NGM (Nematode Growth Media) Plates
Media Protocol (Makes 2 Liters)
1) Clean benchtop station by magnetic stirrers with 70% ethanol
2) Retrieve materials (ALL MUST BE STERILE IE. MARKED WITH BLACKENED AUTOCLAVE TAPE):
[1] 4 Liter Flask (Top Glassware Shelf) OR [2] 2 Liter Flasks
[3] Weigh Boats (Drawer 61)
[3] Spatulas (Drawer 59)
[1] Stir Bar (Drawer 59)
[1] 500 milliliter graduated cylinder OR 1 Liter graduated cylinder (Top Glassware Shelf)
Electronic Scale (Drawer 61)
3) With graduated cylinder, measure out 500 milliliters of milliQ water.
4) Add water and stir bar to 4L flask and place on magnetic stirrer.
5) Begin stirring on low speed. Replace autoclave foil cover on 4L flask.
6) Mass out 6 grams NaCl (Stored on chemical shelf alphabetically as “Sodium Chloride”) and add carefully to 4L flask. Replace foil cover when transfer is complete.
7) Mass out 5 grams BD Peptone (Stored alphabetically as “Peptone”) and carefully to 4L flask. Tap weigh boat gently as necessary to transfer powdery, sticky peptone. Replace foil cover on flask when finished.
8) Mass out 40 grams Agar (NOT AGAROSE) and add SLOWLY to 4L flask. Replace foil cover once more.
9) Turn stirring speed up and allow all added ingredients to dissolve completely.
10) Prepare 4L flask for autoclave sterilization by covering with new foil and marking with green-and-white-striped autoclave tape
11) Clean benchtop by stirrers. Dispose of weigh boats in trash can, rinse spatulas (1x tap water, 3x Distilled Water), and wipe down workstation with 70% ethanol.
12) Autoclave on cycle 4 (around 55 minutes + 10 minutes decompression)
13) After retrieving the 4L flask from autoclave, place flask on magnetic stirrer and set stirrer to moderate speed.
14) Assemble the following materials and supplies:
[3] 5 milliliter Disposable Pipets (Rack Left of Monitor Row)
[1] 50 milliliter Disposable Pipet (Rack Left of Monitor Row)
[1] Pipet Aide (Throughout Workstations)
5 milligram/milliliter Cholesterol Solution (Door of the 4 Degree Celsius Refrigerator)
1 M MgSO4 Solution (Shelf Right of milliQ Water)
1 M CaCl2 Solution (Shelf Right of milliQ Water)
1 M KPHO4 Solution (Shelf Right of milliQ Water)
15) Pipet 2 milliliters cholesterol solution into 4L flask using Pipet Aide and 5mL pipet. Replace foil cover on flask when transfer is complete.
16) Add 2 milliliters MgSO4 solution with clean 5mL pipet and Pipette Aide. Replace flask foil cover.
17) Add 2 milliliters CaCl2 solution with clean 5mL pipet and Pipette Aide. Replace flask foil cover.
18) Finally, use 50mL pipet and Pipette Aide to add 50 milliliters KPHO4 solution to flask. Replace foil cover and allow media to stir briefly.
19) Dispose of pipets in orange biohazard waste bucket.
20) Clean benchtop with 70% ethanol.
Plate Protocol
1) Clean a benchtop with 70% ethanol
2) Prepare to pour NGM Plates by assembling the following materials:
[80] Large Plates (Boxes on Floor Left of Monitor Row)
50 milliliter Disposable Pipets (2-3 per person pouring)
Pipet Aides (1 per person pouring)
3) Lay out 40 plates on one side of a Bunsen Burner and 40 plates on the other side. Each plate must be laid out with the lid (wider part) up.
4) Transfer 4L flask of media from magnetic stirrer to plate benchtop. If more than one person is pouring plates, pouring some media into a 2 liter beaker may be necessary for benchtop logistics.
5) Light Bunsen Burner with flint.
6) Using 50mL pipet and Pipet Aide, pipet 55 milliliters (-5 line near top of pipet) into tube
7) Dispense 25 milliliters media into a plate, covering after filled. Avoid bubbles. If bubbles occur, hover over the bubble with pipet tip. The heat of the air should allow the bubble to rise into the tube and out of the plate.
8) Fill a second plate
9) With 5 remaining milliliters, dispense into main media container and refill to 55 milliliters. Repeat Steps 7-9 until all plates are filled or media runs out.
10) If some media remains, dispense into a spare plate. Mark with an “X” in Sharpie.
11) Dispose of pipets in biohazard waste bucket.
12) Rinse glassware, taking care not to allow agar to wash down the drain. Filter solidified agar remnants with fingers and dispose of in biohazard waste bucket.
13) Allow plates to cool and solidify completely before attempting to move or stack. Eventually, transfer stacked plates to plastic sleeve, label with tape, and place in 4 Degree Celsius Refrigerator.
14) Clean benchtop with 70% ethanol.