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INSTRUCTOR
M. Bastiani, Room 430 ASB (ext. 1-8605)
Teaching Assistant:David Estes
SCHEDULED LAB PERIODS
Tues-Thurs from 12:25-3:20 PM.
Equipment, supplies, and solutions for Developmental Biology 3235.
Each Student should have a station consisting of:
1 stereo dissection microscope with focusing 10 or 15X eyepieces, and 1 or 1.5X objective.
1 Trans illumination base with fiber optic illuminator.
1 alcohol burner
1 clear plastic ruler (mm/inches)
1 stop watch
1 platinum wire pick (made from platinum wire and glass Pasteur pipette. (platinum wire O.01 Dia, or 32 ga. Omega Engineering, Inc. www.omega.com)
1 Box of Standard glass microscope slides and 22mm square cover slips
COMMOM Equipment for Class
3 compound scopes with epiflourescence, cameras, and computer workstation
3 compound scopes with DIC, cameras and computer workstations
immersion oil for oil lenses
incubator 15-37 degree C. range
2 hot blocks for melted agar
pipetteman, about 5 P20s and tips for adding azide solution to slides.
2 flat nosed “micro” pliers to flatten platinum pick ends
about 2000 NGM agar plates seeded with OP50 (60X15 mm plastic)
Box of tooth picks
laser printer with paper
Solutions
Anesthetic Mount 30 mM solution of sodium azide (NaN3) in M9 or S basal
Agar for mounting pads 4-6% Agar or Agarose in water (about 20 ml, aliquoted in 0.5-1ml amounts in small plastic test tubes that fix in hot block holes)
Sodium hypochlorite (Chlorox): one gallon bottle
M9 buffer (Worm book recipe):
KH2PO4 3.0 g
Na2HPO4 6.0 g
NaCl 5.0 g
1M MgSO4 1.0 ml
dH20 to 1liter
PROCEDURE FOR POURING PLATES
1. In a 6 liter flask mix:
12.0g NaCl
64.0g* Agar (Difco Bacto-Agar)
10.0g Bacto-peptone
4.0L dist. water
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2. Be sure there is water in the syringe and in the tubes of the media pump. Remove the syringe mounting screws and pull off the syringe and attached tubes. Wrap the last 16 inches of the intake tube (lower tube) in aluminum foil, and wrap the glass tip of the dispensing tube in foil. Place in a wire basket.
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3. Autoclave the 4L of media, the pump syringe, and an erlenmeyer flask containing 1L of distilled water to use to clean the pump when you are done. Use the liquid cycle for 45 minutes. Allow the media to cool for a couple of hours in the autoclave or at room temperature for one hour. Place the flask of water in the media oven until you are ready.
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4. Arrange stacks of 10 plates on bench for easy access. If you are feeling exceptionally steady, arrange stacks of 20 plates. 4L will make 300 small plates (15 bags) with 10 plates per stack or 16 to 17 bags with 20 plates per stack. Set up a bunsen burner to flame plates. Place two large beakers nearby, one is used to squirt waste agar into, the other to squirt water into.
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5. AFTER AUTOCLAVING DON'T FORGET TO ADD:
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4.0ml cholesterol (5mg/ml in 95% EtOH).
4.0ml 1M MgSO4
4.0ml 1M CaCl2
100.0ml 1M KPO4 (pH 6.0)**
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These solutions are added after autoclaving because they are temperature sensitive or because the salts will precipitate if autoclaved.
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6. Pour plates. Reattach the syringe to the pump. Remove the foil and insert the intake tube in the flask. Remove foil from the dispensing tip. Set the speed at 32. Flip toggle and run the agar into the beaker until the water has been removed from dispensing lines. Lift a stack by lifting the lid of the bottom plate and fill plate with one squirt, replace and move up stack in a similar fashion. At the top plate turn the pump off by quickly hitting the toggle switch down. Slide stack away, slide a new stack forward, lift stack and flip toggle on again. Until you get proficient you may need to let this first squirt go into the waste bucket.
DO NOT LET AGAR RUN DOWN THE SIDE OF THE PLATES, this will grow mold and then when you grab these plates later you will spread mold through your entire collection.
One squirt (11ml) for the small (60mm) plates; three squirts for the large (10mm) plates.
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7. Flame plates. Once you have poured about 4 stacks, stop and flame the surface of each plate with a bunsen burner to eliminate bubbles. Pouring hot media seems to reduce the number of bubbles. However, adding the phosphate buffer to hot media causes it to precipitate badly.
8. STOP when you get close to the bottom of the flask of media. Don't let air run through the tubes. This will cool the agar and jam the valves. Remove the tube from the media and move it to the beaker of very hot water. Run a liter of water through the pump into the waste water beaker. Be sure and leave water in the syringe and tubing.
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9. Pour the waste agar into the waste basket. DO NOT POUR HOT AGAR DOWN THE SINK, it will congeal and plug the pipes. Clean up. Slide the plates out of the way so that someone else can use the pouring space. Label and date the plates so others know who they belong to. If it is after 8 pm, close the door and turn on the UV sterilization lamp.
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*If you let your plates dry at room temperature for a couple of days before seeding them, the agar surface will form a skin which the worms cannot penetrate. If you use these plates very soon after pouring them, the worms will bore under the surface. If this happens frequently you will need to boost the total agar to 80 grams per 4 liters.
**Note: When the pH of the plates is higher than 6.8 the seeded bacterial pellets get crusty. I.e., when the plates are ~pH 7.5 the bacteria is too crusty for the worms to penetrate and they aren't too happy. Addition of pH 6.0 KPO4 is CRITICAL at this step (Knobel), although calcium or magnesium may be the cause of thick and granular bacteria (see Eric Lambie's notes on his MYOB and NGM-lite recipes). Also use fresh K2HPO4 dibasic anhydrous, when it gets old it won't go into solution (Mike Hermann).
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B. solutions
1M KPO4 (6.0)
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517g KH2PO4 monobasic(FW136)
207g K2HPO4 dibasic anhydrous (FW174 -Make sure this is new or it won't go into solution)
or [271g K2HPO4•3H20 dibasic hydrate (FW228.2)]
Bring to 4L with distilled H20
Aliquot into six 1L flasks
Autoclave
Big stock:
2.5kg KH2PO4 monobasic(FW136)
1.0kg K2HPO4 dibasic anhydrous (FW174 )
23.5L dH20
=24.2L @pH6.0
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Nematode Plates
Applied Scientific #AS-4051, 60X15mm; nonvented; 29.50/cs
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