. Why we need to dilute Let's say a culture contains about a million CFUs. At 1 CFU per second, that would take 11.6 days to count. Assuming you didn't stop to eat or sleep.
Clearly a better way is needed. And clearly, this module is going to pick that method apart and pound it into the figurative whiteboard.
So let's get started already. First of all, we're going to use the Viable Plate Count method - the only method that can tell a live cell from a dead one (for a rundown on counting methods, look ).
So, we're going to take a little bit of our culture, put it on a plate, and grow it up. But we still have a problem - too many colonies to count. For example, even 500 colonies on a petri dish would look something like this: What if you are trying to count a population in the thousands or millions? You could literally have a carpet of colonies (also known as a 'confluent lawn') growing on your petri dish. This is where dilution saves the day. Not just dilution, but serial dilution.
Jun 29, 2018 A serial dilution is the repeated dilution of a solution to amplify the dilution factor quickly. It’s commonly performed in experiments requiring highly diluted solutions, such as those involving concentration curves on a logarithmic scale or when you are determining the density of bacteria. Apr 27, 2018 - You can use serial dilutions of a solution of known concentration to calibrate lab equipment and ensure its accuracy.
Meaning dilution over and over again. Why do we dilute? To have less to count Why do we do it repeatedly? Because we don't know how much dilution we need. Every time we dilute, we'll also make a new plate to incubate. So we might do 5 dilutions and grow up 5 plates. Then we'll end up throwing away 4 of them.
Sound wasteful? Well, dilution and plating is quick and easy compared to the pain of starting your experiment all over again.
To perform a serial dilution, a small amount of a well-mixed solution is transferred into a new container and additional water or other solvent is added to dilute the original solution. The diluted sample is then used as the base solution to make an additional dilution.
Doing this several times results in a range of concentrations. The initial concentration and target range needed for a given assay determines the size and number of dilution steps required. Often, serial dilutions are performed in steps of 10 or 100. They are described as ratios of the original and final concentrations.
For example, a 1:10 dilution is a mixture of one part of a solution and nine parts of additional solvent. To make a 1:100 dilution, one part of the solution is mixed with 99 parts of additional solvent. Mixing 100 µL of a stock solution with 900 µL of water makes a 1:10 dilution. The final volume of the diluted sample is 1000 µL (1 mL) and the concentration is 1/10 that of the original solution. This is commonly referred to as a 10x dilution.
The illustration above follows the relationship between volume of solvent, number of molecules of solute and concentration of a solution over a set of 4 dilutions. The concentration can be tracked in M which is a common unit for chemistry, or particles per ml, which is common when diluting bacterial cultures to low concentrations. With molar concentrations it is safe to assume that the solute is evenly dispersed through the solution such that the concentrations change predictably with each dilution.
With particles, like bacterial cells, the solution becomes patchy at low concentrations and actual concentration can diverge from the expected concentration. Test your understanding with the Video Overview Related Content.
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