BIO 32- Environmental Science Lab
Raskoff
Water Quality Testing
Purpose
In this lab you will use various tools to learn how to measure a large number of
commonly tracked biological and physical parameters involved with water quality.
Materials
pH meter
Thermometer (built in to DO probes)
Pasco unit w/ turbidity sensor
Pasco (2) with conductivity meters,
Pasco temperature probes (2)
Conductivity meter
Distilled water wash
Dissolved oxygen meters (YSI)
Beakers, bottles, etc for H2O collection
Hach kits for Nitrate and Phosphate
Fecal Coliform kits and plates
Lab Laptop computers
Background
In this lab we will investigate many of the most commonly used methods for monitoring
and examining water quality. We will use some of the techniques and tools we worked
with in the first lab of this class, and some new ones that you’ll find to be pretty
interesting.
We will look at both the live aspects of the water (the biotic) and the non-living, physical
properties if the water (or the abiotic).
Sampling location
Our location of study will be the catch basin pond on the west side of campus, between
the entrance to MPC and the car wash at the corner of Iris Canyon Road and Fremont
Street. The pond, known as Via Miranda pond serves an important function as it captures
and collected the water which flows through several different streams around this area of
Monterey before it flows into the larger El Estero Lake. As a result, a lot of things can
concentrate in this small area and therefore it is especially vulnerable to pollution and
toxins.
MPC
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Experimental design
We will break into groups which will sample two different areas of the pond (the
location). Each area will be sampled at the top of the water and close to the bottom (the
tests), and each of these tests will be done two times (the replicates), and we may also
need to do different dilutions of water for the bacterial tests.
So, numbers of samples can really add up quick! : 2(area) x 2(depths) x 2(replicates) x
2(dilutions) = 16 samples taken total for the class. Recall the importance of repeating
your measurements and data from our earlier labs. Exact instruction on the design will be
discussed when we are out at the site of the study.
Procedures
pH determination
Recall that pH, is a measure of the acidity of a solution in terms of activity of hydrogen
ions (H+). Using the pH meters we will determine the pH of the various water samples.
Salinity
Salinity, the amount of dissolved solids in a solution can be measured in several ways,
including: electrical conductance and it is usually measured in “parts per thousand (‰).”
Using the conductivity measurement devices you will record the salinity of the water
samples.
pH meter
Conductivity (salinity) meter 1
Pasco meter
Temperature
Recall that there were several different temperature measurement devices. We will use
the sensors built into the dissolved oxygen sensors to read the temperature of our water
samples.
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Dissolved Oxygen
Of all the elements that surround living things, oxygen has a very important role. Most
living organisms use oxygen to ‘burn’ their food for energy inside their cells. Like most
gasses, oxygen can be dissolved into water and O2 levels have a big impact on the things
which live in those waters. Using the old-school, but still reliable YSI dissolved oxygen
meters we will take both the temperature and the dissolved oxygen concentrations [DO]
of the water samples. Since the probes are on long water-proof cables we can also put
them deep in the water to see how DO changes with location and depth in the area.
YSI Dissolved oxygen and temperature sensor
Nutrient Test Kits
Using the two Hach Kits we will measure the levels of two very common and important
elements found in aquatic systems, nitrogen and phosphorous. It turns out that the
amount of these elements it not all we need to know, because in their pure elemental form
they are not usable to most living things, so we actually look other forms of these
elements. For nitrogen we usually look at Nitrate (NO3-), and for phosphorous we look at
phosphate (PO43−). Using the two different kits we will add chemicals to the water
samples to cause a color change that is in proportion to the amount of these compounds.
Then using a small colorimeter we will measure the amount of color and therefore the
amount of these nutrients. Exact directions for each kit and the tests are in the kits.
Follow the instruction exactly.
Hach Test Kit
Pasco Turbidity meter
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Turbidity Testing
Turbidity is a cloudiness or haziness of water (or other fluid) caused by individual
particles (suspended solids) that are generally invisible to the naked eye, thus being much
like smoke in air. Using the Pasco Explore GLX multimeter, sample the turbidity of your
water samples. This is done using a tool called a nephelometer, which is a simplified
spectrophotometer that shoots a beam of light through the sample to see how much passes
through and how much is reflected. The word nephelometer comes from the Greek word
for cloud, nephos. Carefully follow the directions on the laminated card for each sample.
Fecal Coliform Bacteria
We will be analyzing water samples for the presence of fecal coliform bacteria.
According to US Environmental Protection Agency (USEPA) drinking water standards,
the number of fecal coliform bacterial colonies allowable in “safe” drinking water is set
at no more than 1 colony per 100 mL of water.
There are several established methods for detecting and enumerating the number of
coliform colonies in a water sample. We will be using the membrane filtration method.
Water samples will be filtered through a filtration membrane. Then, the membrane will
be incubated on mFC growth medium, which is selective for fecal coliform bacteria.
After the bacteria have grown for 24 hours, we will count the coliform colonies.
NOTE: It is imperative that all testing be done in a safe manner. Gloves must be worn
while collecting water samples and while conducting lab work on samples.
Steps:
1. Wearing gloves and using a sterile sample bottle, collect water from your designated
site at each depth twice (for the replicates). Cap sample bottle tightly and make sure it is
carefully labeled.
2. With your instructor’s help, figure out what volume/s of sample you will be using to
inoculate your mFC plate (ex: 0.01mL, 0.1mL, 1.0mL, 10mL, etc.) You may have more
than one water sample to filter. If this is the case, you will process your samples one at a
time. Record the volume of sample we decide on so you know what volume to use the
next time we test.
3. Obtain a mFC plate and label it with the date, your names, your site location, and the
sample size that you will use to inoculate your plate. Take the red tube of mFC growth
media and place all the liquid in the plate.
4. You will vacuum filter your water sample through a sterile filtration setup using the
following procedure:
a. Make sure that the sterile filtration unit is attached securely to the vacuum.
b. Check to make sure that your instructor has placed a sterile 0.45 µm filter
inside the filtration device. If you need to place one on the plate, use one of
the sterile-wrapped filters.
c. Using sterile technique (which we will demonstrate), pour some sterile water
up to the neck, then pour your water sample from the sample bottle into the
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d.
filter reservoir and swirl the filtration unit to mix up your sample. ** Pour
some sterile water, then your sample, then up to neck of filter unit with more
sterile water.
Turn on the vacuum to filter your water sample through. Once your water
sample has flowed through the filter unit, turn off the vacuum before the filter
is totally dry. Release the vacuum hose then put it back onto the vacuum
nozzle. This will make removing your filter much easier.
Filter, media, and filter plate.
Bacteria filtering setup
5. Once the vacuum is off and you have your labeled mFC plate, you will
transfer your 0.45 µm filter from the filtration unit to your labeled plate. This is a
little tricky. Your instructor will demonstrate.
a. First, you will sterilize a filter forceps by dipping it about 1.5” deep into 95%
ethanol and holding it over a flame. (NOTE: Do not place the hot forceps
back into the alcohol!) Do not set the forceps on the bench top after they have
been sterilized until you have finished transferring the filter! Hold the forceps
while they cool – don’t touch the tip – it is hot! **Make sure your hair,
clothing, jewelry, etc. are not dangling near the flame. Do not leave the flame
unattended. One person in your group should watch the flame – turn it off
when you are done.
b. Once the forceps have stopped flaming, unclamp the filter setup and pick up
the edge of the filter with the forceps.
c.
Remove and hold the lid of the mFC plate while you transfer the filter face up
to the center of the plate. To avoid air pockets, lay the filter down from one
edge to the other edge. You want to do this quickly, but carefully.
d. Once you have the 0.45 µm filter in the mFC plate, replace the lid on the plate.
Set it on the shelf in the 37˚C incubator to incubate for about 24 hours.
e. Repeat entire process for other samples. Rinse with DI water before starting
the next sample.
6.
Clean up your work area and dispose of any waste as directed!
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PROCEDURE – Day 2
After 24 hours, you will count the colonies that have grown on your mFC plates. The
cells with a silver metallic sheen or that are very dark red started from single cells of
fecal coliform species that have grown and multiplied into separate colonies on your
incubating plate. The colonies that are just light red are not from fecal coliform species.
Example Fecal Coliform plate.
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Data Sheet
Location sampled _________________________ Bottom Depth ___________meters
pH
Replicate 1
Replicate 2
Replicate 3
Average
Surface
Bottom
Salinity
Surface
Bottom
Temperature
Surface
Bottom
Dissolved Oxygen
Surface
Bottom
Nitrates
Surface
Bottom
Phosphates
Surface
Bottom
Turbidity
Surface
Bottom
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Fecal Coliform Bacteria Counts
SAMPLE
#
WATER SAMPLING
LOCATION
DATE
SAMPLE
COLLECTED
TIME SAMPLE
COLLECTED
SAMPLE
#
VOLUME
COLONIES
FILTERED PER PLATE
# COLONIES/
100 ML
SAMPLE
Assignment
1. Using internet resources and local agencies, look up nitrate, phosphate, turbidity,
and fecal Coliform safe limits. You can use state or federal standards.
2. Explain why these are often measured and tracked in aquatic systems. In other
words, what about them are of concern for public and ecosystem health?
3. Start thinking about what sort of question and hypothesis you might have about
how all these biological and physical parameters might vary in different systems
and areas. Your group will be coming up with a plan for a water quality testing
from beginning to end. You’ll design the experiments, carry them out, and report
the results to the class. Start thinking about what you find interesting now so that
you can meet with your groups soon and have ideas to kick around.
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