Practical - Lab reports 1-8 PDF

Preview

Summary

Experiment 1 BIOC1010: Introduction to Microbiology Laboratory report: experiment 1 Culture medium preparation Introduction 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 liquid or gelatinous substance that contai...

Description

Experiment 1

BIOC1010: Introduction to Microbiology

Laboratory report: experiment 1 Culture medium preparation Introduction 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 liquid or gelatinous substance that contains essential nutrients, to cultivate target microorganisms or tissues, for further purposes. A culture medium must be sterilised before use so that no unwanted microorganisms grow, which may contaminate the growing sample. The aim of this experiment is to demonstrate that culture media are contaminated naturally (i.e. untreated). Two possible sterilisation techniques – autoclaving & filtration are also tested.

Methodology Briefly summarise what you did in the lab. 1. A nutrient broth was made up by adding 2.5 g of Oxoid Nutrient Broth powder (contains 1% beef extract, 1% peptone, 0.5% sodium chloride, at pH around 7.5) to 100 mL sterile distilled water in a 250 mL bottle. The broth was swirled until the powder dissolved. 2. The broth was split between three 100 mL pre-sterilised conical flasks. Flask A was left untreated; flask B was then autoclaved; flask C’s content was filtered through a 0.22 µm filter before adding to the flask. 3. The flasks were incubated for 2 days at 30°C, then stored in a refrigerator at 4°C for 5 days to stop further growth. 4. The extent of growth (indicated by the broth’s turbidity) of the flask was examined.

Results 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? After incubation, the broth in flask A appeared to be turbid (yellowish in colour) while the broths in flask B & C remained clear. This means that there are bacterial growths in the untreated flask, while no bacterial growth in the autoclaved & filtered flasks. From the class results, out of 67 groups, all groups had bacterial growth in the untreated flask; only one group had bacterial growth in the autoclaved flask; 12 groups had bacterial growth in the filtered flask. Our results agree with the majority of the class results.

1

Experiment 1

BIOC1010: Introduction to Microbiology

Discussion Discuss what your results tell you about the autoclaving and filtration methods. Discuss the relative merits of a) doing nothing; b) autoclaving; c) filtering as strategies for preparing sterile media. Discuss possible reasons for the variation seen in the class results. Can you suggest alternative techniques for media sterilization? The absence of growth of bacteria in the autoclaved and filtered flasks indicated that these two methods maybe effective methods of sterilisation (removing/killing all microbes). Growth in the untreated flask also indicated that natural culture media is contaminated. The unsuccessful cases in autoclaving & filtering may due to several reasons. Firstly, nutrient broth maybe contaminated after treatment, it may not be properly sealed so that bacteria can enter the broth from air and grow. Secondly, filtering requires great precision and unfiltered broth may enter the flask by chance. The higher success rate by autoclaving from the class results suggests that autoclaving maybe a more reliable way of sterilisation. Autoclaving requires less human control, so imposes less human error. Other ways to achieve sterilisation include heating in oven, using radiations (X-rays, gamma rays etc.) or adding antimicrobial agents (bactericidal, bacteriolytic) into the medium.

2

Experiment 1

BIOC1010: Introduction to Microbiology

Question 1. Liquid media used for culturing microorganisms in the lab is typically sterilized using a small autoclave (essentially, a programmable pressure cooker). I prepare 5 litres of medium, split this into five one-litre bottles and put these into the autoclave. The autoclave is programmed for a 20 minute/121oC cycle. My lazy colleague also makes 5 litres of the same medium, but instead puts the whole lot into a single large bottle and autoclaves this using the same programme. A day-or-so after autoclaving, the medium in my colleague’s bottle has gone cloudy indicating that contaminating bacteria have survived the autoclaving and have grown in the medium. In contrast, the medium in all five of my bottles is clear (i.e. no bacterial growth). Why do you think we have different results? The 5-litre bottle is so large that heat transfer to the interior of the solution in retarded. As a result, the entire solution is not heated up to 121oC for sufficient time. By contrast, if splitting them into 1-litre bottles, the total surface area is larger such that the contents of the bottles are sufficiently heated to the required temperature within the 20-minute cycle. What would you do differently if you wanted to autoclave a large volume (such as 5 litres) in a single bottle? I would either programme the cycle of autoclave to be longer or set it to a higher temperature. This either allows more time for heating up the bottle, or the bottle can be heated up faster due to larger temperature difference. 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 45 minutes, what is the ratio of S. red to S. green after 24 hours incubation (assuming unrestricted division of both bacteria throughout the 24 hours)? In 24 hours, S. red divides 24 times while S. green divides 32 times. Hence, the ratio of S. red to S. green after 24 hours incubation is 224 : 232 = 1 : 28 = 1 : 256 End of report.

3

Experiment 2.

BIOC1010: Introduction to Microbiology

Laboratory report: experiment 2 Reducing bacterial contamination Introduction Briefly explain why it is important to use control measures to limit bacterial contamination of food; surfaces within the home; your hands, etc., and then outline the experimental strategy you used. Bacteria are present all around us. It is virtually impossible for us to achieve sterility (i.e. absence of bacteria) as it is not attainable and practical, for example in fresh food. When sterility is not possible to achieve, we aim to achieve disinfection or decontamination where bacterial growth is limited. If bacterial growth is not limited, food may easily get spoiled and we are more prone to getting food-borne diseases and food poisoning. Bacterial growth can also help to spread diseases by other ways (e.g. air, water, direct contact) if the environment around us is abundant in bacteria. In this experiment, some household treatments were tested for their potential antibacterial effects. Our group used Escherichia coli as the test microorganism. The following reagents were used – household bleach, TCP (an antiseptic), isopropanol, liquid soap, saturated salt solution, vinegar and honey. After overnight incubation, the presence of any clear zones and their sizes were noted. The presence of clear zones means absence of bacterial growth, hence effective inhibition of the reagent. As the reagent diffuses away from the centre, its concentration decreases radially – a larger clear zone means that the reagent works at lower concentration. Methodology Briefly summarise the experimental method. 1. 8 nutrient agar plates were prepared. 50 µL of Escherichia coli bacteria suspension were spread across each plate using a sterile plastic spreader. 2. The plates were left to dry for a few minutes, and then 10 µL of each of the following test solutions were added to the centre of a corresponding plate, water was used as a control. 3. The plates were left to dry again, then the plates were incubated at 30°C for overnight, then stored at 4°C to stop further growth for a week. 4. The diameter of zone of inhibition (“clear zones”) of each plate was measured (if any). 5. Another set of data from a group using Micrococcus luteus was obtained.

1

Experiment 2.

BIOC1010: Introduction to Microbiology

Results Report your findings. What did the bacterial growth look like on the control (water only) plate? Which of the various treatments were effective in reducing the amount of bacteria? Was there a difference between the Gram positive M. luteus and the Gram negative E. coli? The size of clear zones (diameter in mm) of different treatments were measured as follows: bleach TCP isopropanol soap NaCl vinegar honey water 11 mm 6 mm 12 mm 6 mm none 6 mm none none The data of another group using M. luteus are as follows: bleach TCP isopropanol soap NaCl 22 mm 9 mm 20 mm none none

vinegar 10 mm

honey none

water none

The class results are as follows (% of groups showing inhibition): bleach TCP isopropanol soap NaCl vinegar 100% 94% 100% 64% 13% 81%

honey 13%

water 0%

Combining the three sets of results above – bleach, TCP, isopropanol are the most effective means of inhibiting bacterial growth (most groups have inhibition). They are all commercial products for disinfections. Soap & vinegar also show good inhibition. Salt solution & honey show some extent of inhibition, but are probably not effective means of bacterial control. All groups had bacterial growth on the control plate; the whole surface of the control plate appeared to be cloudy. Comparing the two sets of data using E. coli & M. luteus, bleach & isopropanol are the most effective treatment for inhibiting growth of two species. TCP & vinegar also inhibit growth of these two species. Soap shows inhibitory effect on E. coli but not on M. luteus, although this maybe due to experimental variations only, so whether the treatments have differences on the two bacteria is inconclusive. Discussion Discuss the significance of your findings in terms of the everyday use of the various chemicals /treatments. The first three reagents – bleach, TCP and isopropanol are all commercial products for disinfections. As most groups show inhibition using these reagents, they are considered to be effective for bacterial control. Bleach and isopropanol are strong disinfectants, judging by their larger sizes of clear zones. TCP is a milder disinfectant. Some household reagents are thought to have bacterial control effect but turns out they do not. For example, only some groups show inhibition of growth using soap, which it is always thought that it kills bacteria. Washing your hands with soap does not necessarily mean that they are clean. Nevertheless, we cannot always use disinfects for bacterial control, for example in food preservation. In this experiment, although vinegar and salt solution do not show remarkable inhibition, we should not overlook their effects in bacterial control in food. When present in higher concentrations, vinegar and salt do really show some effects of preservation.

2

Experiment 2.

BIOC1010: Introduction to Microbiology

Question: Disinfectant products such as Dettol claim that it “kills 99.9% of bacteria”. Discuss your interpretation of this claim, and how you opinion changes if it means “99.9% of all the different types of bacteria” or “99.9% of any bacterial species”. From the first glance, it seems that it really means “killing 99.9% of all bacteria”. However, given the variety of bacteria is so diverse, I believe it is actually impossible to have a formula that could kill so many bacteria at room temperature and is still safe to be touched. I believe such claim is only based on selected bacterial species, perhaps easier to be killed. I would suggest the statement to be changed to “killing 99.9% of common houseshold bacteria”, to make it more sensible. If the latter is true, and E. coli doubles its numbers every 30 minutes on the treated surface, how long before the effects of Dettol are nullified? Suppose there were 1000 bacteria initially, after the treatment of Dettol, only 1 bacterium remained. The bacterium replicates itself in 30 minutes. As this goes on, there will be 2n bacteria after n×30 minutes. As 29 = 512 < 1000 and 210 = 1024 > 1000, the effects of Dettol are nullified after approximately 5 hours (10 divisions). End of report.

3

Experiment 3.

BIOC1010: Introduction to Microbiology

Laboratory report: experiment 3 Colony growth and analysis of two bacterial strains Introduction The use of solid media for the culturing of microorganisms allows the isolation of clonal lines in which all the cells in a colony are derived from a single progenitor cell. Furthermore, the ability of different bacterial isolates to grow on solid media can be used as a diagnostic assay, as can the morphology of the resulting colonies. Finally, individual colonies can be picked from a nutrient agar plate and various diagnostic tests performed to further characterise the isolate. In this experiment, we streaked two strains (Escherichia coli and Micrococcus luteus) onto standard nutrient agar (NA) plates, and onto a selective medium (EMB agar) to test which strain was capable of growth. We then carried out a Gram stain on the colonies from the NA plates to determine the cell morphology and Gram type of the two strains. The results of the Gram stain were compared with that from the Gregerson Test, which also discriminates between the two Gram types. Our findings allow us to describe the colony and cell morphology of Escherichia coli and Micrococcus luteus; to state the Gram type of each, and to state which strain is capable of growth on the selective medium.

Methodology 1. Two nutrient agar plates and two EMB plates were prepared as described in the Coursebook and each was inoculated using overnight cultures of either Escherichia coli or Micrococcus luteus by streaking to single colonies using sterile loops. 2. Following incubation at 30oC for 3 days, individual colonies were picked from the NA plates and the cells stained using the Gram stain procedure as detailed in the Coursebook. The cells were viewed at x1000 magnification using an oil-immersion lens. 3. An individual colony was picked and mixed with 3% KOH to test for lysis and DNA spillage as per the Gregerson test.

1

Experiment 3.

BIOC1010: Introduction to Microbiology

Results Report your findings. Which strain grew on which solid medium? How good was your streaking (were single colonies evident)? What was the appearance of the colonies (did all the colonies have the same morphology)? What were the results of the Gram strain and the Gregerson test? Any unexpected results? Both strains grew well on the pure nutrient agar medium, with cloudiness covering around a quarter of the plate of M. luteus and three quarters of the plate of E. coli. Around 20 single colonies (each of diameter ~2 mm) are found on each plate. However, there are differential growths on the EMB plates – E. coli had well growth on the EMB plate, 15 single colonies were observed (diameter ~2 mm); M. luteus showed only limited growth on EMB plate, only around 20 small single colonies were observed (diameter...