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ANTIMICROBIAL SUSCEPTIBILITY TEST DISCS
Antimicrobial susceptibility test discs are used for determination of susceptibility of bacteria to
antimicrobial agents, applying in vitro diffusion method.
In accordance with the WHO requirements, the disc content is:
Fluka
42456
16527
01601
08541
68601
52856
68611
30321
89867
90424
92241
07651
08587
93646
38139
67237
14683
69531
55941
01403
19607
05577
55376
39782
56758
44543
42079
54357
16244
19951
11435
56383
56973
75139
75141
73477
74794
75156
Antimicrobic
Amikacin
Amoxicillin
Amoxicillin +
Clavulanic acid
Ampicillin
Azithromycin
Cefaclor
Cefalexin
Cefotaxim
Ceftazidime
Ceftriaxone
Cefuroxime
Chloramphenicol
Ciprofloxacin
Clindamycin
Enoxacin
Erythromycin
Fleroxacin
Fusidic acid
Gentamycin
Imipenem
Kanamycin
Lincomycin
Meticillin
Metronidazole
Nalidixic acid
Neomycin
Netilmicin
Nitrofurantoin
Norfloxacin
Ofloxacin
Oxacillin
Penicillin G
Penicillin H (HD)
Pipemidic acid
Piperacillin
Rifampicin
Roxithromycin
Streptomycin
Teicoplanin
Tetracycline
Trimethoprim
Trimethoprim +
Sulfamethoxazole
Vancomycin
Disc content
Colour
20 + 10 g
10 g
15 g
30 g
30 g
30 g
30 g
30 g
30 g
30 g
5 g
10 g
10 g
15 g
5 g
90 g
30 g
10 g
30 g
15 g
10 g
25 g
30 g
30 g
30 g
300 g
5 g
5 g
5 g
6 g
60 g
20 g
100 g
5 g
15 g
30 g
30 g
30 g
5 g
blue/white/orange
red/blue
green/orange
gray/pink
blue/beige
blue/dark green
beige/yellow
pink/dark blue
white/blue
white
yellow/gray
dark blue/white
beige/white/orange
green
beige/white
gray/dark blue
red/dark green/white
orange/blue
red
blue/dark blue
red/blue/white
cyclamen red
orange
dark green/white
white/yellow/dark green
dark green/orange
pink/gray
red/green
tourquize/white
blue
blue/green
beige/orange
green/red/blue
red/dark green
green/red
pink
blue/yellow/beige
yellow
gray/white
1.25 + 23.75 g
30 g
gray/orange/white
beige/white/dark blue
30 g
25 g
1
yellow/green
blue/white
Industriestrasse 25
CH-9471 Buchs
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Regression lines for each antimicrobial have been prepared using agar dillution method for Minimum
Inhibitory Concentrations (MIC) to obtain specific zone sizes.
Antimicrobial susceptibility test discs are 9 mm in diameter and each disc (with specific
antimicrobial agent) is colour coded for safe identification. The discs are manufactured under aseptic
conditions by dry process, resulting in extra stability even at normal room temperature (3-4 years).
Direction for use
Media
Mueller-Hinton agar supplemented with Ca++ and Mg++ ions for real sensitivity of Pseudomonas
strains to aminoglycoside antibiotics. Low level of thymidine, below 50 g/ml of medium, to obtain
false resistance to trimethoprim with sulfamethoxazole.
Inoculum
Select four to five similar colonies and transfer into suitable broth or saline to obtain turbidity
equivalent to 0.5 McFarland barium sulphate standard.
The standard ready-made inoculum requires agitation on a vortex mixer before each use. For proper
turbidity adjustment, it is helpful to use a white background with contrasting black lines. Inoculate
the agar plates directly from the suspension, spreading the inoculum as evenly as possible with sterile
swab. Antimicrobial susceptibility test discs are then placed with the aid of flamed tweezers to the
inoculated medium. Incubate the test materials at 37˚ C for 18 hours and then measure the diameters
of the inhibition zones surrounding the discs in millimeters. The sensitivity of bacteria to
antimicrobials are detected from the table overleaf.
Measurement of inhibition zones
Measure only zones of complete inhibition by visual inspection and record in millimeters. Measure
from the bottom of the plate, or open the plate if blood medium was used.
The sensitivity of bacteria to antimicrobial agents are determined from the Table No. 1.
Table No. 1:
Inhibition Zone Diameter Interpretation
Fluka
Antimicrobic
42456
08541
Amikacin
Ampicillina
Staphylococcus & Streptococcus
Enterococcus
G - bacteria
Amoxicillin
Amoxicillin + Clavulanic acid
for Staphylococcus
for other bacteria
Azithromycin
Cefalexin
Cefotaxim
16527
01601
68601
68611
30321
Inhibition Zones
Susceptible
19
2
Intermediate
16-18
Resistant
15
26
18
21
28
23-25
16-17
19-20
22-27
22
15
18
21
25
18
19
26
23
24-20
17-14
18-15
23-25
18-22
19
13
14
22
17
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CH-9471 Buchs
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89867
90424
92241
07651
08587
67237
93646
14683
69531
55941
01403
19607
05577
39782
56758
44543
54357
42079
16244
56383
11435
56973
75139
75141
73477
74794
75156
Ceftazidime
Ceftriaxone
Cefuroxime
Chloramphenicol
Ciprofloxacin
Erythromycin
Clindamycin
Fleroxacin
Fusidic acid
Gentamycin
Imipenem
Kanamycin
Lincomycin
Meticillin
Nalidixic acid
Neomycin
Netilmicin
Nitrofurantoin
Norfloxacin
Oxacillin
Staphylococcus
Streptococcus pn.
Ofloxacin
Penicillin Ga
Staphylococcus & Streptococcus
Enterococcus
Piperacillin
Pipemidic acid
Rifampicin
Roxithromicin
Streptomycin
Teikoplanin
Tetracycline
Trimethoprim
Trimethoprim + Sulfamethoxazole
Vancomycin
19
16
23
26
21
26
26
19
26
26
20
26
26
21
26
26
22
26
18
29
16
26
22
26
26
12
19
26
26
24
26
17
26
16
26
12
a
17-18
13-15
14-22
23-25
15-20
23-25
23-25
18-16
23-25
23-25
17-19
23-25
23-25
20-17
23-25
23-25
19-21
23-25
16-17
11-18
23-25
23-25
21-23
23-25
16
12
13
22
14
22
22
15
22
22
16
22
22
16
22
22
18
22
15
11
13
21
15
22
22
11
10
22
22
20
22
23-25
11-15
23-25
10-11
22
10
22
9
16-21
23-25
23-25
The susceptible category for penicillin or ampicillin implies the need for high-dose therapy for
serious enterococcal infections. If possible, this should be noted in the footnote of the susceptibility
report form. Enterococcal endocarditis require combination therapy with high-dose penicillin or
ampicillin plus gentamycin or streptomycin for antibacterial action. Since ampicillin or penicillin
resistance among enterococci due to -lactamase production is not reliably detected using routine
disc or dillution methods, a direct, nitrocefin-based -lactamase test is recommended. Synergy
between ampicillin, penicillin, or vancomycin and an aminoglycoside can be predicted for
enterococci by using a high level aminoglycoside screening test.
3
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Interpretation of results (for Table No. 1. and Table No.2.):
S – Susceptible
I – Intermediate
R – Resistant
The infection due to the strain tested may be expected to respond to usual
dosage of this antimicrobic.
The intermediate category implies clinical applicability in body sites where
drugs are intermediate categories, also indicates “buffer zone”, which prevent
small uncontrolled technical factors from causing major discrepancies,
especially for drugs with narrow pharmacotoxicity margins.
The antimicrobic cannot be used in this case.
Table No. 2:
Inhibition Zone Diameter Interpretative, Standards for Streptococcus pneumoniae
Fluka
Antimicrobic
Disc Content
Zone Diameter in mm____
S
I
R___
54357
Oxacillin
5 g
26
21
16244 A Penicillin*
6 g
34
33-27
26
67237
Erythromycin
15 g
23
22-16
15
74794
Trimethoprim +
sulfamethoxazole 1.25 + 23.75 g 26
25-19
18___
75141
Tetracycline
30 g
31
30-24
23
75156 B Vancomycin
30 g
17_______________________
07651 C
Chloramphenicol 30 g
27
26-22
21___
90424
Ceftriaxone**
30 g
26
19
30321
Cefotaxime
30 g
25
18
*Susceptibility to penicillin should be tested with Oxacillin disc. Minimum Inhibitory Concentration
of penicillin should be determined on isolates of the Streptococcus pneumoniae with oxacillin zone
sizes of 21 mm. As it is impossible for many laboratories to do MIC determination, it is recommended
to use susceptibility test with Penicillin disc too.
**Diffusion test for Cefotaxime and Ceftriaxone should be determined as screen test.
A
B
C
Group for primary test and report
The group of antibiotics for primary test, but report selectively (when the organism is resistant
to agents of the same class in Group A)
Represents alternative or supplemental antimicrobial agents.
Quality Control Procedure
The stock culture of Staph. aureus (ATCC25923), E. coli (ATCC25922), Ps. aeruginosa
(ATCC27853) and St. faecalis (ATCC29212), should be obtained from reliable sources.
Factors that seriously affect the accuracy and precision of the disc susceptibility assay must be
rigorously controlled. These include: preparation of the media, thickness of the agar, inoculum density,
or any change in test procedure, for example, a new lot of disc, medium, or new personnel. It is
recommended that the tests be performed at least once a week.
4
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Table No. 3.
Quality Control of Zone Diameter Limits for Individual Tests
on Mueller-Hinton Agar (Fluka 70191) without Blood and other Supplements
Zone diameter in mm
Fluka
Antimicrobic
Staph. aureus E. coli
Ps. aeruginosa
ATCC25923 ATCC25922 ATCC27853
20-27
23-28
23-28
42456
Amikacin
15-20
24-35
08541
Ampicilin
01601
Amoxicillin +
22-27
28-36
Clavulanic acid
15-20
23-30
68611
Cefalexin
30-38
15-24
30321
Cefotaxime
26-32
29-33
32-34
19-21
89867
Ceftazidime
19-22
27-30
19-24
90424
Ceftriaxone
28-32
34-38
27-30
08587
Ciprofloxacin
10-15
28-32
67237
Erythromycin
21-27
25-32
25-32
69531
Gentamycin
23-29
23-29
07651
Chloramphenicol
19-21
27-28
37-38
55941
Imipenem
26-32
93646
Clindamycin
28-32
19607
Lincomycin
25-29
05577
Meticillin
23-28
39782
Nalidixic acid
20-24
26-29
31-34
Netilmicin
26-28
23-27
Nitrofurantoin
19-26
28-35
17-26
44543
Norfloxacin
18-24
54357
Oxacillin
18-25
30-33
25-31
42079
Ofloxacin
26-37
16244
Penicillin G
25-33
24-30
30-32
56383
Piperacillin
25-27
11435
Pipemidic acid
29-31
56973
Rifampicin
18-21
Teicoplanin
18-23
75141
Tetracycline
22-28
74794
Trimethoprim +
26-34
Sulfamethoxazole 30-36
15-18
75156
Vancomycin
Ent. faecalis
ATCC29212
25-27
34-36
19-22
27-29
16-22
25-27
27-29
23-25
13-15
18-20
18-20
24-26
16-18
18-20
17-19
29-31
16-18
Note: The sussceptibility of most patogenic bacteria is usually determined by the diffusion method.
But, some clinically important bacteria grow slowly, need special media and atmosphere, and it is not
possible to use the diffusion method. In these cases the best method is the Minimum Inhibitory
Concentration, and the ability to produce -lactamase.
5
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CH-9471 Buchs
Alternative disc test. Presence of Broad-Spectrum
-lactamase.
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Detection of Broad Spectrum - lactamases in Enterobacteriaceae
-lactamases hydrolize penicillins and oxyminocephalosporins (cefotaxime, ceftazidime and
ceftriaxone). They are susceptible to -lactamase inhibitors, such as clavulanic acid.
The presence of such -lactamases can be detected by performing a synergy test: inoculate a MuellerHinton plate as for sensitivity testing and apply Antimicrobial susceptibility test discs containing
Cefotaxime, Amoxicillin + Clavulanic acid, and Ceftazidime, in line, at approximately 1.5 cm from
each other. Incubate overnight at 37º C. Extension of the zone of inhibition (synergysm) towards the
disc containing Amoxycillin + Clavulanic acid suggests the presence of broad spectrum -lactamases.
6