Introduction  [top]

Bacteria can be found on just about every material and in virtually every environment on the planet. Impressively enough they have even been found in the arctic and the core of volcanoes. Our own bodies also play host to about 100,000 bacteria per square centimeter. However, very few bacteria actually cause disease.

In order for bacteria to infect a host, in the same species or otherwise,  three things must happen. First, the bacteria must maintain a reservoir long enough for the bacteria to be spread. Second, it must leave the host and actually enter a new host. Third, it must manifest itself within the new host.

The scenario from which we are working is as follows: A deadly bacteria has broken out and it has been determined that pill bugs are the vector for that disease. This scenario, if you will remember, is similar to that presented in the motion picture Outbreak as well as the novel The Hot Zone. The assignment for the group was to determine which antibiotics are most effective against the infectious bacteria. Eight different antibiotics were tested with varying results. This web site is a first hand account of the experiment and ultimately the stopping of “an epidemic”.

Materials and Methods   [top]

 (1) Do bacteria exist on Pillbugs? If so, how many?

The 5 pillbugs were collected in 3 places: 2 of which were in the Science Center Courtyard, 2 from the planter on the right as you exit the Science Center, and one from the left hand planter.  Each pillbug was placed in a separate 1.5mL microcentrifuge tube and labeled (1,2,3,4,5).  .5 mL of distilled water was then placed in 5 other plastic  microcentrifuge tubes. One at a time each bug was placed into each  microcentrifuge tube for approximately 30 seconds. Then each “mixture” (including the pillbug) was poured onto 5 separate agar plates, the pillbug removed with sterilized tweezers, and spread using a sterilized triangle. They were then closed, taped, and sat overnight to see if bacteria would grow.

(2) Isolated bacteria for future testing procedures

 From the first method of collecting pillbugs to determine an existence of bacteria, two bacteria were highly prevalent: orange and yellow. From these agar plates we used 2 toothpicks (one for orange and one for yellow) to isolate the bacteria. One plate was dedicated to the growth of yellow bacteria and one plate dedicated to the growth of orange bacteria. A colony of orange bacteria was scraped by the toothpick and streaked onto a different agar plate. The same thing was done for the yellow bacteria. They were then closed, taped, and sat overnight to be able to grow.

 (3) Testing the technique of swabbing vs. dunking

 Two techniques were tested among the lab groups in class to be able to decipher which technique would provide the most accurate results. A total of 6 pillbugs were collected from the Science Center Courtyard. Two were found under a rock, two were found under another rock, and the last two were found in the moist ground cover. Three pillbugs were dunked into 3 separate  microcentrifuge tubes filled with .3 mL of distilled water. The remaining three were swabbed with a Q-tip which was first dipped into .3 mL of water. Each solution was spread onto 6 separate agar plates.

 (4) Testing of antibiotics on the isolated bacteria

 From the two isolated plates of bacteria, two liquid cultures were grown.  A toothpick was used to sample the orange and yellow bacteria and then each toothpick containing each bacteria was placed into a test tube containing liquid broth.  From that, two plates of orange and two plates of yellow bacteria were grown on four agar plates. Each of the 4 plates was divided into four sections, testing 8 different antibiotics: 1) Ampicillin 2) Ciprofloxacin 3) Tetracycline 4) Vancomycin 5) Erythromycin 6) Doxycycline 7) Carbenicillin 8) Kanamycin. Each antibiotic came in the form of a small paper disk and was placed onto each of the 8 sections on both the orange and yellow bacteria plates. The plates were then closed, taped, and incubated overnight to see whether or not these antibiotics would kill the bacteria present. To measure the amount of bacteria which was killed the ring surrounding the antibiotic disk was measured in centimeters.

 (5) Creating a pillbug habitat

Ten pillbugs were collected on the south side of Colton Avenue in the orange groves near the tennis courts. The pillbugs were placed into a large (approximately .33 L) plastic container including dirt, leaves, sticks, and a rock from the same location that the bugs were found. Four squirts of distilled water was then sprayed into the habitat to moisten the environment, thus resembling the natural habitat of a pillbug. The habitat was then examined a week later by counting living pillbugs to determine whether or not it provided an adequate living condition for the pillbugs.

 (6) Creating a habitat to test tetracycline (in order to stop the spread of bacteria on pillbugs)

 Twenty pillbugs were collected in the same area as in method #5. Ten pillbugs were placed into 2 separate large (approximately 10x6x6 cm) plastic containers to create a habitat (each filled with dirt, leaves, and a rock from the same area the bugs were collected). One habitat was sprayed with 5 mL of tetracycline and the other with 5 mL of distilled water (to create a control for comparison).

 (7) Checking for bacteria

 One week later the 10 alive pillbugs from each of the 2 habitats (created in method #6) were dunked and shaken in a mini-plastic test tube for approximately 5 seconds in .5 mL of distilled water. The solution was then spread onto 20 separate agar plates using a sterilized triangle, closed, taped, and incubated to determine whether or not the tetracycline was effective in killing the bacteria present on the pillbugs.

Results [top]

(1)    Pillbugs were collected and then sampled to determine whether or not bacteria were present. Sampling was done by dunking bugs in water and spreading this water onto agar plates. The bacteria were not counted, only the colonies were observed. Colonies are groups of millions of bacterial cells together.  Picture: As shown, bacteria are present. The yellow, orange, and white colonies indicate the presence of bacteria.

(2)    Two forms of bacteria, orange and yellow, were isolated for future experiments to determine whether different types of antibiotics would be effective in eliminating either form of bacteria. They were isolated by removing a colony of bacteria from the original plate with a toothpick . They were then spread onto two new plates.  Picture: Here are the two isolated bacteria: yellow and orange; the bacteria grew wherever the toothpick was streaked.

(3)    The pillbugs were both swabbed and dunked to determine which technique provided the best results. The swab method was done by picking up the pillbug with sterile tweezers and swabbing the bug in its entirety with a damp q-tip. Then spread onto two agar plates.  The dunk method was done by dunking bugs into 1.5 mL   microcentrifuge tubes and shaken. The water was then spread onto agar plates. The dunk method was found to produce more colonies of bacteria. Thus, the dunk method was found to be the most useful technique.

Picture: Below are two agar plates, one containing results from the dunk method (left), while the other is of the swab method (right).  The dunk method, as shown, provided more colonies of  bacteria.    Graph 1: Below is a graph of the class’ results from this experiment.

Table to right: These are the results of the T-Test (swab vs. dunk). The value is below .05, stating that the different techniques had different effects on the number of colonies in our lab group.

(4)    Four agar plates, two of the orange bacteria and two of the yellow bacteria, were grown to test the effectiveness of the eight different antibiotic disks.  Overall, the Doxycycline antibiotic proved to have the most effective response to both types of bacteria. Picture: Here are the plates containing both bacteria and antibiotic disks. The rings present around the disks indicate the effectiveness of the antibiotics. Table 1: Shown below the pictures are the eight antibiotics and their rings of effectiveness in centimeters.

(5)    This habitat was created as a space to maintain the lives of pillbugs for further testing of antibiotics in such a habitat. The dirt, rocks, and leaves were collected from the orange grove previously referred to on Colton Ave.  After one week, eight pillbugs were alive; after two weeks, seven were alive.  One new pillbug was also found. Picture: Pillbug habitat including leaves, dirt, and rocks.  Table2: Results of dead and alive pillbugs.

(6)   A new group of twenty pillbugs were collected and sprayed with tetracycline and sampled a week later to test the strength of this antibiotic in a pillbug habitat.  Found in the control habitat were ten live bugs; in the tetracycline habitat were eleven live bugs.  Table 3: Below are the results of dead and alive pillbugs in each of the habitats.

(7)    The live pillbugs were tested to determine the effectiveness of the antibiotic tetracycline. To test the bugs we sprayed the two habitats, one with water and one with Tetracycline.  Overall, it appeared that there was a higher prevalence of bacterial colonies from the plates that contained the solutions of pillbugs having been treated with the antibiotics. Graph 2: Below are the number of bacterial colonies from the control habitat (water) and the antibiotic habitat.

Table to right: These are the results of the T-Test (Tetracycline vs. Water). The value is above .05, stating that the different treatments had no effect on the number of bacterial colonies.

Discussion of Results  [top]

Bacteria that cause human disease can be found in almost any area of the world, including within or on organisms as small as pill bugs.  To investigate this hypothesis, we tested various groups of pill bugs to see whether bacteria exists within their habitat and thus on the surface of their bodies.  And from this, we tested eight different antibiotics to determine whether one would be effective in eliminating a bacterium that could potentially lead to human disease.

Using two techniques, swabbing and dunking, to test the prevalence of bacteria, dunking was found to be a better way to asses to number of bacteria on the surface of a pillbug.  We arrived at this conclusion through the fact that more colonies were created using the dunk method. In future experiments, we recommend using this method.  However, please use precautionary techniques such as not dunking a pill bug for more than 30 seconds as pill bugs may drown.

Many antibiotics were tested on both bacteria to determine which was most effective. As shown in our results (table 1), the antibiotic Ampicillin gave a result of 1.4 cm range of effectiveness on the orange bacteria, and 0 cm on the yellow bacteria. The antibiotic Doxycycline, however,  was found to be the most effective in controlling the orange and yellow bacteria on the surface of a pillbug; with an effective  radius of 2 cm and 2.3 cm where the bacteria had been killed. Given that the antibiotics were more effective against the yellow bacteria, it can be presumed that if human disease occurred the orange bacteria would be responsible. This can be concluded because of its resistance to the antibiotics.

Treating the pill bug habitats with the antibiotic Tetracycline did not decrease the number of bacteria on the surface of pill bugs.  In fact, it may have increased the number of bacterial colonies.  We feel that it was not effective because in our earlier experiment (method #4, table 1 in Results) Tetracycline was not found to have any effect on the orange bacterium and only 1.7 cm of effectiveness on the yellow bacterium.  Thus, when adding it to the habitat it did not have a significant effect.

If we had the chance to continue this experiment with more resources and funding we would test more effective antibiotics, such as Doxycyline which was found by us to be the most effective. However, we were unable to test it in the terrarium given the time and money constraints.

In conclusion, if a human disease were to be found on the surface of a pill bug there would be many factors which would play a role in the selection of a medication. For example, we chose Tetracycline for its availability and price.  If a world wide epidemic did break out, these factors, as well as many others, would certainly play a role in eliminating the disease.