To gain intuition for the very large numbers of bacterial in a culture.
To review the technique of serial dilution for counting bacteria.
- Electronic Pipettor
- Pasteur Pipette
- Vortex Mixer
- 1.5 mL Eppendorf Tubes
- Disposable Tips
- Electronic Balance
- 50 mL Falcon Tubes
- Kim Wipes
- Petri Dish
Plain yogurt has just two ingredients: milk and bacteria. If you look at a yogurt label, you’ll often see something like “contains live yogurt cultures” and sometimes even the name of the species of bacteria, but it’s rare that you’ll see how many bacteria are in yogurt.
Yogurt can contain up to several hundred billion bacterial colony forming units, or CFUs, per serving. A CFU is basically a live bacterial cell that can grow and multiply into a colony if you put it onto a petri dish.
If we wanted to count how many bacteria were in this yogurt, counting up to 100 billion colonies would take a really long time. To estimate the number of colony forming units we have, we need to first dilute it to get a more manageable number of cells.
Let’s say that in one gram of yogurt, we have 10 million CFUs. If we want to have around 10 colonies instead of 10 million on a petri dish, we would need to dilute that one gram of yogurt 1 million times. To do this we could take that gram and mix it with 1000 liters of water. Instead of wasting so much water, we’re instead going to do serial dilutions until we get the concentration that we want.
Instead of taking a very small volume and diluting it in a huge volume, serial dilutions multiply together many smaller dilutions. Starting with a high concentration, you first dilute 1:10. For example, by mixing 1 milliliter of your solution and 9 milliliters of water for a total volume of 10 milliliters. If you take 1 milliliter of that and then mix it with 9 milliliters of water again, then you have 10•10, or a hundred-fold dilution. If you do that six times you have a one in a million dilution that you had wanted but with much less water.
First, let’s weigh out 1 gram of yogurt and mix it with 10 milliliters of water. That’s our first 1:10 dilution.
Next, we can take 100 microliters of this dilution and mix it with 900 microliters of water. That makes a total volume of 1 milliliter and dilutes our solution 1:10. That’s 10 times 10 and a final dilution of 1:100. Next we take 100 microliters of this solution and mix it with 900 microliters of water, to give us 1:1,000.
We keep doing this until we get 1:10,000, 1:100,000, 1:1,000,000, and 1:10,000,000.
Once we have our dilutions, we want to plate 100 microliters of each dilution onto our tomato milk agar plates. Let’s approximate that we have somewhere between 100,000 and 100,000,000 CFUs per gram. Because we don’t know for sure what our concentration is, we’ll plate a range of dilutions so we can make sure that at least one plate has an easily countable number of cells.
After a few days in the incubator, we can count how many colonies we have on each plate and estimate the total number of CFUs per gram in the original yogurt.
Since we only plated 100 microliters, first we multiply the number of colonies we have times 10. Then we multiply times the dilution factor. If we count 50 cells on the 100,000-fold dilution plate then we estimate that we have 500 times 105 or 5 times 107 – about 50,000,000 cells per gram. In the notes and exercises that go with this video you can try out some different calculations based on dilution factors.
That’s all for now, happy diluting!
Optional Links and Further Reading