Preparation of antibiotic stocks
| Antibiotic |
Stock concentration |
Protocol |
| Kanamycin |
25 mg/mL |
Weight 0.56g of Kanamycin powder, solubilization into 20mL of miliQ water.
Vortex 5min and filtration with a 200nm filter. Stock : -20°C (aliquots of 1mL)
|
| Streptomycin |
100 mg/mL |
Weight 1g of Streptomycin powder, solubilization into 20mL of miliQ water.
Vortex 5min and filtration with a 200nm filter. Stock : -20°C (aliquots of 1mL)
|
| Amplicilin |
50 mg/mL |
Weight 1g of Amplicilin powder, solubilization into 40mL of miliQ water.
Vortex 5min and filtration with a 200nm filter. Stock : -20°C (aliquots of 1mL)
|
| Tetracyclin |
15 mg/mL |
Weight 0.6g of Tetracyclin powder, solubilization into 20mL of ethanol 50%.
Vortex 5min and filtration with a 200nm filter. Stock : -20°C and hiden from light(aliquots of 1mL)
|
| Chloramphenicol |
34 mg/mL |
Weight 0.34g of Chloramphenicol powder, solubilization into 10mL of ethanol 100%.
Vortex 5min and filtration with a 200nm filter. Stock : -20°C and hiden from light(aliquots of 1mL)
|
08-16-2014
Tests of antibiotics' stocks
Six plates of LB agar were made.
Five of them contained one of those antibiotics in the dilution 1:1000:
- Chloramphénicol
- Kanamycin
- Erythromycin
- Ampicilin
- Tetracyclin
(The last one was the control of the growth of our bacteria without antibiotics)
We sowed 10µL of a liquid LB culture of Bl21, and we let them incubate overnight at 37°C.
Survivability tests
Two plates of MB 1X and M9 1X were made and divised in two parts.
Each plate was sowed with 5µL of a liquid MB 1X culture of Pseudovibrio denitrificans(dated 12th August) in a part and with 5µL of a liquid M9 1X culture Pseudovibrio denitrificans (dated 12th August) in the other part.
Pre-cultures
Cultures in MB 1X and M9 1X of Pseudovibrio were passed by a 1/10 dilution and let incubate overnight at 30°C.
New cultures of different E.Coli were also started from glycerol or plate and let incubated overnight at 37°C.
From glycerol stocks:
From plates:
- DH5a tranformed with pCB1C3
- Top10 transformed with pQexp
- DH5a pyr tranformed with pMK2
Bacteria tranformed with plasmid were cultivated with the corresponding antibiotic for the selection.
For each medium we make a negative contrôle without bacteria.
08-17-2014
Tests of antibiotics' stocks - Results
| Plates |
LB Agar only |
LB+Cam |
LB+Kan |
LB+Amp |
LB+Erm |
LB+Tet |
| E.coli |
X |
0 |
X |
0 |
X |
0 |
There was a problem with the stock of Kanamycine so we made a new one at 25mg/mL.
For the erythrompycine, we learn that E. Coli is not really sensitive to this antibiotic and that it's better to use it with a dilution at 1:100
Survivability tests - Results
| liquid cultures/Plates |
MB |
M9 |
| MB |
++++ |
++ |
| M9 |
++ |
+ |
Any liquid culture can be plated and will grow up. Of course, it works better on MB medium, wich is a rich medium than on M9.
08-18-2014
Tests of antibiotics' stocks - Results(2)
| Plates |
LB Agar only |
LB+Kan |
LB+Erm (1:100) |
| E.coli |
X |
0 |
0 |
New stocks are ready for antibiotics' tests on Pseudovribrio
Antibiotics's tests in liquid M9 1X
For each tested antibiotic we made the following range of concentrations:
| 0 |
1:500 |
1:1000 |
1:50 000 |
1:100 000 |
We made these range two time to have a replicate and we added a controle tube without antibiotic for each bacteria.
Each tube is made like in the following table in Falcon tubes (15mL):
| / |
0 |
1:500 |
1:1000 |
1:50 000 |
1:100 000 |
| Medium+agar |
3mL |
3mL |
3mL |
3mL |
3mL |
| Antibiotic |
0 |
6µL of stock |
3µL of stock |
6µL of stock diluted 1/100 |
3µL of stock diluted 1/100 |
| Pre-culture |
300µL |
300µL |
300µL |
300µL |
300µL |
For each range, we test Pseudovibrio denitrificans but also E.Coli and E.Coli tranformed with a speicific resistance cassette (NM:The E.Coli strain for the control case is the same strain that is tranformed for the cassette efficiency test)
The initial OD at 600nm of each bacteria has been mesured to after be able to calculate the ΔOD at 600nm, during three days.
| / |
Pseudo |
Bl21 |
DH5a |
Top 10 |
DH5a pyr+KanR |
DH5a+CamR |
Top 10+ErmR |
| OD init |
0.02 |
0.795 |
0.873 |
0.693 |
0.408 |
0.687 |
0.319 |
Plates for antibiotics's tests
For each tested antibiotic we made the same range of concentrations than in liquid.
We also made these range two time to have a replicate and we added a controle plate without antibiotic on which we showed all our tested bacteria. Plates will be checked during three days.
We made these same ranges of tested antibiotic's concentrations for three media:
- MB agar 1X
- MB agar 0.5X
- M9 agar 1X
Each plate is made like in the following table:
| / |
0 |
1:500 |
1:1000 |
1:50 000 |
1:100 000 |
| Medium+agar |
20mL |
20mL |
20mL |
20mL |
20mL |
| Antibiotic |
0 |
40µL of stock |
20µL of stock |
40µL of stock diluted 1/100 |
20µL of stock diluted 1/100 |
And plates are organised like shown in the following picture:
| A |
B |
C |
D |
| Pseudovibrio denitrificans |
Pseudovribrio denitrificans* |
E. Coli |
E. Coli** |
*Bacteria cultivated in media with antibiotic
**Bacteria tranformed with a plasmid containing a resistance cassette for the antibiotic
08-19-2014
Antibiotics' tests in liquid M9 1X - Results Day1
- Chloramphenicol
- Kanamycin
- Ampicilin
- Tetracyclin
- Erythromycin
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