Susceptibility (Sensitivity)
Testing: Results
Pharmaceutical Microbiology – Practical Course
Semester One_2014-2015
Sensitivity Results & MIC Broth Dilution/2014-2015
1
Measure diameter (in mm)
of the zones of inhibition
and compare to diameters
in antibiotic table.
Comment:
E. coli is sensitive to Amikacin, resistant to Gentamicin and
has intermediate sensitivity to Novobiocin and Penicillin.
Sensitivity Results & MIC Broth Dilution/2014-2015
2
Determination of Minimum
Inhibitory Concentration (MIC)
by Broth Dilution Method
Pharmaceutical Microbiology – Practical Course
Semester One_2014-2015
Sensitivity Results & MIC Broth Dilution/2014-2015
3
Minimum Inhibitory Concentration:
Definition
• Definition
 It is the lowest concentration of an antimicrobial
agent that will inhibit the visible growth of a
microorganism after overnight incubation.
• Why?
 Clinically, MIC is not only used to determine the
amount of antibiotic that the patient will receive but
also the type of antibiotic used. This will lower the
opportunity for microbial resistance to specific
antimicrobial agents.
Sensitivity Results & MIC Broth Dilution/2014-2015
4
Common Methods used for MIC
Determination
• MIC determination by Broth Dilution Method
• MIC determination by Agar Diffusion Method
Sensitivity Results & MIC Broth Dilution/2014-2015
5
MIC Determination by Broth Dilution Method
(Serial Dilution Method)
• Principle





Add double strength broth media in all tubes
Serial dilution of the selected antibiotic
Inoculation with the isolated pathogenic microorganism
Incubation for 24 hour
Observation of the turbidity as a parameter of growth
Sensitivity Results & MIC Broth Dilution/2014-2015
6
Detailed procedure
Steps:
Antibiotic
C
1.
Label the test tubes (# 1-6 and C)
2.
Aseptically distribute 1 ml double strength nutrient broth into each wasserman tube with a
5 ml pipette.
3.
Add 1 ml antibiotic into the first wasserman tube. Mix and transfer 1 ml from tube # 1 to
tube # 2. Repeat till tube # 6 using 1 ml pipette. Remove 1 ml from tube #6 after mixing and
empty it in the sink.
4.
Inoculate all tubes with 0.5 ml of the microorganism using a new 1 ml pipette.
5.
Place in incubator at 37°C overnight and inspect for turbidity of media in test tubes.
Sensitivity Results & MIC Broth Dilution/2014-2015
7
Calculations
Antibiotic
Sample
Test Tube
Test Tube
#1
Test Tube
#2
Test Tube
#3
Test Tube
#4
Test Tube
#5
Test Tube
#6
Control
Test Tube
Concentration
after addition
of AB (mg/ml)
200
100
50
25
12.5
6.25
3.125
-
Final
Concentration
(mg/ml) after
addition of
microorganism
-
66.7
33.3
16.7
8.3
4.17
2.083
-
Antibiotic
Sensitivity Results & MIC Broth Dilution/2014-2015
8
Precautions
•
•
•
•
•
Mark carefully all tubes (#1-6 and C)
Adjust the volume in all tubes
Don’t open the broth tube more than 2 times
!!DO NOT ADD AB TO THE CONTROL TUBE!! 
Mixing should be at the bottom of the tube? To avoid air
bubbles ?? Contamination !!!!!!!!
• YOU MUST MUST MUST AVOID ANY CONTAMINATION
Sensitivity Results & MIC Broth Dilution/2014-2015
9
Expected Results
1
2
3 4
5
6 C
• Minimum Inhibition Concentration is equal to the
concentration of the antibiotic in test tube #4 = 8.3 ug/ml
(Case #1)
Sensitivity Results & MIC Broth Dilution/2014-2015
10
Other Expected Results
2
4
1
6
3
C
5
• All tubes are CLEAR (CASE #2)
Comment: MIC is less than concentration of the antibiotic in tube # 6
(i.e. < 2.083 ug /ml)
1
2
3
4
5
6
C
• All tubes are TURBID (CASE #3)
Comment: MIC is higher than concentration of the antibiotic in tube # 1
(i.e. > 66.7 ug/ml)
Sensitivity Results & MIC Broth Dilution/2014-2015
11