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YOGURT CELL CULTURE - MOLECULAR BIOLOGY FOR HIGH SCHOOLS

By Aaron Ortiz,2014-07-04 08:38
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YOGURT CELL CULTURE - MOLECULAR BIOLOGY FOR HIGH SCHOOLS

What would Jamie Lee Curtis say?

    Question Are all yogurts created equal? Several television commercials

    lead you to believe that one particular type of yogurt will solve all of your medical woes because it is the best pro-biotic. Are there really any differences between brands? Pre-Lab Question - What is a pro-biotic? (Use your root words for help).

    Prediction What do you think the answer to the question will be? Explain your opinion. Part 1 - Isolate bacterial cultures from different yogurt brands.

    Day 1 Media Preparation

    1. Make skim milk media.

    a. Media: Weigh 20 g dehydrated nonfat milk. Place in a 250 ml graduated cylinder

    and bring the volume up to 200 ml.

    b. Pour back and forth into a 250 ml beaker to mix well.

    c. Pour 100 ml of this media into a second beaker and save the rest for your agar.

    d. Heat this second beaker to a low boil while stirring on a hot plate. Boil for 5

    minutes, cool, repeat. Be careful to not let the media boil over.

    e. Label 10 sterile culture tubes with your initials, the date and “milk media.”

    f. While the media is still warm, pipette 5 ml into each of 5 sterile culture tubes and

    15 into the other 5.

    2. Make the skim milk agar plates.

    a. Weigh 1 g of agar powder. Add the 1 g of agar to the 250 ml beaker from your

    media (which should still contain 100 ml of un-boiled milk).

    b. Boil while stirring on the hot plate for 5 min, cool slightly, repeat. Be careful to not

    let the agar boil over.

    c. While it cools, label 5 sterile petri dishes with your initials, the date and “milk

    agar.”

    d. While the media is still warm, pipette 20 ml into each of 5 sterile culture tubes.

    3. Place the media and the agar in the designated place for your class.

    4. Bring your group’s choice of yogurt to class tomorrow.

    Day 2 Grow a culture of your yogurt bacteria.

    1. You are going to inoculate one plate of your agar from a store bought yogurt culture. So

    retrieve one of your plates. Label it

    with the brand of yogurt you are

    using.

    2. Get a sterile toothpick. Dip this

    toothpick into your yogurt and

    streak a plate as shown. Do not

    poke the plate, but barely touch the

    agar as you follow the pattern.

     ?C incubator to grow overnight. 3. Place these in the 37

    BCH590 C Frame 1

Part 2: The differences between bacteria

    Day 3 Isolate a colony and grow a monoculture.

    Many brands of yogurt contain more than one kind of bacteria. We want to isolate just one type to grow.

    1. Observe your plate.

    a. Count the number of colonies and record it.

    b. Try to see if there are any differences between the colonies. Are some shiny and

    others dull? Are they different colors? Different shapes or sizes? How many of

    each type are there?

    c. Make detailed notes about the differences.

    2. Choose a colony to sample. Circle it with sharpie on the bottom outside of the plate.

    a. Retrieve one of your plates. Label it with the brand of yogurt you are using and

    the word “monoculture.”

    b. Get a sterile toothpick. Pick up part of the colony you circled, trying to leave part

    of it behind so it can continue to grow.

    c. Streak your monoculture plate the way you did yesterday.

     ?C incubator to grow overnight. d. Place this in the 37

    Day 4 Bacteria Identification and Comparison

    For steps 2-4, you do not have to do them in order, because we are sharing Gram Stain Kits.

    1. Observe your monoculture plate.

    a. Count the number of colonies and record it.

    b. All of the colonies should look very similar color, texture, etc.

    2. Retrieve one of your 15 ml media tubes.

    a. Label it with the brand of your yogurt and the word “monoculture.”

    b. Get a sterile toothpick. Pick up a colony and swirl it in the milk media.

    c. Place this in the 37 ?C incubator to grow overnight.

    3. Get a Gram Stain Kit. Follow the instructions to stain your bacteria. Make detailed

    observations about what you see under the microscope. Color, shape of cells.

    4. Make a wet-mount slide.

    a. Obtain a slide and place 1-2 drops of saline on your slide.

    b. Get a sterile toothpick. Pick up a colony and swirl it in the drop of water.

    c. Observe your slide at 4, 10, and 40x. Try to darken the background to see the

    bacteria better. You should be able to see them pretty well at 40x.

    d. Make detailed observations about what you see under the microscope. Do they

    move? How? What shape are they?

    5. You plate will be stored in the refrigerator. Tape it closed.

BCH590 C Frame 2

    Day 5 Bacteria Identification and Comparison (cont.)

    1. We will perform a serial dilution so we can estimate the number of bacteria in your

    culture.

    a. Retrieve your monoculture tube. -1-3-5-6, 2. 10, 3. 10, 4. 10 and b. Retrieve 5 tubes of 5 ml of media. Label these 1. 10-75. 10.

    c. Get a sterile plastic pipette.

    d. Mix your monoculture tube well. Use the pipette to very carefully remove 0.5 ml

    of the culture and place in Tube 1. Mix Tube 1 well by pipetting liquid in and out

    of the pipette.

    e. Very carefully remove 0.5 ml from Tube 1, but only place 1 drop in Tube 2.

    Return the rest of the liquid to Tube 1. Mix well by putting the cap on the tube

    and inverting it several times. Rinse the pipette in sterile water. f. Very carefully remove 0.5 ml from Tube 2, but only place 1 drop in Tube 3.

    Return the rest of the liquid to Tube 2. Mix well by putting the cap on the tube

    and inverting it several times. Rinse the pipette in sterile water. g. Very carefully remove 0.5 ml from Tube 3 and place it in Tube 4. Mix well by

    pipetting in and out several times.

    h. Very carefully remove 0.5 ml from Tube 4 and place it in Tube 5. Mix well by

    pipetting in and out several times. Rinse the pipette in sterile water. -6 i. Retrieve an agar plate. Draw a line across the middle. Label one side 10and -8the other side 10. -6 j. Using a rinsed pipette, draw up liquid from Tube 4 and place 1 drop on the 10-8 side. Rinse and repeat for Tube 5 on the 10side.

    k. Using a sterile toothpick spread the drop on its side of the plate. Do not cross the

    line and use a separate toothpick for each of the two drops.

    l. Place these in the 37 ?C incubator to grow overnight. Place your original culture

    in the refrigerator.

     0.5 ml

    1 drop 1 drop 0.5 ml 0.5 ml

    5 ml 5 ml 5 ml 5 ml 5 ml

    1 drop 1 drop

    BCH590 C Frame 3

Day 6 Evaluation

    1. Calculating the cell density in your overnight monoculture.

    a. Retrieve your plate and count the number of colonies on each side. Record this. -6 -7 side. For the 10side, just change the b. Use the following calculation for the 10-6 -710in the calculation to 10.

    -6 Number of colonies 10= Number of cells in 1 drop of original culture

    15 ml of original culture x 20 drops per ml = Number of drops in the culture Number of cells in 1 drop of original culture x Number of drops in the culture

    = Number of cells in the original culture.

    Write out these calculations in your notebook.

    We will make a class table of the brands of yogurt each group brought in and the various data we collected.

Data Table

Brand Colony Gram Shape Movement pH of an Number of Cells in

    Appearance Stain Overnight an Overnight Culture -6 -7Culture 10 10

Bar Graph

    Create a bar graph of the brand of yogurt (x) versus the average number of cells in the overnight culture (y).

Conclusion

    You are expected to write a concluding paragraph about your response to the original question: Are all yogurts created equal? Are there really any differences between brands?

Planning:

    You will pick an additive for your yogurt culture to apply in different amounts. This you should predict whether this additive will help (promote) or hurt (inhibit) the growth of the bacteria you isolated. For tomorrow, you need to bring your additive, have planned the amounts you will use in each 15 ml tube and have your prediction of what it will do to the growth of the bacteria.

Lab by: Cinnamon Frame

    Biology, chemistry, physics teacher, Cleveland High School

    Clayton, NC

    e-mail: cinnamonframe@johnston.k12.nc.us

    BCH590 C Frame 4

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