Science Buddies: "Ask an Expert"

Dear Science Buddies Expert,

I am replicating an experiment in which researchers inoculated sterile water with E.coli, then poured the contaminated water into 2.0 liter copper pots and glass jars (controls). After 16 hours, the researchers removed 100 micro liters of the contaminated water from each container, plated it, and found out how much bacteria was still present. I plan to "make this experiment my own" by finding out what the bacterial levels are after 4 hours, 8 hours, and 12 hours.

I have read the procedure of the original experiment, but I cannot understand what the researchers did to get the E.coli into the sterilized water. I have looked at other Science Buddies experiments that deal with E.coli, but none have been helpful. I know that I can purchase E.coli, but I have no idea how much to purchase and how to inoculate the water. The original experiment states that the culture "was serially diluted in normal saline (NaCl 0.85%) for inoculation in water." Then, "the sterilized water was inoculated to ~500 colony-forming units with the serially diluted culture."

Does what I describe make sense? I am grateful for any guidance you can provide. I had planned to do this experiment at our local university with help from a professor who mentored me on another project. Unfortunately, the university changed its procedures and doesn't allow minors to do research in any of its labs anymore. I was able to find a college that would supply me with agar plates (at a cost), and access to an incubator.

Thank you very much for your time.

Hi there,

Thanks for checking out Science Buddies.

Inoculation of a water sample is usually done by simply putting the E.coli in! It sounds overly simple, and that's partly because it is. But I'll go into further detail below.

Before that, though, it's worth mentioning that if it's hard to get access to an incubator, you can also find a place in your home (or elsewhere) with constant, elevated temperature. It doesn't have to be exactly 37ºC, and it doesn't have to have all the fancy CO2 and moisture controls that a fully stocked lab's incubator might have. Of course, it will add to your reproducibility and precision, but if it's too much trouble, understand that it is a part of your procedure you can afford to sacrifice.

I'm assuming you have already figured out a way to find out what the bacterial levels are from a water sample. In that case, I suggest you also add a "0 hours" timepoint to your data. It's important to record how much bacteria you started with, because that could be varying between samples. (And later, in statistical analysis, you can take percent increase/decrease from this initial measurement rather than raw numbers.)

Anyways! Regarding inoculation. If you have a way to record initial (0-hours) bacterial levels, then that makes it even easier to inoculate, since you don't have to worry about how much you're putting in.

Here's how I've done it in a university lab - you should amend the steps to fit your project specifically:
1) Take the E.coli sample and streak it on an agar plate. (For streaking info: https://www.sciencebuddies.org/science- ... plates.pdf , or look up your own resources) You don't need an "inoculating loop"; you can use a cotton-tipped applicator, as long as it's sterile, i.e. comes in its own individually wrapped package.
2) Incubate the plate for a day or two (it could take longer, or shorter. Depends on incubation conditions and sometimes, just luck.)
3) You should see colonies of your bacteria formed on your agar plate. I believe for E.coli they should be pale white spots. If you have multiple colors or shapes, your plate was contaminated...
4) You now want to scrape a little bit of those E.coli colonies and put them into your water. When I say "scrape," I mean extremely gently. Don't dig into the agar at all; literally all you have to do is to make sure you get some bacteria on the tip of your scraping tool. You could probably even do it just by touching the tip to the colony a few times. Now, as for your tool, I've always used a micropipette tip, but that's just something we have a lot of in labs. You could use a toothpick or something similar...something sterile is best, but maybe not always feasible.
5) Dip your tool into the water.
6) That's pretty much it. Your water has now been inoculated.

"Serial dilution" just means the researchers decreased the bacteria concentration in their water to a really low level, probably to allow room for more growth. The second part about ~500 units is their recorded "initial" or "0-hours" bacterial levels. Again, if you record your initial bacteria concentration, you should be covered.

I hope that helps. I'm happy to answer more questions in detail!

Dear Science Buddies Expert,

I am very grateful for your quick reply and information! Thank you. After reading your explanation, I realize that I did not ask the right question.

I have found a place to do my research. This college has an incubator, autoclave, and spectrometer (along with supplies like agar plates and sterile pipettes that I can purchase). The lab supervisor is very kind and will provide the e.coli. She will be culturing it in LB broth and incubating it for me.

For this project, I want to inoculate sterilized water with about 500 colony-forming units/mL of e.coli. This is the amount that the researchers of the project that I am replicating used. If I have a flask filled with e.coli in broth, I was thinking that I need to first figure out how much total bacteria is in the flask by plating the bacteria. I would use a variety of serial dilutions (1:10 dilutions) to end up with one plate with an appropriate number of colonies (between 30 and 300). If I know the total bacteria in the flask, could I then dilute that until I have a solution that is about 500 cfu/mL?

From your explanation I now understand how to get the bacteria into the water (yes, that sounds very simple!). But how do I know it is about 500 cfu/mL of bacteria that I am putting into the sterilized water?

Does this make sense, or do I sound hopelessly confused? Your guidance once again will be appreciated. Thank you very much.

It's good to hear that your project is coming along! Let me just jump right into the questions.

First of all, let's define "colony-forming units" (cfu). As far as I can tell, that pretty much refers to the raw number of colonies observed; going off of my earlier response, you would literally count the number of pale white fuzzy spots on your petri dish in step 3).

How do we convert this into cfu/mL, which is a measure of concentration? For that, you'll have to go back to whatever solution you used in step 1) to streak the plate itself. How many mLs did you use to inoculate? For a standard petri dish, I would suspect 1mL is plenty. Then all you have to do is take the cfus you counted earlier, and divide that by the number of mLs you used to get those cfus. Then you have your cfu/mL number.

Most often, this inoculation solution has been serially diluted specifically to inoculate the plate. If that is the case for you, then you simply take your calculated cfu/mL and multiply it by your dilution factor - as if to "undo" the dilution mathematically.

In summary:
- you start with a stock solution of bacteria, with X cfu/mL;
- dilute this down (with a dilution factor F) to (X/F) cfu/mL;
- use Y mL of this dilute solution to streak the plate;
- the plate shows Z cfus in total.

(X/F) = Z/Y
X = F x (X/F)

I hope that helped...writing that down in text form can be confusing, so feel free to ask additional questions.