Materials:The materials used in each part of the procedure varied. Different materials were needed for different parts. The materials needed to perform Part 1: Making LB Agar were: four 250 milliliter media bottles and four black caps, a 1000 milliliter beaker, a scoopula, weigh boats, LB Agar base, a balance, distilled water, a hot plate, a magnetic stirrer, autoclave tape, label tape, and a marker. The next part was Part 2: Making LB Broth. The materials needed were: a 125 milliliter media bottle and a black cap, LB Broth base, a balance, weight boats, distilled water, a hot plate, autoclave tape, a scoopula, a 200 milliliter beaker, a magnetic stirrer, label tape, and a marker. After that, was Part 3: Pouring Plates. The materials needed were: fourteen Petri plates, four bottles of LB Agar, a hot hand, a microwave, parafilm, and a timer. The materials in Part 4: Making a Plate Culture were: two LB Agar Petri plates, E. coli 101, B. subtilis, a flame stick, an inoculating loop, parafilm, and an incubator set at 37oC. Next came Part 5: Making a Broth Culture. The materials needed were: the E. coli 101 plate culture, the B. subtilis plate culture, two 15 milliliter centrifuge tubes, a test tube rack, LB Broth, an inoculating loop, a flame stick, a p-1000 micropipette, micropipette tips, and an incubator set at 37oC. The materials needed to carry out Part 6: Making Concentrated Salt Water were: sodium chloride (NaCl), distilled water, a balance, weigh boats, a scoopula, six 200 milliliter beakers, and 36 filter paper disks. Lastly, the materials needed in Part 7: Making Test/Experiment Plates were: the salt water soaked filter paper disks, twelve LB Agar plates, L-spreaders, the E. coli 101 broth culture, the B. subtilis broth culture, parafilm, a marker, and an incubator set at 37oC.
Methods: In this experiment, there were two independent variables: the type of bacteria used (E. coli 101 & Bacillus subtilis) and the concentration of the salt water (0%, 1%, 3.5%, 7%, 15%, 25%). The dependent variable was the zone of inhibition caused by the salt water. The constants were temperature, light, amount of LB Agar on plate, and amount of bacteria spread on plate. There were three trials, and each salt concentration for each bacterium had three replicates. The procedure was broken up to alleviate much confusion, and it consisted of seven parts.
Part one was making the LB Agar. To decrease the time it took to create the agar, instead of making 125 milliliters at a time, 500 milliliters was created at once. Four 250 milliliter media bottles and four black caps were washed thoroughly to rinse off any remaining substances and lessen the chances of contamination. Then, the desired amount of the LB Agar base was weighed out on a standard tabletop balance in a weigh boat. (See calculations, page) A 1000 milliliter beaker was filled with 500 milliliters of water. The LB Agar base was poured in slowly while the mixture was being gently stirred. This was to prevent any clumps that could have formed. Then, a magnetic stirrer was gently slid down the side of the beaker into the mixture. This prevented any splashing that could have occurred if the stirrer was just dropped in. The beaker was placed on a hot plate that was set to ¾ heat and the magnetic stirrer was set to stir at a slow-medium speed. This was done until the mixture cleared up. However, the mixture was not allowed to boil. If it had boiled, it would have been ruined. After the LB Agar cleared, it was divided into 125 milliliters in the four media bottles that were cleaned previously. The bottles were loosely capped and labeled with name, date, section, and LB Agar. One inch of autoclave tape was applied to the caps, and the bottles were placed in the designated spot to be autoclaved.
Part two was making the LB Broth. A 125 milliliter media bottle and a black cap was washed thoroughly because there might have been some substances remaining in the bottle. The desired amount of LB Broth base was measured out on a standard table-top balance in a weight boat. (See calculations page) Then, fifty milliliters of water was obtained in a 200 milliliter beaker. The LB Broth base was slowly poured into the water while it was being gently stirred to avoid clumping. A magnetic stirrer was slid down the side of the beaker into the mixture, so the mixture wouldn’t splash out. The beaker of LB Broth was heated on a hot plate at low heat while it was being stirred at a slow pace. It did not take very long for the mixture to clear up. After it cleared, the LB Broth was poured into the previously cleaned 125 milliliter media bottle and then loosely capped. The bottle was labeled with name, date, section, and LB Broth. Lastly, one inch of autoclave tape was applied to the cap, and the bottle was placed in the designated spot to be autoclaved.
Part three was to pour the LB Agar that was previously made into Petri plates. To do so, fourteen large Petri plates and four bottles of LB Agar were obtained. The LB Agar was heated in the microwave one bottle at a time. They were heated at 15 second intervals to prevent the LB Agar from boiling. When the LB Agar was fully melted with no clumps, it was left to cool for about one or two minutes. After the LB Agar was cool enough to touch, the lip of the bottle was flame sterilized with a flame stick, and the agar was poured into the Petri plates. The dime size technique was used to attain the optimal amount of agar in the plates. Once there was no more agar left in the bottle, it was washed with water and a new bottle was heated and poured. After all the plates had been poured, they were covered and left in a dry area for about 15 minutes so the LB Agar could solidify. Once they were cooled, all the plates were parafilmed, stacked into threes and left in a dry, undisturbed area. The plates needed to sit for about 24 to 48 hours before they could be used.
Creating plate cultures was part four of the procedure. Two LB Agar Petri plates that were made beforehand were obtained. The laboratory supervisor provided E. coli 101 and Bacillus subtilis, the two bacteria needed to make the plate cultures. An inoculating loop was flame sterilized before it was used. It was used to obtain about one milliliter (about one colony) of the desired bacteria. Using the Z-technique, the bacteria was applied to the Petri plate. After the streaking was finished, the plate was covered and parafilmed and placed in the incubator at 37oC. Then, the other bacteria plate culture was made following the same steps. The plate cultures needed to sit for at least 24 to 48 hours before use.
Making broth cultures out of the plate cultures was part five of the procedure. The pre-made plate cultures for E. coli 101 and Bacillus subtilis was obtained from the incubator, and the LB Broth was obtained from the autoclave machine. The lip of the LB Broth media bottle was flame sterilized to prevent contamination. Using a p-1000 micropipette, 10 milliliters of LB Broth was inserted into each of the two centrifuge tubes. Then, the inoculating loop was sterilized and one milliliter (about one colony) of the desired bacteria (E. coli 101 or B. subtilis) was obtained. The bacteria was inserted into one of the centrifuge tubes, and the loop was swirled around in the LB Broth to ensure that there were no chunks of bacteria floating in the LB Broth. The centrifuge tube was capped and incubated at 37oC for 24 to 48 hours. Then, the other bacteria species was added into the other centrifuge tube and the same steps were followed.
Part six of the procedure was making different concentrations of salt water. Sodium chloride, which is common table salt, was obtained from the prep room in the laboratory. Then, the amount of sea salt needed for 100 mL of each concentration (0%, 1%, 3.5%, 7%, 15%, 25%) was calculated. (See calculations page) Six 200 milliliter beakers were filled with 100 milliliters of distilled water. The desired amount of sodium chloride was weighed out using a standard table top balance. The salt and the water were mixed together to create the desired concentrations of salt water. Six paper filter disks were added to each beaker, and they were soaked overnight.
The final part of the procedure was creating the experimental test plates. The remaining twelve previously made Petri plates, E.coli 101 and B. subtilis broth cultures and the salt water soaked filter paper disks were brought out to be used in this final part. The Petri plates were labeled with the type of bacteria that was going to be applied, the date, name, and then, it was sectioned into three. For each bacteria, there were six plates. The sections on three of the plates were labeled with 0%, 1% and 3.5%, and the other three were labeled with 7%, 15%, and 25%. One milliliter of bacteria was applied onto each designated plate. There were left, undisturbed for about 10 to 15 minutes. Then, the soaked filter paper disks were applied onto their designated section on the Petri plates. The lids were closed and the plates were left to sit for about five minutes. Then, they were parafilmed and placed in the incubator at 37oC.
The experimental test plates were observed over the course of three days, and the zones of inhibition in each section of the plates caused by the salt water was measured and recorded daily. The zones of inhibition were measured using a standard metric ruler in millimeters. Also, pictures of each individual plate were taken and qualitative observations were written down. After the course of three days, all the necessary data was collected and as a method of statistical analysis, the mean, median, mode, and range were calculated into a chart. The tables, graphs, and charts that display the information can be found on page.
Calculations:LB Agar
4.38 grams/125 mL = x grams/500 mL
125x = 500(4.38)
125x = 2190
x = 2190/125
x = 17.52 grams
LB Broth
Salt Water Concentrations
0% = 100 mL + 0 grams of NaCl
1% = 100 mL + 1 gram of NaCl
3.5% = 100 mL + 3.5 grams of NaCl
7% = 100 mL + 7 grams of NaCl
15% = 100 mL + 15 grams of NaCl
25% = 100 mL + 25 grams of NaCl
Results:Figure 1.1: E. coli 101 Day 1
Figure 1.2: E. coli 101 Day 2
Figure 1.3: E. coli 101 Day 3
Figure 1.4: E. coli 101 Statistics (of Zone of Inhibition)
Figures 1.1, 1.2, and 1.3 display the daily records of the zones of inhibition for E. coli 101. Each day of observation, the zones of inhibition were measured and pictures were taken of each individual plate. The largest zone of inhibition for the E.coli 101 was measured to be 17 millimeters which was on Day One. The smallest zone of inhibition was 0 millimeters. Multiple sections and trials had no zone of inhibition particularly on Day Three.
The statistics, provided in Figure 1.4, display the mean, median, mode and range. All of these components are an essential part of the statistical analysis. The mean shows the average zone of inhibition for each day, and the median displays neither the smallest nor the largest, sort of like a happy medium. The mode exhibits the most common size of the zone of inhibition for each day, and the range shows how far apart the smallest and the largest zone of inhibition for each day was. The largest mean zone of inhibition for Day 1 was 13 millimeters (by 0%), for Day 2, it was 12 millimeters (by 0%), and for Day 3, it was 8 millimeters (by 0%).
Some qualitative observations were also recorded and pictures were taken to display these observations. The pictures shown below in Figure 5.1 are of Trial 1: E. coli 101 on Day One. There was a definite lawn of growth with inhibition displayed around the filter paper disks that had been previously soaked in different concentrations of salt water. Although, it is not clear in the picture, there was definitely a zone of inhibition.
Figure 5.1: E. coli 101; Trial 1; Day 1
Figure 2.1: B. subtilis Day 1
Figure 2.2: B. subtilis Day 2
Figure 2.3: B. subtilis Day 3
Figure 2.4: B. subtilis Statistics (of Zone of Inhibition)
Figures 2.1, 2.2, and 2.3 display the daily records of the zones of inhibition for Bacillus subtilis. Each day of observation, like the E. coli 101, the zones of inhibition were measured and pictures were taken of each individual plate. The largest zone of inhibition for the Bacillus subtilis was measured to be 20 millimeters which was on Day One. That is three more millimeters than the largest zone of inhibition for E. coli 101. The smallest zone of inhibition was 0 millimeters. Like the E. coli 101, multiple sections and trials had no zone of inhibition particularly on Day Three.
The statistics, provided in Figure 2.4, display the mean, median, mode and range. The largest mean zone of inhibition for Bacillus subtilis on Day 1 was 15.7 millimeters (by 15%), for Day 2, it was 12.7 millimeters (by 7% & 15%), and for Day 3, it was 8 millimeters (by 15%). From these results, it can be concluded that 15% salt concentrated water is most effective in inhibiting the growth of Bacillus subtilis.
Some qualitative observations were also recorded and pictures were taken to display these observations. The pictures shown below in Figure 6.1 are of Trial 1: Bacillus subtilis on Day One. There was a definite lawn of growth with inhibition displayed around the filter paper disks that had been previously soaked in different concentrations of salt water. Although, it is not clear in the picture, there was definitely a zone of inhibition.
Figure 6.1: Bacillus Subtilis; Trial 1; Day 1
Figure 3.1: E. coli 101 (Avg. Zone of inhibition vs Concentration of salt water) Day 1
Figure 3.2: E. coli 101 (Avg. Zone of inhibition vs Concentration of salt water) Day 2
Figure 3.3: E. coli 101 (Avg. Zone of inhibition vs Concentration of salt water) Day 3
These graphs show the trend of lower concentrations of salt water inhibiting bacterial growth to higher concentrations of salt water inhibiting bacterial growth. From Figure 3.1, it can be concluded that 25% concentrated salt water inhibited more bacterial growth than any other concentration. Of course, this is only day one. Looking at Figures 3.2 and 3.3, it can be concluded that the bacterial inhibiting effects of the 25% concentrated salt water lessens each day by quite a bit, whereas the 0% concentrated salt water (also known as distilled water) consistently stayed higher up meaning that it consistently inhibited bacterial growth without dropping in its effects by a large amount. In Figure 3.2, the bacterial inhibiting effects of the 3.5%, 7% and 15% concentrated salt water seem to be on the same level. But in Figure 3.3, it is visible that only the 3.5% concentrated salt water kept its bacterial inhibiting effects.
Figure 4.1: B. subtilis (Avg. Zone of Inhibition vs. Concentration of salt water) Day 1
Figure 4.2: B. subtilis (Avg. Zone of Inhibition vs. Concentration of salt water) Day 2
Figure 4.3: B. subtilis (Avg. Zone of Inhibition vs. Concentration of salt water) Day 3
These graphs display the trend of which the average zone of inhibition follows. From Figure 4.1, it can be concluded that 15% concentrated salt water was more effective in inhibiting bacterial growth than any other concentration. But it is only day one. The next two days afterwards are shown in Figures 4.2 and 4.3. Looking at Figures 4.2 and 4.3, it can be concluded that the bacterial inhibiting effects of the 15% concentrated salt water lessens each day little by little, but not so much that it would have an immense impact. The 3.5% concentrated salt water seems to lessen in its ability to inhibit bacterial growth quite a bit each day. In Figures 4.1, 4.2, and 4.3, the 3.5% concentrated salt water is consistently the lowest for inhibiting bacterial growth. 3.5% concentrated salt water is about the same salinity of sea water. With these results, it can be concluded that Bacillus subtilis may be a potential candidate for helping vegetation cope with salt stress, especially near oceanic ecosystems.
Discussion:In this lab, the purpose was to determine whether or not higher concentrations of salt water would not inhibit the growth of bacteria, specifically E. coli 101. The results proved that higher concentrations of salt water would still inhibit bacterial growth. However, although higher concentrations of salt water still inhibited the growth of the bacteria, the Bacillus subtilis was able to grow in 3.5% concentrated salt water which is the most common concentration associated with seawater. As displayed in Figures 4.1, 4.2, and 4.3, the average zone of inhibition for Bacillus subtilis for the 3.5% concentrated salt water decreased significantly each day. In the E. coli 101, the 7% concentrated water was the least effective in inhibiting bacterial growth. As displayed in Figures 3.1, 3.2, and 3.3, the average zone of inhibition for the 7% concentrated salt water decreased significantly each day. The concentration that was most effective in inhibiting bacterial growth in E. coli 101 was 0%, or distilled water. This was unexpected because, normally, water would not inhibit any bacterial growth. In the Bacillus subtilis, the most effective concentration of salt water was 15%. The average zones of inhibition were consistently higher than the other concentrations of salt water.
To answer the research question, the E. coli 101 could withstand higher concentrations of salt water than the Bacillus subtilis. The E. coli 101 could withstand twice the concentration of salt water that Bacillus subtilis could. (E. coli 101 = 7%; B. subtilis = 3.5%) However, the hypothesis was not supported because, in fact, the concentration of salt water that least inhibited the bacterial growth in E. coli 101 was 7% concentration of salt water. 7% is a much lower concentration than 25%, and yet it did not inhibit the bacteria as much as the 25% concentration did. On the final day of observations (Day 3), the average zone of inhibition for 25% concentrated salt water was 3.3 millimeters, whereas for 7%, it was 3 millimeters. This is displayed in Figure 1.4. There is not a big difference between the two measurements, but when it comes to deciding on which salt concentration would inhibit more bacterial growth, the 25% concentration would definitely be the answer.
However, for the Bacillus subtilis, on the final day, there were two concentrations that least inhibited the growth of the bacteria, 3.5% and 25%. One concentration was on the lower side, whereas the other concentration was on the much higher side. In Figure 2.4, the average zone of inhibition for both concentrations was 3.3 millimeters. In the E.coli 101 bacteria, the 25% concentrated salt water had a zone of inhibition of 3.3 millimeters. The highest concentration of salt water seems to be the most consistent, although not the least inhibiting, with the zones of inhibition.
Some sources of errors that may have occurred during this lab are contamination and not adding the correct amount of salt in each salt solution. There was definitely contamination in the test plates. There were small red colonies growing on almost all the plates. Neither E.coli 101 nor Bacillus subtilis is known to have red colonies. However, the cause of the contamination is yet to be determined. The incubator, in which the plates were placed, might have been contaminated by the other bacteria plates that were being grown inside it. However, the contamination can be concluded to not have a significant effect on the experiment. The red colonies did not grow near the zone of inhibition except on one plate, but that may have been a fluke. Another source of error may have been not making the correct concentration of salt water. Multiple times, the scoopula used to obtain the salt was not rinsed after mixing one solution. The salt water could have adhered to the scoopula, and then it would have been transferred into the other salt water mixtures. This may have not affected the concentration of the water significantly, but it would have skewed the results at least by a little. Also, when the salt was measured out in the weigh boats, the salt adhered to the sides of the weigh boat and not all of the salt was poured into the water. This would also alter the concentration of the salt water.
To improve the project, some steps that could be taken are to reduce the amount of contamination risk and make exact concentrations of salt water. These little steps could greatly affect the outcome. To reduce contamination, the inside of the incubator could be bleached to kill of any lingering bacteria and only one experiment should go on inside the incubator. This would lessen the chances of there being contamination in the test plates. Also, to make exact concentrations of salt water, the salt could be measured in the beakers themselves. When water is added, all the salt will be accounted for. This would help to make exact, if not accurate, concentrations of salt water.
In this experiment, it was discovered that Bacillus subtilis would be the better candidate for helping vegetation cope with salt stress from the ocean water. Although E. coli 101 can also cope with higher concentrations of salt water, the Bacillus subtilis was able to withstand the salt concentration that is most commonly associated with the ocean water. Sea water is usually 3.5% concentrated salt water and the Bacillus subtilis was able to grow even in this concentration. However, the bacteria was inhibited by the salt water for the first two days. But on the final day, B. subtilis flourished in the plate.
To better this experiment, the next step would be to expand this experiment and add in vegetation as a factor. It has been concluded from this experiment that Bacillus subtilis would be the prime candidate for helping vegetation cope with salt stress. So, if there was a way that would allow the Bacillus subtilis to flourish on the vegetation of choice, then the vegetation could be watered with the salinity of sea water (3.5%) or could attempt to be grown in the sea water itself. This would further test the coping abilities of the Bacillus subtilis to deal with a saline environment. However, a couple flaws in this experiment would be that it would be difficult to measure the amount of bacteria growing on the plant, and it is probably not possible to measure of zone of inhibition. The experiment is still a work in progress, but if further research was done, the results may better the environment.
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