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.
|
0
|
|
-1
|
|
-2
|
|
-3
|
|
-4
|
|
-5
|
|
-7
|
|
-6
|
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.
