Mueller Hinton Results
Introduction
The choice of the proper media is important to ensure reproducibility
of the performed tests.
Many laboratory techniques may be used to in vitro evaluate bacterial
resistance to different antimicrobial agents. Among these techniques,
the disk diffusion susceptibility test is the most common, as well as
the dilution tests in broth and agar. To obtain reliable and uniform
results, the techniques used and the media and procedures
employed must be standardized.
The use of standardized culture media and careful control of all test
conditions are fundamental requirements in the microbiological
sensitivity testing in order to achieve satisfactory results.
An antibiogram allows to in vitro subject a standard bacterial inoculum to one or different antibiotic concentrations and,
after analyzing results, predict in vivo effectiveness. The interpretation of results (Sensitive, Intermediate or Resistant) is
carried out on the basis of parameters previously established by a number of committees, such as The Clinical and Laboratory
Standards Institute in the U.S., or The European Committee on Antimicrobial Susceptibility Testing. The standardized procedures are based on the Bauer-Kirby method. The following results are done according to The European Committee on
Antimicrobial Susceptibility Testing. Routine and extended internal quality control as recommended by EUCAST. Version 5.0,
2015. http://www.eucast.org
Procedure
1.- MEDIUM: the standard culture medium for the production
of the antibiogram is Mueller Hinton (Cat. 1058) and Mueller
Hinton II (Cat. 1055) , intended for the growth of many aerobic
organisms. For fastidious organisms, such as streptococci and
gonococci, 5% defibrinated blood must be added to Mueller
Hinton and Muller Hinton II. Ready-to-use plates must be
prepared following the data sheet instructions for each
medium.
2.- PREPARATION OF MATERIAL: 4-5 colonies that show
similar morphology are taken from the primary isolation
medium with a flamed loop, and suspended in 4-5 ml broth:
Mueller Hinton Broth (Cat. 1214), Trypticasein Soy Broth (T.S.B.)
(Eur. Pharma)(Cat. 1224) or Brain Heart Infusion Broth (Cat.
1400). They are incubated at 37ºC for 2-5 hours. If there is
turbidity at the end of this time, adjust the turbidity with a Mc
Farland Standard Tube 0.5 (equivalent to a uniform suspension
of 1.5x10 bacterial cells/ml). If the bacteria concentration is
low, continue incubating for 3 more hours and adjust.
3.- INOCULATION INTO PETRI DISH: prepared bacterial
suspension is homogenously mixed and streaked, turning the
plate into the Mueller Hinton Petri Dish, with the help of a
sterile cotton swab. Another technique is to pour several ml
into the agar, distribute homogeneously and remove excess
fluid. After inoculation, leave the plates unsealed for 10-15
minutes to allow for any surface moisture to be absorbed
before applying the drug- impregnated discs.
4.- ANTIBIOTIC DISC APPLICATION: Allow containers to
reach room temperature before opening. Open the
CONDA Antibiotic discs cartridge under the flame and
discharge the disc with the trigger or flamed and cooled
forceps. Press the disc gently into the agar with the help
of the forceps. The space between the discs must not be
narrower than 24 mm, and the distance to the edge no
less than 1 cm. CONDA Special Dispenser 6x and 8x may
also be used. It is preferable to deposit penicillin and
cephalosporin discs no less than 10 mm from the edge of
the Petri dish, with their centers at least 30 mm apart.
5.- INCUBATION: Place the Petri Dish into the incubator
inversely at 37ºC for 18-24 hrs. For Staphylococcus spp.,
testing at temperatures above 35°C may not detect methicillin- resistant staphylococci (MRS); for N. gonorrhoeae,
incubate at 36 ± 1°C. Haemophilus spp., N. gonorrhoeae, S.
pneumoniae and other streptococci should be incubated in
an atmosphere enriched with 5% CO2.
6.- RESULTS: The diameter of the inhibition zone is read
from the underside of the disc. If Mueller Hinton with
Blood has been used, inhibition areas must be read from
the surface with a compass or ruler. Zones are measured
to the nearest whole millimeter. The inhibition areas
must be evaluated according to the Table provided by
CONDA.
Escherichia coli
ATCC 25922
Antibiotics
EUCAST Range
Competitor/mm
MH/mm
MH-II/mm
Amikacin 30 µg
19-26
20
21
22
Ampicillin 10 µg
16-22
17
18
19
Cefoxitin 30 µg
23-29
24
24
24
Chloramphenicol 30 µg
21-27
26
27
Ciprofloxacin 5 µg
30-40
35
36
35
Gentamicin 10 µg
19-26
19
19
21
Levofloxacin 5 µg
29-37
32
35
34
Moxifloxacin 5 µg
28-35
29
30
30
Nitrofurantoin 100 µg
17-23
20
20
17
Norfloxacin 10 µg
28-35
30
34
31
Ofloxacin 5 µg
29-33
30
33
30
Tigecycline 15 µg
20-27
20
24
21
Tobramycin 10 µg
18-26
17
17
20
Trimethoprimsulfamethoxazole 1,25 - 23,75 µg
23-29
23
23
24
Cefotaxime 5 µg
25-31
25
26
27
Cefpodoxime 10 µg
23-28
22
25
26
Ceftriaxone 30 µg
29-35
27
30
30
Cefuroxime 30 µg
20-26
20
24
25
Doripenem 10 µg
27-35
27
30
30
Ertapenem 10 µg
29-36
31
33
32
Imipenem 10 µg
26-32
35
32
32
Meropenem 10 µg
28-34
30
35
33
Ticarcillin 75 µg
24-30
21
26
25
Piperacillin-Tazobactan 30-6 µg
21-27
21
23
25
Cefalexin 30 µg
15-21
13
16
18
Cefadroxil 30 µg
24-20
13
15
17
Aztreonam 30 µg
28-36
29
34
34
Amoxicillin-Clavulanic acid 20-10 µg
18-24
18
21
20
Cefixime 5 µg
23-27
20
23
23
Ceftazidime 10 µg
23-29
22
26
27
Piperacillin 30 µg
21-27
23
25
25
Ampicillin-sulbactam 10-10 µg
19-24
20
22
21
Ticarcillin-clavulanic acid 75- 10 µg
24-30
25
27
30
Mecillinam 10 µg
24-30
26
26
27
Ceftibuten 30 ug
27-35
25
27
28
Pseudomonas aeruginosa
ATCC 27853
Antibiotics
EUCAST Range
Competitor/mm
MH/mm
MH-II/mm
Amikacin 30 µg
18-26
25
26
26
Ciprofloxacin 5 µg
25-33
35
33
33
Levofloxacin 5 µg
19-26
24
26
26
Tobramycin 10 µg
20-26
22
21
24
Cefepime 30 µg
24-30
26
29
30
Doripenem 10 µg
28-35
34
35
35
Imipenem 10 µg
20-28
28
28
28
Meropenem 10 µg
27-33
35
33
33
Piperacillin-Tazobactan 30-6 µg
23-29
30
29
29
Aztreonam 30 µg
23-29
35
29
29
Ceftazidime 10 µg
21-27
23
27
27
Ticarcillin-clavulanic acid 75-10 µg
20-28
20
28
28
Antibiotics
EUCAST Range
Competitor/mm
MH/mm
MH-II/mm
Amikacin 30 µg
18-24
20
20
21
Ampicillin 2 µg
15-21
17
19
16
Cefoxitin 30 µg
24-30
25
25
27
Chloramphenicol 30 µg
20-28
23
23
21
Ciprofloxacin 5 µg
21-27
20
22
21
Clindamycin 2 µg
23-29
28
27
26
Erythromycin 15 µg
23-29
20
24
25
Fusidic Acid 10 µg
26-32
26
26
26
Gentamicin 10 µg
19-25
19
20
19
Levofloxacin 5 µg
23-29
30
28
27
Linezolid 10 µg
21-27
25
24
22
Minocycline 30 µg
23-29
22
24
23
Moxifloxacin 5 µg
25-31
30
30
26
Mupirocin 200 µg
31-37
31
34
Nitrofurantoin 100 µg
17-23
17
17
20
Norfloxacin 10 µg
18-24
24
24
23
Ofloxacin 5 µg
21-27
24
25
25
Quinupristin-dalfopristin 15 µg
21-27
21
23
21
Rifampicin 5 µg
30-36
30
30
30
Tetracycline 30 µg
23-31
20
25
25
Tigecycline 15 µg
19-25
17
20
20
Tobramycin 10 µg
20-26
20
20
23
Trimethoprimsulfamethoxazole 1,25 - 23,75 µg
26-32
25
26
26
Staphylococcus aureus
ATCC 29213
Haemophilus influenzae
Haemophilus influenzae
NCTC 8468
Antibiotics
EUCAST Range
Competitor/mm
MH/mm
MH-II/mm
Chloramphenicol 30 µg
30-38
28
28
30
Ciprofloxacin 5 µg
31-39
35
35
34
Erythromycin 15 µg
12-18
14
15
15
Levofloxacin 5 µg
32-38
36
36
34
Minocycline 30 µg
27-33
30
33
32
Moxifloxacin 5 µg
29-35
34
35
34
Ofloxacin 5 µg
30-36
32
34
32
Rifampicin 5 µg
20-26
23
25
26
Tetracycline 30 µg
28-34
32
34
34
Trimethoprimsulfamethoxazole 1,25-23,75 µg
26-34
10
26
26
Cefaclor 30 µg
24-30
38
30
30
Cefepime 30 µg
29-35
32
34
33
Cefotaxime 5 µg
29-35
24
29
29
Cefpodoxime 10 µg
28-34
30
32
33
Ceftriaxone 30 µg
33-41
28
33
34
Cefuroxime 30 µg
35-31
35
31
31
Doripenem 10 µg
26-32
36
32
32
Ertapenem 10 µg
27-33
30
33
33
Imipenem 10 µg
25-31
40
31
31
Meropenem 10 µg
28-34
35
34
34
Telithromycin 15 µg
15-21
30
21
21
Amoxicillin-Clavulanic acid 2-1 µg
17-23
35
23
23
Cefixime 5 µg
27-33
17
27
27
Antibiotics
EUCAST Range
Competitor/mm
MH/mm
MH-II/mm
Nitrofurantoin 100 µg
18-24
17
20
20
Tigecycline 15 µg
20-26
20
25
22
Imipenem 10 µg
24-30
25
30
30
Teicoplanin 30 µg
15-21
16
21
21
Enterococcus faecalis
ATCC 29212
Streptococcus pneumoniae
ATCC 49619
Antibiotics
EUCAST Range
Competitor/mm
MH/mm
MH-II/mm
Chloramphenicol 30 µg
24-30
29
30
30
Ciprofloxacin 5 µg
22-28
28
28
28
Clindamycin 2 µg
22-28
23
24
24
Erythromycin 15 µg
26-32
27
29
29
Levofloxacin 5 µg
21-27
25
26
26
Linezolid 10 µg
23-29
27
28
28
Minocycline 30 µg
25-31
25
26
26
Moxifloxacin 5 µg
24-30
27
30
30
Nitrofurantoin 100 µg
25-31
26
28
28
Norfloxacin 10 µg
18-24
24
24
26
Ofloxacin 5 µg
18-24
21
21
21
Rifampicin 5 µg
26-32
28
30
30
Tetracycline 30 µg
28-34
29
32
33
Tigecycline 15 µg
24-30
23
24
24
Trimethoprimsulfamethoxazole 1,25-23,75 µg
20-26
30
25
26
Cefaclor 30 µg
25-31
33
30
30
Cefepime 30 µg
31-37
41
35
35
Cefotaxime 5 µg
28-34
35
32
32
Cefpodoxime 10 µg
29-35
36
33
33
Ceftriaxone 30 µg
32-38
36
38
38
Cefuroxime 30 µg
28-34
36
33
32
Doripenem 10 µg
31-37
32
31
32
Ertapenem 10 µg
28-34
33
30
30
Imipenem 10 µg
34-42
39
40
40
Meropenem 10 µg
30-38
36
34
34
Oxacillin 1 µg
8-14
9
10
10
Teicoplanin 30 µg
18-24
19
22
22
Telithromycin 15 µg
27-33
31
31
31