.. g the results of the tests, we made graphs to determine the mean generation time. My group was in charge of measuring the effects that the nutrients had on growth rate of E. coli. We started by getting all our flasks together.
They were already prepared with the medium because of our limited lab time. We had six different flasks. Three of them were for the three types of media that we added to the medium (E. coli). The other three flasks contained blank medium. These blanks were used to zero the spectrophotometer, and each different nutrient had it’s own blank flask.
So, the first thing we did was place a tube of the blank medium in the spectrophotometer and set the absorbance needle on zero. Then we added MSG (4 mL), by a use of a pipet, to a cultured flask and took the absorbency reading and recorded it. For the next media, we used a different blank flask to zero the spectrophotometer, and again added 4 mL to a cultured flask and took the absorbency reading at 600nm. We did this for all three of the nutrients; MSG, MSGT, and MSGTYE. It was very important that we remembered to use the corresponding blank flask for each type of media. After taking our initial readings, at 0 minutes, we waited for 15 minutes, 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. For the temperature and aeration trials, the flasks of E. coli with tryptic soy broth (TSB) were measured the same way that the nutrient trials were tested. Before every sample, we had to re-zero the spectrophotometer with a blank flask, and then we took the reading from the cuvettes.
As we did for the nutrient experiments, we also took the readings from the temperature and aeration samples every 15 minutes. The temperature flasks were prepared by exposing one flask to room temperature (25 degrees Celsius), another flask in a heated compartment (37 degrees Celsius), and the final flask in a lab oven at 33 degrees Celsius. In the aeration samples, one flask was stationary exposed to the atmosphere. The second flask was put in a regular flask, and placed in a wavering water bath. The third cuvette, which was irregular shaped, 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: Every 15 minutes, my fellow students and I measured the light absorbency (at 600nm) of the growing E. coli and it’s varied conditions. From these findings, we were able to calculate the mean generation time (MGT), by graphing our spectrophotometer results and deduced an approximate time that correlated with the doubled initial absorbency. For example, the flask with the temperature of 37 degrees Celsius had an initial absorbency of .13. After doubling this number (.26), we looked at our growth curve (Figure 1.) and found at what time would the growth curve of 37 degrees reach an absorbency of .26.
In this example, the mean generation time (MGT) was approximately 73 minutes. In simpler terms, for the growth of E. coli to double it’s initial population, at the given temperature of 37 degrees Celsius, it would take 73 minutes. Our results of all spectrophotometer readings are listed in Tables 2 through 4. Table 2 displays the outcome of the effects of different types of aeration on E.
coli. Table 3 exhibits the readings for three different temperatures (25 , 33 , and 37 ), while Table 4 shows the absorbance variation with three types of media (MSG, MSGT, and MSGTYE). Following the tables, are Figures 1, 2 and 3. These graphs show the growth curves of E. coli after treatments. Although the curves may look like one absorbs more and one absorbs less, it is very difficult to make a judgment based on the appearance of the graph. Only when we calculated the MGT’s, were we able to realize which variable had the greatest effect and which had the least. Table 5 presents all of the approximate MGT’s for each variable, for each different trial of the variable.
Discussion: Figure 1 shows the growth curve of E.coli in regards to different temperatures. The MGT for 37 was about 73 minutes. The MGT for 33 was about 75 minutes. The MGT for 25 showed that the population was still growing beyond 90 minutes. I hypothesized that the higher temperatures would produce the most growth, but from the calculated results, my hypothesis was refuted. Figure 2 shows the growth curve of E. coli with the nutrients.
I predicted that MSGTYE would have the highest growth rate. I was proven wrong again. The MGT of MSG, MSGT, and MSGTYE were about 5 minutes, 56 minutes, and 50 minutes, respectively. Even though I believed that MSGTYE was going to have the greatest growth, I also predicted that MSG was going to have the lowest growth and this proved to be true. Figure 3 represents the effect of aeration on the growth of E.
coli and I hypothesized that the baffled flask would have the highest growth rate and that the control flask would have the lowest growth rate. As scene in Table 5, the MGT for the normal flask in the shaky water bath was about 43 minutes. For the baffled flask, the MGT was about 50 minutes. Surprisingly, the control flask, the stationary flask, does not have a MGT because its population is still growing. From this lab, I learned that temperature, for the growth of E. coli increases with lower the temperatures. For E.
coli affected by nutrients, I deduced that carbon and nitrogen help make better conditions for the E. coli to grow, but it does not respond as optimality to yeast extract. For the effects of aeration, we can say that E. coli doesn’t necessarily need oxygen for growth. Some errors in this experiment could have occurred at the spectrophotometers readings. Students may have forgotten to re-zero the spec 20 before each new sample.
Another source of error could be how temperature affects the bacteria. A student could have held a cuvette long enough to make the solution a little bit warmer, which could effect the growth curve for that sample. For future research in this field, I would someday like to see if they could make a substance that would remove bacteria from skin. I could also see a substance that a human could digest to make the body resist all bacterial diseases. Although this seems crazy, bacteria can be limited by many factors.
We live in the age of technology, and I believe that at this point, anything is possible. Animal Science.