Lab Assignment #3
Kirby-Bauer Antimicrobic Sensitivity Testing
Background Information – (Please open and use the LAB NOTES for Kirby-Bauer Sensitivity Test from the weekly notes), also refer the appropriate chapter in the book.
The Kirby-Bauer Test is routinely used in clinical labs to test the efficacy of various antibiotics against microbes. In this procedure, using a sterile swab, a large size agar plate is inoculated with a predetermined known culture. The agar is called Mueller-Hinton Agar, and it is specifically used for this kind of testing. Small paper discs impregnated with an antibiotic are then placed around the plate and the plate is incubated overnight. There may be up to 12 different discs, each is abbreviated with the name and dosage of an antibiotic. The next day one will see a blanket of bacteria over the plate and possibly clear circles around some or many of the discs where the bacteria has been inhibited by the given antibiotic. These circles are referred to as a “Zone of Inhibition”.
Each of the zones are measured in millimeters and recorded. Then one must look up each antibiotic alphabetically on the Sensitivity charts (3 pages) within the chapter and compare the measured result to the reference numbers to determine which column (Resistant, Intermediate or Sensitive) that each particular antibiotic falls under. You will see that every antibiotic has different reference zone size numbers so that’s why you need to do them individually. When you are done you can easily compare the efficacy of the different antibiotics to see which are the most or least effective. Doctors will routinely order this kind of test for their patients, especially if there is a recurring infection and if they need to switch antibiotics for other reasons.
Please pay attention to “the factors which affect zone size” in the Notes because it is very important that the inoculations are done properly for this procedure.
Questions:
1) Here are some hypothetical results for the following antibiotics. Use the charts in the book to determine the zone ratings (Resistant, Intermediate, Sensitive) for the following disc measurements first for Staphlococci and second for Enterobacteriaceae.
Antibioitic Measured Zone Size Rating- Staph/ Enterobac
Am- 10 14mm
CF-30 24mm
E-15 18mm
Va-30 23mm
S-10 17mm
P-10 13mm
G-10 11mm
B-10 0 mm
2) If you have a plate with no zones what does that tell you about the importance of this organism? Consider your response from both the patient and an epidemiological perspective. Have you heard of such a thing? Yes…..so give an example.
3) If you left the plates about 8 or more hours in the incubator, what would it do to the zone sizes and results?
4) What zone effects would you get by putting too heavy or light amount of bacterial inoculum on the plates?
5) Why is this method universally used for epidemiological studies across the country?
6) Your patient has a bad infection but is allergic to Penicillin so therefore the doctor would categorically reject which other antibiotics for the patient? Why?
Below is an actual Mueller-Hinton Agar plate already incubated so you can see the growth and the zones of inhibition. For simplicity the discs are actually alphabetized not abbreviated.
These are the Kirby Bauer results for Escherichia coli isolated from a urine culture.
A. Novobiocin
B. Amikacin
C. Chlorophenicol
D. Vancomycin
E. Ciprofloxacin
F. Cephalothin
Using the sensitivity charts in the chapter, please determine whether this member of the Enterobacteriacae is Susceptible, Intermediate, or Resistant to the previous drugs?
