May 01, 2026
Experimental Induction of a New Infusoria Strain via Ultraviolet Mutagenesis
- Evhenii Hordiienko1,2,3
- 1Independent Researcher;
- 2Member of the American Society for Microbiology;
- 3Member of the Microbiology Society
- Microbiology
Protocol Citation: Evhenii Hordiienko 2026. Experimental Induction of a New Infusoria Strain via Ultraviolet Mutagenesis. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwwr7dvmk/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: In development
We are still developing and optimizing this protocol
Created: April 30, 2026
Last Modified: May 01, 2026
Protocol Integer ID: 316086
Keywords: Infusoria, Paramecium caudatum, Tetrahymena thermophila, UV–C mutagenesis, hay infusion, protozoology, ciliate genetics, new strains of infusoria, ultraviolet mutagenesi, new infusoria strain, ultraviolet mutagenesis this paper, generating new strain, ciliate genetics, isolation of individual mutant, infusoria, individual mutant, irradiation of cell, photoreactivation, environmental microbiology, using uv, irradiation, hay infusion
Abstract
This paper presents a detailed and reproducible protocol for generating new strains of infusoria using UV–C mutagenesis (254 nm). The protocol includes the preparation of a hay infusion supplemented with 1% sucrose as a selective substrate, irradiation of cells in the exponential growth phase at LD90–LD95 lethality levels, dark incubation to minimize photoreactivation, microscopic screening, and the isolation of individual mutants. It is proposed that phenotypic stability be confirmed over a minimum of 7 generations. This method is designed for laboratories with basic equipment and is applicable in experimental protozoology, ciliate genetics, and environmental microbiology.
Materials
- Model organisms**: Paramecium caudatum or Tetrahymena thermophila (cultures in exponential growth phase, density 1–5 × 10^3^ cells/mL).
- Dry meadow hay**: (untreated).
- Sucrose**: (analytical grade).
- UV lamp**: 254 nm (15–30 W).
- Microscope**: Phase-contrast or stereo microscope.
- Micropipettes**: For single-cell isolation.
- Counting chamber**: Goryaev chamber or equivalent.
- Irradiation buffer**: Chalkley’s solution or 10 mM phosphate buffer (pH 7.0).
Safety warnings
UV–C (254 nm) is hazardous to the eyes and skin. Wear UV–C protective goggles, gloves, and protective clothing. Avoid direct exposure.
Methods
Add 10–15 g of dry hay to 1 L of dechlorinated water; boil for 20–25 min. Cool, filter through gauze, adjust volume back to 1 L, and autoclave (121 °C, 15 min). Adjust pH to 7.0–7.5.
Add sucrose to the base infusion to a final concentration of 1.0% (10 g/L). Sterilize by filtration through a 0.22 μm filter.
Gently centrifuge the exponential phase culture (600 g, 5 min), wash twice with irradiation buffer, and resuspend to a density of 1–2 × 10^3^ cells/mL. Pour the suspension into Petri dishes in a thin layer (no more than 2–3 mm).
A survival curve must be pre-established for the specific strain and lamp used. Approximate parameters: Distance to lamp 20–25 cm; irradiation time 30–180 seconds to achieve 90–95% lethality. Immediately after irradiation, close the dishes and incubate in complete darkness for 12–24 h at 20–25 °C.
Following dark incubation, transfer surviving cells into the selective medium (hay infusion + 1% sucrose) and incubate at 20–25 °C.
Perform microscopic screening after 3–10 days. Isolate cells exhibiting altered traits (morphology, size, swimming speed/pattern, cytostome behavior) using a micropipette into individual wells of a 96-well plate containing selective medium.
Cultivate each clone for a minimum of 7 generations (10 recommended). Formula: Nt = N0 × 2^^n^^. Where n is the number of divisions. Regularly monitor for phenotype retention.
Control Experiments
Non-irradiated control.
Irradiated culture without selective medium.
Survival count immediately post-irradiation using a counting chamber.
Protocol references
1. Cassidy-Hanley D.M. Tetrahymena in the Laboratory: Strain Resources and Methods. Methods Cell Biol. 2012.
2. Pennock D.G. Selection of Motility Mutants in Tetrahymena. Methods Cell Biol. 2000.
3. Russo J.A. et al. Protocol for the generation of Symbiodiniaceae mutants using UV mutagenesis. STAR Protocols, 2023.
4. Kobayashi T. et al. Abortive conjugation induced by UV–B irradiation in Paramecium. 1998.
5. Standard hay infusion methods for Paramecium cultivation.
Acknowledgements
Data availability: The full protocol will be available on Protocols.io. Raw data regarding survival curves and screening will be provided upon request.