TITLE: Salmonella spp. antibiotic susceptibility testing by the Kirby-Bauer disk diffusion method
AUTHORS: Enrique Delgado

[STEPS]
?. [Refreshment of pure isolates]
Pick growth from pure isolates of samples to be analyzed and from quality control organisms to be included in the AST test. Transfer to sterile assay tubes containing 10 mL of TSB and place the tubes in a shaking incubator at 35 ºC / 18-24 h / 150 rpm.
?. [Refreshment of pure isolates]
Using a sterile loop, collect TSB broth and streak onto TSA agar plates. Incubate TSA agar plates at 35 ºC / 18-24 h.
?. [Inoculum standarization and plating in Muller-Hinton (MH) agar]
Prepare assay tubes containing 4 mL of 0.85-0.9% (w/v) NaCl solution. Sterilize tubes by autoclaving at 121 ºC / 20 min.
?. [Inoculum standarization and plating in Muller-Hinton (MH) agar]
Using a sterile cotton swab, pick growth from an isolated colony on the TSA agar plate and mix it in the tube containing the saline solution. Discard the used cotton swab.
?. [Inoculum standarization and plating in Muller-Hinton (MH) agar]
Adjust inoculum turbidity to 0.5 McFarland units with the aid of a spectrophotometer. Use an automated pipette to add more saline solution (if turbidity is higher) or a second cotton swab to add more growth (if turbidity is lower). Standardized  inoculum shall be processed immediately (or within the first 15 min post-standardization).
?. [Inoculum standarization and plating in Muller-Hinton (MH) agar]
Bring 150-mm MH agar plates and SensiDics to room temperature before inoculation. To avoid leaving the standardized inoculum standing for more than 15 min, place MH agar plates in an incubator at 35 ºC before starting inoculum standarization. Likewise, take the Sensidiscs out of the refrigerator 2-3 h before standardizing inoculum and let them stand on the benchtop. In that way, both the plates and the disks will be at room temperature timely.
?. [Inoculum standarization and plating in Muller-Hinton (MH) agar]
Use a sterile cotton swab and dip it into the tube containing the standardized inoculum. Squeeze the cotton swab against the tube's walls to avoid bringing excess liquid to the MH plate. Streak the 150-mm MH plate horizontally, making sure the whole agar surface is covered. Then, rotate the MH plate 60 º and streak again. Rotate the MH plate 60 º a second time and streak again.
?. [Inoculum standarization and plating in Muller-Hinton (MH) agar]
Place the discs cartridges in the disk dispenser. Open a MH plate and place it below the dispenser. Press the dispensing knob firmly and all the way down to dispense the disks. After dispensing, visually check the plates to make sure all the disks are in full contact with the MH agar surface. If necessary, use a sterile tweezer to press the top of the disk until the whole disk area touches the agar. Close the MH plate and let it stand for 2-3 min before incubation.
?. [Inoculum standarization and plating in Muller-Hinton (MH) agar]
Invert MH plates and incubate at 35 ºC / 16-18 h. Incubation shall start not later than 15 min after dispensing the disks.
?. [Inoculum standarization and plating in Muller-Hinton (MH) agar]
Read the inhibition zone diameter around each disk with a ruler and against a dark background. Record results for each sample and for quality control organisms. The inhibition zones of quality control organisms must comply with CLSI quality control ranges for nonfastidious organisms. Otherwise, the test must be repeated.
?. [Inoculum standarization and plating in Muller-Hinton (MH) agar]
Compare results with CLSI clinical breakpoints and classify each strain as susceptible, intermediate (I) or resistant (R). The table below summarizes the clinical breakpoints of the antimicrobial panel used in our lab according to the 30 Edition  of CLSI M100 Supplement.  ABCD1AntimicrobialConcentration, ugZone diameter breakpoint, mm (R)Zone diameter breakpoint, mm (I)2Amikacin30≤1415-163Gentamicin10≤1213-144Carbenicillin100≤1920-225Amoxicillin-clavulanic acid20/10≤1314-176Cefotaxime30≤2223-257Ceftriaxone30≤1920-228Cefepime30≤1819-249Imipenem10≤1920-2210Ertapenem10≤1819-2111Meropenem15≤1920-2212Chloramphenicol30≤1213-1713Trimethoprim-sulfamethoxazole1.25/23.75≤1011-1514Ciprofloxacin5≤2021-3015Tetracycline30≤1112-14
ABCD1AntimicrobialConcentration, ugZone diameter breakpoint, mm (R)Zone diameter breakpoint, mm (I)2Amikacin30≤1415-163Gentamicin10≤1213-144Carbenicillin100≤1920-225Amoxicillin-clavulanic acid20/10≤1314-176Cefotaxime30≤2223-257Ceftriaxone30≤1920-228Cefepime30≤1819-249Imipenem10≤1920-2210Ertapenem10≤1819-2111Meropenem15≤1920-2212Chloramphenicol30≤1213-1713Trimethoprim-sulfamethoxazole1.25/23.75≤1011-1514Ciprofloxacin5≤2021-3015Tetracycline30≤1112-14