Sunday, September 1, 2013

Discussion

Key findings
In the results, there were many dilutions. We would use the 10^-3 dilution to compare due to the fact that it has the least TMTCs and would be the most reasonable and accurate to compare the data between the various beverages. Basically, ALL COMPARISONS would be done in the 10^-3 dilution.

We will go over results of each of the beverages individually.

1. Coffee
In 10^0 Dilution, the lactobacillus colonies grown on the MRS plates were too much to count.
In 10^-2 Dilution, the lactobacillus colonies grown on the MRS plates were too much to count.
In 10^-3 Dilution, there was an average of 3185 lactobacillus colonies grown on the MRS plates.
In 10^-4 Dilution, there was an average of 154 lactobacillus colonies grown on the MRS plates.
Compared to Milk, Coffee had an average of 3185 lactobacillus colonies grown on the MRS plates while Milk had 3781. A difference of 596 colonies. (Milk has more)
Compared to Sterilized Water, Coffee had an average of 3185 lactobacillus colonies grown on the MRS plates while Sterilized Water had none. A difference of 3185 colonies.
Compared to Tea, Coffee had an average of 3185 lactobacillus colonies grown on the MRS Plates while Tea had only an average of 8 colonies. A  difference of 3177 colonies.
Compared to LB Broth, Coffee had an average of 3185 lactobacillus colonies grown on the MRS Plates while LB Broth had only an average of 239. A difference of 2946.
Compared to Cola, Coffee had an average of 3185 lactobacillus colonies grown on the MRS Plates while Cola had none. A difference of 3185 colonies.
Compared to Energy Drink, Coffee had an average of 3185 lactobacillus colonies grown on the MRS plates while Energy Drinks had none. A difference of 3185 colonies.

2. Milk
In 10^0 Dilution, the lactobacillus colonies grown on the MRS plates were too much to count.
In 10^-2 Dilution, the lactobacillus colonies grown on the MRS plates were too much to count.
In 10^-3 Dilution, there was an average of 3781 lactobacillus colonies grown on the MRS plates.
In 10^-4 Dilution, there was an average of 569 lactobacillus colonies grown on the MRS plates.
Compared to Coffee, Milk had an average of 3781 lactobacillus colonies grown on the MRS plates while Coffee had 3185. A difference of 596 colonies. (Milk has more)
Compared to Sterilized Water, Milk had an average of 3781 lactobacillus colonies grown on the MRS plates while Sterilized Water had none. A difference of 3185 colonies.
Compared to Tea, Milk had an average of 3781 lactobacillus colonies grown on the MRS Plates while Tea had only an average of 8 colonies. A  difference of 3773 colonies.
Compared to LB Broth, Milk had an average of 3781 lactobacillus colonies grown on the MRS Plates while LB Broth had only an average of 239. A difference of 3542.
Compared to Cola, Milk had an average of 3781 lactobacillus colonies grown on the MRS Plates while Cola had none. A difference of 3781 colonies.

3. Sterile Water
In 10^0 Dilution, the lactobacillus colonies grown on the MRS plates were too much to count.
In 10^-2 Dilution, the lactobacillus colonies grown on the MRS plates were too much to count.
In 10^-3 Dilution, there were no lactobacillus colony growth on the MRS Plates.
In 10^-4, there were no Lactobacillus colony growth on the MRS Plates.
Compared to Coffee, Sterile Water had no lactobacillus colony growth while Coffee had an average of 3185 Lactobacillus colony growth. A difference of 3185 colonies.
Compared to Milk, Sterile Water had no lactobacillus colony growth while Milk had a average of 3185 Lactobacillus colony growth. A difference of 3781 colonies.
Compared to Tea, Sterile Water had no lactobacillus colony growth while Tea had an average of 8 Lactobacillus colony growth. A difference of 8 colonies.
Compared to LB Broth, Sterile Water had no lactobacillus colony growth while LB Broth had an average of 239 Lactobacillus colony growth. A difference of 239 colonies.
 Compared to Cola, both of them did not have any Lactobacillus growth. There were no difference.
Compared to Energy Drink, both of them did not have any Lactobacillus growth. There were no difference.

4. Tea

In 10^0 Dilution, the lactobacillus colonies grown on the MRS plates were too much to count.
In 10^-2 Dilution, the lactobacillus colonies grown on the MRS plates were too much to count.
In 10^-3 Dilution, there was an average of 8 lactobacillus colonies grown on the MRS plates.
In 10^-4 Dilution, there was an average of 1 lactobacillus colonies grown on the MRS plates.
Compared to Coffee, Tea had an average of 8 lactobacillus colony growth while Coffee had an average of 3185 Lactobacillus colony growth. A difference of 3177. (Coffee has more)
Compared to Milk, Tea had an average of 8 lactobacillus colony growth while Milk had a average of 3781 Lactobacillus colony growth. A difference of 3773.
 Compared to Sterilized Water, Tea had an average of 8 lactobacillus colony growth while Sterilized Water had none. A difference of 8 colonies. (Tea has more)
Compared to LB Broth, Tea had an average of 8 lactobacillus colony growth while LB Broth had an average of 239 Lactobacillus colony growth. A difference of 239 colonies.
Compared to Cola, Tea had an average of 8 lactobacillus colony growth while Cola had none. A difference of 8 colonies. (Tea has more)
Compared to Energy Drink, Tea had an average of 8 lactobacillus colony growth while Energy Drink had none. A difference of 8 colonies.

5. LB Broth
In 10^0 Dilution, the lactobacillus colonies grown on the MRS plates were too much to count.
In 10^-2 Dilution, the lactobacillus colonies grown on the MRS plates were too much to count.
In 10^-3 Dilution, there was an average of 239 lactobacillus colonies grown on the MRS plates.
In 10^-4 Dilution, there was an average of 45 lactobacillus colonies grown on the MRS plates.
Compared to Coffee, LB had an average of 239 lactobacillus colonies grown on the MRS Plates while LB Broth had only an average of 239. A difference of 2946.
Compared to Milk, LB Broth had only an average of 239 while Milk had an average of 3781 lactobacillus colonies grown on the MRS Plates. A difference of 3542.
Compared to Sterlized Water, LB Broth had an average of 239 Lactobacillus colony growth. while Sterilized Water had none.. A difference of 239 colonies.
Compared to Tea, LB Broth had an average of 239 Lactobacillus Colony growth, while Tea had only 8. A difference of 231 colonies. (LB Broth had more.)
Compared to Cola, LB Broth had an average of 239 Lactobacillus Colony growth while Cola had none.
Compared to Energy Drink, LB Broth had an average of 239 lactobacillus colony growth while Energy Drink had none. A difference of 239 colonies.

6. Cola
In 10^0 Dilution, there were no Lactobacillus colony growth on the MRS Plates.
In 10^-2 Dilution, there were no Lactobacillus colony growth on the MRS Plates.
In 10^-3 Dilution, there were no lactobacillus colony growth on the MRS Plates.
In 10^-4, there were no Lactobacillus colony growth on the MRS Plates.
Compared to Coffee, Cola had no lactobacillus colony growth while Coffee had an average of 3185 Lactobacillus colony growth. A difference of 3185 colonies.
Compared to Milk, Cola had no lactobacillus colony growth while Milk had a average of 3185 Lactobacillus colony growth. A difference of 3781 colonies.
 Compared to Sterilized Water, both of them did not have any Lactobacillus growth. There were no difference.
Compared to Tea, Cola had no lactobacillus colony growth while Tea had an average of 8 Lactobacillus colony growth. A difference of 8 colonies.
Compared to LB Broth, Cola had no lactobacillus colony growth while LB Broth had an average of 239 Lactobacillus colony growth. A difference of 239 colonies.
Compared to Energy Drink, both of them did not have any Lactobacillus growth. There were no difference.

7. Energy Drink
In 10^0 Dilution, there were no Lactobacillus colony growth on the MRS Plates.
In 10^-2 Dilution, there were no Lactobacillus colony growth on the MRS Plates.
In 10^-3 Dilution, there were no lactobacillus colony growth on the MRS Plates.
In 10^-4, there were no Lactobacillus colony growth on the MRS Plates.
Compared to Coffee, Energy Drink had no lactobacillus colony growth while Coffee had an average of 3185 Lactobacillus colony growth. A difference of 3185 colonies.
Compared to Milk, Energy Drink had no lactobacillus colony growth while Milk had a average of 3185 Lactobacillus colony growth. A difference of 3781 colonies.
 Compared to Sterilized Water, both of them did not have any Lactobacillus growth. There were no difference.
Compared to Tea, Energy Drink had no lactobacillus colony growth while Tea had an average of 8 Lactobacillus colony growth. A difference of 8 colonies.
Compared to LB Broth, Energy Drink had no lactobacillus colony growth while LB Broth had an average of 239 Lactobacillus colony growth. A difference of 239 colonies.
Compared to Cola, both of them did not have any Lactobacillus growth. There were no difference.

Explanation of Key Findings
We would explain the results of each beverage individually.

1. Coffee. The reason why there were so much Lactobacillus growth on the MRS Plates after being put with Coffee is that there were milk in the Coffee.
Lactobacillus is one of the Lactic Acid Bacteria. These Lactic Acid bacteria are found in fermented food and probiotic products. (Lactic Acid Bacteria, 2013, Wikipedia) And the fact that when we consume the probiotic product, the Lactobacillus is still there, it shows that the Lactobacillus can thrive in milky conditions.
2. Milk. As said above, Lactobacillus thrive in Milky Environments, and thus they can survive, and in fact grow and multiply in conditions like Milk.
3. Sterile Water. Everything was going fine for Sterile Water until 10^-3 dilution. As the word implies, Sterilized is about killing all fungi/bacteria. This means that Sterilized Water does not contain any Bacteria/Fungi, or any nutrients for the Lactobacillus to survive on. We guess that when we first put the Lactobacillus into the Sterilized Water, there were a lot of Bacteria inside. As time went by, the Lactobacillus did not have nutrients to survive and eventually died. This was our prediction since we did 10^-2, 10^-3 and 10^-4 later than the one we did on 10^0.
4. Tea. There were too much to count amount of bacteria for Tea when it was in 10^0 dilution. As the dilution increased the amount of lactobacillus colonies decreased. The reason for this is because as dilution implies, it is the reducing of concentration in the chemical. Thus, the concentration of the bacteria decreased and as the amount of dilution increased, the amount of concentration decreased and hence there were less bacterial colonies.
5. LB Broth. LB Broth itself is a nutrient for bacteria. However, Lactobacillus does not seem to grow on it and its effectiveness on cultivating bacteria is not that high. However, as it contains nutrients, it is a suitable environment for the Lactobacillus to grow in and thus, there were an satisfying amount of Lactobacillus while put in LB Broth.
6. Cola. According to Greenwood(2011), said in her survey, Caffeine kills bacteria and sometimes can be stronger than an antibiotic. Cola contains caffeine and might have inhibited or killed of the Lactobacillus in the solution. Due to the caffeine in the Cola, the caffeine might have affected the growth of Lactobacillus.
7. Energy Drink. Similarly, Energy Drinks contains caffeine and would affect the Lactobacillus similarly as it affected the Cola. Energy Drinks also contains other chemicals that might have affected the growth of Lactobacillus colonies.
In overall, the factors that affected the amount of Lactobacillus grown in the MRS plates for the various beverages was decided by the ingredients in the beverage in question. For the beverages that contained different ingredients, the amount of Lactobacillus grown varies.

Evaluation of hypothesis
Coming back to our hypothesis, we predicted that :All of the tested beverages would affect the Lactobacillus. Casei bacteria, but the petri dishes which comprised of beverages other than sterile water in them would cause there to be an average of 30% less colonies than the plates which only sterile water.
From our experiment and results, it is not that accurate.
For Sterile Water, there was no growth in Lactobacillus colonies. It is like it can be compared to Cola and Energy Drink, which both also do not have growth in their respective MRS Plates. However, MIlk and Coffee had a very big increase in Lactobacillus, compared to Sterile Water.
Compared to LB Broth, it is a bit more accurate. However, it is still shocking that the Milk and Coffee values were so high.
From this( Our experiment results), we can conclude that Cola and Energy Drinks would inhibit the growth of Lactobacillus Casei, and that it is not healthy for consumption together with probiotic drinks.
For Milk and coffee however, there was a positive effect, and benefited the Lactobacillus Casei.
Referring to our hypothesis, the amount of Lactobacillus for Milk and Coffee is obviously 30% more than the controls, Sterilized Water and LB Broth. Also, for Cola and Energy Drink, both of them had 30% less bacteria than our control, LB Broth. Which affects too much.

Areas for improvement
The areas of improvement in our tests are as follows:

1. Bacteria from microtubes (containing mixed solution of L.case and beverage) were used over the span of weeks and this bacterial health and count could have deteriorated over multiple freezes. These host microtubes with beverages mixed with lactobacillus (of 0% dilution) from lesson 4 and follow on diluted microtubes from later dates were used, then transferred back into the freezer for later reference. This freezing undoubtedly have helped maintain the bacterial growth status of the bacteria in the microtubes by slowing down life processes occurring inside. However, this freezing does not stop these processes. Therefore, the freezing, itself, would definitely have killed of a certain percentage of bacteria. Consequently, the numbers of living bacteria in each plate could vary unexpectedly from earlier tests and later tests. If results were to be cross-compared, the tests that were taken last would have been most likely to have the results with the least living bacteria to earlier tests. However, these bacteria terminated are usually of small percentages and their effect are usually insignificant. Moreover, this is by far the most feasible method of lab preservation.

2. Contents in perishable food (beverages) like milk are prone to spoilage and beverage spoilage could affect the experiment as there is a change beverage’s contents. As a result, levels of other factors (of decomposition) are increased, which includes bacteria, mold, yeast and enzymes. Even with constant refrigeration, these beverages are capable of becoming spoiled due to having a fixed shelf life, thus creating inaccurate results and ultimately, an unreliable experiment. We can solve this problem by conducting the experiment efficiently within the beverage’s shelf time with little refrigeration for preservation and little intervals in between.

3. For the tests involving the 100 and 1000 dilution sets, the accuracy of the results and consistency of the procedure were reduced as we were able to only do two repeat sets for each beverage for both dilution sets. This meant that there were only two sets to find an average instead of three in previous tests. This failure to carry out a full three sets was due to a mistake made when doing one of the dilution sets, but we only troubleshooted and rectified the error later. As a result, several MRS agar plates had already been wasted, which lead to this eventual deficit and lack MRS agar plates. We should be more carefully in the future and not allow this careless mistake to happen again.

4. As the entire experiment was hinged on the mixture of the 3 Lactobacillus colonies and sterile water which formed the original mixture and the basis of our experiment, any errors would have been voided and nullified the entire experiment. Therefore, we could instead have had redone the entire experiment from the start 3 times to ensure the highest chances of getting accurate results.

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