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

Get from Peg

Grow in LB

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 Agar Fisher 212122 (Difco)

to distinguish Lac+ and Lac- bacterial strains.

50 g/L

Total plates needed =~625

in 2 L flasks

• 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

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

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

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

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

(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

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

Jan 29 & 31, 2013

• 200 LB plates

• 200 MacConkey plates

• 200 sterile swabs

• Biohazard waste beaker at each table

• 20 MacConkey plates per student

• sterile swabs

• gloves

• spray ethanol

• markers

• 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

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

(Fisher A550328M050 50 mg, $34.32)

MW = 212.68

soluble up to 100 mg/mL in water

Working Concentration = 10^-5M

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

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

Artificial Pond Water for Mussels

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

Use for partial water changes in mussel "aquaria"

pH 7

100 X from stock solutions

80 mL aliquots to dilute to 8 L in carboy.

3 boxes changed daily for 3 weeks

pH 7

Filter sterilize (NaHCO3 may not be stable during autoclaving)

Chaos inorganic medium from stock solutions

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

Growth medium of axenic P. multimicromucleatum culture

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

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)

Paramecia multimicronucleatum from Parameciavap

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

Get PO from Charlene

To increase population size:

• http://wahoo.nsm.umass.edu/content/parameciavap

• 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)

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

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)]