线虫冷藏--Freezing Worms

 

<center> <h2> <font><font>Freezing Worms</font> </font></h2> </center>

 

<center> <font>by Michael Koelle</font></center>

 

 

 

I. The preferred method

1. Wash worms off of 1 large plate or 3 small plates in 3 mls S Basal into a sterile 15 ml disposable centrifuge tube. Worms should be harvested off of just starved plates which are predominantly L1 and L2. This should be 1 day after the bacteria have been exhausted. The worm book says dauers don't freeze, but E. Jorgenson says he freezes from really old plates and it works fine. Rumor has it that if the plates aren't completely starved, the freezing won't work - i.e. food in the gut somehow prevents the worms from surviving. To wash the worms off plates, add ~2 mls S Basal to each small plate (w/sterile 5 ml glass pipette and a pasteur pipette bulb), swirl briefly to dislodge worms, and suck off the suspension with the sterile glass pipette and place into a 15 ml sterile plastic centrifuge tube.

2. Spin down in a clinical centrifuge for ~30 sec. Remove all but 1.5 ml of the supernatant.

3. Add 1.5 ml freezing solution, mix well, and aliquot 1 ml each into 3 sterile freezing vials (Nunc CryoTubes #363401).

4. Freeze slowly to -80°. This is accomplished by placing the vials in a styrofoam rack (the kind that 15 ml disposable sterile centrifuge tubes come in), placing another inverted such rack on top of the first, fastening the two racks together with rubber bands, and placing in a -80° freezer.

5. The next day, move two vials to a permanent location. Record the strain number, genotype, and comments in a computer database. We use "FileMaker Pro" software. Na An tells me that freezing in a liquid N2 freezer truly is preferable -80°; she gets excellent thaws from 15 year old vials stored in liquid N2, but less good thaws from old -80° vials.

6. The third vial should be used for a test thaw. Take the vial out of the freezer, thaw quickly by holding in your hand or in a 37° water bath (leave in the bath only until the ice is gone so as not to heat it up). Can dump the whole vial out in a seeded large plate, or use a sterile 200µl pipetteman tip to withdraw the bottom 200µl (containing the settled worms) and place it on a small plate. The next day, pick live worms to a new plate.

II. The agarose method. This gives much lower viability than the above method in most people's hands, but has the advantage that one doesn't need to thaw an entire vial at a time. On balance, I don't think it is worth risking losing strains (especially risky with heterozygous mixtures) to get this minor savings. However, some people seem to have better luck with this method than me. Leon Avery says the agar method works better for him than the non-agar method. He suggests being especially careful when thawing not to break the worms by scraping, but rather to be sure to actually take an unbroken frozen chunk of worm suspension out of the tube.

1. Melt freezing solution + agarose in microwave (carefull to fully melt but not boil over).

2. Put FS + agar bottle on the bench to cool. Some people place it in 50° water bath for ~15'. The idea is to have it cooled to ~50° by the time you use it, but to use it while it is still liquid.

3. Wash worms off of 1 large plate or 3 small plates in 3 mls S Basal into a sterile 15 ml disposable centrifuge tube. Worms should be harvested off of just starved plates which are predominantly L1 and L2. This should be 1 day after the bacteria have been exhausted. The worm book says dauers don't freeze, but E. Jorgenson says he freezes from really old plates and it works fine. To wash the worms off plates, add ~2 mls S Basal to each small plate, swirl briefly to dislodge worms, and suck off suspension with a sterile glass pipette and place into a 15 ml sterile plastic centrifuge tube.

5. Chill on ice 15' or longer.

4. Spin the worms down (30 sec. in a clinical centrifuge), and remove all but 3 mls of supernatant with a sterile pipette.

6. Pipette 3 ml FS + agar into a tube of worms and immediately mix by inversion of tube 8-10X.

7. Pipette 1.8 ml of the mixture into 3 prepared freezer vials.

8. Repeat steps 6 and 7 for each strain.

9. Freeze slowly to -80°. This is accomplished by placing the vials in a styrofoam rack (the kind that 15 ml disposable sterile centrifuge tubes come in), placing another inverted such rack on top of the first, taping them tightly together, and placing in a -80° freezer.

10. The next day, move two vials to a permanent location. (Or, to a box in the -80°, and when the box is full, move all the vials to a liquid N2 freezer). Record the strain number, genotype, and comments in a computer database.

11. The third vial should be used for a test thaw. Can either thaw the whole vial and dump on a large plate. Or, using a flamed spatula, dig a ~0.1 ml chunk of frozen worms onto a small plate, and put the remainder of the vial back in the freezer. Leon Avery says to be sure not to scrape flakes of frozen stuff out of the tube; this will break all the frozen worms. Instead, run the spatula around the edge of the tube and break out an intact chunk of worm suspension.

 

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Freezing Solution

200 ml 1M NaCl

100 ml 1M KPO4, pH 6.0

600 ml glycerol

Bring to 2 liter w/ dH20

Distribute to 200 ml bottles; autoclave

Add 0.06 ml sterile 1M MgSO4 per 200 ml bottle

(N.B. in Horvitz lab the stuff Na An supplies does not have MgSO4 added yet!)

Freezing solution with agarose

freezing solution with 0.4 g agarose added per 100 ml and reautoclaved for 20 min.

M9 buffer (Horvitz lab version)

Na2HPO4 5.8 g

KH2PO4 3.0 g

NaCl 0.5 g

NH4Cl 1.0 g

dH20 to 1l

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