Etest for antibiotic susceptibility

Summary

This video demonstrates the process of performing antibiotic susceptibility testing using the E-test system. The procedure involves creating a bacterial lawn on a Mueller Hinton agar plate enriched with sheep blood. Bacteria are inoculated on the plate using a swab, and an antibiotic strip is placed at the center. After incubation, the zone of inhibition around the strip is measured to determine the effectiveness of the antibiotic. This method provides valuable insight into bacterial resistance and sensitivity to various antibiotics.

Takeaways

• 😀 Make a lawn of bacteria on a Mueller Hinton agar plate with 5% sheep blood.
• 😀 Use the proper inoculation technique by lightly touching two or three bacterial colonies with a wand.
• 😀 Prepare a saline blank and mix the bacteria into it by flicking the tube to achieve the correct turbidity for testing.
• 😀 Use a sterile swab to collect the bacterial suspension and ring out the excess before streaking it on the plate.
• 😀 Streak the plate in three different directions, rotating it 90 degrees each time to ensure full coverage.
• 😀 After streaking, ring the edge of the agar plate with the swab to promote bacterial growth up to the edge.
• 😀 Prepare the antibiotic strip using sterile forceps and flame the forceps to ensure sterility before handling.
• 😀 Place the antibiotic strip upright in the center of the plate and work out any air bubbles from the strip.
• 😀 Label the plate appropriately before incubating it for 24 hours to allow for bacterial growth.
• 😀 After incubation, read the results by examining where the growth line intersects with the zone of inhibition on the strip.
• 😀 The E test system helps to determine the susceptibility of bacteria to specific antibiotics based on the inhibition zones.

Q & A

What is the first step in antibiotic susceptibility testing using the E test?

The first step is to make a lawn of the bacteria to be tested on a Mueller Hinton agar plate, which is supplemented with 5% sheep blood.

Why is it important to use a Mueller Hinton agar plate for antibiotic susceptibility testing?

Mueller Hinton agar is used because it is a standardized medium that supports the growth of many bacteria and allows for reliable antibiotic testing.

How do you prepare the bacterial suspension for inoculating the plate?

You prepare the bacterial suspension by lightly touching two or three colonies of the bacteria with a sterile wand, mixing it with saline in a blank tube, and flicking the tube 10-15 times to achieve the appropriate turbidity.

Why is it important not to make the plate too wet with bacteria?

It is important to avoid excess moisture to ensure even distribution of the bacteria and proper growth patterns on the plate.

How should the swab be used to streak the plate?

The swab should be inserted into the bacterial suspension and then lightly rung on the edge of the tube. The plate should be streaked in three directions, rotating the plate 90 degrees between each streak, covering the entire surface.

What is the purpose of ringing the edge of the agar plate with the swab?

Ringing the edge of the agar plate ensures that the bacterial growth reaches the outer edges of the plate, providing a complete zone of growth for the antibiotic testing.

How is the antibiotic strip applied to the plate?

A sterile forceps is used to place the antibiotic strip upright in the center of the plate, and any air bubbles on the strip are worked out by gently pressing the side.

What is the next step after applying the antibiotic strip to the plate?

After placing the antibiotic strip on the plate, the plate should be labeled and incubated for 24 hours to allow bacterial growth and antibiotic interaction.

How are the results of the antibiotic susceptibility test interpreted?

The results are interpreted by examining where the line of growth or the zone of inhibition intersects the antibiotic strip on the plate, which indicates the bacteria’s susceptibility to the antibiotic.

What role does the incubation period play in the antibiotic susceptibility test?

The incubation period allows the bacteria to grow and interact with the antibiotic on the plate, creating a visible zone of inhibition if the bacteria are susceptible to the antibiotic.