Lab (6)

Antimicrobial Susceptibility
Testing
Tube (Broth) Dilution Method
Objectives:
• To determine the MIC (Minimum Inhibitory
Concentration) and MBC (Minimal Bactericidal
Concentration) of antibiotics against certain
microorganisms.

Notes: There is report (for this exp.)

Next week: there is quiz
Introduction:
• MIC: the lowest concentration of antimicrobial
agent required to stop the growth of certain
microorganism
• MBC: the lowest concentration of antimicrobial
agent required to kill certain microorganism or
stop bacterial growth permanently.
• Antimicrobial agents are classified into two
types; Bacteriostatic or Bactericidal.
• Bacteriostatic Agents: inhibit the growth of
bacteria at the normal therapeutic
concentrations. These agents exhibit both
characters (-static and –cidal) at different levels.
• Bactericidal Agents: kill bacteria at the
normal therapeutic levels. There is little
difference between MIC and MBC.
 Bactericidal Agents Bacteriostatic Agents

Gentamicin Chloramphenicol

Bacitracin Clindamycin

Amoxicillin Ethambutol

Isoniazid Erythromycin

Metronidazole Nitrofurantoin

Polymyxins Novobiocin

Pyrazinamide Sulfamethoxazole

Ciprofloxacin Trimethoprim

Rifampin Tetracycline

Vancomycin
Why are MIC & MBC required to be checked?

1. If susceptibility cannot be reliably predicted from

knowledge of microorganism’s identity, especially for
highly resistant microorganisms (with continuously
changing profile of resistance).
Such microorganisms include: Staphylococci,
Streptococcus pneumoniae, Viridans Streptococci,
Enterococci, Enterobacteriaceae, Pseudomonas
aeruginosa and Acinetobacter.
There is no constant parameters (MIC, MBC)
identified for such microorganisms, and thus, their
MIC and MBC should be checked routinely.
2. To confirm the susceptibility of bacteria,
especially for narrow-spectrum non-toxic
antibiotics.
Penicillins are an example, they have low human
toxicity (mainly GI manifestations, since allergy is
an idiosyncratic reaction) and if a microorganism,
such as Streptococcus pneumoniae, is resistant to
them; the MBC can be measured by increasing the
dose without causing much of human side effects.
Procedure: (under aseptic conditions)

1. Using a set of test tubes containing 1 mL NB, prepare

a series of dilutions for the two antimicrobials by
serially diluting 1 mL of the AB stock solution
through the set of test tubes. Notice that a single
test tube would not contain an antimicrobial
dilution (this test tube is called the control).
2. Add 1 mL of the bacterial inoculum (bacterial
suspension of 1*106 cells/mL) to each test tube,
including the control.
3. Incubate the test tubes at 37°C for 18-24 hours.
Interpretation of Results:

• After incubation: Compare each AB dilution test tube

with the control test tube; to decide whether it is
turbid (T) or clear (C).
• The AB concentration of the first clear test tube is
considered as the MIC.
• To determine the MBC, the first 2 to 4 clear test
tubes are subcultured on agar plates (using the
surface inoculation technique). This will dilute the
antibiotic to levels below the MIC, and if there were
still surviving bacteria; growth on the plate would be
seen as colonies.
Example:
1. An antimicrobial susceptibility testing – the
tube (broth) dilution method was performed as
following:
2. 1mL broth solution was first added to 8 test
tubes.
3. 1mL of Chloramphenicol was added from a stock
solution 512 µg/mL to the 8 test tubes by
following a serial dilution.
4. 1mL of bacterial inoculum is added after that to
each test tube.
Report
 Report Sheet
Group #:
Students’ Names:

Tube (Broth) Dilution Method

1.Antibiotic Name:

Tube # 1 2 3 4 5 6 7 8
Conc.
After
serial
dilution
Conc.
After
additio
n of
bact.
Turbid
or
Clear

MIC Determination:
B. Antibiotic Name:

Tube # 1 2 3 4 5 6 7 8
Conc.
After
serial
dilution
Conc.
After
additio
n of
bact.
Turbid
or
Clear

MIC Determination:
Thank You