Living in the Microbial World: How Many Microbes?

Dilution Plating Technique

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Dilution and plating of samples is a common technique used to try to approximate the number of bacteria present in a sample. We will be using samples of soil, compost, mulch, and others to try to get an idea of how numerous and diverse bacteria are in these environments. In order to count how many bacteria are in a sample, we first need to dilute the sample by a defined amount using serial dilution. Then we will grow a portion of the sample on a medium such as tryptic soy agar (TSA) or nutrient agar.

For this activity, you should work in groups of two. You will receive the following:
  • 4 plates of 1/10th strength TSA
  • 7 scintillation vials containing 9 ml of sterile 1/10th strength TSB (tryptic soy broth)
  • 6 1ml disposable, sterile graduated pipettes (Fisher #371120)
  • 4 disposable sterile fine-tip graduated pipettes (Fisher #014-003-11)
  • 4 sterile microcentrifuge tubes containing sterile 5mm glass beads (Fisher #110312C)
    1. Label the tops of vials 1-7 with a Sharpie marker.
    2. Weigh out one gram of your sample (compost, soil, mulch, leaf litter, etc.) and place it into vial #1. Shake the vial vigorously approximately 100 times.
    3. Use the sterile graduated pipette and place 1 ml of the mixture from vial 1 into vial 2. You may have to let the larger pieces of your sample settle to the bottom first.
    4. Using a new pipette, take 1 ml of liquid from vial 2 and place it in vial 3. Take a new pipette and transfer 1 ml of liquid from vial 3 into vial 4. Transfer 1 ml of liquid from vial 4 into vial 5. Continue this set of transfers, using a new pipette each time, until you have transferred 1 ml into each of the vials.
    5. Take the last four dilutions that you made (vials 4-7). We will be plating samples from each of these onto the four plates. Label the bottom of each plate with the numbers 4-7. Using the fine tip sterile pipettes, take a sample from each vial and place it onto the appropriately labeled plate. Place only 7 DROPS onto each plate. (This is tricky; you can practice by pipetting some liquid onto a paper towel). 7 drops from these pipettes are equivalent to approximately 1/10th of a milliliter (100(1). Take a small plastic tube and dump the sterile beads onto the plate—don't worry if one of the beads is stuck on the bottom of the tube. Close the plate and GENTLY swirl the bead across the surface of the agar so that they spread the liquid all over the plate. Do not swirl the plate too hard, or the beads will only go around the edge of the plate. When done, dump the beads into a beaker of bleach solution (they can be reused).
    6. Repeat for the remainder of the samples, 5-7.
    7. Tape the plates together with masking tape and return them to us. We will incubate at 30C for two days.

    Recipes (Recommended Vendors)
    1/10th strength TSB: from premixed powder (Difco 0370-17)
    • 3 g TSB
    • 1 liter wate
    1/10th strength TSA
    • 3 g premixed Tryptic Soy Broth powder (Difco 0370-17)
    • 15 g agar
    • 1 liter water
    Tryptic Soy Broth-full strength ("from scratch")
    • Tryptone 17 g/l
    • Soytone 3 g/l
    • Dextrose 2.5 g/l
    • NaCl 5.0 g/l
    • K2HPO4 2.5 g/l
    • Agar 15 g/l

    One thing to keep in mind is that when doing dilutions, we want to place the samples into a liquid that will not lyse or kill the bacteria in a sample, so we want to use a BUFFERED solution of appropriate pH and ionic strength. One of the simplest dilution fluids to use is liquid media (as we did here). You can also use 10 mM Tris buffer, 10mM phosphate buffer, or soil extract. If you are diluting marine samples, you can place them directly into sterile seawater, since this is already buffered.

    How to Count Your Plates

    After the plates have incubated and colonies have grown, pick a plate that has between 50 and 200 colonies on it. Count the number of colonies on the plate (include small colonies, but be sure to count large colonies that are composed of spreading bacteria only once). Each colony is assumed to have arisen from a single cell. After counting the number of colonies, write that number in your notebook. Note the number of the plate from which you made the count. You should add this number of zeroes to the end of the number you wrote down. Add one more zero to your total number. (**Each of these zeroes represents the factor of ten by which you diluted your original sample. Placing one gram in 9 ml represents one factor of ten dilution. Each successive transfer is another factor of ten dilution, by taking 1/10th ml of the final ml you transferred into vial 7)

    Also note the different characteristics of each colony. Colonies can be characterized based on their color, shape, consistency, and type of edge they have. These characteristics aid toward identifying the type of bacterium that made the colony. You can use your dissecting scope, or the dissecting scope with the video camera attached, to get a closer look at your colonies.

    This technique is not perfect—we will only be able to get an estimate of how many there are, and this will be a very low estimate. Why?? Once you go through the procedure, you may start to see the limitations of these techniques. (Think about the media we are using, what kind of organisms can grow on it, and what you know so far about the diversity of the microorganisms on Earth. Do you think we are culturing everything present in the sample?)