Dandekar & Chandler:Selective media

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Selective Antibiotics

• Trimethoprim (for pseudomonas selection)

Working concentration [100 ug/ml] from DMSO stock [100 mg/ml]

• Triple antibiotic (for burkholderia selection from co-culture)

150,000 IUs/L Polymixin B (=17.3 mg/L)

5 mg/L gentamicin

100 mg/L ticarcillin

Pa selective media options

• PC agar

Columbia agar made as directed on bottle and sterilized

Add:

30 mg/L 1, 10 phenanthroline

30 mg/L C-390

• PIA

Follow directions on the bottle, man

Selection for Protease Production

- Useful for selection of LasR mutants from WT Pseudomonas aeruginosa

- After competition against another species, first spread dilute co-culture onto antibiotic plate and grow overnight, then patch isolated pseudomonas onto milk plates

- WT colonies will create clearings, allowing determination from LasR mutants

Skim Milk Plates

• 25% LB

• 4% milk

• 1.5% agar

Directions:

Do not combine LB and milk solutions before autoclaving unless you enjoy cleaning up large messes

Do not forget to put a stir bar in/leave the stir bar in the skim milk autoclave bottle

1. Prepare the following solutions:

• 150 ml H2O
• 150 ml LB (can be previously autoclaved)
• 9 g bacto-agar

- Stir and autoclave; keep molten!

• 300 ml H2O
• 24 g powdered milk (2%)

- Stir well, leave stir bar in flask and autoclave

2. Autoclave solutions separately

3. Start milk solution stirring on stir plate

4. Slowly pour molten LB agar into milk solution

Recipe above pours one sleeve of plates