Lab - Antibiotic Resistance
Today we will test the sensitivity of four organisms to various antibiotics. The
purpose of the day’s lab will be to evaluate sensitivity of each organism to the
various antibiotics tested.
Organisms used are listed in the following table:
ORGANISM
Gram Reaction & Morphology
Bacillus cereus
G+ Rod
Escherichia coli
G- Rod
Staphylococcus aureus
G+ Cocci
Klebsiella pneumoniae
G- Rod
Antibiotics tested include:
DISK CODE
ANTIBIOTIC
(printed on the paper disk)
Ampicillin
AM
Gentamicin
GM
Penicillin
P
Tetracycline
TE
Ciprofloxacin
CIP
Nalidixic Acid
NA
Procedure:
Obtain a Mueller Hinton plate. Using aseptic technique open the culture tube. Insert a
sterile swab into the liquid broth to saturate. With close strokes in a back & forth motion,
swab the entire surface of your plate, being sure to use the cover in an ‘umbrella fashion’ to
keep unwanted environmental organisms from the medium. Rotate the plate 90 degrees and
re-swab – DO NOT reinsert your swab into the culture. The purpose of this dual direction
swab is to create a ‘lawn’ of bacteria over the entire surface of the medium.
After inoculation apply the six antibiotic disks to the media by touching them lightly to the
surface, label side up so that they may be read. Press LIGHTLY onto the surface and the
disk will adhere to the media. You do not need to press the disk down into the media.
Arrange the disks about ¾” in from the outer edge of the media in a regularly spaced pattern
similar to 2, 4, 6, 8, 10 & 12 on a clock face. (see figure)
Allow the culture to incubate at 37oC for 48 hours.
Measure the zone of inhibition (if any) for each antibiotic using a metric ruler. Measure from
the edge of the paper to the edge of the zone of inhibition. Record the results in the table on
the following page.
P
TE
AM
NA
GM
Day 1
Inoculation
&
Antibiotics
CIP
P
TE
AM
Measure this
distance (mm) for
each antibiotic
exhibiting inhibition
NA
Day 2
Evaluation
GM
CIP
Group 1 Organism
Group 2 Organism
Bacillus cereus
Bacillus cereus
Resistant/Sensitive
(circle R or S)
Zone in mm
(0 if resistant)
Resistant/Sensitive
(circle R or S)
Zone in mm
(0 if resistant)
AM – R/S
AM – R/S
GM – R/S
GM – R/S
P – R/S
P – R/S
TE – R/S
TE – R/S
CIP – R/S
CIP – R/S
NA – R/S
NA – R/S
Group 3 Organism
Group 4 Organism
Staphylococcus aureus
Staphylococcus aureus
Resistant/Sensitive
(circle R or S)
Zone in mm
(0 if resistant)
Resistant/Sensitive
(circle R or S)
AM – R/S
AM – R/S
GM – R/S
GM – R/S
P – R/S
P – R/S
TE – R/S
TE – R/S
CIP – R/S
CIP – R/S
NA – R/S
NA – R/S
Zone in mm
(0 if resistant)
Group 5 Organism
Group 6 Organism
Escherichia coli
Escherichia coli
Resistant/Sensitive
(circle R or S)
Zone in mm
(0 if resistant)
Resistant/Sensitive
(circle R or S)
Zone in mm
(0 if resistant)
AM – R/S
AM – R/S
GM – R/S
GM – R/S
P – R/S
P – R/S
TE – R/S
TE – R/S
CIP – R/S
CIP – R/S
NA – R/S
NA – R/S
Group 7 Organism
Group 8 Organism
Klebsiella pneumoniae
Klebsiella pneumoniae
Resistant/Sensitive
(circle R or S)
Zone in mm
(0 if resistant)
Resistant/Sensitive
(circle R or S)
AM – R/S
AM – R/S
GM – R/S
GM – R/S
P – R/S
P – R/S
TE – R/S
TE – R/S
CIP – R/S
CIP – R/S
NA – R/S
NA – R/S
Zone in mm
(0 if resistant)