GOING TO EXTREMES:
AN INQUIRY INVESTIGATION INTO THE BIODIVERSITY OF EXTREMOPHILES
Background          Methods         Results        Discussion       Possible Extensions         Classroom Applications

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Old Faithful Yellowstone National Park	Black Smoker, Juan de Fuca Ridge Dr. Michael Perfit, University of Florida

    However, do we have to go to exotic locations to see extremophiles?  We cultured materials that may provide extreme microhabitats and tried to grow these organisms in enriched nutrient broth.  We cultured ham and bologna which are both high in salt, milk that has been pasteurized (possibly leaving thermophilic organisms after pasteurization), hot tap water, vinegar, skin (where sweat makes it a saline environment), and soil to see if extremophiles were present.
    Extremophiles and there products have a lot of scientific and industrial applications.  For instance, extremophiles affected OJ Simpson's life:  PCR, a technique used in DNA fingerprinting, relies on Taq polymerase, which is an enzyme derived from thermophilic bacteria.  Detergents, used at high temperatures, may also benefit from enzymes found in thermophilic organisms.

Methods:
    We prepared nutrient broth and enriched the broth to make solutions of 15% salinity using NaCl to culture halophiles, and high acidity using HCl and titrated to pH 3 to culture acidophiles.  We then swabbed each sample with a sterile swab and inoculated the various broths with swabs from each sample.  We incubated  acid and saline broth at room temperature for 72 hours.  We also inoculated unenriched nutrient broth and incubated it at 55 C for 72 hours to culture thermophiles.  We performed a Gram stain analysis on each culture to determine the presence of bacteria.

Results:
    We did see evidence of bacterial growth in extreme environments.  We saw growth in an acidic environment in cultures from milk, soil, and ham.  We saw growth in a saline environment in cultures from skin, and we saw growth at 55 C in cultures from ham and milk.  Click on the pictures below to see the full size image.
 

Ham culture in acid medium	Ham culture incubated at 55 C	Milk culture incubated at 55 C

Discussion:
    We did find some evidence of extremophiles in our samples.  However, our results were not what we expected.  We expected to see halophiles growing on ham and bologna, because they are saline environments.  We also expected to see some thermophiles growing in tap water, which can sit in a storage tank for long periods of time.
    The incubator we used did  not maintain 55 C, but usually settled at 49-51 C.  This temperature range will promote mesophilic (normal range) bacterial growth and, therefore, the bacteria we saw may not have been thermophilic.  An additional difficulty we had was that, when heat fixing the saline enriched samples for Gram staining, the salt crystallized and obstructed our view under the microscope.  We might have had more success enriching agar plates with NaCl.

Possible Extensions:
    To continue our investigation of extremophiles, the next question that we may want to answer is, "What is growing in the cultures?"  While the Gram stain may help up identify the organisms, it is in no way a definitive indicator.  In order to do so, we would have to isolate the bacteria and grow a pure culture.  Once we have a pure culture, we would perform a battery of biochemical tests, such as tests for various sugar digestion, or casein digestion.

Classroom Applications:
    I see many ways of integrating this activity into a general biology class.  First, any microbiology activity must emphasize basic lab techniques, such as measuring, lab safety, following directions, formulating hypotheses and  observation.  It will also introduce students to more sophisticated lab procedures, like aseptic technique and Gram staining.
    By having students conduct an investigation like this, they will develop their own understanding of microbial diversity.  While microbial diversity is not a common unit in high school general biology classes, unity and diversity among species is.  By studying the rain forest, a popular theme among high school students, as well as other biomes, students may understand biodiversity.  But, what better way for them to learn than by observation and self discovery?  If they can see that some organisms can live in one environment but not another or that some can digest a sugar but another cannot,  they have shown themselves biodiversity.  Of course, after such a project, we could have a discussion about the why and how of biodiversity, incorporating evolutionary theory
    I would not begin a microbiology unit with this lab.  I would introduce my students to microbiology with a much more simple investigation, like rubbing their fingers on agar to see bacterial growth.  I would also have students stain known cultures to train them to look at stained organisms.