Jan 23, 2023
Measuring fungal anti-E. coli activity using the zone of inhibition (ZOI) assay
- 1University of Auckland
Protocol Citation: Shara Van De Pas, Siouxsie Wiles 2023. Measuring fungal anti-E. coli activity using the zone of inhibition (ZOI) assay. protocols.io https://dx.doi.org/10.17504/protocols.io.261gen7qjg47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 07, 2022
Last Modified: January 23, 2023
Protocol Integer ID: 66230
Keywords: Zone of Inhibition, Escherichia coli, ZOI, fungi, antibacterial
Abstract
In this protocol, we describe our use of the zone of inhibition assay to assess the anti-E. coli activity of fungi grown on different media, including Czapek Solution Agar (CSA), Czapek Yeast Extract Agar (CYA), Malt Extract Agar (MEA), Oatmeal Agar (OA), Potato Dextrose Agar (PDA), Rice Extract Agar (REA), and Water Agar (WA).
Guidelines
Ensure you have the appropriate biosafety approvals for the work.
Materials
Plasticware
| Description | Catalogue number | Supplier | |
| 90mm Petri Dishes | LAB-021MR | Medi'Ray | |
| Falcon 50mL Conical Centrifuge Tube | BDAA352070 | In vitro technologies | |
| Technoplast 5 mL flat bottom yellow screw cap tube | S5016SU | Mediray, New Zealand | |
| BRAND Semi micro cuvette | BR759015 | Sigma-Aldrich, New Zealand | |
| Sterile cotton swab 150mm | LABMC2744S | Jackson Allison Medical and Surgical Ltd | |
| Pipette tips |
Growth media and chemicals
| Description | Catalogue number | Supplier | |
| Mueller Hinton II Broth Cation Adjusted | 212322 | Fort Richard, New Zealand | |
| Difco Potato Dextrose Broth | 254920 | Fort Richard, New Zealand | |
| Difco Oatmeal Agar | 255210 | Fort Richard, New Zealand | |
| Difco Malt Extract Agar | 211220 | Fort Richard, New Zealand | |
| Difco Czapek Solution Agar | 233910 | Fort Richard, New Zealand | |
| BBL Rice Extract Agar | 211567 | Fort Richard, New Zealand | |
| Bacto Yeast Extract | 212750 | Fort Richard, New Zealand | |
| Agar, Granulated | 214530 | Fort Richard, New Zealand |
Equipment:
- Pipettes – various sizes
- 6mm Biopsy punch (we use Paramount)
- Spectrophotometer (to measure optical density of bacterial culture)
- Sterile scalpel handle and blade
- Germinator
- Biological Safety cabinet (Herasafe KS12)
- Ruler
Initial culturing of fungus from stocks
Initial culturing of fungus from stocks
Subculture fungus onto a Potato Dextrose Agar (PDA) plate from either frozen stocks or an existing culture.
Seal the plate with parafilm and store it in a plastic box at room temperature. Allow fungus to reach at least 50% radial growth before subculturing onto respective media plates.
Culturing of fungus onto different media
Culturing of fungus onto different media
Using a sterile scalpel plate, cut a small section (0.5cm) from the growing edge of the mycelium and inoculate this segment onto fresh media. In our experiments, we routinely subculture fungi onto a selection of different media.
Monitor radial growth of the fungus and start the ZOI assay when it reaches 20, 50, and 100% growth.
Setting up E. coli 25922 lux and ZOI plates
Setting up E. coli 25922 lux and ZOI plates
Inoculate 10 mL of Mueller Hinton II broth in a 15 mL tube with E. coli and grow overnight at 37 degrees C with shaking at 200 rpm. We use a bioluminescent derivative of the antibiotic-testing E. coli ATCC 25922 strain designated 25922 lux.
In these assays, we perform the ZOI on the same media the fungus has been growing on after confirming that E. coli 25922 lux growth is not impacted. Prepare the necessary number of plates of each media depending on how many fungal isolates, and biological and technical replicates of each isolate, you need to test. Once dry, section the back of each plate into six equal segments. Label each with the fungal isolate name/number and the date the fungus was cultured (age).
Measure the optical density of the overnight bacterial culture at 600nm (OD600). To do this dilute overnight cultures 1:10 in a 1.5 mL cuvette with Mueller Hinton Broth (MHB) ( 720 µL broth + 80 µL bacteria). Dilute the bacterial culture with MHB to give a final OD600 of 0.01 which is equivalent of ~106 bacteria per mL.
Add bacteria to each Petri dish. This can be done by pipetting 50ul of diluted overnight bacterial culture and spreading it over the plate using an L-shaped spreader. Alternatively, dip a sterile cotton swab into the diluted bacterial solution and gently streak the agar plate half a section at a time rotating 45 degrees each time to ensure an even lawn of bacteria.
Allow plates to dry before applying fungal plugs.
Adding fungal plugs to ZOI plates
Adding fungal plugs to ZOI plates
Use a 6mm punch biopsy to remove mycelium from the growing edge of the fungus. Place the plug fungus-side down on your labelled plate. Repeat for all biological/technical replicates.
Use a sterile punch biopsy when changing fungi to avoid cross-contamination.
Also prepare a no-fungi control by adding an agar-only plug to the bacterial lawn. Ensure the agar is the same as that the fungus was grown on.
Incubate the plates upside down at 37 °C in a standing incubator overnight.
ZOI measurements
ZOI measurements
The following day, use a ruler to measure the diameter of any zones of inhibition formed. You may need to hold some plates up to the light to get accurate measurements. A diagram of the zone of inhibition results is shown in Figure 1.
Figure 1: Schematic of a zone of inhibition plate