The Kirby Bauer disk diffusion method of susceptibility testing has been standardized primarily for testing rapidly growing bacteria. To perform the test, filter paper disks containing a specific amount of antimicrobial agent are applied to the surface of an agar medium that has been inoculated with a known amount of the test organism. The drug in the disk diffuses through the agar. As the distance from the disk increases, the concentration of the antimicrobial agent decreases, creating a gradient of drug concentrations in the agar medium. At the same time as the drug diffuses through the agar, the bacteria try to multiply and grow across the agar. In areas where the concentration of drug is inhibitory, no growth occurs, forming a zone of inhibition around each disk.

Criteria currently recommended for interpreting zone diameters and MIC results for commonly used antimicrobial agents are published by CLSI. Results are reported categorically as Susceptible (S), Intermediate (I), or Resistant (R) based on the size of the zone of inhibition around each disk.

The antibiotic panel is based on the antibiotics commonly used on San Cristobal island, the antibiotic resistance genes found in the sequence data from last summer, and the CDC NARMS program.

!! Safety !!

1. Follow universal lab safety precautions: wear a lab coat and gloves when working in the lab, no open-toed shoes, no eating and drinking.
2. Wash all surfaces every day with freshly made 1% bleach followed by 70% isopropanol.
3. Immediately clean any spills or drips with 1% bleach. Surfaces must stay wet for at least 1 minute.
4. Wash your hands after handling infectious material and before leaving the lab.
5. Minimize generation of aerosols (e.g. uncap and pour slowly, minimize splashing, etc.).
6. Dispose of liquid waste by adding bleach to a final concentration of 10% bleach. Let sit for at least 30 minutes and then decant down the drain while running the tap.
7. Dispose of solid waste by adding 5 ml of 10% bleach to agar plates. Let sit for 30 minutes and then decant any liquid waste down the drain while running the tap. Dispose of solid waste in the trash.
8. For consumables (e.g. tips, loops), soak in 10% bleach in a disposable plastic bottle for at least 30 minutes. When the bottle is full, drain the bleach solution down the drain while running the tap, then cap the bottle and discard in the regular trash.

Protocol Overview

1. (Day 1) Collect water and plate on E. coli & Coliform MC Media Pads. Alternatively, dilute sewage in sterile saline and plate 1 ml on MC Media Pads.
2. (Day 2) Count the total number of coliform (light blue) and E. coli (dark blue) colonies on the MC Media Pads, targeting 25-250 colonies/pad. Subculture E. coli (dark blue) on MacConkey II agar plates for purity testing.
3. (Day 3a) Start the disk diffusion assay on isolates from the MacConkey II agar plates (Disk Diffusion Assay).
4. (Day 3b) Create subcultures in LB broth for banking of isolates (glycerol stocks and DNA).
5. (Day 4a) Record results of the disk diffusion assay (photos and measurements) for later analysis.
6. (Day 4b) Extract DNA and create glycerol stocks to freeze at -20C.

Reagents

Equipment and Consumables

Protocol

Prepare Tubes for Disk Diffusion Assay and Banking

1. Using sterile technique, create tube sets consisting of the following:
1. CULTURE TUBE (for Disk Diffusion Assay): One (1) 5 ml culture tube containing 2 ml sterile saline.
2. LB TUBE (for Banking and DNA): One (1) 2 ml screw-top tube containing 1 ml sterile LB.

You’ll need n + 3 sets of tubes, where n is the number of isolates being tested. The extra three tubes are for the negative control and for the two E. coli control isolates (ATCC 35218 and ATCC 25922).

2. Using a sterile loop, touch 3-5 colonies of E. coli (pink to rose-red) from the MacConkey II agar plate, and then immediately swirl in the 5 ml culture tube (containing normal saline) followed by the 2 ml screw-top tube (containing LB). Make sure the loop doesn’t touch anything except for the insides of each tube.
3. Tightly cap both tubes and vortex to homogenize.
4. Repeat with all isolates PLUS the control E. coli strains (ATCC 35218 and ATCC 25922) PLUS a negative control. For the negative control, swirl a sterile loop sequentially in the culture and screw-top tubes without touching any isolates.
5. CULTURE TUBE (For Disk Diffusion Assay): Immediately proceed to the Disk Diffusion Assay protocol below.
6. LB TUBE (for Banking): Loosely cap and place on the thermomixer overnight at 35°C with shaking at 1000 rpm.

Disk Diffusion Assay

1. Adjust the density of the CULTURE TUBE to a 0.5 McFarland standard by adding more sterile saline and then vortexing to mix. This is done by visually by comparing the inoculation tube and the 0.5 McFarland standard tube against a card with a white background and contrasting black lines.

2. Dip a sterile cotton swab into the adjusted suspension. Rotate the swab several times and press firmly on the walls to remove excess liquid.
3. Inoculate the dried surface of a Mueller Hinton II agar plate by streaking the swab over the entire surface.
4. Repeat the swabbing two more times, rotating the plate approximately 60° each time.
5. Swab the rim of the agar.
6. Repeat steps 2-5 for a second plate, for a total of two plates per isolate.
7. Leave the lid ajar for 3-5 minutes.
8. Dispense the antibiotic disks, one at a time, onto the surface of the plate using forceps that have been cleaned with an isopropanol-soaked kimwipe and allowed to dry. Add six discs per plate according to the template.

9. Press each disk down to ensure complete contact with the surface.
10. Invert plates and incubate at 35°C for 16-20 hours.
11. Inspect plates after 16-20 hours. There should be a confluent lawn of growth containing uniformly circular zones of inhibition. If individual colonies are apparent, the inoculum concentration was too low and the test must be repeated.
12. Measure and record the zones of inhibition (including the disk) to the nearest whole millimeter (rounding up) using a ruler held on the back of the inverted plate held a few inches above a black, non-reflective surface illuminated with reflective light. Measurements should go on the GERA Metadata and QPCR Results document.

Data Interpretation

These diameters (in mm) are from Table 2A. Zone Diameter and MIC Breakpoints for Enterobacterales of

CLSI M100 ED35:2025 Performance Standards for Antimicrobial Susceptibility Testing, 35rd Edition