Protist Labs

Protist Labs kdorfman Fri, 02/03/2012 - 16:26

Chaos

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

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

Maintenance

  • 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

Feeding

  • 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 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

Autoclave

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

Paramecia

Paramecia kdorfman Sat, 02/04/2012 - 17:00

Culturing Paramecium multimicronucleatum to feed Chaos

Carolina 130871 Paramecium multimicronucleatum Demoslide Set $13.50

Make medium from Carolina concentrate 132350 Liquid Protozoan Nutrient Concentrate, Culture Medium, Makes 1 gal, 100 mL $11.00

1 mL LPNC, 9 mL sterile water, 1 sterile wheat seed in a 25 mm2 vented culture flask.

1 mL P. m. culture.

20C in the dark.

Check concentration.

  • 10 10µL drops of culture
  • on a clean (wipe it first!) slide
  • count with dissecting scope and side illumination.

Initial concentration ~40/mL

Plot growth.

Scale up.

P. m. medium

P. m. medium kdorfman Sat, 02/04/2012 - 17:40

Growth medium of axenic P. multimicromucleatum culture

from http://protistology.ifmo.ru/num4_2/tan.pdf

Ingredient conc MW supplier
L-a-Phosphatidylethanolamine (=cephalin) 0.2 g/L Sigma P7943-100MG $352, Fisher 50-750-5972 50g $125
Proteose peptone 10 g/L
Tryptone 0.5 g/L
Ribonucleic acid 1.0 g/L Sigma R6625-25G $48.90
Magnesium sulfate heptahydrate 4.2 mM
Stigmasterol 0.006 mM 412 =0.00247 g/L Fisher 50-014-46501 1 g $39.30
D_Pantothenic acid hemicalcium salt 0.02 mM 238 =4.76 mg/L 50mg/mL H2O SigmaP2250-5G 24.50
Nicotinamide 0.04 mM 122 Fisher AC12827-1000 100g $14.63
Pyridoxal hydrochloride 0.25 mM 203.63 Fisher AC35271-0010 1g $34.69 need 51 mg/L
Pyridoxamine dihydrochloride 0.01 mM 241.12 Fisher AC43625-0010 1g $80.94 need 2.41 mg/L
Riboflavin 0.01 mM 376.37 Fisher AC13235-0250 25 g $24.18 need 3.7 mg/L
Folic acid 0.06 mM 441 =0.026g/L Fisher 10g AC21663-0100 $13.54 soluble 50mg/mL in 1M NaOH
Thiamine hydrochloride 0.04 mM 337.27 Fisher AC14899-0100 10 g $29.79 need 13.5 mg/L
Biotin 0.01 μM 244.31 =0.0000024g/L; 100mg/0.41L = 1mM. soluble 50 mg/mL in 2M NH4OH Fisher 50-014-20193 100 mg $30.12
Lipoic acid 0.24 μM 206.33 Fisher 1g BP2682-1 $45.92 50mg/mL EtOH

NH4OH 500mL Fisher ICN19385490 MP Biomedicals No.:0219385490 $34.49 ~28% MW = 35 so solution is ~1M

In other studies that require stringent, defined conditions, stigmasterol and either oleate, monolein, or synthetic dioleoyl PsE are the only lipids included in the chemically defined medium

Paramecium Starter Kit

The Kit includes

  • 250 mL of the fresh Paramecium Culture at ~3,000 cells/ml in a diluted Tübingen E3 medium for Zebrafish embryos
  • 250 mL of Paramecium Culture Medium concentrate for preparation of 20.0 L of growth medium
  • 50 mL of Paramecium Food for high density cultures, sufficient for preparation of 5.0 L

  • Suggested schedule of feeding Zebrafish larvae with paramecium

    1 kit $75 (shipping and handling included)

paramedium_lipids.pdf

P.m. (KD)

P.m. (KD) kdorfman Sat, 02/18/2012 - 15:52

Paramecia multimicronucleatum from Parameciavap

http://parameciavap.com/Paramecia.aspx ($45/250 mL)

Get PO from Charlene

To increase population size:

To prepare for student use:

  • Sterile Erlenmyers with foam plug
  • Chaos saline (http://wahoo.nsm.umass.edu/content/chaos-saline) to volume that gives maximum surface area/volume ratio)

  • Add sterile wheat seed (~1/25 mL)1

  • Transfer P.m. from PC to Chaos saline + sterile wheat seed (~1:100)

  • Store at 22C in the dark
  • Transfer ~10 mL culture to tissue culture flask with new seed (1 flask per pair of students)

  1. Wheat seeds support dense rotifer populations. Start the Paramecium culture only with cultures that have no rotifers. Take multiple samples to determine rotifer content. ↩︎

Parameciavap

Parameciavap kdorfman Thu, 02/16/2012 - 16:25

Culturing Paramecia ala Parameciavap

PC - 250 mL Paramecia Culture 3000/mL in diluted Tubingen E3 medium for zebrafish embryos

PCM 100X Paramecium Culture Medium concentrate (250 mL)

PF - 200X Paramecium Food for high density cultures (50 mL)

Per Liter:

  • PCM 10 mL
  • H2O 1 L
  • PF 5 mL (can be added after initial incubation)
  • PC 33 mL

Keep at 25-27C

To make mL PCM + Food
add: mL PCM
add: mL PF
add: mL PC

After 1 week, add 5 mL PF/L

Culture 1 more week

[For zebrafish, filter and resuspend in Tubingen E3 (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4)]

Supplies

Supplies kdorfman Wed, 01/01/2014 - 18:37

From Carolina: 131324 Chaos (Pelomyxa) carolinensis, Living $7.30

A simple mass culture of the amoeba Chaos carolinense:

http://protistology.ifmo.ru/num4_2/tan.pdf

(attached below)

Culturing Chaos is not really so simple. Order repeatedly?

131558 Paramecium multimicronucleatum, Conjugating, Living $14.60

130871 Paramecium multimicronucleatum Demoslide Set $13.50

Dishes

Amoebae must have tissue culture treated bottom to attach to.

If you use a culture flask, you can't pick up the amoebae.

Culture flask (Krackeler 40-229331 Celltreat Polystyrene Tissue culture flask 50mL Triangular TC-treated 25cm2 Angled Vent $103.00/200)

If you use culture dishes (Krackeler 40-229670 Tissue culture dish with lid Polystyrene 8mL 70 x 15mm 36.3cm2 With gripping ring $115.00/500), you can more easily see and handle them, but you risk spilling.

Bigger dishes for mass cultivation Krackeler 40-229620 Tissue culture dish with lid Polystyrene 16mL 100 x 15mm 55cm2 With gripping ring $103.00/300