Introduction:  Since the beginning of mankind we have been plagued by infectious diseases. Even in today's modern world we are still troubled. For many of these there is not a cure or treatment available. Scientists spend many long hours discussing and studying these diseases. In this study we are pretending that an epidemic is starting through the spread of rolliepollie bacteria. This scenario is realistic because the spread of disease through animals or insects is entirely possible. For example mosquitoes are the most common vector insect on the planet.  The purpose of our experiment was to study the natural amount of bacteria that is found on rolliepollies. The rollipollies were all sampled from the University of Redlands campus. This would be done through taking cultures from various specimens. They were collected in a specific area and then kept in a superficial habitat. Each bug in a habitat was sampled only once. Even though they were sampled only once each bug was found to have large amounts of bacteria present in the sample. Each culture was grown in a separate agar plates. Each agar sample was observed each individual colony was counted and recorded. Then the cultures were tested with various antibiotics in the attempt to find if they were affected. Some of the antibiotics were found to be effective while others had no effect at all. If effective the antibiotics would prove to help in reducing the chances of an outbreak. If an outbreak did occur the tests would help to fight the spread of disease. This somewhat simple experiment could be used as a basis for a new form of preventive research when dealing with bacteria. This experiment could be used to test other insects or animals that carry bacteria, that could cause disease in humans.  Once cultured it can then be tested with antibiotics for a cure. We were able to find that tetracycline was effective at killing both types of bacteria that we cultured from the rolliepollies. We also found that the best way to culture these bacteria was to use the dunk method on the rolliepollies.

Material and Method: The pill bugs were taken from a flower bed in the University of Redlands Science Center Court Yard.  In order to find the rolliepollies, we sifted through the dirt and debris moving rocks, sticks, leaves and other objects until finding a "rolliepollie haven".   Using latex gloves, one at a time they were placed in a single plastic test tube and taken to the lab to be tested for bacteria.  While in the lab, the next step was to isolate each pill bug.  Each pill bug was then placed in individual 1.5 ml test tubes of their own through the use of sterilized tweezers (dipped in ethanol and then passed through a flame). 

Swab Method:   On one occasion what we have titled as “the swab” method was used.   Using this method, with latex gloves, each pill bug was placed on a clean paper towel.  Next, a Q-tip was swabbed along the tops and bottoms each bug.  The Q-tip was then placed in a new 1.5 ml plastic tube which contained .5 ml of water.  The Q-tip was submerged in the water and shaken forcefully.  Next, all .5 ml of the water was poured onto an agar plate.  Each plate was covered and left to grow any and all of the bacteria taken form each pill bug. 

Dunk Method:  Using the second method entitled “the dunk” method; 1.0 ml of water of pure water was then added to each of the test tubes (with the bugs already in them).  The test tubes were then shaken to submerge each pill bug in the water in order to extract any and all bacteria from them.   Next, all of the water from each test tube was taken and poured on an agar plate and spread with a cell spreader.  The agar plates were then covered and left to grow any and all bacteria.

Isolation of Bacteria:  There were many different kinds of bacteria that grew on the agar plates.  In our next experiment we chose to isolate two of the different kinds of bacteria that developed from our previous experiment.  In this case we chose to isolate "yellowish" and "whiteish" bacteria.  In order to do so, we used one of the previous plates from our last experiment and with a toothpick we scraped away a large colony of  "yellowish" bacteria.  Then we took the toothpick and scraped it directly onto another agar plate.

The same was done for the "whiteish" bacteria.  All the steps were the same.  Once both the "yellowish" and the "whiteish" bacteria were each isolated one time, we repeated the procedures a second time for both bacterias.  This was done enabling us to test eight different antibiotics for each bacteria.

Testing Bacteria with Antibiotics:  On each of the bacteria cultures (two per color / kind) four small disks were placed evenly apart from each other.  The eight disks (four per agar plate / eight total per bacteria) each contained a different kind of antibiotic.  The names of the eight different antibiotics are (Plate #1): kamamycin--place 1; carbenicillin--place 2; doxycycline--place 3; erythromycin--place 4.  Plate #2 antibiotics were placed as follows: vancomycin--place 1; tetracycline--place 2; ciprofloxacin--place3; ampicillin--place 4. 

It is important to stress that the "yellowish" and "whiteish" bacteria colonies each were tested with 8 antibiotics using two agar plates apiece.  Once again it is important to understand that each bacteria had two plates with the antibiotics placed in the identical spots in each of their plates. 

Rolliepollie Habitat:  On another occasion, we three created a "rolliepollie habitat" with the hopes of testing tetracycline which is an antibiotic to see if it had any effect on the bacteria found on the rolliepollies and their living environment..  In order to create this habitat we took a Tupperware container and ventured off into the Orange Tree Grove Orchard located on the East Colton  directly across from Gregory Hall.  The first step we took was to find rolliepollies.  The dirt on the Orchard grounds was hard and dry; finding a large colony of rolliepollies took some time.  After digging off the surface layer (a few inches) we finally found a large colony of bugs on our fourth attempt under an shaded area of an orange tree.  Before actually taking any rolliepollies form their natural habitat, we took a good amount (approx.4 in.) of damp soil and placed it in the Tupperware container.  Next we added some rocks, sticks, leaves and other debris to the container in an attempt of making the habitat as close to the real things as possible.  Once we were satisfied with our habitat we gathered 10 rolliepollies and placed them in their new home and proceeded to the lab.  Once in the lab we then sprayed the habitat with Tetracycline with the hopes of killing any and all bacteria that was extracted with the rolliepollies and their new habitat. 

In order to test the validity of this experiment we repeated all the steps we took with the previous habitat and created a second one.  Every aspect of the creation was kept the same.  The only difference the second time was that we sprayed the new habitat (the second one) with pure water instead of Tetracycline.  This step was our groups "control" method.  If the amounts of bacteria were the same after sampling both habitats then we would have know that the Tetracycline was of no use to us in this experiment. 

Results:

In order to determine if rolliepollies carry bacteria on their bodies when in their natural environment, we took sample rolliepollies and dunked half of them, and then swabbed the other half. The above pictures are examples of the results that were found from using the dunk vs. the swab technique. As you can see there is a wide variety of bacteria that was found to be present on the sampled rolliepollies. They ranged in color from almost see through white, to a dark almost orange yellow. The dunk technique was simply to dunk the rolliepollies. in a small sampling of 1.5ml of water and then to spread the water across the dish. This left the bug in the water for a longer period of time and it was believed that there would then be more bacteria in the water that was spread on the dish. The swab technique was to take a small swab and wipe down the rolliepollie and then to swirl the swab stick around in the sample of water. Then the water was spread on a dish just as the sample into which the bug was dunked. As you can see the dunk method transferred a much larger amount of bacteria from the sample bug to the dish where it was spread.

This graph shows the amount of bacteria found from all the dishes that the separate methods were used on. The dunk method shows a much larger amount of bacteria present then the swab method. This is most likely because in the dunk method the bug was put in direct contact with the sample of water. In the swab method the bug was quickly swabbed and then the swab was put in the water, not the actual bug. This graph demonstrates that the dunk method was much more effective at transferring bacteria from the bug to the agar plate. After doing the "TTest" on the above information a value of .000542 was found. This indicates that the rolliepollies were not taken from the same master population. In other words the differences in the amount of bacteria found on each individual rolliepollie can be explained.

Because there were so many different types of bacteria found to be present on the sample rolliepollies we decided to take individual samples. This was so that we could more easily test the bacteria for vulnerability to antibiotics. These two dishes are samples of the bacteria that were cultured from the rolliepollies using the dunk and swab methods. A sterile toothpick was taken and individual samples of bacteria were spread across an unused agar plate. The samples were cultured from individual colonies of white and yellow bacteria.

Once we has two separate cultures of individual bacteria we were able to test them to a variety of antibiotics at the same time so as to more quickly where the bacteria's weaknesses lay. The above dishes are examples of when cultures from the white and yellow bacteria cultures were spread and then tested for vulnerability to certain antibiotics. The antibiotics where put onto small disks which were placed evenly across the dishes, and then left to see if they would have any effect. On this first set of dishes the antibiotics kenamycin. carbenicillin, doxycycline, and erythromycin. On the white bacteria none of these antibiotics seemed to have any effect except for doxycycline, which is in the third place. On the yellow bacteria it appears that both doxycycline and kanamycin had a positive role in killing it off. Kanamycin was in the first space on both of the above dishes.

These two dishes are as seen above but four separate antibiotics were used. On this set of plates the antibiotics vancomycin, tetracyline ,ciprofloxacin, and ampicillin where used. On the white bacteria it seems that tetracycline and ciproflaxin had a positive effect on it. It appears that the same two bacteria had a positive effect on the yellow bacteria as well. Tetracylcine was in the second spot on both dishes, while ciproflaxin was in the third space.

After testing the individual bacteria with multiple antibiotics, we found that tetracycline seemed to have a positive affect as far as killing off both types of bacteria. These two dishes were used to test the yellow bacteria to see if the antibiotic tetracycline would be effective at killing it. On the first dish we spread the bacteria and then placed a disk in the middle and put the antibiotic on it. The second dish was simply used as a control to make sure that it was the antibiotic that was killing the bacteria and not something else that would have happened anyways. From the results shown above it can be clearly seen that tetracycline was indeed effective at killing off the yellow bacteria.

These two dishes are as seen above, but with the white bacteria. They show that tetracycline was also effective at killing off the white bacteria.

After finding that the bacteria which we had cultured off of the rolliepollies could be killed by the antibiotic Tetracycline, we decided to see if this antibiotic could lower the bug's bacteria counts when introduced into their natural environment. To do this we had to make superficial habitats and collect random rolliepollies to live in them. This is a picture of a  habitat that was used to keep rolliepollies in. This is one of two habitats that we made. Before testing the antibiotic on these superficial environments we took counts after a couple of days to see if our environments were created well enough for at least a percentage of the selected rolliepollies to survive in. After seeing that our habitats were sufficient, we tested the antibiotic. One of these habitats we sprayed with water mixed with the antibiotic tetracycline. The other one was sprayed with simply water, to use as a control. The rolliepollies from both habitats were then tested for bacteria, and the results where inconclusive. It was hard to tell if the antibiotic, when applied to the rolliepollies natural environment, would lower the amount of bacteria found on them. This was because the amount of bacteria found on the rolliepollies who's environment was sprayed with tetracycline were more or less in the same range as the numbers found on those who's habitat was sprayed only with water.

This graph shows the difference in the average amount of bacteria found on the rolliepollies that were in the tetracycline environment versus the amount found on the water sprayed rolliepollies. The graph however is slightly misguiding in its appearance. The difference between the tetracycline rolliepollies and the water rolliepollies is only 120 bacteria. The graph however makes the difference look much bigger. However, in the real world, a difference of 120 bacteria is basically inconsequential. Also, there were ten rolliepollies used for the water average, and only six used for the tetracycline average. This is because four of the rolliepollies from the tetracycline habitat died or went missing before we could use them as a source of data. Thus, it is difficult to tell whether or not tetracycline could have been used to lower the bacteria counts of wild rolliepollies in their natural habitat. Upon doing the TTest on the above data, the result was a percentage difference of 0.738471. This shows that these rolliepollies were much more likely taken from the same sample population.

Discussion of Results: From this study we were able to find that, in their natural habitat, rolliepollies do in fact carry bacteria. We also found that dunking a rolliepollies is a much more effective way of sampling bacteria then swabbing them.  This is displayed through the above graph that showed that the dunking technique brought about much more bacteria in the cultures that were made using these techniques. Also, through the testing of antibiotics on the bacteria that we cultured, we found that tetracycline is very good at killing off most if not all of the bacteria that was cultured off of the rollipollies that we captured. These results imply that if we were to spray tetracycline on rolliepollies in their natural habitat, that it might be able to lower bacteria counts across the board in that population. The limitations of this are that not even one hundred rolliepollies were studied total in this chain of experiments. Also, they were all collected more or less from the same general location, just outside the science building on the campus of the University of Redlands. Another limitation is that this experiment, although it was done with the best of intentions, was done by college students, and not scientists. This means that the results may not be as reliable as they could be. The future direction of this experiment is possibly to give a way for people to test the natural level of bacteria present in certain bugs and animals, to culture that bacteria, and then to test it for vulnerability to antibiotics, and thus come up with a cure.