Oct 17, 2025
Version 6
Axenic Diatoms cultures protocol V.6
This protocol is a draft, published without a DOI.
- Francesco Manfellotto1,
- Andrea Broccoli1,
- Monia Teresa Russo1,
- Antonella Ruggiero1,
- Rossella Annunziata1,
- Mariella Ferrante1
- 1Stazione Zoologica Anton Dohrn
Protocol Citation: Francesco Manfellotto, Andrea Broccoli, Monia Teresa Russo, Antonella Ruggiero, Rossella Annunziata, Mariella Ferrante 2025. Axenic Diatoms cultures protocol. protocols.io https://dx.doi.org/Version created by Francesco Manfellotto
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: October 17, 2025
Last Modified: October 17, 2025
Protocol Integer ID: 230059
Keywords: axenic diatoms cultures protocol axenic cultures protocol, axenicity of culture, adding antibiotic, axenicity, treatment, culture, coltures with new medium
Funders Acknowledgements:
DISCO
Abstract
Axenic cultures protocol
Axenicity of cultures is obtained by multi-antibiotic treatment: by adding antibiotics directly to the medium in which the strain will be inoculated. After a variable amount of days we refresh the coltures with new medium.
antibiotics concentration
The antibiotics used are:
Ampicillin, streptomycin, penicillin, and kanamycin.
Prepare F/2 medium with the following final antibiotic concentrations:
- Ampicillin: 0.7 mg/mL
- Streptomycin: 0.1 mg/mL
- Penicillin: 0.5 mg/mL
- Kanamycin: 0.1 mg/mL
Filter the F/2 medium with antibiotics (“F/2 + APSK”) through a 0.22 μm pore-size filter before use.
The F/2 + APSK medium can be stored for up to 4 weeks at 4 °C (avoid light exposure).
Before use, allow the medium to reach room temperature to prevent thermal shock.
Start with 20,000–50,000 cells in 25 mL of F/2 + APSK medium.
Refresh the cultures three times, every 3 days, using fresh F/2 + APSK medium
Transfer the cultures into a variable higher volume flask containing F\2+APSK.
Arrive at the desired final volume.
contamination test
Assess axenicity of the strains by fluorescence microscopy using DAPI staining (4',6-diamidino-2-phenylindole-a DNA stain).
(DAPI stains DNA; if bacteria are present in the culture, bacterial nucleoids can be visualised as fluorescent spots).
To fix cells, use neutralized formaldehyde 1.6%
DAPI final concentration 1:1000
Pseudo-nitzschia multistriata fluorescence microscopy images using DAPI staining.
A. Normal culture with bacteria
B. Axenic culture without bacteria.