Differential stain differentiating between Gram-positive

Assignment Detail:- MI2011 Microbial Diversity - James Cook University Practical 1: Bacteriology Part 1: Agar Plate Preparation Please ensure this procedure is completed with the supervision of a staff member- 1- Label the base of 3 petri dishes provided with your name and the date-2- Obtain 3 x 20ml bottles of molten nutrient agar from the water bath provided- The agar medium is maintained in a molten state at 45° C-3- Pour the molten agar into the 3 petri dishes within the C2 cabinet-4- Immediately mix the molten agar to ensure a continuous distribution within the petri dish by sliding the agar plate on the bench in a figure of 8 motion-5- Replace the agar plate lid, slightly ajar, and allow for the agar to solidify- Part 2: Selective and Differential Media Specialised media have been devised to facilitate the isolation and identification of bacteria- Selective media contains components which selectively inhibit the growth of certain microorganisms- Differential media typically have a pH indicator which allows the differentiation between various chemical reactions during growth- Nutrient agar supports the growth of all conventional bacterial organisms- MacConkey agar is a selective and differential medium that inhibits Gram positive organisms and allows for the differentiation of Pseudomonas -appears translucent- from E- coli -appears pink in colour-- Cetrimide agar is a selective medium that contains cetrimide to inhibit the growth of most organisms except Pseudomonas which produces a green pigment- Mannitol salt agar -MSA- is used to selectively isolate Staphylococcus- Pathogenic Staphylococcus -coagulase positive Staphylococcus- will form small yellow colonies on the MSA plates, as the organism ferments mannitol- However, non-pathogenic Staphylococcus -coagulase negative Staphylococcus- will form small colourless colonies on the MSA plates as it does not ferment mannitol- A broth containing a mixture of 3 bacteria -Staphylococcus aureus, E- coli andPseudomonas aeruginosa- is provided- 1- Streak the mixed broth onto the following media:• Nutrient agar -NA-• MacConkey agar -MAC-• Cetrimide agar -CET- Part 3: Microbes in the Environment: Airborne1- Expose 1 plate -prepared in Part 1: Agar Plate Preparation- to the air for 20 minutes- Select various positions inside and outside of the laboratory building- Body Surface -External-:1- Use a sterile swab moistened with sterile water to swab the interdigital spaces of the left hand of one individual in your pair-2- Inoculate a nutrient agar plate -prepared in Part 1: Agar Plate Preparation-, using the lawn plating method -refer to Practical 1, Part 3 for method--3- Wash hands of the subject with soap and water OR disinfectant and water -as directed by the instructing staff members-- Lightly dry hands with a paper towel-4- Use a sterile swab moistened with sterile water to swab the interdigital spaces of the opposite hand as completed previously-5- Inoculate a nutrient agar plate -prepared in Part 1: Agar Plate Preparation-, using the lawn plating method- Part 4: Motility Test: Observe live unstained P- aeruginosa and S-aureus by completing the wet preparation method- Ensure that you observe a positive control initially, and then progress onto the sample- 1- Place a drop of liquid culture onto the centre of a glass slide-2- Cover with a coverslip-3- Observe under the microscope, setting it up as previously instructed- Part 5: Gram Stain Gram staining is the most widely used staining procedure in bacteriology- It is a differential stain differentiating between Gram-positive and Gram-negative bacteria- Bacteria that stain purple are termed Gram-positive; those that stain pink are Gram- negative bacteria- Gram-positive and gram-negative bacteria stain differently because of differences in the structure of their cell walls; bacterial cell walls contain peptidoglycan- Gram- positive bacterial cell walls appear thick and consist of numerous interconnecting layers of peptidoglycan- Typically, 60% to 90% of the cell wall is peptidoglycan- Gram-negative bacterial cell walls contain a much thinner, single layer of peptidoglycan only 2 or 3 layers thick which forms only 10% to 20% of the cell wall- Prepare a Gram stain of S-aureus and E-coli from the agar plate cultures provided using the instructions below- Fill in observations in table 2-3 Preparation of bacterial film1- Label the slide, with your name and the organisms that you are preparing -Figure 2-1--2- Place the slide as close to the Bunsen burner as possible3- Remove the lid of a sterile bottle of saline and retain in the hand avoiding contamination-4- Sterilise the top of the bottle; by waving it through the hottest part of the flame-5- Sterilise a loop in the Bunsen burner and transfer a loop of the water on the slide for each sample- Re-sterilise the wire loop-6- Place the previously cultured agar plate upside down on the bench so that the lid is on the bench-7- Pick up the plate leaving the lid on the bench-8- Sterilise a loop in the Bunsen burner and pick a single colony of bacteria-9- Replace the agar plate lid-10- Smear the loop, containing bacteria, in the saline that you have placed on the slide and spread over an area of approximately 1cm2-11- Re-sterilise the loop-12- Allow the film to air dry, or if you are pressed for time, pass the slide over the Bunsen burner flame- However ensure the slide does not become too hot, you can assess the temperature of the slide by touching it to the back of your hand- If it is too hot to touch, allow the slide to cool down in temperature- Gram StainingStaining kits are on the side benches under the windows at the stainless steel sinks- 1- Cover the entire bacterial film with crystal violet-2- Leave the stain on for 1 minute- Drain off the crystal violet, do not rinse with water-3- Cover the entire bacterial film with iodine solution4- Leave the stain on for 1 minute- Rinse the slide with tap water5- Cover the entire bacterial film with decolouriser for 5-10 seconds- Please ensure that you do not over-decolourise, by leaving the decolouriser on the slide for an extended period of time-6- Immediately rinse with tap water- Cover the entire bacterial film with safranin-7- Leave the stain on for 1 minute then wash gently with water and pat dry with paper towel or tissue- Part 6: Viable count of water samples: This is the only result that will be used in your practical report along with week 3 practical resultsYou will need to use the water that you have collected prior to arriving to the practical session-1- Gently shake the water sample container to ensure the sample is well distributed through the container-2- Pipette 1ml of the water sample aseptically into 9ml of saline-3- Mix well- This is a 10-1 dilution-4- Transfer 1ml of the 10-1 dilution to 9ml of saline-5- Mix well- This is a 10-2 dilution-6- Label the base of the 3 empty petri dishes provided, with your name, the date and dilution-7- Plate 1ml of each of the 10-1 dilution and 10-2 dilutions onto 2 of the petri dishes-8- Within close proximity to the Bunsen burner, pour the molten agar into these petri, avoiding direct contact with the sample-9- Additionally, pour molten agar into the third, empty petri dish, within close proximity to the Bunsen burner- This will be your negative control plate; which will determine the sterility of the plate formation-10- Immediately mix the molten agar by sliding the agar plate on the bench in a figure of 8 motion-11- Replace the agar plate lid, slightly ajar, and allow for the agar to solidify- Practical 2: Bacteriology Catalase Production1- Place a glass slide, your cultured plate and your tootpick container within the zone of sterility-2- Select a colony from a previously cultured plate using a toothpick- Take care to avoid agar- Smear this onto the surface of a glass slide3- Place a drop of catalase reagent -hydrogen peroxide- onto the deposited bacteria on the glass slide-4- Observe the bacteria for the production of bubbles Note: If the organism is catalase positive, bubbles will develop around the inoculum almost immediately- Oxidase Production1- Place a filter paper onto the surface of a glass slide-2- Add 2 to 3 drops of the oxidase reagent to the filter paper3- Place the previously cultured agar plate upside down on the bench so that the lid is on the bench-4- Hold the plate within the zone of sterility leaving the lid on the bench-5- Using a sterile toothpick pick a single colony of bacteria from a previously cultured plate- Do not use a metal loop-6- Replace the agar plate lid7- Smear the bacterial colony onto the filter paper on the surface of a glass slide • Examine the plates exposed to the airborne and external body surfaces bacteria- Count the number of colonies formed by the different microorganisms- Describe the bacterial colonies, as outlined in Appendix 1 Macroscopic Examination of Bacteria- Consider What effects do the different hand washing treatments have on the microbial flora???? • Examine the plate inoculated with the nasal swab collection and external body surface swab- Describe the bacterial colonies, as outlined in Appendix 1 Macroscopic Examination of Bacteria- Does your plate indicate the person sampled is carrying a pathogen???? • Examine the plates prepared for the water quality assessment- Count the number of colonies for each dilution- Note: colony morphology is not required for pour plates- How much bacteria was in the original water sample???? - include this section only in Practical 3 results Attachment:- Microbial Diversity-rar

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