Disk Susceptibility Testing Step By Step

• McFarland turbidity standards - When turbidity of test inoculum matches McFarland 0.5 standard, test inoculum contains approximately 1.5 x 10⁸ CFU/mL
• Use growth method or direct colony suspension method; for the latter, colonies must be no older than 18 to 24 hours

Step 2:  Inoculum standardization

• Vortex McFarland 0.5 standard and test inoculum, and hold tubes side by side against a white card with heavy black lines
• If lines look same through tubes, inoculum is satisfactory
• If lines appear less sharp through inoculum tube, dilute with sterile saline or broth until turbidity is comparable
• If lines appear sharper through test inoculum, reincubate (growth method) or add more cells (direct colony suspension method)

Step 3:  Agar plate inoculation

• If testing nonfastidious organisms (such as E.coli or staphylococci), use unsupplemented agar
• If testing fastidious organisms, use Mueller-Hinton agar that has been supplemented with blood products
• Dip a sterile cotton swab into inoculum; raise swab above liquid and rotate against wall of tube to remove excess liquid
• Apply inoculum to plate, covering entire surface; rotate 60 degrees and swab; rotate 60 degrees and swab again

Step 4:  Antimicrobial disk application

• For assistance in disk selection refer to Tables 1 and 1A; be sure tables are current
• Place no more than 12 disks on a 150 mm plate
• Place no moe than five disks on a 100 mm plate

Step 5:  Plate incubation

• Inver plate (agar side up) and incubate at 35^oC
• With Streptococcus pneumoniae, Haemophilis species or Neisseria gonorrhoeae, incubate in 5 to 7% CO₂
• For most tests, incubate 16 to 18 hours

Step 6:  Measurement of zone of inhibition

• After 24 hours, check agar for confluent lawn of growth and uniform, circular zones of inhibition
• Hold plate (agar side up) over a black, nonreflective surface using reflected light directed from above
• Measure diameter of xone of inhibition to nearest mm
• If examining blood-supplemented Mueller-Hinton agar, remove lid and measure from above agar surface
• if measuring hemolytic bacteria, read zone of growth of inhibition, NOT zone of hemolytic inhibition

• Use zone measurements with Table 2; be sure table is current
• Interpretive categories
• Susceptible - infection may be treated with antimicrobial agent at recommended dosage
• Resistant - organism not likely to inhibited by usual antimicrobial agent doses
• Intermediate - therapy handled in variety of ways

Quality control

• Use Table 3; be sure table is current
• Quality control records must include date of testing; microbiologist's initials; lot numbers, expiration dates of disks, and media; zone measurements for each quality control strain and antimicrobial agent

Procedure modification for fastidious bacteria

• Streptococcus pneumoniae
• Use Mueller-Hinton agar supplemented with 5% sheep blood
• Incubation 35^oC, 5 to 7% CO₂^, 20 to 24 hours
• Haemophilus species
• Use Haemophilus test medium (HTM) - Incubation 35^oC, 5 to 7% CO₂^, 16 to 18 hours
• Neisseria gonorrhoeae
• Use supplemented GC agar -Incubation 35^oC, 5 to 7% CO₂^, 20 to 24 hours