Preparing NGM-lite Plates

Recipe for NGM-lite Plates:
This recipe makes enough solution for about 35 plates. Now, since one can mess up plates by either contaminating them or allowing the OP50 to run to the edge of the plate, the recipe usually yields 30 pristine plates. Mistakes happen.

1. 1g NaCl
2g Bactotryptone
1.5g KH2PO4
0.25g K2HPO4
10g agar
.5ml of 5 mg/mL cholesterol (dissolved in 100% ethanol)

2. Add to 1 liter flask.
3. Add 500m of deionized or distilled water.
4. Stir to dissolve then autoclave for 15 minutes.
5. Let the solution cool for 10 minutes or so and then pour the solution into 6 cm petri dishes.

Seeding NGM-lite Plates with OP50:
Prepare the OP50 by extracting 100 ul and transferring to a tube of Luria broth. Allow the culture to incubate overnight at room temperature. We are now ready to seed the NGM-lite Plates with OP50, the bacterial food source for the C. elegans. Using aseptic technique, transfer 100 ul of the culture to each plate and spread by using what I like to call the "Labryinth Game Technique" (see http://en.wikipedia.org/wiki/Labyrinth_(game). I spread the plates very precisely, tipping the plate at different angles, careful not to allow any of the OP50 culture to touch the sides of the plates. OP50 that is separate from the single spread of OP50 and/or is touching the sides of the plate jeopardizes the sanitary integrity of the C. elegans environment.



Proper Aseptic Technique:

Maintaining proper Aseptic Technique is absolutely crucial for ensuring uncontaminated plates. If the plates are uncontaminated, the worms can live for as long as it takes them to eat up their food source, OP50. It is important to keep everything you are working with sterile. You generally keep the lab instruments sterile by using alcohol and/or a bunson burner (fire). In seeding the plates, you can't flame the micropipetter tips as they are plastic and will melt. Maintaining the tips sterility comes from keeping the box of tips closed. One should only have the box open when he or she is ready to pipette. The broth needs to be flame opened and flame closed. This means you have to run the top of the tube through the flame every time you remove the cap and every time you put the cap back on.

Aseptic technique for chunking the worms is detailed in the next section.

Transferring worms from NGM-lite plate to NGM-lite plate: Chunking
Touching the sides of the plate jeopardizes the sanitary integrity of the C. elegans environment. Transferring the worms onto 'fresher' plates is crucial for preventing the worms from dying whether they run out of their food source, OP50, and starve, or the NGM-lite medium dries out. An easy and effective way to transfer the worms, and the way that I have transferred the worms, is called "chunking." Chunking entails dipping a scalpel in alcohol and burning off the excess alcohol. Once the scalpel is cooled off, cut a chunk from the NGM- lite medium about 1 cm square and flip the chunk over unto a seeded plate. Simply enough, the worms crawl out from the chunk onto the 'fresh' plate. The N2 strain of worms have now been transferred effectively. After about every 3-5 days, I repeat the chunking process onto seeded plates. One must be sure to put the seeded plates, being stored in the refrigerator, into the incubator, making sure the medium is near room temperature.


Healthy Worms:
fri78N2worms.jpg
A Very Informative Source:
http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html
With this source, you can discover other techniques of transferring worms, techniques of seeding with OP50, and an alternate recipe for the NGM-lite plates.