Introduction:    [top]

It was a warm sunny day in Southern California when University of Redlands biology students were sitting outside in the grass having class. Shortly after, all of the students including the teacher, became ill with very similar symptoms. Slowly, these flu-like symptoms became fatal and one by one students were dying. Immediately scientists, doctors and research experts gathered to collect data and investigate the one variable the group had in common; the soil. The entire class was sitting outside and exposed to the same grass, soil and those creatures living in the soil. An abundance of pillbugs were found in the soil and immediately tested for the amounts of bacteria they carried. These pillbugs helped scientists expose the presence of bacteria and allowed them to work towards a collection of data and consequently a solution or treatment through use of antibiotics. This scenario was not a new one; scientists that have worked with the Hanta virus had seen these terrifying occurrences before. The Hanta virus affected those in Japan, primarily those whom worked in the rice fields in the country side. The virus was spread through the urine, and fecal matter of field mice. Just as the rice farmers were exposed to the mice and plagued with the Hanta virus, those in the biology class that sat outside were exposed to pillbugs and consequently an illness caused by the bacteria of those pillbugs. These scenarios are strikingly similar in means of transmission however, the Hanta virus does not have a cure and pillbugs contain bacteria which is treatable. After acknowledging the difference of treatment, data was collected, manipulated and tested against various antibiotics to find the most effective treatment. The following materials and methods explain where the pillbugs were found, and how the bacteria were collected. Conclusions on the effectiveness of antibiotics were then made after understanding the collection of data, and graphs that portray our results.

Materials and Methods:  [top]

Collection of Pillbugs

We first went out to collect ten pillbugs.  We found our ten pillbugs by randomly picking them out of the soil at first sight. We gathered these ten pill bugs in the courtyard between Hedco and Gregory Hall. Each bug was pulled out of the soil and placed in an individual tube. We then took the ten tubes back to the lab for further testing. In each tube holding a pill bug, we added .5ml of distilled water. The tube was shaken up so the bacteria may be able to spread off the bug and into the water evenly.  Using a pipette, we took 100 micro liters (one drop) from each tube and placed it on their own separate agar plate. In order to spread the drop fairly, we took a metal spreader that was soaked in alcohol and ran through a burner which kills off all germs not associated with the pillbug solution.  The metal spreader distributed the drop of water to all places on the plate. We closed and labeled the plates, which were set aside for later observation.

Pillbugs' Environment

We journeyed out to the same location described in trial one.  Again, the pill bugs were selected at random from soil environments. This time the bugs were placed together in a small plastic container filled with the environment they were found living in. This consisted of dirt, rocks, tree sticks, and other natural elements found in any ordinary planter box.  The pill bugs in the container were taken back to the lab for further observation. (see figure 1)

 
fig.1 Small plastic container filled with the soil environment the
           pillbugs were found living in.

Swab and Dunk Method

 We tested two different methods in hopes of solving for a better method of finding bacteria on pill bugs. These two methods being tested are known as Swab and Dunk. We used six pill bugs for our testing. Our group found these random pill bugs using the exact same procedure described in trial one. Back in the lab, we used the swab method first. We grabbed a pill bug from its container using sterilized tweezers. The tweezers were sterilized using alcohol and a burner. A dry Q-tip was rubbed all over the bug, ensuring the best probability of collecting bacteria. The Q-tip was then placed in a tube filled with .5ml of distilled water. We then used a pipette to take 100 micro liters. We dropped this amount of hopeful bacteria infested water onto an agar plate. Once again, we spread the solution using the same technique explained in trial one. This procedure was repeated for two more pill bugs; giving us a total of three pill bugs being tested with the swab method. We still had three pill bugs remaining to be tested with the dunk method. For the dunk method we placed .5ml of distilled water in to a test tube with a pill bug. . The tube was shaken up so the bacteria may be able to spread off the bug and into the water evenly.  Using a pipette, we took 100 micro liters (one drop) from each tube and placed it on their own separate agar plate. In order to spread the drop fairly, we took a metal spreader that was soaked in alcohol and ran through a burner which kills off all germs that were not associated with the pillbugs.  The metal spreader distributed the drop of water to all places on the plate. We closed and labeled all plates, which were set aside for later observation.

Orange and Yellow Bacteria

 For the following we used a previous agar plate containing a numerous amount of bacteria which had been gathered from pill bugs earlier in the week. The bacteria was cultured using the dunk method which produced yellow, orange, white and off white colonies of bacteria. We chose one distinctive orange and one yellow bacteria colony from the plate. Each colony was lifted off the plate with a toothpick, and then knocked off in to a test tube filled with Luria Broth (3ml). The solution was left to sit for a week. From the yellow solution we put two drops on two different agar plates and spread the solution evenly. We then placed one paper disk in the middle of each plate. Following that, we put a drop of water on one of the paper disk. On the other disk we put a drop of the antibiotic Kanamycin. We repeated the same steps for the orange solution, leaving us with four plates in all. (see figure 2)

Using Antibiotics (more information on antibiotics)

We then took two drops from the orange solution, placed each drop on to two agar plates. The solution was spread evenly on the plate using the same disinfectant method described in the previous trials. We repeated this process on to two more agar plates using the yellow solution. We labeled all four agar plates (two yellow solutions, two orange solutions) on the bottom, indicating their different regions with North, South, East and West. This was done to eliminate any confusion of later results. On one orange and one yellow plate the following antibiotic disks were placed in their own individual regions; kanamycin, carbenicillin, doxycycline and erythromycin. On the other orange and yellow plate, we place the following antibiotics; vancomycin, tetracycline, ciprofloxecin and ampicillin.  The labeled plates were closed and set aside for later observation.

 
Fig2 The result of the yellow and orange bacteria colonies
(after being spread onto two agar plates, and sitting for approx. a week)

Spraying of Antibiotic

 We went out to the previous area explained in trial one and randomly gathered twenty pill bugs. We placed these pill bugs into two different plastic containers; very similar to the container mentioned in trial one. Ten pillbugs at random were placed into the first container, and the other ten were placed in to the second container. The containers were filled with their environment. Back at the lab, we sprayed one container (chosen at random) with 6ml of distilled water. The other container was sprayed with 6ml of tetracycline antibiotic. These containers were placed aside for later observation. One week later, we tested ten bugs from the water sprayed container using the dunk method. We also tested ten bugs from the antibiotic sprayed container using the dunk method.

 Results: [top]

Collection of Pillbugs:

6 Plates were used in the demonstration of the collection of pillbugs. Our objective was to test the amount of bacteria found on each pillbug.  . In each tube holding a pill bug, we added .5ml of distilled water. The tube was shaken up so the bacteria may be able to spread off the bug and into the water evenly. Using a pipette, we took 100 micro liters (one drop) from each tube and placed it on their own separate agar plate. In order to spread the drop fairly, we took a metal spreader that was soaked in alcohol and ran through a burner which kills off all germs not associated with the pillbug solution.  The metal spreader distributed the drop of water to all places on the plate. We closed and labeled the plates, which were set aside for later observation.
All six plates were left at room temperature for approximately thirty six hours. Each plate had an average of 20-60 colonies. The bacteria found consisted of many different shades of yellow, orange, and white.

Swab and Dunk Method:

We tested two different methods in hopes of solving for a better method of finding bacteria on pill bugs. These two methods being tested are known as Swab and Dunk. We grabbed a pill bug from its container using sterilized tweezers. A dry Q-tip was rubbed all over the bug, it was then placed in a tube filled with .5ml of distilled water. We then used a pipette to take 100 micro liters. We dropped this amount onto an agar plate. For the dunk method we placed .5ml of distilled water in to a test tube with a pill bug. The tube was shaken up so the bacteria may be able to spread off the bug and into the water evenly.  Using a pipette, we took 100 micro liters (one drop) from each tube and placed it on their own separate agar plate. For both methods we took a metal spreader that was soaked in alcohol and ran through a burner which kills off all germs that were not associated with the pillbugs. The metal spreader distributed the drop of water to all places on the plate. The average number of colonies found from the swab method on each plate consisted of a range between 50-100. The dunk method had a range of 500-700 colonies. These results showed us that the dunk method is a more successful tool in finding the bacteria on pillbugs. 
 

  fig3 results of the swab colonies of bacteria (left) compared to the dunk method (right).
                            These results are typical; the dunk method has proved to be a more successful technique                             in gathering bacteria.

 
Chart 1 above represents the average amount of colonies collected during swab
            versus dunk techniques for the entire class.

Orange and Yellow Bacteria:

In this experiment we looked at the possibility of different colored bacteria reacting to an antibiotic differently. We decided to test the yellow and orange colonies. The antibiotic kanamycin was used. Each colony was lifted off the plate with a toothpick, and then knocked off in to a test tube filled with Luria Broth (3ml).  From the yellow solution we put two drops on two different agar plates and spread the solution evenly. We then placed one paper disk in the middle of each plate.  We then put a drop of water on one of the paper disk. On the other disk we put a drop of the antibiotic kanamycin. We repeated the same steps for the orange solution, leaving us with four plates in all. This is what we found in the results: The yellow bacteria treated with kanamycin left a 1cm ring around the disk which had a very clear appearance. The orange bacteria treated with kanamycin left a distorted circle with the smallest radius .5cm and the largest just a bit over 1cm. The appearance of the ring was cloudy. We concluded that the kanamycin had a stronger effect on the yellow bacteria. There was a consistent 1cm ring that was very clear showing that all the bacteria was eliminated in the region where antibiotic was applied.

Using Antibiotics

We now know that different colored bacteria reacts differently to an antibiotic. We decided to test eight different antibiotics on the yellow and orange bacteria. We took two drops of  orange solution, the solution was spread evenly on an agar plate. We repeated this process using the yellow solution. We labeled all four agar plates (two yellow solutions, two orange solutions) on the bottom, indicating their different regions with North, South, East and West. This was done to eliminate any confusion of later results. On one orange and one yellow plate the following antibiotic disks were placed in their own individual regions; kanamycin, carbenicillin, doxycycline and erythromycin. On the other orange and yellow plate, we place the following antibiotics; vancomycin, tetracycline, ciprofloxecin and ampicillin. Looking at the orange plates, the antibiotics vancomycin, ampicillin, poxycycline left a ring with a range of 1-2cm. They all had very clear appearances with little trace of bacteria. The antibiotic ciprofloxecin had no effect on the orange bacteria. Doxycycline, tetracycline, and vanconycin had the strongest effect on the yellow bacteria. They all left clear rings with radius' of 1cm. The antibiotic kanamycin showed very little effect killing the bacteria.



Spraying of Antibiotics:

Here we tested the effect of tetracycline on pillbugs in their environment. We went out and randomly gathered twenty pill bugs. We placed these pill bugs into two different plastic containers. Ten pillbugs at random were placed into the first container, and the other ten were placed in to the second container. The containers were filled with their environment. We sprayed one container with 6ml of distilled water. The other container was sprayed with 6ml of tetracycline antibiotic. One week later, we tested ten bugs from the water sprayed container using the dunk method. We also tested ten bugs from the antibiotic sprayed container using the dunk method. The dunk method was used because of it's proving effectiveness from past experiments. The container and pillbugs sprayed with the antibiotic had a range of 20-500 colonies found on the agar plates. The pillbugs sprayed with water had a range of colonies between 200-900. The results showed that the antibiotic had some effect on killing bacteria. However, bacteria was found in both the treated and untreated. The antibiotic lowered the amount of bacteria found on the pillbugs. Since the amount of colonies found had such a wide range, perhaps the technique of applying the antibiotic was unsuccessful. The data proves that the spraying of the antibiotic failed to eliminate all bacteria.
 

  Chart 2 above represents the average amount of colonies collected after applying antibiotics
 compared to the control group which was left untreated

Discussion of Results: [top]

 

After analyzing the results shown, one can conclude that bacteria is able to be collected, manipulated and controlled with the affective use of antibiotics. In early stages of the experiment, once pillbugs were collected, the most effective method of bacteria collection was hypothesized and found. Our results and visual data in figure 3 prove the dunk method was more effective than the swab when examining numbers of colonies of bacteria on each agar plate. The dunk method may be more accurate in collection of bacteria because the swab method requires more human contact which consequently, could lower the amount of bacteria that will spread to the q-tip. Additionally, the dunk method requires water which may serve as a conductor or lubricant for the bacteria to be emerged in. With less human interaction, pillbugs are able to be dunked into water and bacteria are then flushed off their exteriors into the water which has an equal amount of bacteria per space. (Change) In addition to graphs, and other data presented we conducted a T-test which is important because it helps determine the likelihood that two groups of data came from the same population. The result of our T-test was .0673, which is very close to 7%. This number has a value higher than .05 which tells us we cannot be completely sure that the two groups of data were taken from the same population.

When looking for a solution to the crisis at the University of Redlands, the data that reflects the amounts of bacteria after being sprayed with antibiotics would be very helpful. It is shown in chart 2 and in the spray test that the use of antibiotics does affect and in fact decrease the number of colonies of bacteria. However, to take this test as an indication that spraying an entire lawn or campus would be successful is not entirely accurate. Using tetracycline on a larger scale might be dangerous because affects of other living organisms in the soil was not measured nor was the half life of the antibiotic. Tetracycline may prove to cause damage, and if this antibiotic is very persistent in ground soil, serious problems could occur.

 The Hanta virus was seasonal in that it affected the most people during crop season when large numbers of farmers were exposed to the urine and fecal matter of the mice. This explanation might also prove that this bacterial illness is seasonal in that people are only affected during warm weather months when closely exposed to the grass and soil. Scientists working in this experiment have an advantage compared to the Hanta virus because an effective treatment can be obtained when dealing with bacteria. Our group would say when comparing and contrasting the data collected, we do not know enough about tetracycline to deem it an appropriate solution. Although it is stated that this antibiotic does decrease the number of bacteria on the pillbugs, the numbers are not substantial enough to risk the side effects that could possibly accompany this use. Until further tests can be conducted and more information gained it is safest to educate the public about this bacterial illness. Warn people to stay off the grass through out the campus, and wear closed toed shoes as possible protection from any bacteria. Most importantly, keep everyone informed as best possible at all times and continue to search for the best antibiotic that will serve as the solution.

 The Hanta virus, being a virus, does not have the luxury of finding a suitable antibiotic or treatment. However, the “baggage” that these pillbugs are carrying can be defeated through more testing and the findings of the most effective antibiotic.