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Experiment 1.

BIOC1010: Introduction to Microbiology

YOUR NAME: Jacopo Gabrielli

Laboratory report: experiment 1 Culture medium preparation Introduction (~ half a page) Briefly describe what a culture medium is, why it needs to be sterile and what the aims of the experiment were. A culture medium is a solid, gelatinous or liquid broth of essential nutrients used to aid the growth of microorganisms such as bacteria. Media can be defined, if we know the chemical content of it (nutrient agar), or undefined, if we only have a rough idea of what it contains (bovine serum). In our experiment, we used Oxoid Nutrient Broth. Sterilization is a fundamental precaution in microbiology to avoid contamination. It is different from disinfection or sanitization because these only remove part of the biological agents on a surface/medium while sterilization kills all of them. Also, there are many ways to carry it out like the use of chemicals, heating, or filtration. During this experiment we tested two techniques of sterilization: autoclaving and filtration, to show that an untreated sample is going to be contaminated, while a sterilized one is not. Consequently, we aim to prove that a growth medium should always be sterilized before any experiment involving microorganisms could be done on it. Moreover, we can then compare the efficacy of the two processes. Results (~ half a page of text, plus diagrams & photos) Report your findings. Which of your flasks showed bacterial growth? (what did ‘growth’ look like; what did ‘no growth’ look like). What were the class results?

Figure 1: The three flasks with the autoclaved on the right, the untreated in the middle and the filtrated on the right

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Experiment 1.

BIOC1010: Introduction to Microbiology

My flasks showed bacterial growth only in the untreated one. A comparison with the class’ results shows similarity, but for a better accuracy, the whole pool of results should be analysed. The table above displays the results obtained by the class after a 48 hours period of incubation at room temperature and five days at 4°C. Our parameter to determine growth was viscosity and cloudiness of the liquids. The first table was generated by assigning a + to cloudy liquids, therefore showing contamination, and a – to clear liquids, not showing contamination. Afterward, results were collected from all the groups to create the second table. The first figure that stands out from the results is that 100% of the autoclaved flasks showed no bacterial growth. Secondly, 90% of the filtered flasks didn’t show any contamination, however, 9 flasks did. Lastly, the untreated sample showed bacterial growth in 83.3% of the flasks. Our parameter to determine growth was viscosity and cloudiness of the liquids.

Discussion (~ half a page) Discuss the relative merits of autoclaving versus filtering as strategies for preparing sterile media. Discuss possible reasons for the variation seen in the class results. Can you suggest alternative an alternative technique for media sterilization, particularly one that would be more appropriate for industrial scale cultivation of microorganisms? From the data above, we can see that both methods inhibited growth to a certain extent. Autoclaving proved itself to be the most reliable technique as 100% of the flasks weren’t found to be cloudy. Filtration was effective in most cases, although not all of them, this is probably due to the fact that some staphylococcal species can pass through the pores. On the other hand, autoclaving kills a wider range of bacteria thanks to the very high temperature and pressure. Filtration is a suitable alternative to autoclaving in case the solution is very sensitive to temperature and pressure changes, in which case, the aforementioned technique could drastically damage its chemical integrity. Furthermore, autoclaving is more expensive and complicated to carry out while filtration only requires a very specific membrane. Even though autoclaving seems to be the best sterilization technique, it is not used in some industrial settings where a very large quantity of medium has to be sterilized. An alternative method in this kind of setting is passing the liquid through tubes containing jet heaters which allows more liquid to be sterilized at the same time. Nonetheless, if the medium is heat sensitive non-ionizing radiations can be used because they don’t cause a significant change in temperature.

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Experiment 1.

BIOC1010: Introduction to Microbiology

The untreated flasks underwent contamination in the greatest majority of the cases, but some groups didn't encounter bacterial growth in their samples. Their unexpected result is most probably due to a mistake in the preparation of the medium by, perhaps, not using all the culture broth or not taking down cloudiness correctly. Otherwise, the only valid explanation would be that they operated in completely sterile conditions, which didn’t occur. To conclude, we should have operated in complete sterile conditions to avoid a few odd results in some groups, for example, autoclaved flasks with significant growth.

Question 1. When I prepare a new batch of sterile liquid or solid media (e.g. flasks or nutrient agar plates) by autoclaving, I typically leave the flasks or plates on the lab bench for a few days before using them. Can you suggest why I do this? Autoclaving kills almost every species of bacteria, but some might survive. Leaving the flasks on the bench for a few days would give enough time to the remaining microorganisms to grow and become detectable, consequently, it would confirm or disprove the complete removal of biological agents. Besides, it would ensure that there is no inlet of air in the flasks and that they are properly sealed.

Question 2 In an imaginary experiment you carefully introduce a single cell of the bacterium Smartie red into a flask of sterile growth medium with the aim of producing a pure culture of this bacterium. Unfortunately, your lab partner sneezes at the critical moment and a single cell of Smartie green flies from his nose and into the flask. If S. red divides every 60 minutes and S. green divides every 40 minutes, what is the ratio of S. red to S. green after 12 hours incubation (assuming unrestricted division of both bacteria throughout the 12 hours)? Total minutes = 12x60 = 720 mins Number of bacteria S. red = 720/60=12 divisions 2^12 = 4096 bacteria Number of bacteria S. green = 720/40= 18 divisions 2^18 = 262144 cells Ratio of S. red to S. green = 4096/262144 = 0.0156 1 : 64

End of report.

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