May 12, 2025

Public workspaceFlynn's Encystment Medium preparation (400 mL)

DOI
https://dx.doi.org/10.17504/protocols.io.kxygxqx14v8j/v1
  • Carrie Flynn1,
  • Andrew Harmez2,
  • Rebecca I Colon-Rios2,
  • Barbara Kazmierczak2
  • 1Harvard University;
  • 2Yale University
  • Kazmierczak lab
Carrie A. Flynn
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DOI: https://dx.doi.org/10.17504/protocols.io.kxygxqx14v8j/v1
Protocol Citation: Carrie Flynn, Andrew Harmez, Rebecca I Colon-Rios, Barbara Kazmierczak 2025. Flynn's Encystment Medium preparation (400 mL). protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxqx14v8j/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: May 11, 2025
Last Modified: May 12, 2025
Protocol Integer ID: 218056
Keywords: amoeba, ameba, Acanthamoeba castellanii, Acanthamoeba, cyst, encystment, encystation, media, medium, synchronous encystment of acanthamoeba castellanii trophozoite, acanthamoeba castellanii trophozoite, novel encystment medium, recipe for fem, mature cyst, medium preparation, higher numbers of mature cyst, fem, preparation
Funders Acknowledgements:
NIAID
Grant ID: R21 AI149628
Abstract
Recipe for FEM (a novel encystment medium) which is used to induce synchronous encystment of Acanthamoeba castellanii trophozoites. Results in significantly higher numbers of mature cysts compared to other media (https://dx.doi.org/10.17504/protocols.io.bvqcn5sw).
Troubleshooting
Dry ingredients
To 500 mL bottle, add:
Amount4.0664 g MgCl2 x 6H2O (50 mM)
Amount3.6032 g glucose (50 mM)
Amount2.9824 g KCl (100 mM)
Amount1.344 g NaHCO3 (40 mM)
Amount0.7888 g MgSO4 x 7H2O (8 mM)
Amount0.5248 g hydroxyproline (10 mM)
Amount2 mL 20 mM MnSO4 (100 µM)
Amount2 mL 10 mM NiCl2 (50 µM)
Amount2 mL 80 mM CaCl2 (0.4 mM)
Amount2 mL 64 mM AMPD (2-amino-2-methyl-1,3-propanediol) (0.32 mM)
  • stir bar
Dissolve
Bring volume toAmount400 mL with dH2O and place on stir plate (unheated).

Stir until dissolved.
pH
Measure pH. Confirm that the pH is between 8 and 8.5 (the optimal range for encystment).
Sterilize
Filter sterilize into plastic bottle using 0.22 µm filtration system.

Note
Storage in plastic, not glass, is essential to maintaining the pH.

Store atTemperature4 °C for up to 2 months.

Protocol references