E. coli Testing Virtual Lab by 6t592n

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INTERACTIVITY OF THE EXERCISE TO WORK
PROPERLY. TO CHANGE TO “SLIDE SHOW” MODE
YOU CAN CLICK ON “VIEW” AT THE TOP OF THE
PAGE AND SELECT “SLIDE SHOW” FROM THE PULL
DOWN MENU. YOU CAN ALSO JUST HIT THE “F5”
KEY.




                              Instructor Terry Wiseth
                                                                                   Click on the blackboard
                                                              Agar Plates
                                                                                   to view a larger board for
Antiseptic Dispenser                         Swabs                    Loops        discussion. You may
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                   also link directly to the
                                                                                   experiment you would
                                                                                   like to perform by using
                                 Bunsen burner




                         Incubator


                                                                                   the links below.
                         37 0C




            Total and Fecal Coliform                                                      E. Coli Detection
               Count Virtual Lab                                                             Virtual Lab
                                       COLIFORM BACTERIA
                                  Agar Plates


Bacterial contamination cannot be detected by sight,
smell or taste. The only way to know if a water supply
pH = 11    pH = 5   pH = 3


contains bacteria is to have it tested. The Environmental
Protection Agency (EPA) requires that all public water
suppliers regularly test for coliform bacteria and deliver
water that meets the EPA standards.

 Public water systems are required to deliver safe and
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 reliable drinking water to their customers 24 hours a day,
      0                          0
-10 C       0 C                  35 C       50 C    0
                                                      100 C     0             0
 365 days a year. If the water supply becomes
 contaminated, consumers can become seriously ill.
 Fortunately, public water systems take many steps to
 ensure that the public has safe, reliable drinking water.
 One of the most important steps is to regularly test the
 water for coliform bacteria.
Pathogens are relatively scarce in water, making them
                                  Agar Plates
difficult and time-consuming to monitor directly. Instead,
fecal coliform levels are monitored, because of the
pH = 11    pH = 5   pH = 3
correlation between fecal coliform counts and the
probability of contracting a disease from the water.

 Coliform bacteria are organisms that are present in the
 environment and in the feces of all warm-blooded
 animals and humans. Coliform bacteria will not likely
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 cause illness. However, their presence in drinking water
 indicates that disease-causing organisms (pathogens)C
-10 C 0
            0 C                  0
                                35 C      50 C      0
                                                     100        0             0


 could be in the water system. Most pathogens that can
 contaminate water supplies come from the feces of
 humans or animals.
Bacterial contamination cannot be detected by sight,
                            Agar Plates
smell or taste. Testing drinking water for all possible
pathogens is complex, time-consuming, and expensive.
pH = 11   pH = 5   pH = 3
It is relatively easy and inexpensive to test for coliform
bacteria. If coliform bacteria are found in a water sample,
water system operators work to find the source of
contamination and restore safe drinking water.

 Fecal Coliform
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 Fecal coliform bacteria are a group of bacteria
 that occur in large numbers in the gut and feces
 of 0C            other
-10 humans and 0 0C mammals. They enter          35 0C       50 0C       100 0C
 streams and lakes with sewage or wastes. Most
 fecal coliforms are normal inhabitants of the
 digestive tract and considered relatively
 harmless. In fact, their absence can lead to
 some types of vitamin deficiencies in humans.
 However, their detection in water may indicate
 the presence of more harmful microorganisms
 found in feces. E. coli is the major species of
 the fecal coliform group.
           TYPES OF TESTS FOR COLIFORMS
                                  Agar Plates


 There are different groups of coliform bacteria. Each has
 a different level of risk. The name "coliform" is given to a
pH = 11    pH = 5   pH = 3


 whole group of bacteria which can occur in water and
 indicate potential health problems. They are divided into
 two groups: Total coliform (TC), which are all of the
 coliform bacteria, and fecal coliforms (FC), which are a
 subset of the TC. The fecal coliforms are considered
 much more serious from a hygiene viewpoint. Positive
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 tests for the presence of TC, and more particularly FC,
      0                          0
-10 C       0 C                                     0           0             0
                                  35 C        water. Total C
 indicate serious hygiene safety issues in 50 C          100

 coliform, fecal coliform, and E. coli are all indicators of
 drinking water quality.
TOTAL COLIFORM                    Agar Plates

 Total coliform (TC) bacteria are commonly found in the
 environment (e.g., soil or vegetation) and are generally
pH = 11    pH = 5   pH = 3

 harmless. Total coliforms include a wide variety of
 bacteria including Escherichia coli and a broad spectrum
 of other bacteria. These other bacteria are able to grow
 frequently in the intestine, but can also grow to a
 variable extent in the environment. The TC count does
 not necessarily relate specifically to fecal pollution.
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 Since the count is dominated by the bacteria which can
 occur in the intestine, it is used as a broader spectrumC
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                                  35 C       50 C   0
                                                         100    0             0



 test for fecal pollution in a water sample.
FECAL COLIFORM                    Agar Plates

 Fecal coliforms are a subset of Total coliform. Fecal
 coliforms (FC) are types of total coliform that mostly
pH = 11    pH = 5   pH = 3

 exist in feces. Fecal coliforms are pretty specialized
 types of bacteria and are dominated by Escherichia coli
 also known as E. coli. This bacterium thrives in the
 healthy human intestine and passes out in high numbers
 in the fecal material. What is important to remember is
 that the presence of Fecal coliform is a widely accepted
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 indicator of the potential pollution of water with fecal
 material. If0 Fecal Coliform is present, then there is a100 C
-10 C 0
               C                 0
                                  35 C       50 C   0           0             0



 much greater risk of infectious microorganisms
 occurring in numbers large enough to cause an infection
 to break out if the water is consumed. These organisms
 include viruses, other bacteria, protozoa and a variety of
 worms.
ESCHERICHIA COLI                  Agar Plates

E. coli is a subset of fecal coliform. Escherichia coli are
common in the human intestine, but it is not usually
pH = 11    pH = 5   pH = 3

harmful. (However, there are some strains which can
cause infections.) The presence of E. coli in a drinking
water sample almost always indicates recent fecal
contamination. E. coli outbreaks receive much media
coverage. Most outbreaks have been caused by a
specific strain of E. coli bacteria known as E. coli
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O157:H7.
-10 0C                        0 0C              35 0C       50 0C       100 0C
 The maximum standard for fecal coliform bacteria in
                                   Agar Plates
 drinking water is zero (or no) total coliform per 100 ml of
 water. If fecal coliform counts are high (over 200
pH = 11    pH = 5    pH = 3
 colonies / 100 ml of water sample), there is a greater
 chance that pathogenic organisms are also present. A
 person swimming in such waters has a greater chance of
 getting sick from swallowing disease-causing
 organisms, or from pathogens entering the body through
 cuts in the skin, the nose, mouth, or the ears. Diseases
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 and illness such as typhoid fever, hepatitis,
 gastroenteritis, dysentery, and35 C infections can be100 C
-10 C 0
             0 C                  ear
                                  0
                                             50 C                   0               0                  0


 contracted in waters with high fecal coliform counts.
 Coliform standards of bacteria per 100 ml for various
 water uses are listed in the table below.
                    Fecal and Total Coliform Standards for Water
                                      Coliform Standards (bacteria (colonies) / 100 ml
            Drinking Water                               0 Fecal Count      1 Total Count
            Total body contact (Swimming)                200 Fecal Count    2000 Total Count
            Partial body contact (Boating)               1000 Fecal Count   10,000 Total Count
     TOTAL COLIFORM AND FECAL COLIFORM TEST
                                  Agar Plates

In this exercise you will analyze different water samples
for bacteria, and, in particular, an indicator group of
pH = 11    pH = 5   pH = 3

bacteria called the coliforms. Although you will be
looking at the total counts of bacteria in the water
samples, the coliform bacteria are the critical test
organisms which are found in improperly treated water
or water that has fecal contamination.
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-10 0C                        0 0C              35 0C       50 0C       100 0C
 This test tends to indicate only the bacteria which grow
 rapidly at higher temperatures (30 – 40 0C) in 48 hours.
                                  Agar Plates




 These bacteria are more likely to have originated from
pH = 11    pH = 5   pH = 3
 warm blooded animals (including humans) rather than
 from the environment where temperatures are commonly
 at less than 30 0C. In this test, the sample is exposed to
 the surface of the agar. The agar is to too rich in
 nutrients and the incubation temperatures are too high,
 for many of the environmental bacteria to survive. All
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 colonies are counted and weighted by the dilution factor
 to give the0 bacterial population per ml. 50 C
-10 C 0
              C                  0
                                   35 C             0
                                                       100 C    0             0


 Each colony is presumed to have been formed from one
 bacterial unit to form a colony forming unit which grows
 distinctly within or upon the agar. There is an indicator
 dye in the agar gel that colors the colonies. Total
 coliform counts are determined by both the red and blue
 colonies. Fecal coliform counts are determined by only
 the blue colonies.
COLIFORM PLATE COUNT
                      violet
The pH indicatorAgar Plates red is incorporated into
the gel, along with bile which inhibits non-
coliform bacteria. Violet red indicates the
presence of acid forming bacteria. Most coliform
pH = 11 pH = 5 pH = 3


bacteria produce acids, thus the coliform colonies
will show the red dye. In addition, there is an
indicator of the enzyme glucuronidase which will
turn blue if the bacterium makes the
enzyme. Most fecal coliform bacteria make
glucuronidase and those colonies will be
blue. Non-fecal coliforms will be red colonies.
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 Our next step is to perform a coliform plate count. Click
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                               35 C       50 C  0
                                                     100 C  0             0


 on the NEXT button below to perform the exercise.
To perform this exercise use the link shown
                                  Agar Plates


below for “Total and Fecal Coliform Count”.
You will be required to fill out Table 1 with your
pH = 11    pH = 5   pH = 3



experiment results and perform sketches of
each of the agar plates with the individual
water samples. You can access these by using
the links given below.
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    Total and Fecal Coliform C
      0                          0
                                                               Table 1
-10 C      0 C             35                       0
                                                               50 C0
                                                                          100    0
                                                                                  C
                                                            Total and Fecal
       Count Virtual Lab                                    Coliform Count

                                     Sketches                Questions
                         Total and Fecal                    Total and Fecal
                         Coliform Count                     Coliform Count
Total and Fecal Coliform Count
The water samples to be tested are as follows:
          Treated Faucet Water
                        Agar Plates


          Untreated River Water
          Boiled Untreated River Water
pH = 11   pH = 5 pH = 3
          Filtered River Water
          Raw Sewage
          Treated Sewage

The Treated Faucet Water has been treated by your local Water Department and
collected from the water faucet.

               River
The UntreatedRefrigerator Water was collected from the local river.
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 The Boiled Untreated River Water was collected from the local river and then
 boiled
-10 0C for 30 seconds.
               0 0C                        35 0C           50 0C           100 0C

Filtered River Water is Untreated River Water collected from the local river that
has been processed through a carbon filter.

Raw Sewage has been collected from the sewage pipe leading from the houses in
the city to the lagoons.

Treated Sewage has been treated by the local Sewage Department and then
dumped into the river.
Procedure                         Agar Plates

 **For this experiment we will be testing our water
 samples for the presence of coliforms and fecal
pH = 11    pH = 5   pH = 3

 coliforms. Use the procedure listed below to perform this
 exercise.
      1) Obtain six Coliform Test agar plates and label
      them with the codes for the different water samples
      to be tested.
      2) Use an eyedropper to add 1 ml of each of the water
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      samples to the surface of each of the respective agar
-10 C plates. C
      0
             0                   0
                                  35 C       50 C   0
                                                        100 C   0             0



      3) Incubate the inoculated agar plates for 48 hours at
      40 0C.
      4) Count the red and the blue colonies in the agar
      plate as total coliforms and only the blue colonies as
      fecal coliforms and record in Table 1. Indicate the
      total coliforms (red + blue colonies) and the fecal
      coliforms (blue colonies only).
                                                                                    Click on the Water Samples test tube
                                                                                    rack to bring the samples to the table.
                                                              Agar Plates
                                                                                    Next click on the Agar Plates to bring
                                                                                    six of them to the table to be labeled.
Antiseptic Dispenser                         Swabs                      Loops
                                                                                    Label the number codes of each of the
                                                                                    water samples on each of the agar
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                    plates by clicking on the pencil. Click on
                                                                                    NEXT when the agar plates have been
                                 Bunsen burner
                                                                                    labeled.           1) Treated Faucet Water
                         Incubator
                                                                                                         2) Untreated River Water
                                                                                                         3) Boiled Untreated River Water
                                                                                                         4) Filtered River Water
                         40 0C                                                                           5) Raw Sewage
                                                                                                         6) Treated Sewage



                                                                                6
                                                                    5
                                                       4
                                         3
                           2                                                               6         4                 2
                 1
                                                                                                                                  1
                                                                                                               3
                                                                                                5
                                                                                    Click on the blue eye-droppers to
                                                              Agar Plates
                                                                                    transfer 1 ml of each of the water
                                                                                    samples to their respective agar
Antiseptic Dispenser                         Swabs                      Loops       plates. When the plates have
                                                                                    been inoculated click on NEXT.
                 Water Samples         Pencil        Lactose Broth Culture Tubes




                                 Bunsen burner

                                                                                                      1) Treated Faucet Water
                         Incubator
                                                                                                      2) Untreated River Water
                                                                                                      3) Boiled Untreated River Water
                                                                                                      4) Filtered River Water
                         40 0C                                                                        5) Raw Sewage
                                                                                                      6) Treated Sewage



                                                                                6
                                                                    5
                                                       4
                                         3
                           2                                                             6        4                 2
                 1
                                                                                                                               1
                                                                                                            3
                                                                                              5
                                                                                    Click on the incubator to place
                                                              Agar Plates
                                                                                    the inoculated plates into the
                                                                                    incubator. We will incubate for 48
Antiseptic Dispenser                         Swabs                      Loops       hours at 40 0C. At this point we
                                                                                    will assume that 48 hours has
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                    transpired, so you can click
                                 Bunsen burner
                                                                                    NEXT once the plates are
                                                                                    secured in the incubator.
                         Incubator




                         40 0C


                                                                                         1
                                                                                         1
                                                                                         1
                                                                                6        1
                                                                    5
                                                       4                                 1
                                         3
                           2                                                             6
                                                                                         1        4         2
                 1
                                                                                                                  1
                                                                                                       3
                                                                                              5
                                                                                    Click on the incubator to place
                                                              Agar Plates
                                                                                    the inoculated plates on to the
                                                                                    table. We will need to view each
Antiseptic Dispenser                         Swabs                      Loops       of the inoculated plates to make
                                                                                    our total coliform and fecal
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                    coliform counts. Click on NEXT
                                 Bunsen burner
                                                                                    to start viewing the plates.
                         Incubator
                            1
                            1
                            1
                            1
                            1
                            1

                         40 0C

                                                                                         1
                                                                                         1
                                                                                         1
                                                                                         2
                                                                                6        1
                                                                                         3
                                                                    5                    1
                                                       4                                 4
                                         3                                               5
                                                                                         1
                           2                                                             6
                 1
                                                                                    Click on each of the incubated and
                                                                                    inoculated plates on the table to view
                                                              Agar Plates
                                                                                    the colonies. Record the total coliform
                                                                                    count (Red + Blue colonies) and the
Antiseptic Dispenser                         Swabs                      Loops
                                                                                    fecal coliform count (Blue colonies only)
                                                                                    in Table 1. You will also need to provide
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                    a sketch for each of the plates
                                                                                    indicating the colony growth.
                                 Bunsen burner

                                                                                                        1) Treated Faucet Water
                         Incubator
                                                                                                        2) Untreated River Water
                                                                                                        3) Boiled Untreated River Water
                                                                                                        4) Filtered River Water
                         40 0C                                                                          5) Raw Sewage
                                                                                                        6) Treated Sewage



                                                                                6
                                                                    5
                                                       4
                                         3
   Click Here if you have
                 1
                           2                                                               6        4                 2

                                                                                                                                 1
   viewed all of the plates                                                                     5
                                                                                                              3
                                                                                    Click on each of the incubated and
                                        1                                           inoculated plates on the table to view
                                                              Agar Plates
                                                                                    the colonies. Record the total coliform
                                                                                    count (Red + Blue colonies) and the
Antiseptic Dispenser                         Swabs                      Loops
                                                                                    fecal coliform count (Blue colonies only)
                                                                                    in Table 1. You will also need to provide
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                    a sketch for each of the plates
                                                                                    indicating the colony growth.
                                 Bunsen burner

                                                                                                        1) Treated Faucet Water
                         Incubator
                                                                                                        2) Untreated River Water
                                                                                                        3) Boiled Untreated River Water
                                                                                                        4) Filtered River Water
                         40 0C                                                                          5) Raw Sewage
                                                                                                        6) Treated Sewage



                                                                                6
                                                                    5
                                                       4
                                         3
   Click Here if you have
                 1
                           2                                                               6        4                 2

                                                                                                                                 1
   viewed all of the plates                                                                     5
                                                                                                              3
                                        2
                                                                                    Click on each of the incubated and
                                                                                    inoculated plates on the table to view
                                                              Agar Plates
                                                                                    the colonies. Record the total coliform
                                                                                    count (Red + Blue colonies) and the
Antiseptic Dispenser                         Swabs                      Loops
                                                                                    fecal coliform count (Blue colonies only)
                                                                                    in Table 1. You will also need to provide
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                    a sketch for each of the plates
                                                                                    indicating the colony growth.
                                 Bunsen burner

                                                                                                        1) Treated Faucet Water
                         Incubator
                                                                                                        2) Untreated River Water
                                                                                                        3) Boiled Untreated River Water
                                                                                                        4) Filtered River Water
                         40 0C                                                                          5) Raw Sewage
                                                                                                        6) Treated Sewage



                                                                                6
                                                                    5
                                                       4
                                         3
   Click Here if you have
                 1
                           2                                                               6        4                 2

                                                                                                                                 1
   viewed all of the plates                                                                     5
                                                                                                              3
                                                                                    Click on each of the incubated and
                                             3
                                                                                    inoculated plates on the table to view
                                                              Agar Plates
                                                                                    the colonies. Record the total coliform
                                                                                    count (Red + Blue colonies) and the
Antiseptic Dispenser                         Swabs                      Loops
                                                                                    fecal coliform count (Blue colonies only)
                                                                                    in Table 1. You will also need to provide
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                    a sketch for each of the plates
                                                                                    indicating the colony growth.
                                 Bunsen burner

                                                                                                        1) Treated Faucet Water
                         Incubator
                                                                                                        2) Untreated River Water
                                                                                                        3) Boiled Untreated River Water
                                                                                                        4) Filtered River Water
                         40 0C                                                                          5) Raw Sewage
                                                                                                        6) Treated Sewage



                                                                                6
                                                                    5
                                                       4
                                         3
   Click Here if you have
                 1
                           2                                                               6        4                 2

                                                                                                                                 1
   viewed all of the plates                                                                     5
                                                                                                              3
                                                                                    Click on each of the incubated and
                                        4
                                                                                    inoculated plates on the table to view
                                                              Agar Plates
                                                                                    the colonies. Record the total coliform
                                                                                    count (Red + Blue colonies) and the
Antiseptic Dispenser                         Swabs                      Loops
                                                                                    fecal coliform count (Blue colonies only)
                                                                                    in Table 1. You will also need to provide
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                    a sketch for each of the plates
                                                                                    indicating the colony growth.
                                 Bunsen burner

                                                                                                        1) Treated Faucet Water
                         Incubator
                                                                                                        2) Untreated River Water
                                                                                                        3) Boiled Untreated River Water
                                                                                                        4) Filtered River Water
                         40 0C                                                                          5) Raw Sewage
                                                                                                        6) Treated Sewage



                                                                                6
                                                                    5
                                                       4
                                         3
   Click Here if you have
                 1
                           2                                                               6        4                 2

                                                                                                                                 1
   viewed all of the plates                                                                     5
                                                                                                              3
                                                                                    Click on each of the incubated and
                                       5
                                                                                    inoculated plates on the table to view
                                                              Agar Plates
                                                                                    the colonies. Record the total coliform
                                                                                    count (Red + Blue colonies) and the
Antiseptic Dispenser                         Swabs                      Loops
                                                                                    fecal coliform count (Blue colonies only)
                                                                                    in Table 1. You will also need to provide
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                    a sketch for each of the plates
                                                                                    indicating the colony growth.
                                 Bunsen burner

                                                                                                        1) Treated Faucet Water
                         Incubator
                                                                                                        2) Untreated River Water
                                                                                                        3) Boiled Untreated River Water
                                                                                                        4) Filtered River Water
                         40 0C                                                                          5) Raw Sewage
                                                                                                        6) Treated Sewage



                                                                                6
                                                                    5
                                                       4
                                         3
   Click Here if you have
                 1
                           2                                                               6        4                 2

                                                                                                                                 1
   viewed all of the plates                                                                     5
                                                                                                              3
                                             6
                                                                                    Click on each of the incubated and
                                                                                    inoculated plates on the table to view
                                                              Agar Plates
                                                                                    the colonies. Record the total coliform
                                                                                    count (Red + Blue colonies) and the
Antiseptic Dispenser                         Swabs                      Loops
                                                                                    fecal coliform count (Blue colonies only)
                                                                                    in Table 1. You will also need to provide
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                    a sketch for each of the plates
                                                                                    indicating the colony growth.
                                 Bunsen burner

                                                                                                        1) Treated Faucet Water
                         Incubator
                                                                                                        2) Untreated River Water
                                                                                                        3) Boiled Untreated River Water
                                                                                                        4) Filtered River Water
                         40 0C                                                                          5) Raw Sewage
                                                                                                        6) Treated Sewage



                                                                                6
                                                                    5
                                                       4
                                         3
   Click Here if you have
                 1
                           2                                                               6        4                 2

                                                                                                                                 1
   viewed all of the plates                                                                     5
                                                                                                              3
        E. coli Detection


                    6
                5
            4
        3
    2
1
                                  Agar Plates
                                                E.coli DETECTION
 E. coli has the rather unique ability to ferment the sugar
 lactose to produce acid and gas products. So if you find
pH = 11    pH = 5   pH = 3

 lactose-fermenting bacteria in a sample the chances are
 high that it is E. coli and that the material is
 contaminated with feces. Because of these
 characteristics E. coli was chosen as the indicator of
 fecal pollution of water. All bacteria are like bags of
 enzymes and they selectively use different foods,
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 particularly sugars. One of the favorite sugars for the
 coliform group is lactose. Strangely, that 50 Cthe main C
-10 C 0
            0 C                  0
                                   35 C       is         100 0           0             0



 sugar in mother's milk and, therefore, the first one that
 the bacteria in the baby's intestine come across after the
 baby has been born and is suckling the milk.
Bile is pretty lethal to bacteria, but the E. coli bacteria
                                  Agar Plates
can tolerate it. Therefore, it was found that a soup rich in
lactose and bile encouraged the E. coli to grow while
pH = 11    pH = 5   pH = 3
discouraging competition (bacteria that cannot use
lactose or tolerate the bile salts). Not only that, but the
E. coli produce quantities of gas when feeding on the
lactose.

 It is possible to use this gas presence to detect E. coli.
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 For example, if you took 100 ml of water and found that
 gas
-10 C was produced from 2 ml sample of water when theC
      0
            0 C                  0
                                 35 C       50 C    0
                                                       100      0             0


 lactose bile broth was added, but none was produced
 from 1 ml, then that would suggest that the water
 contained 50 coliforms per 100 ml (ie. enough for just 1
 cell in every 2 ml of water).
Bacteria can make the water go cloudy, taste peculiar,
                                  Agar Plates
generate odd odors and the population can harbor some
potentially dangerous organisms to human health.
pH = 11    pH = 5   pH = 3
Traditionally, the coliform test has been used to
determine whether there are any dangerous bacteria
present, since the coliforms are thought to be a good
indicator of fecal pollution.

 The test for presence of E. coli involves adding a portion
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 of the water sample to lactose broth tubes which contain
 small inverted tubes for trapping any gas50 C
-10 C 0
            0 C                  0
                                35 C        formed. 100 C
                                                    0           0             0


 Following a 24 hour at 37 0C incubation, inverted tubes
 that contain at least 10% gas are determined to test
 positive for E. coli.
                                  Agar Plates   On the left is a filtered water sample that
                                                did not contain any E. coli. On the right is
                                                a non-filtered water sample which is
pH = 11    pH = 5   pH = 3
                                                cloudy and has a gas bubble in the inner
                                                tube indicating E. coli were present in the
                                                unfiltered water.




 Freezer                     Refrigerator                Incubator     Incubator     Incubator




-10 0C                        0 0C                      35 0C          50 0C         100 0C




A Chinese man wades
through water polluted with
fecal matters in China's
Shandong province.
The water samples to be tested are as follows:
          Treated Faucet Water
                        Agar Plates


          Untreated River Water
          Boiled Untreated River Water
pH = 11   pH = 5 pH = 3
          Filtered River Water
          Raw Sewage
          Treated Sewage

The Treated Faucet Water has been treated by your local Water Department and
collected from the water faucet.

               River
The UntreatedRefrigerator Water was collected from the local river.
Freezer                                         Incubator        Incubator   Incubator




 The Boiled Untreated River Water was collected from the local river and then
 boiled
-10 0C for 30 seconds.
               0 0C                        35 0C           50 0C           100 0C

Filtered River Water is Untreated River Water collected from the local river that
has been processed through a carbon filter.

Raw Sewage has been collected from the sewage pipe leading from the houses in
the city to the lagoons.

Treated Sewage has been treated by the local Sewage Department and then
dumped into the river.
PROCEDURE
                                  Agar Plates
For this exercise we will be testing our water samples for
the presence of E. coli. We will perform the detection
pH = 11    pH = 5   pH = 3
experiment by using the procedure listed below.

           1) Obtain six lactose - bile broth tubes and label them
           with the codes for the various water samples to be
           tested.
 Freezer                     Refrigerator       Incubator   Incubator   Incubator


           2) Use an eyedropper to add 1 ml of each of the water
-10 0C     samples to their respective broth tubes.
                 0 C             0
                                     35 C       50 C0
                                                           100 C0             0




           3) Incubate at 37 0C for only 24 hours.

           4) Determine which samples have the required gas
           content in the inverted tubes to be considered a
           positive test for E. coli. Indicate the positive or
           negative tests in Table 2.
To perform this exercise use the link shown
                                  Agar Plates


below for “E. coli Detection Virtual Lab”. You
will be required to fill out Table 2 with your
pH = 11    pH = 5   pH = 3



experiment results and perform sketches of
each of the broth tubes with the individual
water samples. You can access these by using
the links given below.
 Freezer                     Refrigerator       Incubator       Incubator   Incubator




-10 0C
                       coli
                    E. 0 C Detection
                                 0
                                                35 0C           Table 2
                                                                50 C0
                                                                            100 0C
                       Virtual Lab                          E. coli Detection


                                     Sketches                 Questions
                                E. coli Broth               Total and Fecal
                               Culture Tubes                Coliform Count
                                                                                    Click on the Water Sample to
                                                              Agar Plates
                                                                                    bring the rack of samples to the
                                                                                    table. Next click on the Lactose
Antiseptic Dispenser                         Swabs                      Loops       Broth Culture Tubes to bring this
                                                                                    rack to the table. Click on NEXT
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                    when both of the test tube racks
                                 Bunsen burner
                                                                                    are on the table.
                         Incubator




                         37 0C




                                                                                6                               6
                                                                    5                                       5
                                                       4                                                4
                                         3                                                          3
                           2                                                                    2
                 1                                                                          1
                                                                                    Click on the blue eye-droppers to
                                                              Agar Plates
                                                                                    transfer 1 ml of each of the water
                                                                                    samples to their respective
Antiseptic Dispenser                         Swabs                      Loops       lactose broth culture tubes.
                                                                                    When all of the broth culture
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                    tubes have been inoculated click
                                 Bunsen burner
                                                                                    on NEXT.
                         Incubator




                         37 0C




                                                                                6                                6
                                                                    5                                        5
                                                       4                                                 4
                                         3                                                           3
                           2                                                                     2
                 1                                                                           1
                                                                                   Click on the incubator to place
                                                              Agar Plates
                                                                                   the inoculated plates into the
                                                                                   incubator. We will incubate for 24
Antiseptic Dispenser                         Swabs                    Loops        hours at 37 0C. At this point we
                                                                                   will assume that 24 hours has
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                   transpired, so you can click
                                 Bunsen burner
                                                                                   NEXT once the plates are
                                                                                   secured in the incubator.
                         Incubator




                         37 0C


  1) Treated Faucet Water
  2) Untreated River Water
  3) Boiled Untreated River Water                                              6                                6
                               5                                                                            5
                          4                                                                             4
                     3                                                                              3
  4) Filtered River Water
          1
               2
                                                                                            1
                                                                                                2
  5) Raw Sewage
  6) Treated Sewage
                                                                                   Click on the incubator to place
                                                              Agar Plates
                                                                                   the inoculated culture tubes on to
                                                                                   the table. We will need to view
Antiseptic Dispenser                         Swabs                    Loops        each of the inoculated broth
                                                                                   culture tubes to determine if
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                   there are positive tests for E. coli.
                                 Bunsen burner
                                                                                   Click on NEXT to start viewing
                                                                                   the culture tubes.
                         Incubator




                         37 0C


  1) Treated Faucet Water
  2) Untreated River Water
  3) Boiled Untreated River Water                                              6                                6
                               5                                                                            5
                          4                                                                             4
                     3                                                                              3
  4) Filtered River Water
          1
               2
                                                                                            1
                                                                                                2
  5) Raw Sewage
  6) Treated Sewage
                                                                                   Click on each of the incubated and
                                                                                   inoculated broth tubes on the table to
                                                              Agar Plates
                                                                                   view tube and determine if there is
                                                                                   sufficient (10% or more) gas collected in
Antiseptic Dispenser                         Swabs                    Loops
                                                                                   the inverted tube inside the culture tube.
                                                                                   Record your results in Table 2. You will
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                   also need to provide a sketch for each
                                                                                   of the culture tubes indicating the
                                 Bunsen burner
                                                                                   presence or absence of gas in the
                         Incubator
                                                                                   inverted tube.

                         37 0C


  1) Treated Faucet Water
  2) Untreated River Water
  3) Boiled Untreated River Water                                              6                                   6
                               5                                                                               5
                          4                                                                               4
                     3
  4) Filtered River Water
          1
               2                                                                                  2
                                                                                                      3
                                                                                              1
  5) Raw Sewage
  6) Treated Sewage



    Click Here if you have
    viewed all of the tubes
                                                                                   Click on each of the incubated and
                                                                                   inoculated broth tubes on the table to
                                                          1   Agar Plates
                                                                                   view tube and determine if there is
                                                                                   sufficient (10% or more) gas collected in
Antiseptic Dispenser                         Swabs                    Loops
                                                                                   the inverted tube inside the culture tube.
                                                          1                        Record your results in Table 2. You will
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                   also need to provide a sketch for each
                                                                                   of the culture tubes indicating the
                                 Bunsen burner
                                                                                   presence or absence of gas in the
                         Incubator
                                                                                   inverted tube.

                         37 0C


  1) Treated Faucet Water
  2) Untreated River Water
  3) Boiled Untreated River Water                                              6                                   6
                               5                                                                               5
                          4                                                                               4
                     3
  4) Filtered River Water
          1
               2                                                                                  2
                                                                                                      3
                                                                                              1
  5) Raw Sewage
  6) Treated Sewage



    Click Here if you have
    viewed all of the tubes
                                                                                   Click on each of the incubated and
                                                                                   inoculated broth tubes on the table to
                                                             2Agar Plates
                                                                                   view tube and determine if there is
                                                                                   sufficient (10% or more) gas collected in
Antiseptic Dispenser                         Swabs                    Loops
                                                                                   the inverted tube inside the culture tube.
                                                            2
                                                                                   Record your results in Table 2. You will
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                   also need to provide a sketch for each
                                                                                   of the culture tubes indicating the
                                 Bunsen burner
                                                                                   presence or absence of gas in the
                         Incubator
                                                                                   inverted tube.

                         37 0C


  1) Treated Faucet Water
  2) Untreated River Water
  3) Boiled Untreated River Water                                              6                                   6
                               5                                                                               5
                          4                                                                               4
                     3
  4) Filtered River Water
          1
               2                                                                                  2
                                                                                                      3
                                                                                              1
  5) Raw Sewage
  6) Treated Sewage



    Click Here if you have
    viewed all of the tubes
                                                                                   Click on each of the incubated and
                                                                                   inoculated broth tubes on the table to
                                                          3   Agar Plates
                                                                                   view tube and determine if there is
                                                                                   sufficient (10% or more) gas collected in
Antiseptic Dispenser                         Swabs                    Loops
                                                                                   the inverted tube inside the culture tube.
                                                           3                       Record your results in Table 2. You will
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                   also need to provide a sketch for each
                                                                                   of the culture tubes indicating the
                                 Bunsen burner
                                                                                   presence or absence of gas in the
                         Incubator
                                                                                   inverted tube.

                         37 0C


  1) Treated Faucet Water
  2) Untreated River Water
  3) Boiled Untreated River Water                                              6                                   6
                               5                                                                               5
                          4                                                                               4
                     3
  4) Filtered River Water
          1
               2                                                                                  2
                                                                                                      3
                                                                                              1
  5) Raw Sewage
  6) Treated Sewage



    Click Here if you have
    viewed all of the tubes
                                                                                   Click on each of the incubated and
                                                                                   inoculated broth tubes on the table to
                                                            4 Agar Plates
                                                                                   view tube and determine if there is
                                                                                   sufficient (10% or more) gas collected in
Antiseptic Dispenser                         Swabs                    Loops
                                                                                   the inverted tube inside the culture tube.
                                                            4                      Record your results in Table 2. You will
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                   also need to provide a sketch for each
                                                                                   of the culture tubes indicating the
                                 Bunsen burner
                                                                                   presence or absence of gas in the
                         Incubator
                                                                                   inverted tube.

                         37 0C


  1) Treated Faucet Water
  2) Untreated River Water
  3) Boiled Untreated River Water                                              6                                   6
                               5                                                                               5
                          4                                                                               4
                     3
  4) Filtered River Water
          1
               2                                                                                  2
                                                                                                      3
                                                                                              1
  5) Raw Sewage
  6) Treated Sewage



    Click Here if you have
    viewed all of the tubes
                                                                                   Click on each of the incubated and
                                                                                   inoculated broth tubes on the table to
                                                             5Agar Plates
                                                                                   view tube and determine if there is
                                                                                   sufficient (10% or more) gas collected in
Antiseptic Dispenser                         Swabs                    Loops
                                                                                   the inverted tube inside the culture tube.
                                                            5                      Record your results in Table 2. You will
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                   also need to provide a sketch for each
                                                                                   of the culture tubes indicating the
                                 Bunsen burner
                                                                                   presence or absence of gas in the
                         Incubator
                                                                                   inverted tube.

                         37 0C


  1) Treated Faucet Water
  2) Untreated River Water
  3) Boiled Untreated River Water                                              6                                   6
                               5                                                                               5
                          4                                                                               4
                     3
  4) Filtered River Water
          1
               2                                                                                  2
                                                                                                      3
                                                                                              1
  5) Raw Sewage
  6) Treated Sewage



    Click Here if you have
    viewed all of the tubes
                                                                                   Click on each of the incubated and
                                                                                   inoculated broth tubes on the table to
                                                            6 Agar Plates
                                                                                   view tube and determine if there is
                                                                                   sufficient (10% or more) gas collected in
Antiseptic Dispenser                         Swabs                    Loops
                                                                                   the inverted tube inside the culture tube.
                                                            6
                                                                                   Record your results in Table 2. You will
                 Water Samples         Pencil        Lactose Broth Culture Tubes
                                                                                   also need to provide a sketch for each
                                                                                   of the culture tubes indicating the
                                 Bunsen burner
                                                                                   presence or absence of gas in the
                         Incubator
                                                                                   inverted tube.

                         37 0C


  1) Treated Faucet Water
  2) Untreated River Water
  3) Boiled Untreated River Water                                              6                                   6
                               5                                                                               5
                          4                                                                               4
                     3
  4) Filtered River Water
          1
               2                                                                                  2
                                                                                                      3
                                                                                              1
  5) Raw Sewage
  6) Treated Sewage



    Click Here if you have
    viewed all of the tubes
1) Treated Faucet Water
2) Untreated River Water
3) Boiled Untreated River Water
4) Filtered River Water
5) Raw Sewage                                                      5
                                                                       6
                                                               4
6) Treated Sewage                                      2
                                                           3
                                                   1




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