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Lab Report for Control by Heat and UV Light....

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Branisha Wilson Biol 2310 Lab Report 2 June 29th, 2018 Control by Heat and UV Light Introduction: The control of microbial growth can be determined by various factors including heat and ultraviolet (UV) light. Heat and UV light will either kill the microorganisms completely or inhibit the growth. Heat has thorough effects on the physiological and structural characteristics of bacteria and is categorized into dry and moist heat sterilization. Dry heat consists of hot air or fire being used to sterilize materials at a high temperature. This process eliminates microbes by destructive oxidation which destroys the proteins, bacterial spores, and necessary factors in bacteria that allow it to survive. Because dry heat uses an environment that has a relative humidity less than 100%, it is less effective compared to moist heat which has a relative humidity of 100% (Fox & Pflug, 1968). Moist heat consists of water being used at high pressure to inhibit the growth and kill bacteria; however, it is at a lower temperature than dry heat. This process denatures and coagulates proteins, destroys enzymes and DNA strands, and causes loss of cell membrane function in the microorganism. Typically, bacterial spores are resistant to temperatures that are 45 degrees Celsius higher than the optimal temperature for their living conditions. The factors causing the resistance are the spore’s level of dipicolinic acid (DPA) which composes 10% of the bacterial spore’s dry weight, the cations affiliated with DPA, and the optimal temperature of the

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microorganism (Coleman et al., 2007). With moist heat, in comparison to dry heat, sterilization occurs at a shorter amount of time with a lower temperature. UV light is non-ionizing radiation which applies a mutagenic effect that ultimately destroys the DNA of bacteria. This process occurs by the mutagenic effect causing exciting electrons in DNA to form excess bonds within thymine. Eventually, the bonds produce pyrimidine dimer that induce malfunctions in the replication process of DNA and destroy the bacteria (Furlong, 2012). UV light is used to sterilize or disinfect the work places of food processing areas, hospital operating rooms, hospital equipment air purification, and aquarium maintenance (Bank et al., 1990). UV light varies in its effectiveness depending on the bacteria used against it. Materials and Methods: First, the effects of temperature on bacteria lab was performed. The three organisms Serratia marcescens, Escherichia coli, and Bacillus subtilis, three TSA plates, an inoculating loop, Bunsen burner, Vortex mixer, and striker were obtained. The three plates were divided into three sections with a marker for each organism. After dividing the plates, inoculation was performed on all three organisms for each section of the plate for all three plates and the inoculating loop was sterilized each time the organism was changed. A zig zag motion of the bacteria was spread across each section of the plate. Once all three plates were inoculated, each was placed in its own designated location. One was placed invertedly in the incubator at 37 degrees Celsius, another in the refrigerator at 10 degrees Celsius, and the last one into a box at room temperature (25 degrees Celsius). The plates were left overnight to allow growth of organisms.

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Second, the effects of UV light on bacteria lab was performed. The two organisms Escherichia coli and Bacillus subtilis were used in this experiment along with two TSA plates, a stainless-steel cell spreader, ethanol, a pipette, pipette droppers, Bunsen burner, striker, and a Vortex mixer. The two plates were divided into two sections with a marker and inoculated with bacteria on the entire plate by using the pipette to retrieve 200 microliters and dropping it onto the center of the plate. One plate was specifically for Escherichia coli and the other was for Bacillus subtilis. When changing to the other bacteria, the pipette dropper was changed to a new one. When inoculating, the bacteria was spread by the stainless-steel cell spreader all over the entire plate. The stainless-steel cell spreader was sterilized by dipping it into ethanol and holding it into the Bunsen burner flame after switching to the opposite bacteria. Next, each plate was placed under the UV light for five minutes each with one half of the plate under the light and the other half covered with paper. Prior to placing the plate under the UV light, the plate’s lid was removed. After both plates were done under the UV light, they were placed in the incubator invertedly at 37 degrees Celsius. Lastly, the control by heat lab was performed. The two organisms Escherichia coli and Bacillus subtilis were used along with eight TSB tubes, a pipette, and pipette droppers. The pipette was used to retrieve 200 microliters of bacteria and drop it into each TSA tube. Four TSB tubes were inoculated with Escherichia coli and the other four were inoculated with Bacillus subtilis. When inoculating the tubes with the pipette, the pipette droppers were changed each time. Each group was assigned a specific time and temperature and each tube was placed into the water bath. The times varied from 10-40 minutes and the temperatures ranged from 40-100 degrees Celsius. After inoculation, the tubes were placed invertedly into the incubator and left overnight.

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Results: After incubation for all tubes and plates were completed, they were removed from the incubator, refrigerator, and room temperature box for observations. For the effects of temperature on bacteria plates, the plate that was in the room temperature box displayed the most growth. The colony morphology of the Serratia marcescens section of the plate consisted of a bright red color with the middle of the colony with a lighter red hue, the form was punctiform in the middle and circular bordering the middle, the elevation was flat, the margin was entire, and there was a significant amount of growth present. The colony morphology of Escherichia coli consisted of a dull tan color with one tiny red dot in the middle of the colony, the form was punctiform in the middle and circular bordering the middle, the elevation was flat, the margin was undulate, and there was growth present, but the most growth occurred at the border around the middle of the colony. The colony of morphology of Bacillus subtilis consisted of a brighter tan color than E. coli and had 5-6 red dots on the far-left side of plate, the form was punctiform, filamentous, and circular, the elevation was flat, the margin was lobate, and there was a significant amount of growth. The plate from the incubator displayed growth; however, it had less growth than the room temperature plate. The colony of morphology of Serratia marcescens consisted of a dull red hue with a border of dull tan color, the form was circular throughout, the elevation was raised at the edges and flat in the middle, the margin was entire, and there was growth present that covered the entire section, but it was not as strong as the growth at room temperature. The colony of morphology of E. coli consisted of a white color in the middle and a yellow/tan color at the edges, the form was punctiform in the middle of the colony and circular on the outside border, the elevation was flat in the middle and raised at the borders, the margin was undulate, and the

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growth of the organism covered the entire section. The colony of morphology of Bacillus subtilis consisted of a light tan color, the form was filamentous and irregular, the elevation was flat, the margin was filamentous and lobate, and the growth of the organism covered the entire section of the plate. The plate from the refrigerator had no growth present. For the UV light plates, the plate with Bacillus subtilis displayed less bacteria growth on the UV light half. The plate with Bacillus subtilis had growth on the control side of the plate and also had growth on the UV light exposed side, but there was less present. The UV side displayed huge, filamentous colonies at the edge of the plate while towards the middle there was no growth present. The plate with E. coli had growth on the control side of the plate and also had growth on the UV light exposed side, but there was less present. The UV side displayed small, round, circular colonies that stopped growing midway of the plate. For the control by heat lab, all results from each group were recorded. The E. coli test tubes set in the water bath at 40 degrees Celsius had growth present at 10, 20, 30, and 40 minutes. At 55 degrees Celsius, the tubes had a turbid solution which showed growth at 10, 20, and 30 minutes, but had no growth at 40 minutes. At 80 degrees Celsius, the tubes had no growth at 10, 20, 30, and 40 minutes in the water bath. At 100 degrees Celsius, the tubes had no growth at 10, 20, 30, and 40 minutes in the water bath. As for the Bacillus subtilis test tubes, there was growth present at 40 degrees Celsius for 10, 20, 30, and 40 minutes. At 55 degrees Celsius, the tubes had growth present at 10, 20, 30, and 40 minutes. At 80 degrees Celsius, the tubes began to display no growth at 10, 20, 30, and 40 minutes in the water bath. At 100 degrees Celsius, there was no growth present in all tubes.

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Figure 1. Effects of Temperature (room temperature) on Gram (+) bacteria, Bacillus subtilis, Gram (-) bacteria, Escherichia coli, and Gram (-) bacteria, Serratia marcescens (Wilson, 2018)

Figure 2. Effects of Temperature (incubator 37 degrees Celsius) on Gram (+) bacteria, Bacillus subtilis, Gram (-) bacteria, Escherichia coli, and Gram (-) bacteria, Serratia marcescens (Wilson, 2018)

Figure 3. Effects of Temperature (refrigerator) on Gram (+) bacteria, Bacillus subtilis, Gram (-) bacteria, Escherichia coli, and Gram (-) bacteria, Serratia marcescens (Wilson, 2018)

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Figure 4. Effects of UV Light on Gram (-) bacteria, Escherichia coli (Wilson, 2018)

Figure 5. Effects of UV Light on Gram (+) bacteria, Bacillus subtilis (Wilson, 2018)

Table 1. Bacterial Growth Based on Temperature

Time (min) Escherichia coli Bacillus subtilis

40℃ 55℃ 80℃ 100℃ 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 + + + + + + + + + + + + + + + -

(+) = growth; (-) = no growth Conclusion:

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Heat, UV Light, and temperature all had major effects on the killing or inhibiting of growth of the various bacteria used in this experiment. In the effects of temperature on bacteria lab, the room temperature plate had the most growth present. This occurred because the optimal temperature for Serratia marcescens, Escherichia coli, and Bacillus subtilis was at room temperature. Optimal temperature for bacteria allows it to survive and grow at its best capacity and is also the temperature where the generation time is the fastest. The generation time is responsible for the total amount of cells present after a certain amount of time. It was intriguing observing the refrigerator plate with no sign of bacterial growth. The temperature in the refrigerator inhibited the growth of bacteria due to the bacteria being unable to survive in the cold temperatures. In this experiment, the bacteria we worked with would be considered mesophilic because the optimal temperature was between 15 and 50℃ (Jenson et al., 2012). In the UV light experiment, the Bacillus subtilis plate displayed more resistance than the Escherichia coli plate. This occurred because Bacillus subtilis contains endospores that make it more resistant and E. coli does not have endospores making it less resistant to the UV light. The different effects of the UV light also occurred because Bacillus subtilis is gram-positive bacteria, while E. coli is gram-negative bacteria. The UV light inhibited the growth of both bacteria by destroying its cell wall. Although the UV light was effective inhibiting the growth of the bacteria, it was not the most effective heat process performed throughout the experiment. The control by heat experiment proved that there are thermal death points and thermal time points for each bacterium used. The thermal death point is the time at which all bacteria is killed at the 10-minute mark and thermal death time is the time at which it takes to kill all bacteria. For E. coli, the thermal death time was 55℃ at 40 minutes and the thermal death point

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was 80℃ at 10 minutes. For Bacillus subtilis, the thermal death time was 80℃ at 10 minutes and the thermal death point was also 80℃ at 10 minutes. This experiment proved that bacteria can be controlled by heat and UV light. The use of these techniques makes it easier to manipulate the harmful bacteria in the world. Knowing which bacteria are resistant at a given temperature aids with keeping people healthy and protected from harmful bacteria. Although the effect of heat is more beneficial than the affect of UV light, both can be used to inhibit the growth of bacteria. However, lack of sensitivity to these procedures can eventually lead to a population of bacteria that is resistant. It is important to test how heat and UV light control these bacteria to prevent that from happening.

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References Bank, H. L., Schmehl, M. K., John, J., & Dratch, R. J. (1990, December 01). Bactericidal Effectiveness of Modulated UV Light. Retrieved June 29, 2018, from http://aem.asm.org/content/56/12/3888 Coleman, W. H., Chen, D., Li, Y., Cowan, A. E., & Setlow, P. (2007, December). How Moist Heat Kills Spores of Bacillus subtilis. Retrieved June 29, 2018, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2168948/ Fox, K., & Pflug, I. J. (1968, February). Effect of Temperature and Gas Velocity on the Dry-Heat Destruction Rate of Bacterial Spores. Retrieved June 29, 2018, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547409/ Furlong, M. (2012). Effects of UV Light Exposure on Bacteria. Retrieved June 29, 2018, from http://www.clayton.edu/portals/438/microsoft word - uv lab.pdf Jensen, D. B., Vesth, T. C., Hallin, P. F., Pedersen, A. G., & Ussery, D. W. (2012). Bayesian Prediction of Bacterial Growth Temperature Range Based on Genome Sequences. Retrieved June 29, 2018, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3521210/...