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Amp quantification

Standard Protocol

1- The one bacterial strain which can be used:

 

E.coli.

 

2- Quantification of the produced ampicillin using the disk diffusion method:

Preparation of the inoculum:

  • We took 3 to 5 colonies of the isolated colonies with a loop, and we added them in 2ml sterile saline (NaCl 0.9%)
  • We Vortex the saline tube to create a smooth suspension.
  •  
  • We adjust the turbidity of this suspension to a 0.5 McFarland standard

by adding more organism if the suspension is too light or diluting with

sterile saline if the suspension is too heavy.

 

  • Use this suspension within 15 minutes of preparation.

 

  • We inoculate the surface of Mueller Hinton agar plate by streaking the swab 3 times over the entire agar surface, we rotated the plate approximately 60֯ each time to ensure an even distribution of the inoculum.
  • We allow the plate to sit at room temperature at least 3 to 5 minutes (but no more than 15 minutes) for the surface of the agar plate to dry before proceeding to the next step.

 

 Preparation of the disks:

 1)      We dilute the standard ampicillin 6 times (Concentrations: 20;15;12;10;8;5 mg/ml) to obtain different concentrations.

2)      We distribute 16 disks in plates.

3)      On each disk, we add 20 µl of each ampicillin concentration obtained before, in order to have three disk with known concentration and one disk with unknown concentration per plate when we shall add our obtained produced ampicillin.

4)      Once all disks are in place, we replaced the lid, inverted the plate and placed it at 37oC for 18 to 24 hours.

 

Quantification of the produced ampicillin:

1)      After the growth time, we measured the zone of inhibition that had appeared using a ruler.

2)       We drew a graph showing the concentration of ampicillin as a function of the diameter in order to be able to quantify the produced ampicillin (diameter as a function of Log C).

 

Trial:

To test the effectiveness of the presented ampicillin a trial with bacterial cultivation was carried out where such volume of liquid ampicillin with known concentration is added to the surface of the agar before adding the bacterial strain. Then the results was reported that the ampicillin doesn't have any activity through E.coli