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Penicillin Quantification for MEGBI-APP

 

I- Proposed experimental

protocol to quantify the produced penicillin:

 Preparation of the turbidity calibration 0.5 McFarland(5):

 

  • we added 0.5 mL of a 0.048 mol/L solution of BaCl2 (1.175% w/v BaCl2 2H2O) to 99.5 mL of a 0.18 mol/L solution (0.36 N) of H2SO4 (1% v/v) and we shook vigorously

 

  • We checked the density of the suspension using a spectrophotometer with a 1 cm beam and matching cuvettes. The absorbance at 625 nm should be between 0.08 and 0.13

 

 

  • We distributed the suspension in tubes of the same size as those used to adjust the inoculum and then we sealed the tubes

 

  • Once sealed, we stored these tubes at room temperature and protected from light. Before use, we mixed the tube vigorously using a Vortex (6 months’ storage)

 

 

 

The three bacterial strains which can be used:

 

E.coli / Klebsella.spp / MRSA

 

 

Quantification of the produced penicillin using the disk diffusion method (Kirby-Bauer Test)(6):

 

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

6)     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: 7)     We dilute the standard penicillin 10 times (Concentrations: 1.5; 1.4; 1.3; 1.2; 1.1; 1.0; 0.9; 0.8; 0.7; 0.6) to obtain different concentrations8)     We dip each of the 10 discs in one of the 10 concentrations of penicillin9)     We dip another one disk in the unknown produced penicillin10) We distribute the 11 disks in plates at a distance of (26) mm apart11) 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 penicillin:

12) After the growth time, we measured the zone of inhibition that had appeared using a ruler 13)  We drew a graph showing the concentration of penicillin as a function of the diameter in order to be able to quantify the produced penicillin (Log C as a function of diameter)

 

   

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Penicillin Quantification (docx)

 

Working Plan Dec 2021

Monday

Tuesday

Wednesday

Thursday

Friday

 

 

1 (عرض الطرق الثلاثة واعتماد واحدة)

2(البحث لوضع مخطط تجريبي موحد)

3  (تحديد المستلزمات والنواقص)

6

7

8

9

10

13

14

15

16

17

20

21

22

23

24

27

28

29

30

31

 

عند تأمين كافة المستلزمات ستتوزع أيام التجربة على الشكل التالي (4-5 أيام):

  • يوم لتحضير الوسط الزراعي+ تحضير المياه المالحة+ تحضير الانبوب الشاهد (0.5MF)
  • يوم لتحضير أقراص الATB و زراعة البكتيريا الكاشفة
  • يوم لتطبيق التجربة من زرع وتطبيق اقراص الATB
  • يوم لتقييم النتيجة واستنتاج الكثافة للأمبيسيلين المحضر

 

ما تحتاجه التجربة:

BaCl2-

Filter paper or penicillin disk with different concentration-

Agar Muller Hinton-

E.coli or S.aureus(SARM)-

Petri dish-