QSB

QSB kdorfman Wed, 01/11/2012 - 21:02

QSB lab syllabus

QSB lab syllabus kdorfman Mon, 02/06/2012 - 14:43

LABORATORY SCHEDULE

  • 01/24 01/26 0 No laboratory
  • 01/31 02/02 1 Welcome to the resistome
  • 02/07 02/09 2 Welcome to the resistome
  • 02/14 02/16 3 Welcome to the resistome
  • 02/21 02/23 4 The ecology of pond scum
  • 02/28 03/01 5 The ecology of pond scum
  • 03/06 03/08 6 The physics of physiology
  • 03/13 03/15 7 Electrical beings: muscle physiology
  • 03/20 03/22 0 No labs – spring break
  • 03/27 03/29 8 Electrical beings: muscle physiology
  • 04/03 04/05 9 Electrical beings: muscle physiology
  • 04/10 04/12 10 Behavioral ecology: niche partitioning in birds
  • 04/17 04/19 11 Behavioral ecology: niche partitioning in birds (FIELD WORK – NO LAB SESSIONS)
  • 04/24 04/26 12 Behavioral ecology: niche partitioning in birds

2018 Biocube

2018 Biocube kdorfman Wed, 03/21/2018 - 13:53

Biocubes: Exploring Biodiversity

Aluminum tubes (8601) @ $0.55 from (speak to David):

K & S Precision Metals

6911 W 59th Street

Chicago, IL 60638

773-586-8503

Antibiotic Resistance

Antibiotic Resistance kdorfman Wed, 01/11/2012 - 21:05

Resistance 2012

Resistance 2012 kdorfman Thu, 01/17/2013 - 18:39

Resistance 1

Resistance 1 kdorfman Wed, 01/11/2012 - 22:06

1/31/12 and 2/2/12

  • 6 tubes of freshly growing E. coli BZB1011 in LB (one on each set of desks) from Peg's lab
  • 6 tubes with 20 mL sterile LB (one on each set of desks)
  • ~150 Sterile swabs (one beaker full per set of desks) (= 2 100-packs, sterilized)
  • 12 Pipetteman and tips (1 per 2 students) (for 10 to 100 µL volumes)
  • ~125 MacConkey plates (about 4 per student with extras available) @ 25 mL/plate = 3125 mL (@ 50 g/L = 62.5 g)
  • 24 black markers
  • shelf in an incubator
  • shelf in a refrigerator
  • camera to photograph plates (or students use iphones)
  • scale for weighing 25 grams
  • weigh boats
  • vortexes

Have on hand for student use outside of class hours:

  • Scales, weigh boats and vortexes
  • 500 sterile capped tubes with 25 mL LB – need about 10/student
  • pipetman and 100 µL tips – need about 10 tips per student
  • BZB1011 for use as positive control – stored in refrigerator
  • 500 MacConkey plates – need about 10 per student
  • 500 sterile swabs – need about 10 per student

E. coli BZB1011

E. coli BZB1011 kdorfman Wed, 01/11/2012 - 22:17

Get from Peg

Grow in LB

LB

LB kdorfman Mon, 01/16/2012 - 19:35

Luria-Bertani broth (Miller)

LB broth (Fisher BP1426500) 500 g

25 g/L

= 20 g/800 mL (in a 1 L bottle)

Autoclave on liquid cycle 45 min

= 87.5 g/3.5L (in a 4 L flask)

Autoclave on liquid cycle 80 min

Aliquot 25 mL/tube 500 tubes (= 12.5 L)

MacConkey plates

MacConkey plates kdorfman Wed, 01/11/2012 - 22:17

MacConkey Agar Fisher 212122 (Difco)

to distinguish Lac+ and Lac- bacterial strains.

50 g/L

Total plates needed =~625

in 2 L flasks

flasks Total vol (L) max # plates
1 1.75 70
2 3.5 140
3 5.25 210
4 7 280
5 8.75 350
6 10.5 420
7 12.25 490
8 14 540
  • 2-L flasks, each with:
    • 87.5 g MacConkey dry mix
    • 1.75 L distilled water
    • magnetic stir bar
  • Stir to mix.
  • Cover loosely with foil
  • Pack Pour-Boy tubing in foil to go in same cycle if it's not already sterile
  • Autoclave 55 min, liquid cycle
  • Cool till handle-able on stir plate
  • Dispense 25 mL per plate with Pour-Boy, stacking 10 tall.

in 4 L flasks

  • 4 L flasks, each with
    • 175 g MacConkey dry mix
    • 3.5 L distilled water
    • magnetic stir bar
  • Stir to mix.
  • Cover loosely with foil
  • Pack Pour-Boy tubing in foil to go in same cycle if it's not already sterile
  • Autoclave 75 min, liquid cycle
  • Cool till handle-able on stir plate
  • Dispense 25 mL per plate with Pour-Boy, stacking 10 tall
flasks total vol (L) max # plates
1 3.5 140
2 7 280
3 10.5 420
4 14 560

Mueller-Hinton agar

Mueller-Hinton agar kdorfman Fri, 01/20/2012 - 22:39

90922 Mueller Hinton Broth 2 from Sigma

22 grams per liter, consisting of:

  • Casein acid hydrolysate 17.5
  • Beef extract 3.0
  • Starch 1.5

Final pH 7.3 +/- 0.2 at 25°C

Add 17 g agar per L

Make 3.5 L in a 4L flask: (yields up to 140 plates)

77 g broth mix

stir till dissolved. Leave stir bar in

59.5 g agar

autoclave 80 minutes

QSB antibiotics

QSB antibiotics kdorfman Thu, 01/12/2012 - 14:40

From Peg's list (weight indicates how much should be on one disk.)

Calculate concentration from volume needed per disk (25 uL)

Make 35 mL of each (because the filter syringe only holds that much)

See recipes in stock solutions.

Make 35 mL each, make 48 0.7 mL aliquots

Antibiotic ug/disk mg/mL category solubility Fisher #
Ampicillin 10 0.4 beta-lactam 50 mg/mL in 1 M NH4OH
Erythromycin 15 0.6 macrolide 50 mg/mL in EtOH ICN19452901 1g $33.68
tetracycline 30 1.2 tetracycline 10 mg/mL in water
cipro 5 0.2 quinolone 30 mg/mL in water
Kanamycin 30 1.2 Aminoglycoside 50 mg/mL in water

cipro solubility http://www.inspiralis.com/go/antibiotics.php 30 mg/mL in water

ampicillin solubility 50MG/ML IN 1M AMMONIUM HYDROXIDE http://www.sigmaaldrich.com/catalog/DataSheetPage.do?brandKey=SIGMA&sym… ampicillin sodium salt is soluble 50 mg/mL in water http://www.bioxys.com/i_gentaur/ampicillin_sodium.htm

erythromycin solubility 50MG/ML IN ETHANOL http://www.sigmaaldrich.com/catalog/DataSheetPage.do?brandKey=SIGMA&sym…

Kanamycin 50 mg/mL in water

Tetracycline 10 mg/mL

The HCl forms are more water soluble

Not using Spring 2012

Antibiotic ug/disk mg/mL category Fisher #
Amikacin 30 BP2643250 250 mg $28
Cefazolin 30 NC9863037 1g/10mL $17
gentamicin 10 ICN1676045 10mg/mL 10 mL $29
ticarcillin 7 50-121-7983 1 g $45
trimethoprim 5 ICN19552701 1 g $34
tobramycin 10 50-146-785 100 mg $56

Media Tally

Media Tally kdorfman Tue, 02/28/2012 - 14:22
Medium started with (approx) left over actually used
LB 25 mL in 50-mL tube 500 489 11
LB 20 mL in 125 mL flask 14 14 0
MacConkey plates 650 162 488
MH plates 560 325 235
Soy-Trypticase plates 32 0 32

Antibiotic started with left over actually used
amp 48 26 22
cipro 48 27 21
erythro 48 34 14
Kan 48 31 17
Tetra 48 30 18

resistance supplies

resistance supplies kdorfman Thu, 01/19/2012 - 15:54

Fisher cotton-tipped applicators 23-400-101 (come in 100-count autoclave bags.)

Fisher 43-400-102 plain wood applicators for colony picking

Fisher 11-312A solid glass beads 3mm for spreading liquid on plates

Krackeler 10-231039 Blank discs 1/4" dia. 6 vials of 50 $23.76

Resistance 2013

Resistance 2013 kdorfman Thu, 01/17/2013 - 18:43

Antibiotics 2013

Antibiotics 2013 kdorfman Mon, 01/28/2013 - 19:48

(weight indicates how much should be on one disk.)

Calculate concentration from volume needed per disk (25 uL)

Make 35 mL of each (because the filter syringe only holds that much)

See recipes in stock solutions.

Make 35 mL each, make 48 0.7 mL aliquots

Antibiotic ug/disk mg/mL category solubility Fisher #
Ampicillin 10 0.4 beta-lactam 50 mg/mL in 1 M NH4OH
tetracycline 30 1.2 tetracycline 10 mg/mL in water
cipro 5 0.2 quinolone 30 mg/mL in water
Kanamycin 30 1.2 Aminoglycoside 50 mg/mL in water

cipro solubility http://www.inspiralis.com/go/antibiotics.php 30 mg/mL in water

ampicillin solubility 50MG/ML IN 1M AMMONIUM HYDROXIDE http://www.sigmaaldrich.com/catalog/DataSheetPage.do?brandKey=SIGMA&sym… ampicillin sodium salt is soluble 50 mg/mL in water http://www.bioxys.com/i_gentaur/ampicillin_sodium.htm

Kanamycin 50 mg/mL in water

Tetracycline 10 mg/mL

The HCl forms are more water soluble

Plate Tally 2013

Plate Tally 2013 kdorfman Mon, 02/04/2013 - 15:03
type poured remaining used
LB 222 145 77
McC 1006 280 726
M-H 1150

Resistance 1 - 2013

Resistance 1 - 2013 kdorfman Thu, 01/17/2013 - 18:52

Jan 29 & 31, 2013

Resistance 2 2013

Resistance 2 2013 kdorfman Mon, 02/04/2013 - 15:01
  • 20 MacConkey plates per student

  • sterile swabs

  • gloves

  • spray ethanol

  • markers

Resistance 3 2013

Resistance 3 2013 kdorfman Mon, 02/04/2013 - 15:14
  • 26 Mueller Hinton plates

  • sterile swabs

  • 26 1-mL aliquots physiological saline

    0.9% NaCl (=0.9 g/100 mL = 9 g/L)

  • sterile sticks

  • parafilm

  • markers

  • gloves

  • micropipettors & tips

  • vortex

  • antibiotic solutions in freezer

  • sterile disks

  • sterile forceps

  • rulers

  • microtube racks

Resistance 2015

Resistance 2015 kdorfman Fri, 01/16/2015 - 18:22

Change antibody concentrations so 10 uL contains the correct number of mg.

Antibiotic ug/disk mg/mL category solubility Fisher #
Ampicillin 10 1 beta-lactam 50 mg/mL in 1 M NH4OH
Erythro 15 1.5 macrolide 50 mg/mL in EtOH
tetracycline 30 3 tetracycline 10 mg/mL in water
cipro 5 0.5 quinolone 30 mg/mL in water
Kanamycin 30 3 Aminoglycoside 50 mg/mL in water
Doxycyline * 30 tetracycline 50 mg/mL in water

*One group wants 1.2 mg/mL for project

Bird Labs

Bird Labs kdorfman Mon, 01/16/2012 - 15:08

Buy Birdseed:

  • 2 25 pound bags of black oil sunflower seeds @ $11.95 (2011)
  • 1 25 pound bag of thistle seed (NYJER seed) @ $23.49

Amherst Farmer's Supply

April 10: bird lab up at student dorm

April 17: students collect data in field

April 24: bird lab analyses in ISB

Mussel Labs

Mussel Labs kdorfman Mon, 01/16/2012 - 15:05

2012 mussel labs schedule

2012 mussel labs schedule kdorfman Tue, 03/12/2013 - 14:13

week of Feb 28

2/29 (9 am): practice using iworx

Try ordering small (~1-2 L) dispensers, one for artificial sea water and one for artificial pond water

week of March 5

run through with mussels

order 12 mussels 2/28 for delivery Friday 3/1

get marine mussels from grocery store. Make sea water with salt from Patek lab (34 g/L). ADD SALT TO FULL VOLUME OF WATER to avoid precipitating salts.

March 6

iWorx introduction "Physics of physiology"

bring balances in from prep rooms

order 48 mussels for delivery Friday 3/8

March 13

mussel lab 1

Patrick gets mussels from grocery store - they work much better than FW mussels. Make artificial sea water from salt obtained from Patek lab.

March 27

mussel lab 2

order ? mussels for delivery Friday 3/30

April 3

mussel lab 3

Drugs

Drugs kdorfman Mon, 01/16/2012 - 15:07

Needed for

March 13 & 15: mussel lab 1

March 27 & 29: mussel lab 2

April 3 & 5: mussel lab 3

Acetyl Choline (Fisher AC159170050 5 gr, $13.92)

Serotonin (Fisher A550328M050 50 mg, $34.32)

Acetylcholine

Acetylcholine kdorfman Thu, 02/23/2012 - 15:57

Fisher AC159170050 5 gr, $13.92

MW = 181.7

Working concentration = 10^-4M = 0.01817 g/L


10 mL 100X stock

0.01817 g acetylcholine

10 mL ringers or seawater


12 mL 10X stock for students to make dilutions

1.2 mL 100X stock

Ringers or sea water to 12 mL

Aliquot 2 mL into 5 tubes


15 mL 1X working concentration

0.15 mL 100X stock

Ringers or sea water to 15 mL

aliquot 1 mL into 14 tubes

Serotonin

Serotonin kdorfman Thu, 02/23/2012 - 15:52

(Fisher A550328M050 50 mg, $34.32)

MW = 212.68

soluble up to 100 mg/mL in water

Working Concentration = 10^-5M

X M mM µM mg/mL
1X 10^-5 0.01 10 0.0021268
10X 10^-4 0.1 100 0.021268
100X 10^-3 1 1000 0.21268
1000X 10^-2 10 2.1268

1.4 mL 1000X stock

=10 mM

3 mg (use the mg scale in 266A)

1.41 mL 1X ringers or sea water


12 mL 10X solution for students to make dilutions

0.12 mL 1000X stock

11.88 mL 1X Ringers or sea water

aliquot 2 mL into 5 tubes


15 mL 1X working concentration for student use

0.015 mL 1000x stock

15 mL Ringers or sea water

aliquot 1 mL into 14 tubes

Mussel saline

Mussel saline kdorfman Mon, 01/16/2012 - 15:06

Mussel Ringers

from http://www.biolbull.org/cgi/reprint/181/1/175.pdf

  • 5 mM NaCl
  • 0.5 mM KCl
  • 5 mM CaCl2
  • 5 mM NaHCO3
  • 5 mM Na2SO4

1X Ringers from 10X stock

100 mL 10X stock per L final volume

Ringers needed for

  • diluting drugs
  • keeping dissected mussels moist

10X Ringers from stock solutions

Stock solution ci (M) cf (M) 50 75 100 200 final vol (mL)
NaCl 5 0.05 0.5 0.75 1 2 mL
KCl 1 0.005 0.25 0.375 0.5 1 mL
CaCl2 1 0.05 2.5 3.75 5 10 mL
Na2SO4 1 0.05 2.5 3.75 5 10 mL
NaHCO3 1 1 0.05 2.5 3.75 5 10 mL
OR dry NaHCO3 MW=84 0.05 2.1 3.15 4.2 8.4 g

pH 7.8

Filter sterilize (NaHCO3 may not be stable during autoclaving)


  1. make fresh - it doesn't keep. ↩︎

Mussels 2 & 3

Mussels 2 & 3 kdorfman Tue, 03/12/2013 - 14:44

Check sea water supply

Make drugs

Pond Water

Pond Water kdorfman Thu, 03/01/2012 - 16:25

Cichlid minerals

Cichlid minerals kdorfman Fri, 03/02/2012 - 15:43

API Natural Trace

African Cichlid naturally derived trace minerals

Use 66 µL/L

Same for crayfish tank.


Calculations

for cichlids (as per bottle):

5 mL/gal x 0.264 gal/L = 0.132 mL/L = 132 µL/L

Divide in half for FW mussels and crayfish

66 µL/L

Water recipe

Water recipe kdorfman Fri, 03/02/2012 - 15:35

Artificial Pond Water for Mussels

from http://www.biolbull.org/cgi/reprint/181/1/175.pdf

Use for partial water changes in mussel "aquaria"

Salt 1X (mM) 10X (mM) 100X (mM)
CaCl2 0.4 4 40
KCl 0.05 0.5 5
NaCl 0.5 5 50
NaHCO3 0.2 2 20

pH 7

100 X from stock solutions

80 mL aliquots to dilute to 8 L in carboy.

3 boxes changed daily for 3 weeks

Stock solution ci (M) cf (M) 80 800 1280 1600 final vol (mL)
NaCl 5 0.05 0.8 8 12.8 16 mL
KCl 1 0.005 0.4 4 6.4 8 mL
CaCl2 1 0.04 3.2 32 51.2 64 mL
NaHCO3 1 1 0.02 1.6 16 25.6 32 mL
OR dry NaHCO3 MW=84 0.02 0.134 1.344 2.15 2.688 g

pH 7

Filter sterilize (NaHCO3 may not be stable during autoclaving)


  1. make fresh; NaHCO3 is only stable for a couple of weeks. OOPS CANNOT GET THIS MUCH INTO SOLUTION! ↩︎

Sea Water

Sea Water kdorfman Thu, 05/24/2012 - 16:16

Make artificial sea water

Get salts from Patek lab.

PUT WATER IN FIRST!

To make L artificial sea water
add: g marine salts (from Patek lab)

Mix in 8L carboy.

iworx lab

iworx lab kdorfman Tue, 03/12/2013 - 14:11

iWorx introduction "Physics of physiology"

  • bring balances in from prep rooms

  • set up

    • power supply
    • transducer with thread and hook
    • ring stand
    • clamp
    • baggies for treats
    • computer (best on the right, power supply, etc. on the left)

mussel equipment

mussel equipment kdorfman Tue, 03/13/2012 - 13:33

Per pair:

  • Dissecting pan
  • Dissecting bowl with black wax at the bottom
  • Ball of modeling clay
  • Dissecting kit, containing:
    • scissors
    • 2 forceps
    • 2 probes
    • scalpel
  • thread
  • fine insect pin

Nature's Notebook 2017

Nature's Notebook 2017 kdorfman Thu, 03/30/2017 - 17:32

I have reserved two vans for each of those days from 730am to noon. Amrita will be driving one van and I was hoping to find someone else to drive the other - Kate do you want to spend time on my farm looking at critters? Just a thought....I can always get my husband to drive the second van if he is free.

Students will be asked to dress appropriately to be out in the meadows and woods. We will drive them to my 90 acre farm - 4 miles from campus - and then they will spend their time learning how to observe nature while contributing to Nature's Notebook, an online phenology program for citizen scientists.

Equipment:

Binoculars - I am hoping that we can have one pair of binoculars per pair of students - which I guess means we need 12 or so. I recall that Sheila Patek ran a bird lab with binoculars - are those still available? I have two pair so if we are in a crunch for them, I can donate mine for those days.

Plastic coated identification sheets - for common birds, mammals, trees/flowers and butterflies. I bought a few sets last year - but we didn't have enough. 

We need 8 of each of these: butterflies Guide to common and Exotic butterflies in New England birds Massachusetts Birds Waterford Press trees and flowers New England Trees and Wildflowers

Clipboards with storage. I have found the perfect clipboard that will also hold the plastic field guides and their papers and pens - we will need about 12 of these:

https://www.google.com/webhp?sourceid=chrome-instant&rlz=1C5CHFA_enUS68…

Protist Labs

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

Chaos

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

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)

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