Mussel Labs

2012 mussel labs schedule

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

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

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

(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 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 1

Buy mussels from grocery store.

get two bags (they last well in the refrigerator)

Make artificial ocean

Dissecting equipment

Mussels 2 & 3

Check sea water supply

Make drugs

Pond Water

Cichlid minerals

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

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

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 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

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