LB5-Kirby-Bauer Method

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Micro

Two Broth Cultures

Escherchia Coli
Staphylococcus Epidermidis

Tubes of Antibiotic Disks

Chloramphenicol
Penicillin
Streptomycin
Tetracycline

Before you streak your plates

Place E. Coli culture next to turbidity standard
observe-are they equal in cloudiness?
A. E. Coli too cloudy-add sterile saline to dilute
B. E. Coli too clear-Incubate to increase growth
Do the same w/ S. Epidermidis

Antimicrobial sensitivity tests are used:

to measure effectiveness of antibiotics on pathogens. Pathogen is susceptible to drug when zone of inhibition appears around paper disk

Kirby-Bauer Test is Standardized

1. Media in plates-uniform thickness
Thick media-antibiotic diffuses slower
Skewed results: falsely assume antibiotic is less susceptible than it really is

2. Media should not have moisture on the surface and be used w/n certain time period
old, dry media -antibiotic diffuses slower
new, moist media-antibiotic diffuses faster

3. Broth cultures must be equal in turbidity to standard and used w/n 30 minutes
0.5 McFarland turbidity standard contains roughly 1.5 x 10^8 cells/ml
30 minutes represents a standard germination time

Slow diffusion

Falsely assume microbe is less susceptible
why?
Zone of inhibition is smaller
Thick, old dry media drug more effective

Fast diffusion

Falsely assume microbe is more susceptible
why?
Zone of inhibition is larger
Thin, new moist media drug is less effective

1a. What might the consequence be of pouring the plates 2 mm deep instead of 4 mm deep?

Thin agar, increased diffusion of drug produces larger zone of inhibition. Falsely think microbe is more susceptible than it is.

1b. The Mueller-Hinton II plates are supposed to be used w/n a specific time after their preparation and should be free of visible moisture. What negative effect(s) might moisture have on the test?

Old plates are dried out-decreased diffusion. Moist plates increase diffusion

In clinical applications of the Kirby-Bauer Test, diluted cultures (for the McFarland standard comparison) must be used w/n 30 minutes. Why is this important?

30 min standard generation time broth culture left standing past 30 min-too turbid-no longer equivalent to standard

E. Coli and S. epidermidis were chosen to represent Gram-negative and Gram-positive bacteria, respectively. For a given antibiotic, is there a difference in susceptibility between the Gram-positive and Gram-negative bacteria? If so, what difference(s) do you see?

Yes drugs that target PG cell wall are less effective on gram (-) cells; cannot get past outer membrane

Suppose you do this test on a hypothetical staphylococcus species w/ the antibiotics pencilllin (p-10) and chloramphenicol (C30). You record zone identical diameters of 25 mm for the chloramphenicol and penicillin disks. Which antibiotic would be more effective against this organism? What does this tell you about comparing zone diameters to each other and the importance of the zone diameter interpretive chart?

Chlorampenicol is the best choice w/ a zone diameter 25 mm, if falls to the right suceptible zone (>18)
Penicillin w/ zone diamter of 25mm, falls to the left of the resistant zone (<28)

How does the antibiotic get from the disk to the agar?

Diffusion-area of high concentration to low concentration

After incubation, does the antibiotic extend into the agar beyond the zone of inhibition? How does your anser related to the concept of MIC?

Yes, edge of zone of inhibition represents the MIC, but not the end of diffusion

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