Reference no: EM133112160

Practical: 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.

Body Surface (Internal)
1. Using a sterile swab, which has been moistened with sterile water, swab the nasal cavity of one individual in your pair.
2. Inoculate a mannitol salt agar (MSA) plate 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.

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.

Attachment:- Microbial Diversity.rar