Antibacterial Activity Test by Disk Diffusion Method_A Complete Procedure (Kirby and Bauer Method)

Summary

This video demonstrates the process of conducting an antibacterial activity test, which involves six key steps: media preparation, bacterial culture preparation, sample placement, incubation, result observation, and reporting. The script walks through the creation of Tryptone Soya Agar, Tryptone Soya Broth, and Muller Hinton Agar, followed by bacterial inoculation and sample preparation. It then details the incubation process and the observation of inhibition zones to assess antibacterial effectiveness. The sample tested showed low antibacterial activity against *Staphylococcus aureus*, compared to the larger zone of inhibition created by the amoxicillin antibiotic disc.

Takeaways

• 😀 The antibacterial activity test is essential for evaluating the effectiveness of antibacterial substances.
• 😀 The test consists of six key steps: media preparation, bacterial culture preparation, sample preparation, incubation, result observation, and reporting.
• 😀 Key equipment needed includes a biosafety cabinet, incubator, autoclave, millimeter scale, cotton swab stick, forceps, scissors, and specific agar media (Muller Hinton, Tryptone Soy Agar).
• 😀 Personal hygiene and good laboratory practices are critical throughout the experiment to avoid contamination.
• 😀 The preparation of Tryptone Soy Agar involves dissolving 40g of dehydrated medium in 1 liter of distilled water, autoclaving at 121°C for 15 minutes, and checking for contamination.
• 😀 For Tryptone Soy Broth, dissolve 30g of dehydrated medium in 1 liter of distilled water, distribute it into test tubes, and autoclave it as well.
• 😀 Muller Hinton Agar is prepared similarly to the other media, using 38g of dehydrated agar powder in 1 liter of distilled water.
• 😀 The bacterial culture is prepared by inoculating a pure culture into Tryptone Soy Broth and incubating at 36-37°C for 24 hours.
• 😀 For inoculation, the bacterial concentration should ideally be matched to 0.5 McFarland turbidity, though this is not strictly necessary for the experiment.
• 😀 The sample is prepared by cutting a square block (6mm x 6mm) and placing it on an inoculated Muller Hinton Agar plate, along with an antibiotic disc (e.g., amoxicillin) for comparison.
• 😀 After incubation, the results are observed by measuring the zone of inhibition, which reflects the antibacterial activity of the sample compared to the control antibiotic.

Q & A

What is the purpose of the antibacterial activity test described in the video?

The purpose of the antibacterial activity test is to screen the effectiveness of any antibacterial substance by evaluating its ability to inhibit bacterial growth.

What are the six main steps involved in the antibacterial activity test?

The six main steps are: 1) Media preparation, 2) Bacterial culture preparation, 3) Collation of culture plate, 4) Sample preparation and placement, 5) Incubation, 6) Result observation and reporting.

What is the importance of sterilization during the test?

Sterilization ensures that there is no contamination of the samples or the bacterial culture, which is essential for obtaining accurate and reliable results in the test.

What materials are required for conducting the antibacterial activity test?

The materials required include a balanced mission bio safety cabinet, incubator, autoclave, millimeter scale, cotton swab stick, forceps, scissors, Muller Hinton agar, Tryptone Soya agar, and Tryptone swab broth.

How do you prepare the Tryptone Soya agar medium?

To prepare Tryptone Soya agar, dissolve 40 grams of dehydrated DSM medium into 1 liter of distilled water, autoclave the mixture at 121°C and 15 pounds pressure for 15 minutes, and then pour the medium into sterile petri dishes.

Why is the McFarland turbidity standard used in this test?

The McFarland turbidity standard is used to match the bacterial concentration in the broth culture to a specific level, ensuring consistent and standardized inoculum density for accurate results.

What is the role of the swab in inoculating the agar plate?

The swab is used to spread the bacterial culture evenly across the surface of the Muller Hinton agar plate in a zigzag pattern to ensure proper inoculation for the antibacterial test.

Why is the amoxicillin antibiotic disc used as a positive control?

The amoxicillin antibiotic disc is used as a positive control to compare the antibacterial activity of the test sample, as it is known to have a well-established zone of inhibition.

What should be observed after incubating the plates?

After incubation, the zone of inhibition around the antibiotic disc and the test sample should be observed. The size of the zone indicates the effectiveness of the antibacterial agent in inhibiting bacterial growth.

How is the zone of inhibition measured, and what does it indicate?

The zone of inhibition is measured using a millimeter scale. The larger the zone, the more effective the antibacterial agent is. The test sample in this case showed a small zone of inhibition (8 mm), indicating low antibacterial activity compared to the amoxicillin disc (34 mm).