18.7.3: Procedure Antibiotic Susceptibility Testing

Videos

Videos reviewing techniques used in this lab:

• Bauer-Kirby Antibiotic Susceptibility Testing Procedure
• Interpreting the Results of the Bauer-Kirby Method of Antibiotic Susceptibility Testing

MATERIALS

• 150mm Mueller-Hinton agar plates (3)
• Sterile swabs (3)
• An antibiotic disc dispenser containing discs of antibiotics commonly effective against Gram-positive bacteria, and one containing discs of antibiotics commonly effective against Gram-negative bacteria

ORGANISMS

Trypticase Soy broth cultures of Staphylococcus aureus (Gram-positive), Enterococcus faecalis (Gram-positive), and Pseudomonas aeruginosa (Gram-negative)

ANTIBIOTIC SUSCEPTIBILITY TESTING PROCEDURE: to be done in groups of 3

The basic steps for the Bauer-Kirby method of antimicrobial susceptibility testing are given below. This outline of procedure is intended to be used as an adjunct to general microbiology laboratory instruction. The procedure is highly regulated and controlled by the Clinical and Laboratory Standards Institute (CLSI) and must be accompanied by a rigorous quality assurance program including performance by certified and/or licensed personnel when the results are to be reported in clinical settings .

1. Take 3 Mueller-Hinton agar plates. Label one S. aureus, one E. faecalis, and one P. aeruginosa.

2. Using your wax marker, divide each plate into thirds to guide your streaking.

3. Dip a sterile swab into the previously-standardized tube of S. aureus. Squeeze the swab against the inner wall of the tube to remove excess liquid.

4. Streak the swab perpendicular to each of the 3 lines drawn on the plate overlapping the streaks to assure complete coverage of the entire agar surface with inoculum.

5. Repeat steps 3 and 4 for the E. faecalis and P. aeruginosa plates.

6. Using the appropriate antibiotic disc dispenser, place Gram-positive antibiotic-containing discs on the plates of S. aureus and E. faecalis; Gram-negative antibiotic-containing discs on the plate of P. aeruginosa.

7. Incubate the 3 plates upside down and stacked on the shelf of the 35°C incubator corresponding to your lab section until the next lab period.

8 . Using a metric ruler, measure the diameter of the zone of inhibition around each disc on each plate in mm by placing the ruler on the bottom of the plate (Fig. \(\PageIndex{1}\)).

Figure \(\PageIndex{1}\): Antibiotic Susceptibility Testing. Antibiotic susceptibility testing plate. You measure the diameter of zones of inhibition in millimeters. The zone seen here measures 20 mm in diameter. (Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0)

• If there is a double zone of inhibition, measure the diameter of the innermost zone (see Fig. \(\PageIndex{2A}\) and Fig. \(\PageIndex{2B}\)).

Fig. \(\PageIndex{2A}\): Double Zone of Inhibition	Fig. \(\PageIndex{2B}\): Double Zone of Inhibition Colonies in Zone, and Feathering
If there is a double zone of inhibition, measure the diameter of the innermost zone (arrowed line).	Examples of double zone of inhibition, colonies in the zone, and feathering.
Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0

If there is a zone containing colonies, measure the diameter of the colony free zone. Measure from the colony closest to the antibiotic disc to the center of the disc (the radius) and double that number to get the diameter. (See Fig. \(\PageIndex{3A}\) and Fig. \(\PageIndex{3B}\)).

Fig. \(\PageIndex{3A}\): Zone Containing Colonies	Fig. \(\PageIndex{3A}\): Double Zone of Inhibition Colonies in Zone, and Feathering
If there is a zone containing colonies, measure the diameter of the colony free zone (arrowed line).	Examples of double zone of inhibition, colonies in the zone, and feathering.
Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0

• If there is a feathered zone, measure the diameter of the point where there is an obvious demarcation between growth and no growth. Measure from the end of the feathering closest to the antibiotic disc to the center of the disc (the radius) and double that number to get the diameter. (See Fig. \(\PageIndex{4A}\) and Fig. \(\PageIndex{4B}\)).

Fig. \(\PageIndex{4A}\): Zone Containing Feathered Growth	Fig. \(\PageIndex{4A}\): Double Zone of Inhibition Colonies in Zone, and Feathering
If there is a zone containing feathered growth, measure the diameter of the growth-free zone (arrowed line).	Examples of double zone of inhibition, colonies in the zone, and feathering.
Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0

• If there is a double zone of inhibition, measure the diameter of the innermost zone (see Fig. \(\PageIndex{2A}\) and Fig. \(\PageIndex{2B}\)).
• If there is a zone containing colonies, measure the diameter of the colony free zone. Measure from the colony closest to the antibiotic disc to the center of the disc (the radius) and double that number to get the diameter. (See Fig. \(\PageIndex{3A}\) and Fig. \(\PageIndex{3B}\).).
• If there is a feathered zone, measure the diameter of the point where there is an obvious demarcation between growth and no growth. Measure from the end of the feathering closest to the antibiotic disc to the center of the disc (the radius) and double that number to get the diameter. (See Fig. \(\PageIndex{4A}\) and Fig. \(\PageIndex{4B}\)).
• When testing Staphylococcus aureus, the haze around an oxacillin should not be ignored. Measure the diameter of the zone free of growth or haze.

9 . Determine whether each organism is susceptible, moderately susceptible, intermediate, or resistant to each chemotherapeutic agent using the standardized table (Table 2) and record your results.

Table \(\PageIndex{1a}\) Zone Size Interpretive Chart for Bauer-Kirby Test

Antimicrobial agent	Disc code	R = mm or less	I = mm	MS = mm	S = mm or more
amikacin	AK-30	15	15-16	-	16
amoxicillin/ clavulanic acid - Staphylococcus	AMC-30	19	-	-	20
amoxicillin/ clavulanic acid - other organisms	AMC-30	13	14-17	-	18
ampicillin - Staphylococcus	AM-10	28	-	-	29
ampicillin - G- enterics	AM-10	11	12-13	-	14

Antimicrobial agent	Disc code	R = mm or less	I = mm	MS = mm	S = mm or more
azlocillin	AZ-75	14	15-17	-	13
aztreonam	ATM-30	15	-	16-21	22
carbenicillin - Enterobacteriaceae	CB-100	17	18-22	-	23
carbenicillin - Pseudomonas	CB-100	13	14-16	-	17
cefamandole	MA-30	14	15-17	-	18
cefazolin	CZ-30	14	15-17	-	18

Table \(\PageIndex{1b}\) Zone Size Interpretive Chart for Bauer-Kirby Test

Antimicrobial agent	Disc code	R = mm or less	I = mm	MS = mm	S = mm or more
cefonicid	CID-30	14	15-17	-	18
cefoperazone	CFP-75	15	-	16-20	21
cefotaxime	CTX-30	14	-	15-22	23
cefotetan	CTT-30	12	-	13-15	16
cefoxitin	FOX-30	13	-	15-17	18
ceftazidime	CAZ-30	14	15-17	-	18

Table \(\PageIndex{1c}\) Zone Size Interpretive Chart for Bauer-Kirby Test

Antimicrobial agent	Disc code	R = mm or less	I = mm	MS = mm	S = mm or more
ceftizoxime - Pseudomonas	ZOX-30	10	-	11	-
ceftizoxime - other organisms	ZOX-30	14	-	15-19	20
ceftriaxone	CRO-30	13	-	14-20	21
cefuroxime	CXM-30	14	15-17	-	18
cephalothin	CF-30	14	15-17	-	18
chloramphenicol	C-30	12	13-17	-	18
cinoxacin	CIN-100	14	15-18	-	19

Table \(\PageIndex{1d}\) Zone Size Interpretive Chart for Bauer-Kirby Test

Antimicrobial agent	Disc code	R = mm or less	I = mm	MS = mm	S = mm or more
ciprofloxacin	CIP-5	15	16-20	-	21
clindamycin	DA-2	14	15-20	-	21
doxycycline	D-30	12	13-15	-	16
erythromycin	E-15	13	14-22	-	23
gentamicin	CN-10	12	13-14	-	15
imipenem	IPM-10	13	14-5	-	16
kanamycin	K-30	13	14-17	-	18

Table \(\PageIndex{1e}\) Zone Size Interpretive Chart for Bauer-Kirby Test

Antimicrobial agent	Disc code	R = mm or less	I = mm	MS = mm	S = mm or more
methicillin - Staphylococcus	DP-5	9	10-13	-	14
mezlocillin	MEZ-75	12	13-15	-	16
minocycline	MI-30	14	15-18	-	19
moxalactam	MOX-30	14	-	15-22	23
nafcillin - Staphylococcus	NF-1	10	11-12	-	13
nalidixic acid	NA-30	13	14-18	-	19
netilmicin	NET-30	12	13-14	-	17

Table \(\PageIndex{1f}\) Zone Size Interpretive Chart for Bauer-Kirby Test

Antimicrobial agent	Disc code	R = mm or less	I = mm	MS = mm	S = mm or more
nitrofurantoin	F/M-300	14	15-16	-	17
norfloxacin	NOR-10	12	13-16	-	17
oxacillin - Staphylococcus	OX-1	10	11-12	-	13
penicillin	P-10	28	-	-	29
Piperacillin/Tazobactum - Enterobactereaceae - Staphylococcus or P. aeruginosa	TZP-110	17 17	18-20 -	-	21 18
sulfamethoxazole + trimethoprim	SXT-25	10	11-15	-	16

Table \(\PageIndex{1g}\) Zone Size Interpretive Chart for Bauer-Kirby Test

Antimicrobial agent	Disc code	R = mm or less	I = mm	MS = mm	S = mm or more
tetracycline	TE-30	14	15-18	-	19
ticarcillin	TIC-75	11	12-14	-	15
ticarcillin/clavulanic acid	TIM-85	11	12-14	-	15
tobramycin	NN-10	12	13-14	-	15
trimethoprim	TMP-5	10	11-15	-	16
vancomycin	VA-30	9	10-11	-	12

Contributors and Attributions

• Dr. Gary Kaiser (COMMUNITY COLLEGE OF BALTIMORE COUNTY, CATONSVILLE CAMPUS)