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RMIT Classification: Trusted Immunology Prac report 1: Infection & Invasion Marked out of 50 (worth 10% of final grade) Introduction & Aim – 10 marks · Provide an overview of the project & give background on key aspects such as cell culture & the virulence factors involved in enteropathogenesis. · Read current literature & reference correctly in-text. · Provide the aim in the last sentence (be clear & concise). Materials & methods – 2 marks · You must refer to the prac manual with a proper IN TEXT citation (one sentence). · DO NOT list all the materials & methods or any alterations. Results – 15 marks · Logical & clear presentation, showing the raw data, calculations and final results. · Include table & figure legends where appropriate. These must be referred to in the text. · All results must ALSO be explained in words! · Explain in words (not just formulas) how the cell dilution was calculated from OD600 reading · Tabulate colony counts · Work out all equations (5 in total) and state the results in words (dot points OK, do not show workings in the text) indicating what each percentage refers to. Discussion – 15 marks · Actually DISCUSS your results · What did you see? What does it mean? In terms the key principles of the prac: · Was it the expected result? Why/Why not? · If not, what would you expect to see? · You MUST discuss any data you included in your results. · Link results back to background provided in the introduction & discuss further. Some appropriate references should be included here. · Consider the immune response that might be induced by the two micro-organisms. · Last paragraph should be the conclusion – summarise the findings. Was the aim achieved? References – 4 marks · Must have AT LEAST 4 different references, follow one formatting style – no Wikipedia or lecture notes. · Prac manual must be one of your references (for the methods only). Formatting – 4 marks · Logical layout, with correct scientific & English expression and spelling. Immunology Practicals 2004 PRACTICAL 1 ADHERENCE and INVASION ASSAYS Background Most pathogenic bacteria causing infection require virulence determinants that enhance their ability to adhere to sites of infection and invade through the membrane. Humans possess several physical and chemical barriers to infectious agents, which are virtually the same in all individuals (unlike adaptive immune responses). The similarity of these barriers has enabled microbes to evolve avoidance mechanisms based on a similar theme. Adherence to cells is the first and most important microbial mechanism for initiating the infectious disease process. Attachment or adhesion requires the involvement of a receptor on the host and a molecule on the surface of the microbe called an adhesin. Generally, host receptors are carbohydrates while adhesins are usually proteins. For example, strains of E. coli possess one type of pilus, referred to as type 1 pili that bind to receptors containing the sugar mannose. Pili are adhesins found in many Gram-negative species. Gram-positive organisms, such as Streptococcus pyogenes, adhere to epithelial cells of the skin and nasopharynx. Epithelial cells are covered with the plasma glycoprotein fibronectin. Fibronectin acts as a receptor for the lipoteichoic acid adhesin of S. pyogenes. Some microbes do not remain on the epithelial surface but instead penetrate to subepithelial layers and into cells. The ability to penetrate below the epithelium is referred to as invasiveness. Salmonella Typhimurium is capable of invading the human intestinal epithelium. Salmonella, and some enteropathogenic E. coli strains, use a terminally differentiated epithelial cell, the M cell of the Peyer’s patch in the terminal ileum and in other gut-associated lymphoid tissue, as a portal of entry into the host. The M cells found in Peyer’s patches are thought to internalise luminal contents for delivery to underlying antigen-presenting cells. M cells possess fewer lysosomes and a sparse mucus layer that makes them a prime target for invasive bacterial pathogens. In this experiment, the invasiveness of Salmonella Typhimurium 82/6915 will be determined. E. coli DH5( is used as a control (this strain of E. coli is not invasive). References: Note these are basic references, in the literature there will be many more recent references to incisiveness. These should be referred to in your practical writeup. 1. Boyd, Robert F., Basic medical microbiology. 5th ed. 1995. Little Brown & Company (Inc.) 2. Neidhardt, Frederick C., Escherichia coli and Salmonella. 2nd ed. 1996. ASM Press. Figure 4. Electron Micrograph of E. coli adhering to the intestinal epithelium (From H. W. Moon, B. Naggy, and R. E. Isaacson, J. Infect. Dis. 136[Suppl.]:124, 1977) Cancer Cells in Culture Both normal eukaryotic cells and cancer cells can be cultured in vitro in the laboratory. However, they behave quite differently. Normal cells pass through a limited number of cell divisions (50 is about the limit) before they decline in vigor and die. This is probably caused by their inability to synthesize telomerase. Cancer cells may be immortal; that is, proliferate indefinitely in culture. For example, INT407 cells are cultured in laboratories around the world. They are all descended from cells removed from the human embryonic intestinal epithelium. Cancer cells in culture produce telomerase and are therefore what we term immortal. Note that although this is an on-line practical, it is written in the way that it would be done in class. This gives you an idea of the steps involved. The results that you need to analyse are given at the end. Day 1 Materials · DMEM (Dulbecco’s Modified Eagle Medium) · Salmonella Typhimurium 82/6915 culture · E. coli DH5( culture · 24-well plate (12 wells seeded with INT407 cells) · PBS (phosphate buffered saline) · Gentamycin (200 (g/mL) · Triton X-100 (0.1%) · LB agar plates · Sterile spreaders · Sterile Eppendorf tubes · Plastic dropper · Incubator (37(C, 5% CO2) Procedure Note: Salmonella Typhimurium 82/6915 is a non-attenuated wild type strain and hence, is virulent and infectious. Wear gloves at all times during the experiment. 1. You are provided with Salmonella Typhimurium 82/6915 and Escherichia coli DH5(. Determine the cell count for both bacterial cultures. You are given the optical density reading of the cell growth at 600 nm. Using the following formula, dilute your bacteria to 5 x 107 CFU in a total volume of 1 mL in 1 x DMEM. (OD600 x 7.65) – 0.3 =? X 109 CFU/mL 2. You are supplied with a 24 well plate, which has 12 wells containing a monolayer of INT407 cells. The cells have been seeded with 105 cells per well. Wash the monolayer in each well gently with PBS 3 times (500 (l of PBS each time). Growing INT407 cells are adherent – that means, they stick to the bottom of the well and multiply. Hence, you can discard growth medium/PBS by carefully tipping the medium off. 3. Label the plate with columns 1-3 and rows a-d (Figure 5). Blank = 1 x DMEM Column 1 – Control (blank – DO NOT ADD ANY BACTERIA) Column 2 – Adhesion and invasion Column 3 – Invasion only 4. Add 200 (L of 1 x DMEM to all wells in column 1. 5. Add 200 (L of 5 x 107/mL bacteria (in DMEM) to columns 2 and 3 according to the diagram shown above. This will equate to 1 x 107 bacteria per well. 6. Incubate at 37 (C in CO2 incubator for 1.5 hours. During this incubation both E. coli and Salmonella will adhere to the INT407 cells. Only Salmonella will invade the INT407 cells, as E. coli DH5( is incapable of invading host cells. During this incubation period, label the tubes you will need for subsequent steps as outlined on the next page. 7. Wash the monolayer gently with PBS three times (500 (l of PBS each time). 8. To columns 1 and 3 add 200 (L of 200 (g/mL gentamycin. To column 2 add 200 (L/well of 0.1% Triton X-100. This reagent will lyse the INT407 cells but will not kill bacterial cells. Note: Gentamycin kills the bacteria that have adhered but not those that have invaded. This is because it can’t enter the INT407 cells. Triton X-100 lyses INT407 cells releasing any bacterial cells that have invaded the INT407 cells. 9. Incubate at 37(C in CO2 incubator for 15 minutes. 10. Transfer 20 (L from wells A2 - D2 into the corresponding 10-1 tubes (mix the well with a pipette first by scraping the cells). Immediately return the plate to the incubator. 11. Make serial dilutions (in 1X PBS) of the samples from the 4 wells of column 2 and plate out 100 (L of 10-2 and 10-3 dilutions onto LB agar. Label these plates adhesion (E. coli or Salmonella). 12. Incubate column 1 and 3 for a further 45 minutes at 37(C in CO2 incubator. 13. Wash the monolayer three times with PBS. 14. Add 200 (L of 0.1% Triton X-100 to columns 1 and 3 and incubate at 37(C in CO2 incubator for 15 minutes. 15. Make serial dilutions (in 1X PBS) of the 4 wells of column 3 and only A1 from column 1 plate out 100 (L of 10-1 and 10-2 dilutions onto LB agar. Label plates from column 3 invasion (E. coli or Salmonella) and that from A1 control. 16. Incubate LB agar plates at 37(C overnight. Table 2. Labelling instructions for Eppendorf tubes for dilutions in steps 11-15. Step Well Sample Dilutions req. for plating Tube labels No. Tubes req. 11 Adhesion and Invasion A2 E. coli 10-2, 10-3 A2 E -2 and A2 E -3 3 B2 E. coli “ B2 E -2 and B2 E -3 3 C2 S. Typhimurium “ C2 S -2 and C2 S -3 3 D2 S. Typhimurium “ D2 S -2 and D2 S -3 3 TOTAL (step 9) 12 tubes 15 Invasion only A3 E. coli 10-1, 10-2 A3 E -1 and A3 E -2 2 B3 E. coli “ B3 E -1 and B3 E -2 2 C3 S. Typhimurium “ C3