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This protocol is designed for use with chloramphenicol and the chloramphenicol acetyltransferase gene, however it may work well for characterising other antibiotic resistance genes, particularly if the antibiotic concentrations are optimised.
Materials
50ml tube
An antibiotic resistant bacterium
Chloramphenicol
LB agar
LB medium
Lots of plates
Spreaders
Method
- Chloramphenicol concentrations used in this assay were; 5μg/ml, 10μg/ml, 20μg/ml, 30μg/ml, 40μg/ml, 50μg/ml, 75μg/ml, 100μg/ml, 150μg/ml and 200μg/ml.
- Make 4 LB agar plates (depending on how many repeats you want) at each chloramphenicol concentration.
- Transfer approxiamtely 10ml of LB into the 50ml tube and chloramphenicol to a concentration of 5μg/ml.
- Innoculate the flask with resistant bacterial strain and grow overnight
- The following morning, plate 50μl of the bacterial solution onto 2 plates per concentration, 100μl onto the third plate at each concentration and 150μl onto the final plates.
- Grow plates in the incubator or on the bench overnight.
- Check plates for growth after 24 hours and note the approximate precentage of the plate covered in cells.
Results
This was used to test chloraphemicol acetyltransferase activity and resistance infered.
These results can be accessed here.
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