Team:Evry/Notebook/CellCharacterization/Antibiotic test

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:

• Bl21
• Top10
• DH5a

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 than in liquid.

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

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. 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	C
  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