Week 2 Formal lab report (Normal and sensitised ileum) Learning Outcomes Develop an appreciation of the use of organ baths to investigate physiological processes. Interpretation and critical analysis of data. To determine the probable identity of the chemical released by mast cells that induces contraction in the ileum of guinea pigs sensitised to egg albumin. The hypothesis is that tissue from an animal that has developed antibodies to egg albumin will respond to antigen challenge by degranulation of mast cells caused by antigen/antibody reaction. This week you will use gut tissue from both control and sensitised guinea pigs. You will set up the ileum as before and carry out the following experiments, using control or sensitised guinea pig ileum as appropriate to determine the probable identity of the molecule in mast cell granules that induces contraction. Each group should carry out at least two of the sets of experiments with separate pieces of ileum for each experiment. Your group will be told which order you should do the experiments. This will ensure that there are sufficient class results for you to write up the report. Labscribe 4 data logger Set up the gut bath and Labscribe 4 as in week 1 of the lab. The only difference is that you should this time set the time base to 2 mins. However, you should ONLY run to 30 sec before washing for all agonists, except egg albumin, where it MAY be necessary to wait up to 2 mins before the response. Remember to use two 30 second wash steps after recording the response of each stage of the experiments below. It is likely that the BSA and egg albumin will cause foaming in the organ bath. If you get a large response with egg albumin before 2 mins, do NOT wait for the entire 2 mins, but wash out when it has reached maximum. This will help prevent desensitisation. The egg response may take some time to return to baseline after the two wash steps. You should continue to wash every 5 mins until the voltage returns to close to the baseline. Remember to record the voltages for each step of each experiment. Please note, that unless you get a response to both histamine and carbachol with your piece of ileum (steps 1 and 2 of each experiment), you should stop at this point and ask the demonstrator for advice. This is more likely to occur for histamine than carbachol, so if you don't get a response to histamine, don't go on to carbachol. Control Experiment (no previous exposure to egg albumin) 1. Obtain response to histamine (0.5 ml of 10-4M) 2. Response to 0.4ml of 10-4M carbachol 3. Response to 1ml of the bovine serum albumin solution 4. Response to 1ml of the egg albumin solution 5. Add 0.3ml of 10-4M mepyramine followed after 30sec by 0.5 ml of 10-4M Histamine. Record the response to histamine. Make sure you do not wash the tissue between the addition of mepryramine and addition of histamine 6. Response to 0.4 ml 10-4M carbachol Sensitised Experiment One (ensure that you use a fresh length of sensitised ileum) 1. Obtain response to histamine (0.5 ml of 10-4M) 2. Response to 0.4ml of 10-4M carbachol 3. Response to 1ml of the bovine serum albumin solution 4. Response to 1ml of the egg albumin solution. You may have to wait longer than 30 seconds for a response (up to 2 mins). Make sure the tissues returns to baseline before continuing to step 5. This could take some time (and several washes) 5. Response to another 1ml of the egg albumin solution, and then without washing add 0.5 ml of 10-4M histamine (record both responses) 6. Add 0.3ml of 10-4M mepyramine followed after 30sec by 0.5 ml of 10-4M Histamine (without washing). Record the response to histamine 7. Response to 0.4 ml of 10-4M carbachol Sensitised Experiment Two (ensure that you use a fresh length of sensitised ileum) 1. Obtain response to histamine (0.5 ml of 10-4M) 2. Response to 0.4ml of 10-4M carbachol 3. Response to 1ml of the bovine serum albumin solution 4. Add 0.3ml of 10-4M mepyramine followed after 30sec by 1ml of egg albumin. Record the response to egg albumin 5. Add 0.3ml of 10-4M mepyramine followed after 30sec by 0.5 ml of 10-4M Histamine. Record the response to histamine 6. Response to 0.4 ml of 10-4M carbachol You should ensure that you obtain a complete set class results for each of the 3 experiments in week 2. These will be posted on NOW and are needed for the lab report. The results in week 2 should be subject to statistical analysis using ANOVA and Tukey test (available in PAST, MINITAB, SPSS and Prism) to determine where the significance lies. It is essential that you ONLY use FINAL bath concentration throughout your report. The bovine serum albumin and egg albumin are supplied at a stock concentration of 125mg/ml. Use mg/ml rather than molarity as concentration for these two albumins only. IMPORTANT: Task specific criteria for the lab report Your formal report should NOT exceed 5 pages (excluding references). The report should be word processed with a minimum font size of 10 and 2 cm margins all round. Aims This should set the report in context. It should be specific to the data obtained from Week 2. Methods (10%) A brief description of the methods used. This should not consider every single little detail (i.e. don't copy out chunks of the schedule listing each step, or show the diagram of the gut bath set up), but should instead give an overview. In effect it should be like the style typical of scientific papers where the methods section is generally concise. If you are unsure how to do this, look at the methods sections of journal papers. It is essential that you only use final bath concentration of drugs throughout your report (e.g. a label of 0.4ml of 10-4M Carbachol on a figure is incorrect) Results (40%) The class data for your group should be downloaded from NOW (in the form of an Excel spreadsheet). You should then treat each horizontal row separately and convert the volts in each row to the % of the first carbachol response for that particular row. It is essential that you treat each row separately, and do not average the responses in volts for each drug. Once you have converted the values to % of the first carbachol response in each row, you can then calculate the mean and a measure of the error (e.g. standard error, standard deviation, or 95% confidence interval) for every column of each experiment. Your results should include three appropriate graphs, one for each of the three experiments from the 2nd week of the lab, together with appropriate statistical analysis. You should statistically analyse each experiment separately (i.e. you will need to conduct three ANOVAS). You therefore need to think very carefully about how best to present the results. Look at journal papers to see how they do this. You should not individually present every single result for every experiment, instead you should show a summary of them (i.e. show means together with an indication of the size of the error for each experiment). Also, you should think about how to show statistical analysis in your figures. MINITAB and other statistical packages may produce pages of analysis and you should not include this. In particular, the MINITAB graphs are very poor. Instead, you need to think about how journal papers report statistical differences. They succinctly indicate statistical differences on the actual figures and their respective figure legends. Make sure that you appropriately label the figures (e.g. never use "A graph to show" or "X" against "Y" as a figure title). Again, look at journal papers to see how they do this. In addition the results should be described (i.e. what do they show). The results section is just to state the results. What the results mean should only be considered in the discussion section. You should not discuss any results in the results section Discussion (40%) This should discuss the significance of the results. What were the experiments trying to show? You will need to consider what mast cells do and suggest which molecule is responsible for the effect on ileal contraction. The discussion section should also discuss the different types of receptors and their intracellular signal transduction pathways. In particular, which drug interacts with which pathway? You will need to critically analyse your data. Is it consistent with previous studies in the literature? There is no need to give full details of how the body produces antibodies to foreign proteins. Instead, you should focus on mast cells and the ileum. References (5%) References are expected and should only be from textbooks and peer reviewed articles. The quality of the references, the citation style used (both in the text and in the reference list) and the correct use of references will be taken into account in awarding marks for the report. In order to get good marks for the report you will need to do a literature search and use relevant papers to inform your report, particularly the discussion. Overall quality of the report (5%) This section includes presentation, including organisation, spelling, grammar, coherence. You are advised to look at the help posted on NOW (statistical help, how to reference correctly etc.) and at a variety of journal papers. Plagiarism/collusion/use of "proof-reading" services etc. You are reminded that the work must be your own individual report. All quotes must be in quotation marks and all sources used should be cited at the appropriate points in the text and reference list (do not use numbered reference lists). Failure to do so risks a potential academic irregularity such as collusion (where two or more work together on the same report) or plagiarism (e.g. taking material from another source without putting it in quotation marks AND indicating the source). Please note, that whilst properly cited text in quotation marks is not plagiarism, it will be ignored in determining your final mark. This is because using quotes does not demonstrate understanding. Students are encouraged to proof-read their own work. You need to be very careful if you use a proof-reading service, as they are very limited in what NTU considers acceptable, before you risk an academic irregularity. Things proof-reading services are not allowed to do (not an exhaustive list) includes: write, or rewrite, any part of the student's work contribute any additional material to the original correct work so that the original errors are not visible correct mistakes where the original intended meaning is reorganise paragraphs correct factual errors, or mistakes in calculations re-label diagrams or figures correct or reformat references etc. clear Further guidance is given in the Quality Handbook supplement 15F proof reading guidance document on the NTU website.