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Critiquing An Experiment

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					Critiquing an Experiment:
(Teacher Information)

Ordering information
Brom Cresol Purple: Wards Catalog # 944 W 6300
100x15mm Plates: Wards Catalog #18 W 7101
500mL Bottletop Vacuum/Filter Unit (Requires you have a vacuum pump): Wards
Catalog # 36W5541

Pre-Lab preparation

Prepare Brom-Cresol Purple (BCP) Plates
These plates contain a pH indicator called BCP. BCP is purple at neutral pH and yellow
at acidic pH. BCP is insoluble in water, so although I keep it in a 10% solution, it is vital
to shake up the solution before taking an aliquot.

Stock solutions
10% BCP: 1g BCP in 10mL sterile water
12.5% Filter Sterilized Glucose: 12.5g per 100mL sterile water, filter sterilized (do NOT
autoclave)

Instructions (makes about 40 100x15mm plates)
    1. Combine the following
           a. 200L 10% BCP
           b. 10g Nutrient Agar
           c. Add water to 300mL
    2. Autoclave
    3. Add 200mL 12.5% filter sterilized glucose
    4. Pour about 10-12 mL per 100mm plate


Autoclave toothpicks
(you can also use any kind of sterile stick, such as the stick end of a cotton tipped swab
[Wards 14 W 5503])

Instructions (makes about 40 100x15mm plates)
    1. Wrap the toothpicks in aluminum foil and autoclave as you would any dry object

Laboratory Activity Preparation

For each group, have the following:
   1 BCP plate
   sterile toothpicks




University of Rochester, Life Sciences Learning Center
                           Student Handouts




University of Rochester, Life Sciences Learning Center
                               The Science Fair Project

You are in a science club, and one of your club members, Tom, has decided that he
wants to do a science fair project that tests the ability of four different drinks to decay
tooth enamel. He has found this protocol online from the Minnesota Dental Association.

        http://www.mndental.org/classroom/grades_7_12/
        Materials include: Several small glass containers with covers, teeth
        (usually available from an oral surgeon, a general or pediatric dentist or
        from the students themselves), and a sampling of soft drinks (include a
        diet soda in your sample).

        1. Pour the pop into the glass containers. (Use one container per brand.)
        Place one tooth in each glass container. Spit in the pop (to add the
        bacteria required for the decay process). Label with brand name and date
        and close the containers.

        2. After about six weeks, the teeth placed in sugar pop will be stained,
        blackened, decayed or broken. If there were food particles on the teeth,
        there may be some fungus-like buildup. The teeth placed in diet pop may
        be white or chalky in appearance, the result of decalcification and loss of
        minerals (due to the acid attack). You should be able to take a dental
        scaler (or a knife) and scrape off some of the decalcified tooth enamel.
        Staining may also be evident.

Tom wants to test four different kinds of soda and asks four club members to each spit
into a different cup, then adds a different soda to each cup. Another club member,
Sarah, mentions that she thinks the spit should all be from one person. Tom likes his
protocol, and doesn’t see why they should be the same. He tells Sarah that everyone
has the same bacteria in their mouths, so it shouldn’t make a difference. Do you think
Tom is right? How might you test Tom’s hypothesis (that everyone has the same kinds
and amounts of bacteria in their mouths)?




University of Rochester, Life Sciences Learning Center
Information that could help you design your experiment:
You know that the bacteria in teeth metabolize sugar and produce acid as a waste
product. You have agar plates that contain nutrients, sugar, and a chemical called BCP
(Brom Cresol Purple). BCP is purple at neutral pH (pH=7) and turns yellow when the
pH goes below 5. You can scrape bacteria off of your teeth, or swab the inside of your
mouth to see what kinds of bacteria are there. Here is how you can put the bacteria
onto the BCP plate so that you will be able to see single colonies of bacteria. A colony
is a group of bacteria that has grown from a single bacterium.




Scrape the surface of your tooth with a sterile toothpick
   1. Figure 1: Gently spread the bacteria on the toothpick onto the BCP plate by
      drawing a line with your toothpick. There should be so many bacteria here, that
      this line should end up growing as a “lawn” of bacteria (a lot of different bacteria
      growing together)
   2. Figure 2: Take a clean sterile toothpick, and draw through your first line and
      spread the bacteria as shown above. This takes some of the bacteria from the
      first line, and spreads it out a little more.
   3. Figure 3: Repeat the previous step, but this time draw a line through the second
      line.

What you might see the next day is this:




                                           The white is bacteria. White circles
                                           are single colonies of bacteria.




University of Rochester, Life Sciences Learning Center
When you see single colonies, you can often get an idea for what kind of bacteria they
are by looking at their morphology (how the bacterial colony looks)

        Colony Morphology of bacteria commonly found in the mouth
        Adapted from http://dentistry.ouhsc.edu/intranet-
        Web/Courses/DMI_8351/Exercise_4.html

        S. mutans -- pinpoint to medium size, wavy margin, circular to irregular
        shape, rough shiny appearance (ground glass), raised to cushion shape or
        irregular pile. If sucrose is in the media there is a watery pool of glucan
        around the colony. The colony is soft to the touch.

        S sanguis -- pinpoint to medium, circular, entire to irregular margin, shiny,
        smooth. May have a very thin edge of moisture resembling dextran.
        Colonies are very hard and rubbery to the touch of an inoculating needle.

        S. salivarius – Looks like a gum-drop. Large, circular, cushion shaped to
        highly raised. Soft to the touch.

        S. oralis -- small to medium, circular. May be rubbery or soft to the touch.
        Flat to slightly raised colonies.

Knowing all this, how might you set up a controlled experiment to test Tom’s hypothesis
that everyone has the same kinds and amounts of bacteria in their mouths?




University of Rochester, Life Sciences Learning Center

				
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