One of the biological parameters that we monitor at Utah Water Watch is for E.Coli.
E.Coli is a bacteria. It's a bacteria that's found in the digestive systems
of all warm-blooded animals. So, humans, cows, pigs, sheep, your dog,
even birds, have E.Coli in them.
The test that we use is called Coliscan Easygel test to monitor for the presence
of E.Coli. There are two parts. There's a field collection, where you
collect the sample from your site and then a indoor portion of it, where you
actually do the incubation and plating of a petri dish.
So I'm going to show you how to do the outdoor field collection portion of it.
Because E.Coli is a bacteria, we have to use a sterile, sanitized container to
capture it. This is called a Whirl-Pak.
When you get yours, you will see that it has a perforation on the top.
When your out, ready to go do your monitoring, you will remove the
perforation, and then you will hold the Whirl-Pak by these white tabs.
And this is how you can open and close it.
You do not want to stick your hands or have anything go in there,
because then you could contaminate it.
So know that I've opened it, I'm going to go into the stream, into my monitoring
location, and this is where I'm going to collect my water sample from.
You're going to go into the stream. You want to collect it in the midpart of
the water column, not right at the surface and you don't want to stick it all the way
through the bottom to get a lot of sediment in there. So I will have it
closed. I'll stick it underwater. I'll open it up, let it fill up some of the
way, close it, and bring it back out of the water. Still holding it by the white
tabs. Never touch the inside of it. Now I'm going to hold it by the yellow tabs.
You wanna make sure that you've filled your water up somewhere to about the
white level. For your test, the maximum that you'll need is only 10 milliliters,
so you don't need to have it filled all the way up.
And what you will now do is you'll seal the bag, flip it over once, flip it over
twice, flip it over three times, and now you will take the yellow tabs and twist
them together, kind of like a bread basket.
So now you have it twisted, sealed.
So now the water is completely sealed.
You can see I can shake it and drop it on the ground. Doesn't happen.
It's completely sealed and sterile, I've never touched the inside of it.
If you collect this, this is the whole part that you do
for the field collection sample, if your going to do the next part
within one hour, you do not need to refrigerate this, or keep it cool.
If your going to stay out here and go some fishing and do some hiking or some other
things and your not going to get to the inside portion until after an hour, this
needs to be either stored on—in a cooler with ice or it needs to be stored in a
refrigerator at a cool temperature. You can keep your water sample for up to eight
hours until you do the next indoor plating part of it with the Easygel medium.
Now we're inside after we've collected our field sample from Temple Fork, from the
stream. So this would be your stream or lake water that you've collected at this
site. The only part of the tier one Utah Water Watch that you have to do inside.
For each sample that you collect, you will do two tests.
So you're doing a duplicate. This is one of our quality control steps.
So you will need your one bag of the water sample, you'll need two pipettes, two
petri dishes, and two of the Easygel E.Coli medium.
Now remember that your E. coli Easygel medium is frozen.
Keep it frozen until you're ready to do the test, and so when you're ready
to do the test, pull out some ones, let them thaw for about an hour, and then you
will use it as a liquid. So how we do the test. We take our bag. We wanna shake it
up to help insure it's very well-mixed, so shake it up.
Then we're going to open it again.
Open the yellow tabs first.
We're going to unroll it.
And then remember that you wanna grab the white tabs
and open it up this way, so we're still not trying
to use our fingers.
We're keeping the inside of it sterile and clean.
So now that you've opened it up, you have it open,
sometimes you can do this with one hand,
or you can have another partner hold it.
If you're doing it by yourself, you can always take it
and put it in a cup like this and it will make it easy to hold.
You'll take your first test, and you'll see on here
you have it marked with— your pipette is marked
in millimeters—or milliliters, excuse me.
So the very top line says 1.0 milliliters, for this test you need to know
how much volume you are putting in here.
So this test is designed to work to be able to place between
one to five milliliters into the Easygel medium.
This test is designed this way so that there is some flexibility.
If you're testing in an area that you would assume would be very high
in E.Coli, you could do a lower amount so there wouldn't be as many
E. coli colonies to count.
So what I do with the pipette is I take it, depress it,
stick it in the water, and then I slowly suck up water until it gets
right at the one milliliter mark.
I'll then take it, open up my thing and depress it a couple times
to get all the water out.
So that's one milliliter, I'm going to put two
milliliters in for this test.
So I'll open it up and take one milliliter
and put it—squirt it a couple of times make sure
all the water gets out, and then I'll recap it.
So, now I have my Easygel E.Coli medium mixed with the sample water.
So now I'm gonna just gently swirl it. That's all you need to do, you just need
to gently swirl it, you don't need to really shake it really hard,
just swirl it around. And now I'm going to place it in my petri dish.
Now before you place it into your petri dish, it's important that you take
good notes as a scientist, and you record on it. So I recorded the name of the creek
that I got this from: Temple Fork. Today's date of when I am doing this test.
How big my sample size was; I put two milliliters in.
And then the time that I am entering, doing this second step.
This is not the time that I collected the sample, this is the time I collected here.
I need to know this because it's important to know how long
we let it incubate for.
And then I wrote S1 for sample one, because we do two samples at a time.
So I'll just go through, and mark my other one same way.
So I'll write down the name Temple Fork. I'll write down today's date. I'll write
down I put in two milliliters because I'm gonna do the same for each one.
Alright 15:00 or 24-hours time military format, and this is my sample two.
Okay. So now that I have this in here,
taken the E.Coli Easygel medium, gently swirled it, I will open it up,
I'll open my petri dish, and I'll pour it out, and evenly move it around all across.
Make sure I get every thing in there.
Once again we wanna keep these things sterile, so I'm just opening and closing
it—that's all I'm going to do for the sample trial.
Once they become a solid gel, then your able to pick them up
and move it around.
We recommend that you do this first part of the incubation
where you're doing the plating.
We recommend that you do this where you're going to leave them.
We're now going to store our two E. coli tests petri dishes
in a warm, draft-free place out of direct sunlight.
So this test is designed to work at—in your home,
it can work at room temperatures, anywhere generally above 65°F.
These can work in there.
And they take a minimum of 48 hours to incubate
at room temperature.
The first 24 hours there's going to be very little
that you're going to see on there.
I have two ones that I've prepared beforehand so you can see that
they're now a gel. I can flip them over and it doesn't run out anywhere.
And these ones are 24 hours old, right around the
24 to 28 hour mark.
You will start to see little tiny fleckings,
and if you look on this one, up close you will see
we'll have these little formation of,
just the beginning formations of our bacteria colonies.
Once you start to see this, that means it needs to have
at least another 24 hours to completely mature.
So to do this test at room temperature, it takes a minimum of 48 hours.
If your house is towards the colder side of room temperature,
it may take longer than 48 hours.
Generally this test takes somewhere between 48 to 60 hours.
We never want to let this test to go longer than 72 hours
because after 72 hours we can start to have the bacteria
colonies just reproducing on the petri dish instead
of being an accurate sample of what it was in the water.
So how do you know when your test is ready to read?
Generally we recommend that you wait another 24 hours
after you start to see the initial flecking of your sample,
but one of the things you can look at is when you open this up
you should clearly defined colonies that have a very strong color
with a nice round circular colony.
Sometimes you will get this examples where you'll get these kind of
oblong splotches on the surface.
Those are indications also that the test has had enough time
for the colonines to mature.
So, it does take a bit of practice, but once you've done this
once or twice you'll get familiar in seeing this.
You wanna make sure that you feel confident, and that your counting
the colors correctly, so if you come and look at your test and say,
"Hmm, I'm not really 100% sure if this is mature," maybe give it another
hour and two and always come back.
Here are two samples that I've done that are 48 hours old.
And we have them in here and as you can see, they are—
they have mini colorations that are on these petri dishes.
The ones that are pink or redish in color, those are total coliforms.
We're not interested in those ones.
The ones that are dark blue or purple, those are E. colis.
So those are the ones that we are counting for our test.
Now hopefully your water coli sample that your doing will not
have as much E. coli as on this.
This is an example of a very high, elevated sample.
And so what we would next do is count up all the colonies that have
E. coli on them.
Sometimes when you have a lot of them, it's easy to place it on a white disk—
on a white piece of paper. Then you can move the paper across—
the petri dish across the paper, so when everything starts to go
and the blue darts start to go across the paper,
you can count all of them.
Slowly move it across and count all of the tests.
This test is designed to be done at home and uses a color guide
to have E. coli specially react.
All of our tier one volunteers have the color guide given to them,
so you compare your chart to the colors of the pictures of
E. coli to see if what your bacteria samples are actually E. coli.
Now that we've let our colonies, our E. coli samples, mature,
we're now ready to fill out the data sheet.
So we have two readings that we did or two duplicate samples.
The first thing that we need to write down is the sample size.
We put in two milliliters for each one.
The sample size is critically important to know
because this test is done as a dilution.
E. coli is measured as the number of
coliforming units per 100 milliliters.
Because we did not place a 100 milliliters into the bottle,
we have to do a little bit of math.
So we placed two in there, so how we do this is
100 divided by two, which is 50 then we times that by
the number of colonies that we counted.
On the first one I counted 38 E. coli—dark blue or purple colonies.
And on the second one I counted 32.
So 100 divided by two is 50.
50 times by 38 will be 1900.
For the second one it would be 50 times by 32,
which is 1600.
We then take the average of them, the average of those two numbers
is 1750, and we report that right here for our average E. coli.
The only other information that we need, we need the incubation time in hours.
We wrote down the time it starts, so you can just count up
the number of hours until you did the reading.
This provides us information about how long the E. coli cells were able
to mature.
And then just the general temperature of the room that they were in.
You have your thermometer, you can leave it out in the area
to get a general temperature.
We don't need to exactly how the temperature fluctuated
and changes throughout the day, just what the general temperature is.
For our room right here, it was at 68°F or 20°C.
So once you record all this information, this is what we need for you to report
for your complete E. coli sampling for the Utah Water Watch tier one program.
Now that we've completely completed our monitoring for E. coli,
we need to dispose of the petri dishes properly.
These are not biohazards, but these are living bacteria colony cells
of E. coli, so we wanna make sure we just properly dispose of it.
How you do that is you can just take some household bleach,
Lysol, cleaner 409, something, and just pour about a tablespoon of it
on the surface of the E. coli petri dish.
So we will do that for each one.
I'll let it sit for one minute, after its sat for one minute,
I'll open them up, I'll take them, gently place them in a ziplock
or other type of plastic bag that I can seal,
close it back up, and then I can just take this and dispose of it
in the trash.
Any household trash would be fine.