Dictyostelium Predation
Protocol
Materials
· Cultures of selected bacteria
· Klebsiella
· Dictyostelium Ax2 grown on E.coli
B/r
· 2.0mL eppendorf tubes
· p200 pipette with tips
· p1000 pipette with tips
· Sterile loops
· Sterile spreaders
· Sterile water
· p20 multichannel pipette with tips
· SM/2 +proper antibiotic plates
Procedure
1.
Label one 2.0 mL eppendorf
tube for each strain of bacteria being used
2.
Add 200 µL sterile H2O
to each eppendorf tubes using a p200 pipette with corresponding tips.
3.
Add one loop of selected
bacteria to the corresponding eppendorf tube.
·
Mix solution well with
vortex or pipette
4.
Pipet 100µL of bacteria solution onto
a SM/2 Chloramphnecol 20 mg/ml plate. Spread the solution evenly with a sterile
spreader to create a bacterial lawn and let dry in the hood for 30 minutes.
5.
Add 500 mL of sterile H2O
to an eppendorf tube and serial dilute 1:10 from 0 to -7 in 8 separate tubes
using a full plate of fruiting Ax2 Dictyostelium grown on E. coli B/r.
·
Do this by adding 450µL sterile water to 7 eppendorf
tubes, then transfer 50µL from the previous dilution.
6.
In a 96 well plate, fill
one row with the 8 dilutions of Dictyostelium with 300µL, and refill the wells when needed.
7.
With a multichannel pipet,
allocate 4 rows of 2 µL
of the dilutions onto each plate.
8.
Let the plate sit upright
for at least 30 minutes to prevent the spots from running together.
9.
Create a titer by adding 800µL of water to a 2.0ml
eppendorf tube and a loop of K.a.
10. Allocate 100µL
of the bacteria solution onto 8 separate plates, and then mark the plates 0
through -7. Add 10 µL
of each dilution to the corresponding plates and spread the liquid out evenly.
11. Observe the plates every day for one week for plaque formation.
12.
Before titer plates are overgrown, calculate
the CFU for available plates
To start, we labeled a 2.0mL eppendorf tube
for each strain of bacteria being used and added 200µL of sterile water to each
tube using a p200 pipette. We added a loop of bacteria to each labeled tube and
mixed well with a vortex. From these bacteria solutions, 100µL were pipetted
onto a SM/2 Amp100 plate and spread evenly with a sterile spreader to create a
bacterial lawn. The plates were dried in a hood for 30 minutes to allow for
optimum Dictyostelium growth. A Dictyostelium
dilution was made by labeling 8, 2.0mL eppendorf tubes 0 to -7 and adding 500µL
of sterile water to the first tube and 450µL to each of the following tubes
using a p1000 pipette. Then a plate of
fruiting D. discoideum Ax2 was added to the first tube and 50µL were
transferred to each following tube creating 1:10 dilutions. 300µL of each
dilution were added to a column in a 96-well plate. Using a p20 multichannel
pipette, 4 rows of 2µL of the dilutions were allocated onto each plate,
changing tips between each row. A K.a.
titer was created by adding 800 µL of sterile water and a loop of K.a. to a 2.0
eppendorf tube. 100µL of the bacterial solution and 10µL of each dilution were
allocated onto 8 separate SM/2 Amp100 plates labeled 0 to -7, and the liquid
was spread out evenly on the plate. The plates and titer were observed for one
week for plaque formation
Summary:
We started by suspended our selected
bacteria in sterile water and created a bacterial lawn on an SM/2 Chloramphenicol 20 mg/ml plate
with 100µL of the bacterial solution. A 1;10 Dictyostelium dilution was made
with 8 total concentrations. Using a p20 multichannel pipet, 4 rows of 2µL of
the dilutions were allocated onto each plate. A K.a. titer was created on 8
plates with a K.a. bacterial lawn and 10µL of each dilution. The plates and
titer were observed for one week for plaque formation.
