Fermentor worm chow

 

Fermentor worm chow

Prep

1. Have autoclaved:
   1. 250 ml straight neck flask
   2. 100 mmfunnel
   3. Fermentor vessel with all openings covered, top loosened, on its side in tub with wedges, after a/c sit upright to seal and allow to cool before use
   4. Eleven liters 2xYT in 4- 4 L flasks, 45 min exposure
   5. Eight 500 ml bottles
   6. Tubing for harvesting
   7. 10% antifoam 289, final concentration in media: 0.01%
2. Start o/n of AMA1004: 2 colonies from a recent plate into 25 ml LB in 250ml flask
3. Check with Montelone lab regarding using the centrifuge

Set up

I. The vessel

1. Secure lid by tightening wing nuts
2. Using autoclaved funnel pour in the 2xYT through addition port, remove funnel
3. Add 10% antifoam 289, to make a final conc of 0.01%, through addition port
4. Add 25 ml of AMA1004 overnight through addition port

II. The fermentor

1. Loosen vessel clamps(2)
2. Get another person to hold black drive shaft extender up while vessel is placed on stand with the water in port on the right, the vessel will "sit" onto two small pegs
3. Tighten vessel clamps never letting go of vessel
4. Remove foil and screw water in and out on to the lid, save foil
5. Remove foil and attach air in to the back port and air out to the front, save foil
6. Plug in temperature probe on right side and place into temp port, fill with ddH₂ 0
7. Place digital temp probe (borrow from Ken) into temp port also, cover port with foil
8. Plug compressor and upper section of fermentor in
9. Make sure drain tube is in sink cup in the hood and turn the water on

Running

I. Drive and temperature control

1. Make sure heat and drive is off and speed control has been turned down (left)
2. Turn on main power
3. Set temp control to 20 and turn on heat switch, this opens water pump and gets all of the bubbles out of the system
4. After 20-30 seconds turn temp control to the 37°C mark realizing fine adjustment will need to be made
5. Turn drive on and adjust rpm to 250-300 rpm
6. Cooling water should already be switched on

II. Air Control

1. Round white gauge should read "20"
2. Turn red knob counter clockwise until the flow meter ball is "sitting" on 3

Notes on running...

• Come back and check every 20-30 minutes to check on temp, rpm, air flow and foaming, it took 1.5 hr the first time to get the temp adjusted
• Made on adjustment to the rpm and flow rate
• With 10 L of media after about 6 hr some foam start to develop, not enough to add 289
• Either run during day or start late and run overnight

Take down

1. Turn off everything in reverse (start at "Running.II.2." and go up to "Running.I.1.)"
2. Remove air tubing
3. With a beaker and paper towels handy unscrew water lines
4. Replace all the foil
5. Transfer the vessel to the cold box or proceed directly to harvest

Harvest

1. Record the weight of six 500 ml bottles
2. Transfer vessel to countertop and mix well
3. Attach tubing, attach l-clamp, and draw suction with pipet bulb
4. Fill bottles leaving 1/2" to 3/4" at top
5. Have another sterile bottle to place end of tubing
6. Adjust volumes of bottles with balance in sets of two
7. Spin 4000 rpm, 16 min at ~10°C in Montelone's centrifuge, pour off s/n
8. Repeat 4-7 until all chow has been harvested

Resuspension

1. Determine wet wt total in grams
2. Number of mls of S medium to add is equal to the wet wt divided by 0.5 g/ml minus wet wt. Divide the S med to each of the six bottles, cap well
3. Shake in secured box for about 1 hr at 250 rpm at RT
4. Measure volume of one bottle, record and transfer contents of other five bottles into first recording volume from each bottle
5. Divide total volume by wet wt and this equals actual g/ml conc.
6. Each cube needs to contain 5 g

Dividing and storage

1. Aliquot amount for 5 g into pre-frozen ice cube trays
2. Label trays
3. Place in -80°C in single layer covered with saran wrap
4. After frozen, transfer to labeled ziploc bags and store at -80°C