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)]