Hey everybody, Dr. O here. This is a very important process in a microbiology lab. This is going to be called the serial dilution. So we'll tie this to a later video about pore plates and spread plates.
But if you want to work with microorganisms, again, it's so important to work with the right amount. And generally that amount is less, not more. So in the case of trying to use a plate, a nutrient auger plate, to count...
The number of colonies or colony forming units to try to determine how many bacteria are in a sample is very difficult unless you dilute it. Like as you can see here from this picture, on the far left there's just so many bacteria growing that you can't even isolate colonies. To me it kind of looks milky almost.
You can tell it's full of microbial contamination, but there is no space for them to actually make colonies. And then in the second dilution here you'd see kind of the same thing. Maybe some small colonies are forming, but they can't form full colonies.
So... There's a reason you have to dilute your samples. So the serial dilution technique, what you're going to do is we are trying to determine how many microbes are in the original sample in colony-forming units per milliliter. So here's the actual process.
Oh, and just so you know, the ultimate goal is to try to get somewhere between 30 and 300 colonies. Too few, and you just can't learn anything from it. Too many, and you have trouble counting them, and it's really hard to tell where kind of one colony stops and the next starts. So you wouldn't know how many serial dilutions to do right away. You basically have to make an estimate based on how contaminated or how much growth you think is in the original sample.
So we'll go through this entire process here, though. In the lab, we do it slightly differently because we're using much smaller samples. But this is the serial dilution technique. So you start with your original sample.
And notice you're taking one milliliter of your sample and adding it to nine milliliters of sterile nutrient agar or broth here. So that's a 1 to 10, so you're just adding a single milliliter of your original sample into this first tube. Then you're going to continue to do a series of dilutions, which is why it's called serial dilution. So you mix it up real well and real thoroughly, then you go back in tube number 1 and take out a milliliter that you're going to transfer into tube number 2. Mix it up really well, go back into tube number 2, and do the same thing again and again and again. one, two, three, four, five times here.
in this example. And then once you've done that, this would actually be the pour plate method. So once you've done that, as this agar cools, you would pour it in these plates.
And then you'd probably do more than one and you would take your plates and do averages. But just you're going to be walking through and you're going to pour all these plates and then incubate them and come back and look at them. And you'll notice the first milliliter, there's just too many microbes to even form colonies in the first sample. But then we're starting to see dilutions. So the second sample here, the 1 to 100 sample, still not very valuable.
The next one, the 1 to 1000 sample, there's still, we're over that 300 number, so this is not a great sample. So we go to the next one, which would be the 1 to 10,000 dilution sample has 50 colonies on it. And if you want, just real quickly, if you go one more time, you only see a couple colonies, so too small to work with.
So out of these five plates, the one I'm most interested in is that 1 to 10,000 dilution that has 50 colonies on it. So you do the math. So notice that when you made the plates, or whether it was a pour or spread plate, you only added 0.1 mL of that original mL that you added in.
So to do the math, you would not take 50 times 10,000. You would take 50 times 10 to get back to how many colonies came from a mL of the original sample, and then take that times 10,000. So if you wanted to know how many colony forming units were in a mL of that original sample on the left.
You would take the 50 colonies you counted, times 10 to get back to that full milliliter, and then times 10,000. There would be 5 million. Again, there's no way to count 5 million colony-forming units per milliliter, so that's why we have to use these serial dilutions. Now, you will notice you would think the one right before it then should be 500, right, if this was perfectly accurate. That's why you don't like to use plates that have more than 300 colonies on them.
So, if it was 30 colonies and 300 colonies, you'd probably see it being a lot closer. The reason it's so far off is that 1,000 plate just still has too much growth, too many microbes. You couldn't get 500 colonies to form on there. So, okay, so that is the serial dilution process.
In the next video in this series, I will talk about what we do with these samples, the pour plate versus the spread plate. So, okay, serial dilution is very important. Hope this helps.
Have a wonderful day. Be blessed.