Abstract/Purpose:
The overall objective of this lab was to analyze patterns of microbial growth in closed system, directly perform measurements of bacterial growth curve through dilution method, and indirectly perform measurement of bacterial growth through spectrophotometer reading
Introduction:
There are two categories that describe the requirements in environment for bacteria growth, which can be divided into physical factors and nutritional factors. The physical aspect are things like temperature, pH, and pressure. Each species of bacteria has its requirements that it needs in order to optimally grow. Certainly, temperature plays a major role and bacteria can be classified according to the temperature in which they grow best in. Psychropiles grow best at -5 to 20 degrees celsius, mesophiles grow best from 20 to 45 degrees celsius and thermophiles grow best at 45 degrees celsius and above. Nutritional factors are things found in nature that the bacteria need in order to grow like carbon, nitrogen, water, and minerals. Each bacterium has its optimal growth conditions which are the environmental requirements that the cell grows optimally and it reproduces the best. Culture media can be classified into two categories based on their composition. An undefined medium is a rich medium that provides all the nutritional components necessary for the bacteria cell to grow and its chemical composition is unknown. A defined minimal medium provides on the essential nutrients and forces the cell to synthesize its metabolites. The Infusion-brain broth is an example of a rich medium and the glucose minimal salt agar is an example of a minimal medium. E. coli can reproduce every twenty minutes under its optimal growth conditions, and the growth curve of E. coli has 4 distinct stages.
The lag phase = Cell is introduced into the medium, and the fact that it has to use energy to adjust to its new environment so it impacts its growth by reducing the level of growth.
The log phase = Starts after the lag phase and in this stage the cell has already adjusted to the current environment and can grow exponentially.
The stationary phase = Is after the log phase and this is where there is an equivalent number of cells being formed and old cells dying at the same rate, so the medium cannot provide nutrients enough for the exponential growth rate.
The last stage is the death phase= Where the medium is nutrient poor, and result in more cell death than actually growth.We can determine the number of cells grown in a liquid medium by using a spectrophotometer to measure optical density. This device sends in a wavelength of light into the medium and records the amount of light scattered in the medium. Another method to calculate the concentration of bacteria in a liquid culture is to use a serial dilution. In a serial dilution you take volume of the original culture, which is called the stock culture, and dispense it into a larger volume of water or medium. This is the first dilution and from there you take a volume of the first dilution and dispense it into a larger volume of water or medium with the same dilution factor. Then you repeat the dilution until you dilute the stock culture so that you can take about 100 microliters of dilution to grow into a countable number of colonies. In order to predict the number of colonies we use the formula cells/mL=number of colonies/(dilution x amount plated). The dilution was determined by which dilution tube corresponded to the plate that we used to determine CFUs.
Methods:
For the first part of the experiment about 2.5 mL of Escherichia coli was transferred from the broth growing at 37 degrees Celsius and transferred into a cuvette and then placed into the spectrophotometer to get an optical density value (the spectrophotometer had to be blanked out with a sterile broth before collecting the reading) being indicated in the picture ahead.
The Escherichia coli from the cuvette was transferred into a tube. Now the tube that contained Escherichia coli from the previous step was used to perform a serial dilution. 5 microtubes were labeled 1-5 and closed caps when finish. The broth culture was mix and carefully transferred 10 microliters to dilution tube 1 and it was mixed well; this was a 10^-2 dilution. Aseptically transfer again 10 microliters from dilution tube 1 to dilution tube 2, mix well, this is a 10^-4 dilution. Aseptically transfer again 100 microliters from dilution tube 2 to dilution tube 3, mix well, this is a 10^-5 dilution. Aseptically transfer again 100 microliters from dilution tube 3 to dilution tube 4, mix well, this is a 10^-6 dilution. Aseptically transfer again 100 microliters from dilution tube 4 to dilution tube 5, mix well, this is a 10^-7 dilution. After mixing, transfer to 100 from dilution tube 2 to a plate which was another 1/10 dilution.
And follow the same technique to finish the plates. Then the plates were incubated from 28 to 48 hours at 37 C. For the second part of the experiment was to analyze and count the colonies and write the data. The plate I choose to count was the one that had colonies between 30 and 300 because some of the less diluted plates had too many colonies to count, and the most diluted plates had very few to count. Therefore, this plate that had the dilution intermediate was the best one to use to count the colonies.
Results:
Optical density of Escherichia coli at room temperature and 37 degrees Celsius
Time Taken (minutes)
OD
Group
0
0.022
25
0.024
45
0.024
65
0.022
85
0.051
105
0.088
125
0.143
135
0.23
149
0.248
175
0.373
G1
Jackie & Luz
193
0.485
G2
Megan & Tircia
224
0.584
G3
Ana & Marisa
245
0.633
G5
Michelle & Karin
268
0.66
G4
Eliane & Natan
302
0.738
321
0.765
341
0.792
365
0.832
386
0.863
410
0.892
440
0.941
463
0.968
487
1.002
509
1.012
532
1.026
Calculations:
OD
To calculate the growth rate constant use the standard formula
And the generation time is simply the inverse of k
N1=0.143
N2=0.248
T=24 min
k=19.09
g=1/19.09
g=0.052 minutes
Time Sampled
Dilution
Factor
CFUs
Cell Density
268 min
10^-8ml
150
 150/10^-8ml
 1.5×10^10cells/ml
193min
10^-7ml
98
 98/10^-7ml
 9.8×10^8 cells/ml
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Discussion:
At 268 minutes was taken the OD reading for our group which had a OD=0.666, and used a dilution factor of 10^-8ml for plate D that was used to get the CFU number of 150 which resulted in the OCD = 1.5×10^10 cells/ml. Alternatively, our class mates Megan and Tircia obtained at 193 minutes a OD reading of 0.485 and they used plate C that had a dilution factor of 10^-7ml to obtain the CFU of 98 which resulted in OCD = 9.8×10^8 cells/ml
Conclusion:
Overall the conclusions that I arrived with the data that I collected was that Escherichia coli grows much better at 37 degrees Celsius and that it also grows better in a rich medium such as the agar plate. The objective of the experiment was fulfilled since we were able to see how the growth of Escherichia coli differed in different environments by using the direct method through dilutions, and the indirect method by simply reading the spectrophotometer Also we are able to see through this experiment that Escherichia coli can be described as a prototroph since it is able to grow in both the medium, and we were we able to clearly observe the distinct phases of bacterial growth by analyzing the graph.
References:
Scheurle, Daniela. General Microbiology. 1st ed. Plymouth: Hyden-McNeil, 2014. Print.
“E. Coli.” E. Coli. Foodsafety.gov, n.d. Web. 01 June 2015.