ChaosChaos kdorfman Sat, 02/04/2012 - 15:43
Chaos cultivationChaos cultivation kdorfman Sat, 02/04/2012 - 16:59
Carolina cultivationCarolina 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.
Better to leave them in the Carolina dish.
Tan ChaosTan 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
- change medium
- 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)
http://www.whatman.com/GlassMicrofiberAccessories.aspx 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 salineChaos saline kdorfman Sat, 02/04/2012 - 16:06
Chaos inorganic medium from stock solutions
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