The Effects of Environmental Factors on the Absorbance of Escherichia Coli
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Introduction
In this experiment, the gram negative bacterium Escherichia coli is being subjected to various environmental factors that affect the rate of growth. These factors scrutinized were the different types of nutrients, the intensity of aeration, or the temperature at which it was stored. The purpose of this lab is to determine which factor affects the Escherichia coli the greatest. It is known that these abiotic factors affect the rate of growth the greatest if they remain at the correct conditions for living.
Escherichia coli and other bacteria will go through four phases; a lag phase, log phase, stationary phase, and a death phase. In the lag phase, the bacteria reproduce fairly slowly, as they are preparing for the rapid division (Doyle). In this lag phase the bacteria is making fats and proteins which will jump-start the log phase (Doyle).
The next phase in the bacterias life cycle is the log (logarithmic or exponential) phase. At this point, the bacteria begin replicating swiftly. Once the culture reaches high densities, their living space and nutrients begin to deplete, and the toxicity levels begin to increase (Doyle).
Due to this rapid growth, the next step is the stationary phase. In this phase, about fifty percent of the new bacteria population will undergo autolysis and become inactive or die, and the other fifty percent will remain and continue replication (binary fission) (Doyle).
The last stage of a bacterias life cycle is the death stage. In the death stage, there are not enough nutrients for the entire culture to survive. This causes the death rate of Escherichia coli to increase, and the division of the living cells will slow down. In other words, the birth rate will be lower than the death rate, and this will be displayed in the graph at the leveling off, or downward slope. From this growth curve that a bacterium produces, the Mean Generation Time (MGT) can be calculated. Because Escherichia coli is a known bacterial organism that thrives in the human body inside the intestines, I hypothesize that the growth rate will be the fastest at 37oC because that is the average internal body temperature. I also hypothesize that the Mean Generation Time will be the lowest without aeration because there is a low amount of Oxygen inside the body. As well, I hypothesize that the MSGTYE will have the most rapid MGT out of the nutrients because that is the most common nutrient found in the true environment of the Escherichia coli.
Methods
After getting a brief introduction and informal “heads-up”, the class was divided into three groups. One group was to observe the effects of aeration on the growth of Escherichia coli; one was to observe the effects of temperature, and another to observe the effects of nutrients. Each group followed the procedures that were laid out for them by our instructor Jason and also the manual. My group was in charge of measuring the effects that the nutrients had on growth rate of Escherichia coli. We began the lab by getting a test tube rack with 6 tubes in it. Three of them were for the three types of media that we added to the medium (Escherichia coli). The other three test tubes contained blank medium, which were used to zero the spectrophotometer. Each different nutrient had its own blank flask because they were at slightly different levels. The next thing we did was put a tube of uninnoculated medium (used as a blank) to set the spectrophotometer at zero before any of the cultures were read. Then we measured the absorbency of MSG, by sticking it into the spectrophotometer at 600nm and recorded it. For the next media, we used a different blank test tube to zero the spectrophotometer, and again used a cultured test tube and took the absorbency reading at 600nm. We did this for the nutrient MSGTYE, with a different blank test tube. It was very important that we remembered to use the corresponding blank test tube for each type of media. After taking our initial readings, at 0 minutes, we waited for 15 minutes, and then took another set of readings. We still made sure to re-zero the spectrophotometer before each nutrient. We took readings every 15 minutes, until the class was over which was at the 90 minute mark.
For the temperature and aeration trials, the test tubes of Escherichia coli with tryptic soy broth (TSB) were measured using identical procedures as the nutrient trials were tested. Before every sample, we had to re-zero the spectrophotometer with a blank test tube, and then we took the reading from the cultured tubes. As we did for the nutrient experiments, we also took the readings from the temperature and aeration samples every 15 minutes. The temperature test tube was prepared by exposing it to room temperature (25oC), another test tube in an oven (33oC), and the test tube in a lab oven which was slightly warmer, at 37oC.
In the aeration samples, one flask was stationary and it was exposed to the atmosphere. The second sample was put in a regular flask, and placed in a shaking water bath. The third sample was placed in an irregular shaped flask, which was also placed in the quivering water bath. After treatment, all the samples were measured on the spectrophotometer, using the same procedure as the nutrient samples and temperature samples.
Results
The results showed that the experimental data did not necessarily display the lowest MGT. For the nutrient data, the MSG had a mean generation time of an hour, the MSGT had a doubling time of less than 13 minutes, and the MSGTYE had a mean generation time of about 20 minutes (Figure 1) (Table 1).
For the aeration experiment, the non-aerated flask had a mean generation time of about 74 minutes (Table 2). The baffled aerated flask had a mean generation of approximately 77 minutes, and the plain shaken flask had a mean generation time of also close to 80 minutes. This experiment actually had the control having the fastest mean generation time (Figure