Paramecia
Paramecia kdorfman Sat, 02/04/2012 - 17:00Culturing 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 - 17:40Growth 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
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)
P.m. (KD)
P.m. (KD) kdorfman Sat, 02/18/2012 - 15:52Paramecia multimicronucleatum from Parameciavap
http://parameciavap.com/Paramecia.aspx ($45/250 mL)
Get PO from Charlene
To increase population size:
Test cultures for rotifers. Split flasks that have only paramecia
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)
-
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 - 16:25Culturing 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
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)]