Posted: May 6th, 2015

Characterisation of Cronobacter resistance to toxic and heavy metals

Paper, Order, or Assignment Requirements

Characterisation  of Cronobacter resistance to toxic and heavy metals
The variation in resistance to toxic and heavy metals will be determined using agar plate disc diffusion methods, and where appropriate with comparison to genomic data
3. The Protocol:  Variation in metal tolerance in Cronobactersakazakii and C. malonaticus.

Metal resistance:
Material and methods:
TSA plates, TSB broth, paper discs.

Solutions: AgNO3 Silver nitrate (1; 0,1; 0,01; 0,001 mol/l)
CuSO4 Copper sulphate (1; 0,1; 0,01; 0,001 mol/l)
Na2TeO3 Sodium tellurite (0,1; 0,01; 0,001; 0,0001 mol/l)
These are just suggested concentrations. You need to change the concentrations according to your results for each strain. Compounds such as arsenic and cadmium are not included, as I prefer you to initially get these thre compounds working.
Procedure:
• Prepare overnight culture of strain.
• Use a swab to inoculate the agar plate surface , using a tight zig-zag pattern.
• Place four discs on each plate, evenly spaced out.
• Pipette 7 µl of metal solution on disc (one dilution on one disc).
• Incubate plates overnight in 37°C incubator and measure radius (in mm) of zone of inhibition (ZOI) in two directions at right-angles.
• Take photos as necessary, and look for interesting appearances on the plate such as halos and colouration
• Incubate the plates for a further 24 hour, and re-record results.