Water Testing 3: Bacteria
Title: Water Testing – E. coli
Overview - Presentation to Students
In order to
determine the amount of E. coli
bacteria in our water sample, we are actually going to help it grow! A common way of determining the amount of E. coli in water is to place a
pre-determined amount of sample water on a special plate. The plate contains a nutritious food source
that helps the bacteria grow into colonies (visible “clumps” of bacteria). It also contains special dyes, so that as the
bacteria “eat” the food, they are also stained a bright color that helps us see
and count the colonies. Other bacteria
may grow on the plate, but the dye will only stain the type of bacteria the
plate is designed for.
All types
of bacteria have an ideal temperature at which they survive and reproduce. Some bacteria live at extreme temperatures,
much too hot or too cold for any other type of life to survive! The ideal temperature for E. coli is much closer to our own body
temperature. We will store the E. coli test close to its ideal
temperature to give it the perfect environment to grow. It takes a couple of days for the E. coli bacteria to develop into visible
colonies, so don’t forget to read your results a couple of days from now!
The plates
we are using will test for both E.coli,
and the larger Total coliform group. Remember from the introductory material that Total coliforms are found
naturally in soil, and that E.coli is part of the larger fecal coliform group,
which is part of the larger Total coliform group.
Grades: High School
Learning Objective: Test the amount of E. coli bacteria in a water sample.
Kansas Grade Level Expectations – Standards/Benchmarks
Standard 3, Benchmark 4
9-12 Benchmark 4: Learners identify and evaluate environmental issues from multiple points of view.
9-12 Indicators:
By the end of the twelfth grade, the students:
1. identify the various uses of soil and water in Kansas.
Example: Watering lawns and irrigating fields.
2. identify the risks and benefits that agriculture, petroleum production, manufacturing, energy production, human communities, and other economic development activities can have on soil and water in Kansas.
Example: Fertilizers used by agriculture and golf courses causing eutrophication of streams.
3. describe the problems that Kansans face in regard to solid and hazardous waste disposal.
Example: Finding suitable areas for solid waste disposal.
4. describe the problems that Kansans face in regard to urban growth.
Example: Good farmland is taken over by urban sprawl.
Materials
1.
Water sample
2.
3M Petrifilm EC Count Plates
3.
Sterile 1-milliliter (mL) pipettes
4.
Distilled water
5.
Incubator or warm place to store
samples
Method
1. If you have more than one water sample, use a
separate plate for each sample and be sure to label them clearly with the
sample site, the date and the time at which you process the sample. Also prepare one plate for your “blank”
sample, which will be explained in the next steps.
2. Make sure you have a sterile 1 mL
pipette for each water sample, including your blank. You should use a new pipette with each sample
- this helps us make sure we’re only growing bacteria from one sample on each
plate. When you remove the pipette from
its sterile covering, open the plastic wrapper at the bulb end. Be sure not to touch the tip or any part of
the pipette that will go into the sample.
3.
Let’s begin with the blank. We run a blank sample with distilled water to
help us make sure there aren’t any problems with our equipment or our
methods. Distilled water has no bacteria,
or any other contaminants associated with natural water. When we place distilled water on a plate and
incubate it with the rest of the samples, it should have no bacterial growth
after incubation. If bacteria grow, it
tells us that our equipment may have had bacteria on it, or we need to be more
careful with our methods.
For your blank sample, carefully fill
your pipette to the 1 mL mark with distilled water. Lift up the cover sheet on the plate, being
careful not to touch anything but the edge. Gently squeeze the water into the center of the pink circle. Then slowly
roll the top cover back down onto the sample, which will help the water spread
over the circle. Gently pick up and tilt the plate from side to side to
distribute the water evenly. Discard the used pipette.
4.
Repeat the same process with your water
sample(s). If your sample has settled,
you may gently shake or roll it to mix it up. Carefully fill your pipette to the 1 mL mark with your sample
water. Lift up the cover sheet on the
plate, being careful not to touch anything but the edge. Gently squeeze the water into the center of
the pink circle. Then slowly roll the top cover back down onto the sample,
which will help the water spread over the circle. Gently pick up and tilt the
plate from side to side to distribute the water evenly. Discard the used
pipette.
5. Repeat until you have all of your
samples processed.
6. The ideal incubation temperature for
this test is 35C (93F). At this
temperature, test plates can be counted in 48 hours (two days). If you don’t have an incubator, you can place
them in a warm spot – on top of your refrigerator for example. Be careful not to place them near a heating
vent or any place where they might reach temperatures higher than 95F, and do
not expose them to sunlight. You can
place the samples in a sealed plastic bag to keep them from drying out. If you don’t have a place to incubate the
plates at 93F, you can incubate them at 80F and read them in three days, or at
70F and read them in four days. Always
note what you did on your field sheet – where you placed them to incubate, what
temperature, and for how many days.
7. After the incubation time, you can
count the plates in a well-lit area. The
pink colonies are fecal coliform colonies and represent several different types
of bacteria that are normal and natural in nature. Count the number of pink dots. Count them at least twice to get an accurate
number. If the entire plate is covered
with colonies, you may write “tmtc” (too many to count) on your data
sheet.
8. Now count the blue dots or
colonies. These are the E.coli colonies that could be harmful to
human health if swallowed. You can
follow the same procedure as above for counting.
9. Be sure to record all of your counts
on your data sheet. The units for these
numbers are colony forming units (cfu) per 1 mL. Most water quality standards use units of
cfu/100 mL, so if you would like to compare to these standards, simply multiply
your counts by 100.
Evaluation
Record your Total coliform and E.coli ratings on your field sheet according to the charts below:
E.coli Rating
Rating
|
1 – Poor
|
2 – Fair
|
3 – Good
|
4 – Best
|
|
|
Description
|
E. coli greater
than 20 colony forming units per plate.
(Not
considered safe for non-contact recreation.)
|
E. coli between
2
and
20 colony
forming
units per
plate.
(Not safe for contact recreation, but
acceptable
for noncontact
recreation,
such as boating.)
|
E. coli detected,
but
less
than 2 colony
forming
units per
plate.
(Safe for contact
recreation,
such
as swimming.)
|
None
detected. (For drinking water, this is
the
only acceptable level.)
|
|
Comments
|
|
|
|
|
|
|
|
Total Coliform Rating
Rating
|
1 – Poor
|
2 – Fair
|
3 – Good
|
4 – Best
|
|
|
Description
|
More
than 200
colonies
or too many
to
count.
|
20
to 200 colonies per
plate.
|
Less
than 20 colonies
per
plate.
|
None
detected. (For
drinking
water, this is
the
only acceptable
level.)
|
|
Comments
|
|
|
|
|
|
Interpreting your results
When you consider your results, first determine the use of the
water you tested. If it is water that
will be used for drinking or household use, the only acceptable level for both
total coliform and E.coli is 0 cfu/1
mL. This includes groundwater (well
water) used for drinking. If bacteria,
especially fecal coliform bacteria, are present in well water, it indicates the
groundwater has been contaminated by surface runoff.
If you are testing surface water, some total coliforms are to be
expected, and simply indicate soil or sediment in the water. However, E.coli
should be 0 or close to 0. The State of
Kansas sets standards for surface water based on what the water is used
for. “Contact recreation” includes all
activities in which you might swallow some water, such as swimming. “Non-contact recreation” includes activities
in which you wouldn’t expect to swallow any water, such as boating or
fishing. You can determine from the E.coli rating chart whether your water
sample is currently safe for contact recreation, non-contact recreation, or
neither.
Notes for teachers
Store unopened Petrifilm plate pouches at temperatures below 46º F. Allow pouches to come to room temperature before opening. Return unused plates to the pouch. Prevent exposure to moisture after opening pouches. Store resealed pouches in a cool dry place for no longer than one month. Exposure of plates to temperatures above 77º F and/or humidities above 50 percent can affect performance of the plates. Do not use plates that show orange or brown discoloration. Expiration date and lot number are noted on each package of Petrifilm plates. After use, Petrifilm EC plates will contain viable bacteria. Put all used Petrifilm in a plastic bag, inside another plastic bag, and take to sanitary landfill or transfer station. Alternatively, mix a mild chlorine bleach solution (10 percent) and put one dropper full on each exposed plate to kill the bacteria before disposal.