:Bacterial count per mL = (120 x 64) / 0.1 = 76,800This means that there are 76,800 bacteria per mL in the original stock solution (1/1).
You have now learned how to perform a four-fold serial dilution and how to calculate bacterial counts from agar plates using the standard formula. You can use this technique to estimate the number of bacteria in different samples or to test their sensitivity to antibiotics or other agents. aa16f39245