Guidelines for Routine Antibiotic Susceptibility Testing (AST)

Last updated on June 11th, 2021

Antibiotic susceptibility testing should be performed only on clinically significant isolates, not on all microorganisms recovered in culture.

World Health Organization (WHO) recommends the use of modified Kirby-Bauer method for routine antibiotic susceptibility testing. According to WHO to avoid errors following guidelines should be used:

1. Antibiotic discs should be of correct diameter (6.35 mm).
2. Antibiotic discs should be of correct potency
3. The stock supply should be stored frozen (-20° C).
4. The working supply should be kept no longer than 1 month in a refrigerator (2–8° C).
5. Only Mueller Hinton agar of performance-tested quality should be used.
6. Correct pH (7.2–7.4) of the finished medium is essential for some antibiotics.
7. The inoculum should be standardized against the prescribed turbidity standard.
8. Zone sizes should be measured exactly.
9. Zone sizes should be interpreted by referring to a table of critical diameters. Zone diameters for each organism should fall within the limits

The three standard control strains are:

1. Staphylococcus aureus (ATCC 25923;
2. Escherichia coli (ATCC 25922);
3. Pseudomonas aeruginosa (ATCC 27853).

• Tests should be carried out with the standard strains:

1. when a new batch of discs is put into use;
2. when a new batch of medium is put into use;
3. Once a week, in parallel with the routine antibiograms.

• Use the quality control chart for recording and evaluating performance tests.