Introduction:

The purpose of this project was to test the hypothesis that pill bugs carry a certain form of bacteria that has the ability to cause human disease. Many studies have been conducted that prove the risks posed by arthropods and other insects to be serious or fatal in the human population, namely the West Nile Virus which is spread by the sting of mosquitoes. Furthermore, our close, day to day contact with these critters increases the risk of infection or contamination dramatically.  The number of tests conducted throughout the study have proved pill bugs carry a sufficient amount of bacteria that is harmful to humans.  Several different antibiotics were tested against these bacteria forms with the hope that some, or one, of them will be effective in controlling the number of disease causing bacteria on a pillbug.

It was found that three varying forms of bacteria are common to pillbugs around the University of Redlands campus. Two methods were used to collect bacteria from the bugs, the dunk and swab methods, and of these two test it was determined that the dunk method was much more effective at collecting the necessary samples required for future tests to determine the effectiveness of antibiotics toward these three forms of bacteria. Furthermore, eight differing forms of antibiotics were used in the test, and results were collected as to the effectiveness of specific antibiotics toward cultures of bacteria.

Materials and Methods:

Starting the Study:

Five pill bugs were collected from the Science Center Courtyard.  Three questions were addressed: How many bacteria are present on a pillbug? How many types of bacteria are present? What type of environment do these pillbugs prefer? In order to determine the number of bacteria on a pillbug all possible dirt was removed from the bug, then the pillbugs were dunked in a test tube with 5ml of water for a few seconds.  Each pillbug was removed from the test tube using sterilized tweezers, then using a pipette, 3ml of water was removed from the test tube and dropped onto the agar plate.  Then the liquid was evenly spread onto the agar plate.  The lid to the agar plate was open briefly while the liquid was being spread.  Each agar plate was labeled accordingly. Pillbugs from plates 1&2 were collected from the flower bed in the center of the Math and Science buildings courtyard, plates 3&4 were collected from  the northwest corner of the Math and Science buildings, and plate 5 was collected from a flowerbed in the northeast corner of the same courtyard. 

Dunk Vs. Swab Experiment:

Later, six more pillbugs were collected. Two types of sampling techniques were used in order to determine which method was best in assessing the number of bacteria present on a pillbug.  The first method was dunking the pillbug into a test tube of water filled with 1.5 ml of ultra-pure water. Next the pillbugs were extracted with sterile tweezers and two drops of the remaining solution was spread over an agar plate. The second method was to thoroughly swab the pillbug with a sterile q-tip and then place the swab in the test tube of water, two drops of the solution was then taken and spread evenly over the agar plate.

The results above are an aggregate amount of information collected from the entire class as a result of the swab vs. dunk methods. An overwhelming amount of bacteria formed under the dunk method as opposed to the swab method. Furthermore, a t-test was created to analyze the data which resulted in a probability of  0.002071, showing that there is a definite difference between the two groups.  The probability that these two groups of data could have been picked from the same master population is not likely.  Therefore it is reasonable to conclude that the dunk method was more effective in assessing the number of bacteria on a pillbug.

Creating the Pillbug Habitat:

Next lab a set of 10 pillbugs was collected from the East Orange groves next to the University tennis courts.  Dirt, rocks, chunks of fallen wood, a small orange, and two small plants were also gathered in order to simulate the pillbugs natural habitat.  This environment was created in a small container roughly 8*8 inches and about 3 inches deep, and the lid to the environment was punctured numerous times to provide oxygen.  Finally, once the pillbug habitat was returned to the lab, a fine spray of water was administered to the environment to provide moisture.  The point of this task was to determine if the pillbugs could survive in the artificial habitat and to tabulate the survival rate.

Specialized Bacteria Cultures:

From the first set of agar plates a toothpick was used to scrape off two types of bacteria colonies, in this case yellow and orange, and each was re-cultured separately.  In order to re-culture, the toothpick used to scrape off the bacteria from the original plate was dropped into a test tube of approximately 3ml of water, it was mixed around and then removed with sterile tweezers.  About two drops of the new solution was then removed and spread evenly over the new agar plate.  This process was completed twice for the yellow and orange bacteria. 

Specialized Antibiotic Testing:

After each of the two colors of bacteria was re-cultured onto two plates they were then tested for antibiotic sensitivity. Paper discs were added to the center of each plate and the antibiotic Kanamycin was carefully dropped onto the center of the paper disc once for each color.  Leaving each color bacteria with one plate untouched by antibiotic to use as a control.

Large Scale Antibiotic Testing:

In order to test the sensitivity of both orange and yellow bacteria cultures, an array of antibiotics were used. Eight small antibiotic discs containing Kanamycin, Carbenicillin, Doxycycline, Erythromycin, Vanomycim, Tetracycline, Ciporfloxacin, and Ampicillin were evenly placed on four different sections of the four agar plates.

Habitat Sterilization Project:

Twenty additional pillbugs were collected from the Science Center Courtyard.  Ten of the pillbugs were placed in a habitat to be used as a test group and the other ten were placed in a separate habitat and used as a control. 6ml of Tetracycline was sprayed into the test group habitat area, covering the dirt and the pillbugs.  6ml of water was sprayed into the control group's habitat. 

Later each of the twenty pillbugs were dunked in 5ml of water, removed with sterile tweezers and then approximately 5ml of the liquid was spread onto the agar plate to determine the effects of the antibiotics on the habitats.

Results:

The numbers of colonies on each of the five agar plates of the first set were counted as follows: plate #1=204 colonies, plate #2=123 colonies, plate #3=15 colonies, plate #4=15 colonies, plate #5=119 colonies. There was evidence of different types of bacteria present on the agar plates, specifically, different colored colonies.  The colors detected were yellow, orange, clear and white colonies. 

Dunk Vs Swab Pictures:

Plates 1,3&5 were completed using the swabbing technique.  Plates 2,4&6 were tested using the dunking technique: plate #1=207 colonies, plate #2=800 colonies, plate #3=114, plate #4=193, plate #5=600, plate #6=completely covered in colonies.

Above and Below: photos taken of the results of the swab vs. dunk method show that the dunk method provides a much more vast colony production.

Results from Pillbug Habitat Project:

Results from the observation of the pillbug habitat after the first week resulted in a count of 10 healthy pillbugs.  Pillbugs replaced back in the habitat and misted with water once again for further observations.

Results from the second week of pillbug habitat observation resulted in 1 death and 9 healthy pillbugs.  This concluded the pillbug habitat project and all remaining pillbugs were returned to the wild. 

Large Scale Antibiotic Testing Results:

Further data was taken from the results of the application of antibiotics to the bacteria cultures. Data was taken by measuring the distance from the edge of the disc to the edge of the area where the antibiotic killed the bacteria; it may be called a kill zone. 

Yellow #1:  Carbenicillin......1/10cm       Yellow #2: Ampicillin.........no effect

                   Kanamycin.......0.5cm                          Vanomycim......8/10cm

                   Erythromycin....1 3/10cm.                    Tetracycline......1 cm

                   Doxycycline......1.5cm                         Ciporfloxacin....1 3/10cm

Orange #1:  Kanamycin.......1cm           Orange #2: Ampicillin.........1cm

                   Erythromycin....1cm                             Vanomycin.......1 1/10cm

                   Carbenicillin......2 1/5cm                       Tetracycline.....1 1/10cm

                   Doxycyclin........2 1/5cm                      Ciprofloxacin....1 6/10cm

Habitat Sterilization Project Results:

These results indicate the total average amount of bacteria present within the control and test groups. The test indicates that the probability of 0.01259 showing that there is a definite difference between the two groups. It is reasonable to conclude that the antibiotics were effective at minimizing the total amount of bacteria overall.

This chart shows the average total of Yellow bacteria present at the end of the Sterilization Project. There is probability of 0.4438 that these two charts are related, meaning it is reasonable to conclude that the treatment had no impact on the total difference in population of yellow bacteria.

The above results show the results in the total average number of orange bacteria in the Sanitation Project. The probability that these two samples are related is 0.048 meaning that there is a definite difference in the results. Although, these results seem inconclusive because there was less orange bacteria present in the control as opposed to the test group, these result conflict with previous tests showing a definite reaction by orange bacteria to Tetracycline.

Discussion of Results:

Dunk Vs Swab:

This test was designed to determine the most effective method for collecting bacteria from the surface of a pillbug. The statistics show that the dunk method as opposed to the swab method is a much better technique to assess the amount of bacteria associated with the typical pillbug.

Pillbug Habitat Project:

This project was designed in order to test the groups ability to create a suitable living environment for captured pillbugs.  Overall the project was successful, however there was one fatality after the second week of observations.  The pillbug habitat was not difficult to maintain, it seemed as though the pillbugs adapted easily to captive environments.

Large Scale Antibiotic Testing:

The purpose of this test was to investigate the effectiveness of multiple antibiotics on two select forms of bacteria.  From looking at the results, the antibiotics that had the most impact as far as eliminating bacteria from the cultures was:

Yellow:

The most effective antibiotics in controlling the number of bacteria on a pillbug that could hypothetically cause human disease are, Doxycycline with an eradication region of approximately 1.5 cm; Ciprofloxacin and Erythromycin at 1 3/10 cm.

The least effective antibiotics are Ampicillin with no effect; Carbenicillin at 1/10 cm.

Orange:

The most effective antibiotics are Carbenicillin and Doxycycline with a diameter of 2 1/5 cm.

The least effective of the antibiotics being Ampicillin, Kanamycin, and Erythromycin at 1 cm.

We found that of the eight antibiotics tested certain ones were more effective at eradicating the bacteria cultures, while some had little to no effect.

Habitat Sterilization Project:

Results from the testing of pillbugs sprayed with Tetracycline show no dramatic differences in efforts to decrease the amount of bacteria present on the surface of the pillbug, but there was some impact made on the whole. These results show that the tetracycline was able to kill regions of both the yellow and orange cultures. The yellow culture showed a kill zone of approximately 1cm and the orange about 1 and 1/10cm. However, when examining the number of colonies that were able to grow on the agar plates there does not seem to be a significant difference from the test chamber, which was sprayed with approximately 6ml of tetracycline, and the control chamber, which was sprayed with the same amount of water. Although when looking carefully at the colonies there does seem to very little evidence of yellow and orange colonies on either plate. Most of the colonies appear to be more of a whitish or clear.