Organic Chemistry II  | Lecture | Laboratory 

Organic Chemistry Laboratory II
Synthesis and Activity of Sulfanilamide
Senstitivity Testing of Synthesized Sulfanilamide
Introduction
Antibiotic sensitivity testing is used to determine the susceptibility of bacteria to an antibiotic. Numerous standardized tests are available to measure the effectiveness of an antibiotic against a specific organism in order to prescribe the most suitable antibiotic therapy.  One of these tests is the Kirby-Bauer Method, a quick and easy assay for evaluating the effectiveness of numerous antibiotics at once.  In the Kirby-Bauer test, a series of antibiotic-impregnated paper disks are placed on a plate inoculated to form a bacterial lawn (even, confluent growth). The plates are incubated to allow growth of the bacteria and time for the antibiotics to diffuse into the agar. If an organism is susceptible to an antibiotic, a clear zone will appear around the disk where the growth has been inhibited. The size of this zone of inhibition depends on the sensitivity of the bacteria to the specific antibiotic and the antibiotic's ability to diffuse through the agar.  Figures 1a and 1b illustrate the Kirby-Bauer Method and measuring zones of inhibition using this method.

Figure 1a:  Plates Used in the Kirby-Bauer Method for Sensitivity Testing

Figure 1b:  Measuring Zones of Inhibition

The Kirby-Bauer method will be used to test the activity of sulfanilamide, synthesized in the organic chemistry lab, against gram positive bacteria (Streptococcus or Enterococcus) and gram negative bacteria (Eschericia coli or Klebsiella).  Commericial sulfanilamide and TMP/SMX(trimethoprim/sulfamethoxazole, 1:5; Bactrim, Septra) a combination sulfa therapy designed to overcome common bacterial resistance in usually susceptible strains of bacteria, will also be evaluated simultaneously.  The sensitivity of the bacteria to the synthesized sulfanilamide will be compared with commercial sulfanilamide and TMP/SMX.. 

Procedure
Preparing Impregnated Disks
Disks for assaying bacterial sensitivity to sulfanilamide will be prepared in the Organic Chemistry lab and the Kirby-Bauer Assay will be done in the Microbiology Lab.  Disks are prepared by immersion of the sterile disk into a solution of sulfanilamide dissolved in a phosphate buffer.

Obtain three, clean sterile tubes and label each tube with your name(s) and Organic Chemistry Lab section.  Label one tube "synthesized sulfanilamide"(1), one tube "commercial sulfanilamide"(2) and the third tube "buffer"(3).  Prepare a solution of sulfanilamide (100mg) in phosphate buffer (3ml) in a small vial.  Heat the solution until the drug is completely dissolved, being careful not to evaporate significant amounts of water.  Sterilize tweezers by holding the tips of the tweezers over a flame until the tips are slightly red.  Using the sterilized tweezers, pick up two disks and immerse the disks into the hot solution of sulfanilamide for 10-20 seconds.  Transfer the disks to tube "1" and seal the tube. Prepare a solution of commercial sulfanilamide (100mg) in phosphate buffer (3ml).  Heat the solution of commerical sulfanilamide until the drug is dissolved.  Sterilize clean tweezers.  Using the sterilized tweezers, pick up two disks and immerse the disks into the hot solution of sulfanilamide for 10-20 seconds.  Transfer the disks to tube "2" and seal the tube.   For a negative control, dispense 3-5ml of phosphate buffer into a small vial.  Heat the solution until almost boiling.  Pick up a two disks using clean, sterilized tweezers and immerse each clean disk into the buffer solution for 10-20 seconds.  Transfer the both disks to tube "3" and seal the tube.  Hand in the tubes to the organic chemistry instructor.  The tubes will be taken to the microbiology lab for the Kirby-Bauer Analysis.