Purpose: Find, select, isolate, and identify extremophiles from everyday sources.

Introduction/Background
When you think of the ideal conditions for your survival, you are probably thinking of a sun drenched
beach with a warm breeze making the air temperature around 24 degrees Celsius.  Of course there would
be a variety of organic food sources available and plenty of cold neutral water to drink. Compared to
these conditions, extremophiles like conditions we detest. Those microbes that thrive in temperatures above
50 degrees Celsius are called thermophiles. Those that thrive in a pH below 5 are called acidophiles. Still
others that thrive in a salt concentration of about 10-20% are called halophiles. Still others prefer the cold,
basic, and sulfur environments. Basically, these extremophiles are capable of living in every possible habitat
on earth.

One of the most well known extremophiles was discovered by Thomas D. Brock while in the hot springs of
Yellowstone National Park. The species Brock discovered was Thermus aquaticus. Unfortunately, he did
not foresee a possible use for this organism which was later utilized for it’s enzyme, Taq polymerase in PCR.
In order to survive in relatively hot environments, the structure the proteins of thermophiles must not denature.
Characteristics that help thermophiles maintain their structural integrity include an increased amount of hydrogen
bonding, hydrophobic bonding, and ionized group interactions within proteins (Herbert and Codd, 1986).
Other examples of thermophiles include Bacillus stearothermophilus, Bacillus coagulans and Clostridium
thermocellom.

In order for acidophiles to survive in their environment, they either maintain a cell surface barrier impermeable
to protons or actively pump protons out of the cell (Herbert and Codd, 1986). Without these adaptations, they
will die due to the denaturing of DNA and other proteins. Acidophiles may be found in nature in acid lakes,
pine forests, soils, acid bogs, volcanic and geothermic vents, coal processing areas, human vagina, and rot in
apples and pears. They may also be found in the food industry in the processing of milk products such as yogurt,
cheese, sour cream, buttermilk, and in the production of vinegar. Examples of acidophiles found in the food
sources listed are Lactobacillus, Stretococcus, Leuconostoc, and Acetobacter.

Halophiles contend with their environment by putting diffusion to work for them. If the membrane permits,
solutes will move from an area of high concentration to an area of low concentration. In this case, in order for
the cell not to dehydrate in a salty environment, the cell must contain a higher concentration of solutes than its
environment. Additionally, halophilic enzymes are stabilized by electrostatic shielding (Lanyi, 1974) and the
maintenance of a hydrated protein surface through the utilization of carboxyl groups in glutamine and aspartate
(Pundak, et. Al, 1981). Examples include Vibrio, Micrococcus, Pseudomonas, Bacillus, and Staphyloccus aureus.
Salted food sources such as soy sauce and miso paste are good places to find halophiles in your neighborhood.

Materials: Sterile nutrient broth, 1M HCl, NaCl, Gram staining materials, test tubes, incubator, pH paper or meter,
sterile water, Bunsen burner, sterile swabs, 1 and 10 ml pipets, Sharpie, and bioassay media as needed

Procedure
1. Finding extremophiles.
To initiate the lab, have students first brainstorm where these extremophiles exist. Since collecting samples
from the hot sulfur springs in Yellowstone National Park and the Dead Sea may not be not practical, look
for mircoenvironments with similar conditions locally. You may use the chart below as a helpful guide to
the whereabouts of extremophiles.
 

Type of extremophile                     Source
Acidophile                                     acid lake
                                                     pine forest soil
                                                     acid bog
                                                     yogurt
                                                      cheese
                                                     sour cream
                                                     buttermilk
                                                     vinegar
                                                     rot in apples and pears

Halophile                                     salt cured meats and fish
                                                     soy sauce
                                                     miso paste
                                                     sauerkraut
                                                     surface of skin

Thermophile                                  milk
                                                     water heater
                                                     canned foods - esp. sugar beets

2. Selecting for extremophiles
Tubes from the sources above were inoculated in each of the following media:
Halophiles: 9 ml nutrient agar + 0.8g of NaCl + 1 ml inoculate
Acidophiles: 9 ml nutrient agar with HCl at a pH = 3 + 1 ml inoculate
Thermophiles: 9 ml nutrient agar + 1 ml of inoculate incubated at 55 degrees Celcius

3. Isolating extremophiles
For all the tubes that show growth after 48 hours, prepare at least three tubes containing
10 ml of nutrient agar plus the selected media for each positive tube. Take 1 ml of original
inoculate and transfer to a fresh tube containing 10 ml of nutrient broth. This will be your
1/10 dilution. Then take 1 ml from this tube and place it in a fresh tube containing 10 ml of
nutrient broth. This will be your 1/100 dilution. Repeat for a third tube containing 9 ml of
nutrient agar. This will be you 1/1000 dilution.
You may try an alternative procedure by streaking a plate and analyizing selected colonies.

4. Identifying extremophiles.
Choose the appropriate bioassays that will help you identify your extremophile using the chart below.

Extremophile         Type         Gram stain         Shape            Urea            H2S           Catalase           Lactose          Sucrose       Other

Halobacterium         Halo                -                 rod                                                          -                        -

Staphylococcus       Halo               +                cocci                -                  -                   +                    acid                acid

Micrococcus            Halo              +                cocci                -                  -                   +                      -                      -

Pseudomonas           Halo              -                  rod                 -                   -                   +                      -                      -

Bacillus                    Halo              +                  rod                 -                   -                   +                      -                  acid

Vibrio                      Halo               -                 rod                                                                                                     acid           maltose+

Bacillus                   Thermo           +                  rod                                                          +                                                            oval
Stearothermophilus                                                                                                                                                                          endospores

Clostridium             Thermo            +                rod                                       -                   -                                          acid          starch +

Lactobacillus           Acido              +                rod                                                                                acid/gas

Streptococcus            Acido           +               cocci                                                          -                    acid                                nonmotile

Leuconostoc             Acido            +               cocci                                                          -                   acid/gas

Acetobacter              Acido             -               rod                                                            +                                                           EtOH+:
 

Bibliography
Herbert, R.A. & Codd, G.A. (1986). Microbes in extreme environments. London: Academic Press.
Holt, J.G., Krieg, N.R., Sneath, P.H., Staley, J.T., & Williams, S.T. (1994). Bergey’s manual of determinative bacteriology, 9th edition. Baltimore:
    Williams & Wilkins.
Lanyi, J. K. (1974). Salt-dependent properties of protiens from extremely halophilic bacteria. Bacteriological Reviews 38, 272-290.
Madigan, M.T. & Marrs, B. L. (1997). Extremophiles. Scientific American.
Madigan, M.T., Martinko, J.M. & Parker, J. (1997). Brock biology of microorganisms. Upper Saddle River, NJ: Prentice Hall.
Pundak, S., Alani, H. & Eisenberg, H.(1981). Structure and activity of malate dehydeogenase from extreme halophilic bacteria of the Dead Sea.
    European journal of biochemistry 118, 471-477.

Back to main page