Microbiology Lab Report 5 Optical Density PDF

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To determine bacterial abundance using optical density and dilutions and to determine the growth curves and generation times of E. coli at different temperatures using optical density...

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Microbiology 2400-L02 Laboratory Report 3 Sarah Kaczor Purpose To determine bacterial abundance using optical density and dilutions and to determine the growth curves and generation times of E. coli at different temperatures using optical density. Results Results – Experiment 5A Table 1 and Table 2: Bacterial abundance, absorbance and plate counts of suspensions of E. coli Test tube

Spectroscopy Bacterial abundance (CFU/mL)

B

Absorbance (660nm) 0.648

Plate counts Dilution 2 Dilution 3 Dilution 3 (100 µL) (1000 µL) (100 µL) TNTC TNTC 114

1.14E+9 C

0.466

TNTC

TNTC

101

0.328

TNTC

TNTC

53

0.225

TNTC

TNTC

54

0.200

TNTC

TNTC

35

0.126

TNTC

204

17

1.01E+9 D 5.3E+8 E 3.4E+8 F 3.5E+8 G 2.04E+6 Calculations for Table 1 and 2: Show the work for the calculations for CFU/mL for test tube B CFU/mL = 114 colonies/0.1 mL x 100/1 x 100/1 x 100/1 = 1.14E+9 CFU/mL Figure 1: Standard curve with my data. Plot Absorbance versus Bacterial Abundance (CFU/mL) using table 1and 2. (Attached).

Class data: Bacterial abundance and absorbance of suspensions of E. coli Test tube B C D E F G

Absorbance (660nm) 0.671 0.475 0.358 0.247 0.181 0.116

Bacterial abundance (CFU/mL) 8.46E+08 6.22E+08 4.17E+08 2.94E+08 2.89E+08 2.80E+08

Figure 2: Standard curve with the class data. Plot Absorbance versus Bacterial Abundance (CFU/mL) using the table above. (Attached). Results - Experiment 5B: Table 3: Optical density (and abundance) of sample of E. coli incubated at 32ºC Incubation time (min) 0 15 30 45 60 75 90 105 120 135

Optical Density (660 nm) 0.163 0.172 0.188 0.196 0.247 0.273 0.305 0.365

Bacterial Abundance (CFU/mL) – obtain numbers from figure 2 (show all work) 2.45E+08 2.55E+08 2.73E+08 2.82E+08 3.38E+08 3.67E+08 4.03E+08 4.69E+08

Class data: Class data for bacterial abundance at 32⁰C and 45ºC at 15 minute intervals. Incubation time (min) 0 15 30 45 60 75 90 105 120 135

Temperature 32⁰C 1.88 E+08 2.03 E+08 2.31 E+08 2.63 E+08 3.03 E+08 3.49 E+08 4.30 E+08 4.88 E+08 5.73 E+08

45ºC 2.01 E+08 2.42 E+08 2.81 E+08 3.58 E+08 4.17 E+08 4.72 E+08 5.85 E+08 6.75 E+08 7.25 E+08

Figure 3: Bacterial abundance (CFU/mL) versus time (min) using the table above – both temperatures on the same figure. (Attached). Table 4 [4]: Generation times of E. coli incubated at various temperatures.

Temperature

32⁰ C 37⁰ C 45⁰ C

Generation Time (minutes)

95 30 57

Figure 4: Generation time (min) versus temperature (°C) using class data. (Attached).

Discussion Questions 1. What differences do you see when you compare figure 1 (your own data) with figure 2 (class data)? Briefly explain what could lead to these differences. The R2 value for the class data is higher than the R2 value for my data. The class data was a better fit for the regression line than my data. There was also less difference between the bacterial abundance between each dilution in the class data compared to my data. This is because averaging the class data helped account for small human errors, such as not vortexing the tubes between dilutions or improper plating techniques. My data has larger differences in bacterial abundance between each dilution because of small errors I might have made during diluting and plating. 2. Compare the results obtained for generation time at the temperature you were assigned to that obtained at the other temperatures. Describe how generation time varies with temperature and explain in terms of cellular metabolism why this would occur. Generation time varies with temperature because all bacterial species have an optimum temperature for growth. Generation time is slowed down when the bacteria grows in a temperature different from their optimum temperature. This is because temperature affects the stability of the cell’s structure. (Kumar and Libchaber, 2013). When cultivated at 32°C, the generation time of E. coli was very slow. This is because its genome altered expression. A few of the genes involved in energy metabolism decreased their expression,

along with genes involved in glycolysis, PTS sugar transport systems and amino acid biosynthesis. Because these genes decreased their expression, energy was metabolized and transported slower, leading to a slower generation time. (Gagdil, Kapur, and Hu, 2005). Generation time was also slowed at 45°C. This is due to the bacteria’s proteins destabilizing and even denaturing. When the proteins denature, the cell is unable to divide. However, some cells can elongate to continue division and growth at 45°C. Other cells can overcome the stress of the high temperature and become resistant to it. Other cells inactivate. Because of the inactive cells, generation time decreases. But because of the resistant and elongated cells, generation does not stop completely. (Van Derlinden, Bernaerts, and Van Impe, 2007).

Works Cited Gagdil, M., Kapur, V., & Hu, W.S. (2005). Transcriptional response of Escherichia coli to temperature shift [Abstract]. Biotechnology Progress, 21(3), 689-699. doi: 10.1021/bp049630l Kumar, P. & Libchaber, A. (2013). Pressure and Temperature Dependence of Growth and Morphology of Escherichia coli: Experiments and Stochastic Model. Biophysical Journal, 105(3), 783-793. doi: 10.1016/j.bpj.2013.06.029 Van Derlinden, E., Bernaerts, K., & Van Impe, J.F. (2007). Dynamics of Escherichia coli at elevated temperatures: effect of temperature history and medium. Journal of Applied Microbiology, 104(2), 438-453. doi: 10.1111/j.1365-2672.2007.03592.x...