LIQUID CULTURE OF WORMS WITH FERMENTOR

Liquid Culture of Worms with Fermentor

This procedure courtesy of - Mimi H. Zhou, Hsiuchin Yang, Bill Walthall Biology Department, Georgia State University, Atlanta, GA 30302

The yield of C. elegans and their food (E.coli) are relatively low in the large liquid cultures growth (Koelle et al.,1994) and the whole growth process is tedious. Here we report the successful growth of C.elegans with a fermentor. The following protocol is based on the protocol by Michael Koelle and Tory Herman (1994) from the Internet.

Day 1:

1. Media preparation for cultivation of E. coil. Superbroth was made up as follows:
Distilled water ---> 18 L
Bactotryptone ---> 150 g
yeast extract ---> 300 g
50% glycerol stock solution ---> 100 ml
Superbroth was prepared in a 40-liter-mobil pilot plant fermentor (Model MPP-40, New Brunswick Scientific) and sterilized by in situ sterilization.

Phosphate Buffer was made up as follows:
Distilled water ---> 2 .0 L
KH2PO4 ---> 46.2 g
K2HPO4 ---> 250.8 g
2. Preparation of seed culture of E. coli. Inoculate single clony of E. coli (HB101) into 50 ml of LB and grow overnight.

Day 2:

1. Cultivation of E. coli. Add 2 liters of the prepared sterile Phosphate Buffer and 50 ml overnight culture of E. coli (HB101) into the prepared sterile medium in the fermentor. The bacteria were grown at 37C with chamber pressure at 2 psi, air pressure at 35 psi and air flow rate at 50 U for 20 hrs.
2. Preparation of media in the 2.5-liter-Batch/continuous fermentor (Model BiofloIII, New Brunswick Scientific). S-basal was prepared as follows in a 2.5-liter batch fermentor and sterilized.

Distilled Water--->1900.0 ml
NaCl --->11.6 g
1 M KH2PO4, pH 6.0---> 100.0 ml
5 mg/ml cholesterol in 95% EtOH ---> 2.0 ml
An extra 2 liters of S-basal was prepared in a flask for washing of bacteria and worm.
Adjust 1 M KH2PO4 to pH 6.0 with 15 g KOH.

Trace Metal Solution (100 X) was prepared as follows and sterilized:
Distilled water --->500.000 ml
FeSO4.7H2O--->0.364 g
Na2EDTA ---> 0.93 g
MnCl2.4H2O ---> 0.098 g
ZnSO4.7H2O ---> 0.144 g
CuSO4.5H2O ---> 0.012 g

Day 3:

1. Preparation of S-Medium for cultivation of C. elegans. S-Medium was prepared by adding the following sterile solution to the sterile batch fermentor containing 2 liters of S-basal:

1 M MgSO4 --->6 ml
1 M CaCl2 ---> 6 ml
100X Nystatin (Gibco) ---> 20 ml
100X Pen/Strp/Neo (Gibco) ---> 20 ml
100X sterile trace metal solution ---> 20 ml
1 M Potassium Citrate ---> 20 ml

2. Harvesting E. coli (HB101) culture. Collect the E. coli (HB101) culture (O. D. 20.0) from the fermentor with sterile flasks and concentrate by filtration with a clean Pellicon Cassette System (Millipore Corp. Model: XX 80 ELO00). After concentration, 20 liters of culture was reduced into about 2 liters. Then the bacteria were harvested by centrifuging in a Beckman J2-M1 centrifuge with JA-10 rotor for 15 min at 8000 rpm at 4C. There should be about 100 g of cell pellet collected in each bottle (six bottles in total). Resuspend the 100 g cell pellet with 10-15 ml of S-basal. Transfer the suspension to sterile 50-ml polypropylene tubes and store at 4C for up to 2 weeks and longer time at -80C. The dissolved HB101 suspension should be very sticky.
3. Preparation of seed culture of the worms. Wash the worms from five almost starved large plates (9 cm petri dishes) with S-basal. Collect the worms by spinning in 15 ml centrifuge tubes at room temperature (about 4000 rpm for 2 mins). There should be about 0.8 ml of worms.

Remove the supernatant.

4. Cultivation of the worms in a fermentor. Add 25 ml of the prepared E. coli HB101 pellet suspension and the washed seed worms to the batch fermentor. The worms were grown at 20C, with 100 rpm agitation and one unit air flow rate for 3-5 days.

Day 4-6:

1. Preparation of the solution for separation of the worms from bacteria and other debris. ICE-COLD 0.1M NaCl (2.5 liters) and ICE-COLD 60% sucrose (100 ml).
2. Examination of the growth conditions. Aliquot 0.5 ml of growing worms every day on an unseeded plate to check the growth conditions of the worms and residue of E. coli HB101. Add additional prepared bacteria if there is not enough food (12.5 ml of bacteria were normally added in the third day of growth).

Day 7:

1. Harvesting the worm culture. Harvest the worm culture after four days growth by spinning down in 6 X 500 ml centrifuge tubes at 3000 rpm for 3 mins in a Beckman JA-10 rotor at 4C. The speed and timing of this and the subsequent spins are fairly critical; Start timing when the speed is up to 3000 rpm and then turn the machine off to start the deceleration when 3 minutes is up (Koelle, Herman, Hengartner, 1994). Remove the supernatant with 10 ml pipette and leave some liquid to prevent the loss of any soft pellets. The supernatant could be cloudy but that is fine. A large worm culture pellet should be obtained.

2. Separation of the worms from bacteria and other debris. Resuspend the worm culture pellet in 0.1 M NaCl and combine them. Bring up to 500 ml with ice-cold 0.1 M NaCl and spin at 2000 rpm for exactly 3 mins in JA-10 at 4=B0C as described above. Carefully reduce the supernatants to 15-20 ml and swirl the flask to resuspend the worm pellet (final volume: ~150 ml ). To each 50-ml sterile centrifuge tube (at least six), distribute 25 ml of the worm suspension, keep them on ice for several minutes to chill, and then add 25 ml ice cold 60% sucrose and mix. Spin the mixture at 3500 rpm for 5 mins in a table top centrifuge at 4C. (The sucrose solution will damage the worms, work fast here.) After centrifugation, bacteria, debris and dead worms should be condensed at the bottom and one side of the tube, and a large light brown layer of living worms should be floating on the top. Use a broken off Pasteur pipette to carefully transfer the worms to a new 50-ml sterile centrifuge tube (it is OK to take the top 30 ml of liquid here, you can determine the level at which you no longer see living worms.)

3. Washing the worms from the sucrose. Distribute 10 ml of worms into several new 50-ml sterile centrifuge tubes and add 40 ml ice-cold 0.1 M NaCl. Centrifuge at 3100 rpm for 3 mins in a table top centrifuge at 4C and remove the supernatant. Repeat the wash step to remove more sucrose. The worm pellet should be clean and light brown. Try to remove as much of the supernatant as you can. About 20 ml worm pellet should be collected by this method from the 2-liter culture in the batch fermentor. Freeze the tubes, containing 1 ml 0.1 M NaCl on the top and 4 ml of the worms in the bottom by liquid nitrogen. If the worms are to be used for a RNA preparation, it is best to have less than 5 ml of worms in a 50-ml tube to allow room to add the necessary guanidinium solution.