|Antimicrobial susceptibility test
|Antifungigram, diffusion method
(c) Costin Stoica
Antibiogram on Muller-Hinton medium
|Beta-hemolytic Streptococcus spp.
Antibiogram on Blood agar
The quantitative method (MIC) - based on dilution: serial dilutions of an antimicrobial
agent of known concentration are prepared in growth medium. All tubes are then
inoculated with the isolated strain and incubated for a defined period of time. The MIC is
defined as the lowest concentration of antimicrobial agent needed to inhibit the growth
of a test organism. Once the MIC is calculated, it can be compared to known values for a
given species or genera, and antibiotic.
Note: before you perform an antibiogram, you need to identify the strain and be sure
you have a pure culture. The antibiotics used for antibiogram are not the same for
different bacteria genera. Antibiotics selection must be correlated with strain'
characteristics (species/genera), patient (human/animal), and costs mostly in animal
The same principle may be applied for Antifungigram by changing antibiotic with
antimycotic impregnated disks.
- Helgomar Raducanescu, Valeria Bica-Popii, 1986, “Bacteriologie veterinara”,
Editura Ceres, Bucuresti
- Francois Jehl, Monique Chomarat, Michele Weber, Alain Gerard, 2010. De
l'antibiograme a la prescription. 3rd ed, bioMerieux.
- Dumitru Buiuc, Marian Negut, 2009. Tratat de microbiologie clinica. Ed. medicala,
- EUCAST - Europeean Comitee On Antimicrobial Susceptibility Testing. Accessed
online 10.01.2013, at http://www.eucast.org/antimicrobial_susceptibility_testing/.
Antibiogram is an in-vitro testing for the sensitivity of an isolated bacteria strain to
different antibiotics. Frequently it follows the strain identification and represents the final
goal of a bacteriolgy analysis. Its main purpose is to help the therapeutic decision, but
may also be useful in epidemiological surveillance or highlighting natural bacterial
The antibiogram is useless/not recommended in some cases: non-pathogenic or
comensal germs (oral streptococci, Proteus spp. from cadavers), culture contaminants
(Bacillus), or low number of germs in clinical sample (urine, saliva, sperm).
Two antibiogram methods may be performed in laboratory: diffusimetric (most used
and easy to do) and quantitative.
The diffusimetric method (Kirby-Bauer) - based on the diffusion property of the
antibiotic from the impregnated paper disks. Disks are dispensed on the surface of an
bacterial strain inoculated media (in one or more Petri dishes). EUCAST recommends
the utilization of Mueller-Hinton agar without supplements for non-fastidious organisms,
including enterococci, and Mueller-Hinton with 5 % horse blood and 20 mg β-NAD/L for
Streptococcus spp. including Streptococcus pneumoniae, Haemophilus spp. and other
Minimum distance between disks must be 30 mm and minimum distance from plate
edge must be 15 mm. Recommended density of inoculum is 0.5 on McFarland scale.
The plates are incubated at 35 ± 1 ºC for 18 ± 2 h within 15 minutes from application
of the disks. Plates are incubated in air for non-fastidious and in air with 5% CO2 for
fastidious organisms. During the incubation the antibiotic will diffuse in the area
surrounding each disk, and a rounded clear area (where the strain's growth is
inhibited) will appear.
Reading for non-fastidious germs: measure zone edges as the point showing no
growth looking from the back of the plate against a black background illuminated with
Reading for fastidious germs (on blood agar or chocolate agar): measure zone edges
as the point showing no growth viewed from the front of the plate after removing the lid
and in reflected light.
The measured diameter (in millimeters) of the clear area is compared with standard
tables. See "Movies" and "Images" sections for more.
- medium quality (nutritivity, pH, humidity), quantity (the thickness of the agar),
horizontality & sterility
- incubation conditions (temperature, humidity, atmosphere composition, time)
- low discs quality or expired
- small distance between discs, or too close to Petri dish margin
- non-standardised inoculum or uneven dispersion in plate
- contaminated culture or bacteriophage infected strain
- reading of the inhibition zones and interpretation (non-standardised measuring