Chaos kdorfman Sat, 02/04/2012 - 15:43

Chaos cultivation

Chaos cultivation kdorfman Sat, 02/04/2012 - 16:59

Carolina cultivation

Carolina cultivation kdorfman Thu, 02/16/2012 - 14:36

Amoebae from Carolina:

131324 Chaos (Pelomyxa) carolinensis, Living $7.30

Order 12 dishes, 1 per group

Culture includes P. multimicronucleatum and other much smaller organisms, as well as the occasional rotifer.

Try washing, as per Tan:

Transfer individual amoebae with sterile transfer pipette to culture dish with Chaos saline

Leave at 22C in the dark overnight

Next day, they don't look any better (floating, not attached, few pseudopodia)

Feed them anyway.

No improvement.

Better to leave them in the Carolina dish.

Tan Chaos

Tan Chaos kdorfman Thu, 02/16/2012 - 14:27

See protocol on Chaos main page


  • Use culture-treated dishes
  • keep in Chaos saline at 22-24C in the dark
  • Every 2-3 days
    • count
    • change medium
    • feed
  • Split every 2-3 weeks, or when the debris becomes detrimental


  • 7-day old Paramecium multimicronucleatum cultures at late log phase or early stationary phase (Fok and Allen, 1979).
  • Remove growth medium:
    • Filter cultures through a 3-piece glass filter funnel (Whatman) 1950-007 Filter Funnel, 3-piece, 70 mm, 115 ml reservoir 1/pk Fisher 09-877-3 $223.58

lined with a layer of GF/A glass microfibre filter (Whatman) 7.0cm dia. 1820-070
Fisher 09-874-16A Pack of 100 for $48.50

  • Wash and concentrate Paramecia in Chaos saline

    • concentrate by allowing the medium to be filtered through
    • collect concentrated Paramecium suspension in a beaker
  • Estimate Paramecium concentration

    • 10 10uL drops
    • count # Paramecium in each
    • average x 100 = cells/mL
  • Feed 36 ± 3 Paramecia per amoeba.

Changing the medium

  • Tilt the dish, let amoebae settle to the bottom
  • Suck out the old medium
  • Carefully squirt fresh medium onto the dish surface
  • Repeat 2 or 3 times
  • Check the culture
    • Fast growing and healthy cultures have little cell debris (dead amoebae, cell fragments) and usually few or no lingering Paramecium left from the previous feeding.
    • slow-growing or sick cultures have more cell debris and a large number of Paramecium left from the previous feeding.

Chaos saline

Chaos saline kdorfman Sat, 02/04/2012 - 16:06

Chaos inorganic medium from stock solutions

salt mM
CaCl2.2H2O 0.5
MgSO4.6H2O 0.05
K2HPO4 0.16
KH2PO4 0.11
To make mL Chaos Saline
add: µL 1M CaCl2.2H2O
add: µL 1M MgSO4.6H2O
add: µL 1M K2HPO4
add: µL 1M KH2PO4


Keep amoebae at 22 - 24C, in the dark.

Each pair of students needs ~60 mL sterile saline

  • 10 mL initial Paramecium culture in culture flask
  • 50 mL to set up further cultures for experiments