The antibiotic disk sensitivity test

One of the most widely used methods to determine the susceptibility of microorganisms to antimicrobial agents is the disc agar-diffusion method. The principle of this method is dependent upon the inhibition of reproduction of a microorganism on the surface of a solid medium by an antimicrobial agent which diffuses into the medium from a filter paper disc. Thus, for an organism which is truly sensitive (susceptible) to an antimicrobial agent (i.e., affected at the target site as explained above), we expect to see a zone of inhibition around the disc impregnated with the agent; beyond this zone an unaffected, area of normal growth (lawn) of the organism is present. However, the degree of inhibition necessary to make each antibiotic effective during use is unique; hence each resistance zone size cutoff is specific to the antibiotic. Accurate measurement of zone diameter is necessary to properly interpret this test.

Period 1

Materials

12 to 18-hour cultures of Staphylococcus epidermidis, Pseudomonas fluorescens, Escherichia coli, Enterococcus faecalis and Bacillus subtilis (all grown in Trypticase Soy Broth). Each person will choose one culture from this list.

1 sterile cotton swab

2 plates of Penassay Agar

2 antibiotic disc dispensers, each dispensing four antibiotic discs

1. Dip the sterile cotton swab into the culture to be tested. With the swab, cover the entire surface of each of the Penassay Agar plates such that a confluent lawn of growth would result if nothing more were to be done on the plates. Let the plates dry at room temperature for several minutes.
2. With the disc dispensers, apply four antibiotic discs to each of the plates, making sure that all eight antibiotics are represented on your plates. The instructor will demonstrate the use of the dispenser.
3. Incubate the plates for 1-2 days at 37°C.

Period 2

Materials

1 ruler with millimeter (mm) divisions

Figure 1 Disk sensitivity test

The results of the disk sensitivity test for P. fluorescens, E. coli, B. subtilis, and S. epidermidis. Notice that the gram-positive bacteria are more sensitive to the antibiotics that we used here. To make your measurements, take a ruler, measure the diameter of the zones of clearing on the screen. Compare the diameters you measure to the table in Figure 10-2.

1. With the ruler, measure the zones of inhibition in millimeters from the underside of the plate. Measure the entire diameter of the zone, including the disc. For any zone which contains one or more isolated colonies, what do these colonies represent? Would you choose to ignore their presence? Is this a zone of inhibition after all?
2. Interpret your findings with the aid of the table below. (The instructor may substitute other antibiotics and indicate their zone diameter data.) Compare the patterns of susceptibility and resistance with respect to gram reaction, potential pathogenicity and natural habitat of each organism.

Table 2 lists many common antibiotics that are used in the disk sensitivity test and their zones of clearing.

Table Zones of clearing for various antibiotics

Antibiotic (and disc identifier)	Disk potency	Inhibition zone diameter to nearest mm
		Resistant	Intermediate	Susceptible
Ampicillin (AM10) Gram-negative rods and enterococci	10 µg	11	12-13	14
Ampicillin (AM10) Staphylococci and highly penicillin-sensitive organisms	10 µg	20	21-28	29
Bacitracin (B)	10 units	8 or less	9 to 12	13 or more
Chloramphenicol (C)	30 µg	12 or less	13 to 17	18 or more
Ciprofloxacin (CIP)	5 µg	15 or less	16 to 20	21 or more
Colistin (CL)	10 µg	8 or less	09 to 10	11 or more
Doxycycline (D)	30 µg	12	13-15	16
Erythromycin (E)	15 µg	13 or less	14 to 17	18 or more
Gentimycin (G)	10 µg	12	13-14	18
Kanamycin (K30)	30 µg	13	14-17	18
Methicillin (ME5)	5 µg	9	10-13	14
Nalidixic Acid (NA)	30 µg	13 or less	14 to 18	19 or more
Oxacillin	1 µg	17	18-24	25
Penicillin G (P)	10 units
staphylococci		28 or less	-	29 or more
most others		11 or less	12 to 21	22 or more
Polymyxin B (PB)	300 units	8 or less	9 to 11	12 or more
Streptomycin (S)	10 µg	11 or less	12 to 14	15 or more
Sulfadiazine (SD) *	300 µg	12 or less	13 to 16	17 or more
Sulfaoxizole (G300)	300 µg	12 or less	13-16	17 or more
Sulfisoxazole (G25) *	25 µg	12 or less	13 to 16	17 or more
Tetracycline (TE)	30 µg	14 or less	15 to 18	19 or more

The zone of clearing varies depending upon the antibiotic and its chemical properties. The medical effectiveness of antibiotics in patients has been correlated with a certain zone size and this is used to decide whether an antibiotic will be useful for treating a test pathogen.
*For this agent, the zone is measured at the margin of heavy growth. Sulfonamides may not inhibit the tested culture until after several generations. Therefore, a band of slight, hazy growth just inside the margin of the heavy growth may be ignored.

Reference:

http://inst.bact.wisc.edu/inst/index.php?module=Book&func=displayarticle&art_id=135