Nov 21, 2019

Public workspaceCultivation protocol for anaerobic ciliates

DOI
dx.doi.org/10.17504/protocols.io.85why7e
  • 1University of Rhode Island;
  • 2Charles University
Johana Rotterova
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DOI: dx.doi.org/10.17504/protocols.io.85why7e
Protocol Citation: Johana Rotterova, Ivan Čepička 2019. Cultivation protocol for anaerobic ciliates. protocols.io https://dx.doi.org/10.17504/protocols.io.85why7e
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: November 08, 2019
Last Modified: November 21, 2019
Protocol Integer ID: 29590
Materials
MATERIALS
ReagentPotassium Chloride
ReagentMagnesium chloride hexahydrateSigma Aldrich
Reagent500g Calcium Chloride,Dihydrate (CaCl2.2H2O)G-BiosciencesCatalog #RC-030
ReagentNaClSigma AldrichCatalog #53014
ReagentDisodium phosphateSigma AldrichCatalog #S7907
ReagentcerophylleWard's Natural Science Establishment, Inc.Catalog #470300-680
Sample isolation
Sample isolation
Cultivation protocol is followed by methods described in Rotterová et al. 2018 and Bourland et al. 2017.


CITATION
Rotterová J, Bourland W, Čepička I (2018). Tropidoatractidae fam. nov., a Deep Branching Lineage of Metopida (Armophorea, Ciliophora) Found in Diverse Habitats and Possessing Prokaryotic Symbionts.. Protist.
LINK
https://doi.org/10.1016/j.protis.2018.04.003

CITATION
Bourland W, Rotterova J, Čepička I (2017). Redescription and molecular phylogeny of the type species for two main metopid genera, Metopus es (Müller, 1776) Lauterborn, 1916 and Brachonella contorta (Levander, 1894) Jankowski, 1964 (Metopida, Ciliophora), based on broad geographic sampling.. European journal of protistology.
LINK
https://doi.org/10.1016/j.ejop.2016.11.002


Isolate 10 ml of targeted sample from environments of interest (e.g., freshwater sulphidic sediment).



Media preparation
Media preparation
Prepare ATCC medium: 802 (Sonneborn's Paramecium medium) for freshwater ciliates or ATCC Medium: 1525 (Seawater Sonneborn's Paramecium Medium) for marine ciliates. Preparations for both below:
Instructions for 500 ml of ATCC #802 Freshwater Cereal Grass Media (Sonneborn's Paramecium medium) preparation:

  • 500 ml dH2O
  • 1.25 g cerophylle
  • Boil for 5 minutes
  • Filter through filter papers
  • Add 0.25 g Na2HPO4
  • Autoclave at 121 °C

Add cerophyll to distilled water and boil for 5 minutes. Add 100 ml distilled water to compensate for evaporation. Filter through Whatman #1 filter paper and add 0.5 g Na2HPO4. Autoclave for 15 minutes at 121 °C.


Instructions for 1 liter of ATCC #1525 Seawater Cereal Grass Media (Seawater Sonneborn's Paramecium Medium) preparation:

4 parts:

Cereal Grass Medium:
500ml bottle: Cerophylle
  • 500 ml dH2O
  • 2,5 g of cerophylle
  • Boil for 5 minutes
  • (it is twice as concentrated as the usual cereal grass media for freshwater cultures, + no Na2HPO4 included)
  • Filter through filter papers

Artificial seawater protocol:
1 L bottle: SWA
  • 300 ml dH2O
  • 24.72 g NaCl
  • 0.67 g KCl
  • 1.36 g CaCl2.2H2O
  • 4.66 g MgCl2.6H2O
100ml bottle: SWB
  • 100 ml dH2O
  • 6.29 g MgSO4.7H2O
100ml bottle: SWC
  • 100 ml dH2O
  • 0.18 g NaHCO3

Autoclave separately at 121 °C, mix after cooling down.

Cultivation establishment
Cultivation establishment
Add each 1 ml of the sediment four times into a 15 ml falcon tube with 10 ml of the designated media.


Cultivation maintanance
Cultivation maintanance
Reinoculate 1 ml of each sedimented culture into a new 15 ml falcon tube with 10 ml of the designated media every two weeks.

Green organic matter from the cereal grass media will be gradually generated over time and the culture will lose anorganic particles, such as sand grains and soil, present in the original sample.

If you have any questions, please contact me here or at johana.rotterova@natur.cuni.cz.


Citations
Step 1
Rotterová J, Bourland W, Čepička I. Tropidoatractidae fam. nov., a Deep Branching Lineage of Metopida (Armophorea, Ciliophora) Found in Diverse Habitats and Possessing Prokaryotic Symbionts.
https://doi.org/10.1016/j.protis.2018.04.003
Step 1
Bourland W, Rotterova J, Čepička I. Redescription and molecular phylogeny of the type species for two main metopid genera, Metopus es (Müller, 1776) Lauterborn, 1916 and Brachonella contorta (Levander, 1894) Jankowski, 1964 (Metopida, Ciliophora), based on broad geographic sampling.
https://doi.org/10.1016/j.ejop.2016.11.002