Dictyostelium lab prepartion prototocols, Jon Moore, Pomona College
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Strains of dicty and bacteria
Preparing fruiting bodies from frozen spores
Seeding a suspension culture of dicty
Propogating of dicty culture
Preparing dicty cells for the under-agarose assay
Preparing Sor buffer plates for the under-agarose assay
SM media and plates recipe
Sorensen's buffer (Sor) recipe
Development buffer (DB) recipe
Folate solution recipes
E. coli concentrate preparation
Preparing frozen spores with silica
A list of equipment and sources used for this lab
Strains of dicty and bacteria
NC4 and Enterobacter aerogenes are common in many Dictyostelium labs and available from the DictyBase stock center.
DH5alpha E. coli are likely the most common E. coli used in molecular cloning and available as competent cells from many biological suppliers including Thermo Fisher.
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Preparing fruiting bodies from frozen spores
Grow Enterobacter aerogenes in SM media (or LB) shaking at 37 C overnight.
Spread Enterobacter culture over SM plates and allow to grow overnight at room temperature or 37 C.
Seed plates with NC4 spores near one edge.
Incubate plates at 18C or room temperature.
Within one or two days, the effects of NC4 eating the bacterial lawn should be seen.
Some fruiting bodies will appear near the seeding site after about 4 days.
These can be kept at 4 C for several months, though fewer spores will be viable after only 2 weeks or so.
Note this is very simlar to the DictyBase protocol.
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Seeding a suspension culture of dicty
Mix 0.25 mL of the E. coli concentrate and 4.75 mL of DB.
Harvest several fruiting bodies of NC4 with a sterilized, wet inoculating loop. Shake into DB/coli mixture.
Shake or tumble at 18 C or a cool, stable room temperture. #
In 2 or maybe 3 days depending on the initial seeding, the culture should be between 50,000 & 2,000,000 cells
per mL.
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Propogating of dicty culture
24 hours before the next split or harvest, cells should be diluted to 10,000 or 20,000 - 25,000 cells per mL
with a mixture of E. coli concentrate diluted 20-fold with DB.
Use 10,000 cells per mL is good if you are just maintaining a culture.
20,000 or 25,000 is better if you are preparing them for the under-agar assay the next day, since you will have more cells per mL.
Cells are incubated with shaking or tumbling at 18 C. #
Under good conditions, NC4 can undergo six doublings in 24 h.
** With this fast doubling rate, keep in mind that 20 h and 28 h are quite different from 24,
so be sure to compensate for this if your times of splitting and harverst are quite different.
# My current preferred method is to grow them in 15- or 50-mL tubes tumbling end-over-end at 24 rpm.
I don't fill the tubes more than 2/3 full so there is plenty of headspace for tumbling.
I use a beverage cooler to maintain the temperture, and simply run the electrical cord for the tumbler right by the insulation of the closed door.
I have also grown them on a shaker in sterile flasks at 18 C.
** Why do you use NC4 and grow them with this approach rather than use an axenic strain and a media like HL5? **
Foremost, two things:
- When running a lab with many students, several instructors, and a tight schedule, everything you can do to behind the scenes to make the lab more reliable is invaluable.
- I do not have a sterile hood near where this lab or its prep takes place.
I sterilize all my solutions and use sterile plastic and glassware, but otherwise I do not worry about sterility using this approach, and it has not been a problem for five years (currently May 2019). Hence, while this appraoch seems awkward and cumbersome, it is more reliable and is easier in the moment.
Also, though I have not been very scientific about this analysis, anecdotally I think the dicty begin migrating faster when fed bacteria.
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Preparing dicty cells for the under-agarose assay
Centrifuge the cells in 50-mL conical tubes at 500 g at 15 C for 5 minutes.
Remove the supernatant and resuspend in Sorenson’s buffer.
Repeat these last two steps to yet remove more bacteria and unwanted metal ions.
Repeat the centrifugation, but now resuspend all the the cells in a smaller quatity of Sor buffer.
You are aiming for
- at least 300 microliters per plate to be run, since each plate takes 4 times 60 microliters
- about 3 to 3.5 million cells per mL.
I usually prepare them at about 9 or 10 AM for a lab that starts at 1:15 PM. During the intervening time, the cells roguhly double.
Cell concentrations at lab time of 1 million to 10 million per mL all yeild fine results in the under agar assay.
Shake or tumble the cells at 18 C until close to lab time so as to keep them from settling, clumping, and becoming less likely to migrate.
Aliquot the cells for each student group shortly before lab. Store them at RT with the tubes sideways so the dicty are less concetrated when they settle.
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Preparing Sor buffer plates for the under-agarose assay
Mix Sor buffer and agarose (1.0% w/v) in a flask with a cap, preferably vented.
You'll need 6 mL per plate plus a little for evaporation and boiling.
Do not try less than 100 mL with this technique. Too much boils off.
Place the flask in a water bath as it warms to 70-75 C. A lead doughnut stabilizer is great for keeping it in place.
Finish melting the agarose by bringing the mixture to a hard boil briefly on hot plate.
Return the flask to the hot water bath.
In my experience, it can stay here indefinitely, but gradually evaporation makes this undesirable.
Add 6 mL of this to each 6-cm Petri dish. A motorized pipette aid is very handy for this.
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SM media and plates recipe
Foremost, this the same as the DictyBase recipe excepting that I use a little less agar.
For 1 L, use
1.9 g KH2PO4
0.6 g K2HPO4
0.5 g MgSO4
10 g glucose
10 g bacto-peptone
1 g yeast extract
distilled water to 1 L.
The pH should be 6.0 to 6.4
Autoclave.
For plates add 15 g of agar.
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Sorensen's Buffer (Sor) recipe
Again, this is pretty much the same as the DictyBase recipe excepting for the concentrate tip.M
Mix 8.00 g KH2PO4, 1.16 g Na2HPO4 and distilled water to 4 L.
The pH should be 6.0 ± 0.1.
Autoclave.
This can also be prepared as a 50x concentrate. Just check the pH when you dilute it.
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Development buffer recipe
Again, this is pretty much the same as the DictyBase recipe.
Prepare a solution of 25 mM each Na2HPO4 and KH2PO4.
Prepare a 10 mM CaCl2 solution.
Prepare a 20 mM Mg Cl2 solution.
Autoclave all three solutions.
To make 1 L Development Buffer, mix 600 mL sterile water, 200 mL of the phosphate solution, and 100 mL of each of the other two. The pH should be 6.5.
Note that a 10x stock of all three together cannot be prepared because of precipitation.
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folate solution recipes
Folate solution (0.2 mM) provided to the students for the assays:
1 part concentrated folic acid
44 parts Sor
Provide about 225 uL per plate run.
Concentrated folic acid, folic acid, 4 mg / mL:
pH’d to 6.5-6.8 with NaOH or KOH.
(Note this is way above the pKa, so it is a a bit of a pain to achieve.
Make a lot; you don't want to do this often.)
Freeze in 1 mL aliquots.
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E. coli concentrate prepartion
Grow a near-saturated 1 L (or more) culture of DH5alpha E. coli overnight in SM (or LB) media with shaking at 37 C.
Distribute this amongst 250 mL centrifuge tubes and centrifuge for 10 minutes at 6000 g.
Carefully pour off the supernatant and resuspend each pellet in approximately 50 mL of DB (or Sor buffer).
Pool the samples into only two centrifuge tubes repeat the centrifugation.
Resuspend these two pellets in a total of 40 mL Sorenson’s buffer per liter of bacterial culture.
Measure the OD600 of a 1:100 dilution of the concentrate and dilute the bacteria to achieve an OD600 of approximately 0.6.
Aliquot into sterile 15mL tubes.
Place at 4 C when not in use.
** Note that concentrates left out overnight can still be used to grow dicty but growth rates will be slower. Concentrates left at 4 C for a year yield roughly the same growth rates as fresh concentrates.
Normal yields are about 50 mL per liter.
This together with the propogation protocol imply that the dicty start feeding on a bacterial suspension at an OD600 of about 3.
OD600's of 1 and likely lower are just fine for propogating cells. This high OD leads to a high cell titer. Consistent ODs lead to highly predictable growth rates.
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Preparing frozen spores with silica
I use identically the DictyBase protocol keeping the final vials at -20 C.
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