Reference no: EM133106473

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 and
Pseudomonas 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:

Airborne
1. 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 film
1. 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 possible
3. 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 Staining
Staining 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 solution
4. Leave the stain on for 1 minute. Rinse the slide with tap water
5. 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 results
You 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 Production
1. 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 slide
3. 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 Production
1. Place a filter paper onto the surface of a glass slide.
2. Add 2 to 3 drops of the oxidase reagent to the filter paper
3. 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 lid
7. 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