Homework answers / question archive / HCS228 – ASSESSED PRACTICAL: BACTERIAL CONJUGATION & ANTIBIOTIC RESISTANCE Please note that all the data (qualitative and quantitative) you need to write your report will be provided – either below or via Canvas

HCS228 – ASSESSED PRACTICAL: BACTERIAL CONJUGATION & ANTIBIOTIC RESISTANCE Please note that all the data (qualitative and quantitative) you need to write your report will be provided – either below or via Canvas

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HCS228 – ASSESSED PRACTICAL: BACTERIAL CONJUGATION & ANTIBIOTIC RESISTANCE
Please note that all the data (qualitative and quantitative) you need to write your report will be provided – either below or via Canvas.
Please write Results and Discussion sections for the practical class on conjugative transfer of antibiotic resistance genes. The Results section should summarize the observations made and data obtained in your study, using a mixture of text, tabulated data (tables) and graphs (figures) as appropriate. Note that the Results section should include sufficient explanatory text to set the data in context and help the reader to understand what you did and why (it is not acceptable to just present data in figures or tables without any explanatory text). You should refer to all figures and tables in this text e.g. “…as shown in Figure 1” or just “…(Figure 1)”. The Discussion section should assess the findings of your study, relate them to relevant scientific literature and to their clinical significance, consider possible shortcomings in the experiments as executed and briefly propose possible next steps / additional experiments. You should consider what these results tell you about inter-species transfer of antibiotic resistance. The Discussion section should cite relevant high-quality references (aim for at least 5 for 1st class marks). The overall word count should not exceed 1500 (not including figures, tables or reference list).
The experiments carried out in laboratory classes examined conjugative transfer between two strains of Escherichia coli with different chromosomal antibiotic resistance marker genes in a qualitative way. We have carried out a further quantitative experiment where we examined the conjugative transfer frequency (number of transconjugants per donor cell) from the same E. coli donor strain (nalR, ampR) to 4 different recipient strains (E. coli as previously, Salmonella enterica, Pseudomonas aeruginosa and Bacillus subtilis) with the same strR antibiotic resistance gene in the chromosome. This experiment was carried out as follows:
Each experimental conjugation was carried-out in triplicate (i.e. 3 replicates) as follows.
1. Grow parental bacterial cultures overnight, with appropriate antibiotics, to the same cell density (approx. 109 cells per ml).
2. Add 0.1 ml of donor culture to 0.9 ml (i.e. excess) of recipient culture (4 tubes for the 4 different recipients).
3. Filter the cells from each tube onto a 0.2 ?m membrane filter and place the filter onto a nutrient agar (NA) plate with bacteria uppermost.
4. Incubate plate with filter at 37 °C to allow mating to occur. Allow mating to occur for 1 hour
5. Aseptically remove filter from plate and resuspend bacteria from filter in 1 ml saline.
6. Perform a 10-fold serial dilution on each mated mixture down to 10-8.
7. Plate 0.1 ml of dilutions neat to 10-8 onto antibiotic free NA and NA supplemented with streptomycin and ampicillin (the latter medium selects for transconjugant cells – recipients that have received a plasmid).
8. Incubate plates at 37 °C overnight. Count colonies.
Please note that your practical report should include analysis of both sets of data: quantitative and qualitative.
Please analyse the raw data presented below using appropriate statistics and present the results graphically in your report, along with the qualitative data you obtained in the practical classes. Some hints on calculations:
1. Calculate the number of donor cells for each mating from the colony counts on NA (Table 1). Remember to take into account in your calculations that you plated 0.1 ml and that 1/10 of that would have been donor cells.
2. Calculate the number of transconjugants resulting from each mating i.e. transconjugants / ml from the colony counts on NA+Str+Amp (Table 2).
3. Calculate the conjugation frequency (i.e. transconjugants per donor)
conjugation frequency = number of transconjugants
number of donor cells
Show one example calculation, using one NA count and one NA+Str+Amp count from the tables below so we can see that you are performing the calculations correctly. Apply appropriate statistics to calculate average values and give an indication of the error associated with each average.
4. Present your analysed data graphically (i.e. using MS Excel or similar software). Think about the best way to report your data so that averages and the differences between them can be seen and assessed for significance, but please note that statistical testing such ANOVA is not required.
Table 1. Colony counts on NA.
Recipient (StrR) Bacteria
Replicate 1
Replicate 2
Replicate 3
Dilution
CFU
Dilution
CFU
Dilution
CFU
E. coli
10-6
99
10-6
97
10-6
91
Salmonella enterica
10-6
104
10-6
98
10-6
89
Pseudomonas aeruginosa
10-6
88
10-6
105
10-6
100
Bacillus subtilis
10-6
117
10-6
93
10-6
111
Table 2. Colony counts on NA + streptomycin + ampicillin.
Recipient (StrR) Bacteria
Replicate 1
Replicate 2
Replicate 3
Dilution
CFU
Dilution
CFU
Dilution
CFU
E. coli
10-4
43
10-3
199
10-3
167
Salmonella enterica
10-3
77
10-3
63
10-3
56
Pseudomonas aeruginosa
10-2
155
10-2
35
10-2
99
Bacillus subtilis
10-1
98
10-1
103
10-0
225
Please note that there is no point pasting the above tables of “raw” data into your results sections – rather you should present your own analysis (reduction) of the data.