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..
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.