Small NGM (Nematode Growth Media) Plates
Media Protocol (Makes 1/2 Liter)
1) Clean benchtop station by magnetic stirrers with 70% ethanol
2) Retrieve materials (ALL MUST BE STERILE IE. MARKED WITH BLACKENED AUTOCLAVE TAPE):
[1] 1 Liter Flask (Top Glassware Shelf)
[3] Weigh Boats (Drawer 61)
[3] Spatulas (Drawer 59)
[1] Stir Bar (Drawer 59)
[1] 500 milliliter graduated cylinder
Electronic Scale (Drawer 61)
3) With graduated cylinder, measure out 500 milliliters of milliQ water.
4) Add about 125mL water and stir bar to 1L flask and place on magnetic stirrer. Label the flask with tape.
5) Begin stirring on low speed. Replace autoclave foil cover on 1L flask.
6) Mass out 1.5 grams NaCl (Stored on chemical shelf alphabetically as “Sodium Chloride”) and add carefully to 1L flask. Replace foil cover when transfer is complete.
7) Mass out 1.25 grams BD Peptone (Stored alphabetically as “Peptone”) and carefully to 1L flask. Tap weigh boat gently as necessary to transfer powdery, sticky peptone. Replace foil cover on flask when finished.
8) Mass out 10 grams Agar (NOT AGAROSE) and add SLOWLY to 1L flask. Replace foil cover once more.
9) Turn stirring speed up and allow all added ingredients to dissolve completely.
10) Prepare 1L 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 1L flask from autoclave, place flask on magnetic stirrer and set stirrer to moderate speed.
14) Assemble the following materials and supplies:
[3] 25 milliliter Disposable Pipets (Rack Left of Monitor Row)
[1] Pipet Aide (Throughout Workstations)
[1] 1000μL micropipette
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 500 microliters cholesterol solution into 1L flask using 1000μL micropipette. Replace foil cover on flask when transfer is complete.
16) Add 500 microliters MgSO4 solution with 1000μL micropipette. Replace flask foil cover.
17) Add 500 microliters CaCl2 solution with 1000μL micropipette. Replace flask foil cover.
18) Finally, use 25mL pipet and Pipette Aide to add 12.5 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:
[100] Small Plates (Boxes Below Printer)
[1] Repeater Pipette
[1] 50mL Repeater Pipette Adapter
50 mL Repeater Pipette Tips
3) Lay out 50 plates in stacks of 5 on one side of a Bunsen Burner and 50 plates in stacks of 5 on the other side. Each plate must be laid out with the lid (wider part) up.
4) Transfer 1L flask of media from magnetic stirrer to plate benchtop. Pour the media into a 600mL beaker.
5) Light Bunsen Burner with flint.
6) Set the repeater pipette to pour 5mL per pour 50mL pipet. Attach the repeater pipette to the 50mL repeater pipette adapter and one 50 mL repeater pipette tip attached.
7) Collect 50mL media in the repeater pipette, then discard extra media back into the 600mL beaker.
8) Dispense 5 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. After a stack of plates has been filled with media, swirl the plates to spread the media.
9) When no media remains in the pipette, collect 50mL media in the repeater pipette. 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 repeater pipet tips 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.
When color-coding small plates for multiple conditions, the color combinations below are typically used to avoid confusion with single similar colors:
