Question

1. In the agar, the highest concentration of your antibiotic is _____.

a)in a ring closest to the edge of the Petri plate

b)in a ring closest to the edge of the disk

c)in a ring halfway between the disk edge and the Petri plate edge

d)in a ring on the lid of the Petri plate

2.Why is it important to spread plate cells from a pure culture?

a)Mixed cultures may cause condensation on the lid of the plate, obscuring the results of the incubation.

b)Pure cultures are used because they respond uniformly to the antibiotic chemical.

c)Pure cultures tend to be non-motile, whereas mixed cultures might “swim” away from the disinfectant.

d)Actually, it doesn’t matter whether the culture is pure or not. Microbiologists opt to use pure cultures because they are easier to manipulate.

3.Why is it important to produce confluent growth, instead of individual colonies, on the Petri plate?

a)Confluent growth ensures that every bacterial cell on the Petri plate touches the Whatman disks.

b)Confluent growth ensures that the zones of inhibition are due to the activity of the antimicrobial chemical rather than a failure to grow.

c)Confluent growth ensures that the microbiologist has used aseptic technique to inoculate plates.

d)Actually, individual colonies are necessary to demonstrate that only one species is represented on the plate.

Based on these lab results, with which antibiotic should this patient with a Staphylococcus aureus infection be treated? Would any of these antibiotics be good candidates?

The antibacterial susceptibility test using disc diffusion method shows the susceptibility of S. aureus to methicilin with an inhibition zone diameter of 17 mm.

The acceptable standard diameter zones of inhibition for sensitive S. aureus for methicillin, ampicillin and vancomycin are >33 mm, >20 mm and >15 mm respectively (NCCLS, 1993).

Why?

(HINT: What would you call this S. aurues?)

Answer 1

1)a) Highest concentration of antibiotic is in the ring closest to the edge of the petriplate as greater the diameter of the zone of inhibition considering the antibiotic containing disc as centre, greater the efficacy of the antibiotic.

2)b) It is important to use a pure culture so that response to the antibiotic is uniform. In case of mixed cultures, different strains will respond differently to the antibiotic and zones of inhibition of different sizes will be obtained resulting in  ambiguous results.

3)b) Confluent growth ensures that zone of inhibition are due to the effect of antibiotic rather than failure to grow. The empty spaces between colonies in case of non- confluent growth will lead to this ambiguity.

4) According to standards only a zone of diameter greater than 33mm indicates sensitivity of the bacteria to methicilin, so the obtained diameter of 17 mm which is lesser than the standard indicates that S.aureus is resistant to methicilin. Since the zone of inhibition is greater than the standard of vancomycin, vancomycin can be tried over S.aureus, however the sensitivity can be determined only upon subjecting it to vancomycin treatment.