1-     Experiment 3: Penicillin quantification

In this section we have realised two trial the first is with an industruel penicillin to know the efficacity of the proposed protocole then the quantification is applied to our produced penicillin.

The first trial of quantification:

Step 1:

• First we dissolute the industruel penicillin (1000000 UI) in 1 ml phosphate buffer(1000000 UI= 600mg penicillin g sodium)
• We filtrate the obtained solution of penicillin to obtain a stock solution containing 1000000 UI/ml.
• From this stock solution we add 1 ml in 9 ml of physiological water (NaCl0.9%). Here the concentration is 100 000 UI/ml and the tube is called tube 2
• From the recent solution (tube 2) we add 1 ml in 9 ml of physiological water (NaCl0.9%). Here the concentration is 10 000 UI/ml and the tube is called tube 3.
• From the recent solution (tube 3) we add 1 ml in 9 ml of physiological water (NaCl0.9%). Here the concentration is 1 000 UI/ml and the tube is called tube 4.
• From the recent solution (tube 4) we add 1 ml in 9 ml of physiological water (NaCl0.9%). Here the concentration is 100 UI/ml and the tube is called tube 5.
• From the tube 5 we took 3 ml and we put them in 7 ml of physiological water (NaCl0.9%). Here the concentration is 30 UI/ml and the tube is called tube 6.
• From the tube 6 we took 5 ml and we put them in 1 ml of physiological water (NaCl0.9%). Here the concentration is 25 UI/ml and the tube is called tube 7.
• From the tube 7 we took 4 ml and we put them in 1 ml of physiological water (NaCl0.9%). Here the concentration is 20 UI/ml and the tube is called tube 8.
• From the tube 8 we took 4 ml and we put them in 1 ml of physiological water (NaCl0.9%). Here the concentration is 16 UI/ml and the tube is called tube 9.
• From the tube 9 we took 2.5 ml and we put them in 1.5 ml of physiological water (NaCl0.9%). Here the concentration is 16 UI/ml and the tube is called tube 9.

Here we have obtained the standard concentration that we will need for drawing the standard curve of concentration.

!!!! The think that the physiological water will be sterile(autoclaved) is important.

Step 2 :

• First we prepared the bacterial inoculate (desired bacterial strain in sterile physiological water) to rich a bacterial concentration of 0.5 MF. In this step a standard 0.5 MF is used to know the bacterial concentration.
• Then we streaked them into a surface of Muller-Hinton agar by using a swab with a rotation of 60^o each time to make sure that the entire medium is covered.
• Here the sterile disk was applied (4 disks per dish) and each of the obtained concentration was added to one of each disk (about 20µl)
• Finally the plates were incubated at  37^oc for 24hours

The second trial of quantification: quantification of our produced penicillin:

The same steps of last quantification protocol are followed and the obtained penicillin is diluted in NaCl0.9%. (we have obtained about 0.1g of penicillin sodium powder ---> diluted in 5 ml NaCl 0.9%).

Results:

For Diameter= 3.5cm ;log C=2.08 ===> C~120 UI. This result show the effectiveness and the applicability of this methode to quantify the produced penicillin

The second trial of quantification: quantification of our produced penicillin: