MC 3 Laboratory
Environmental Influence
Antimicrobial Susceptibility Testing (AST)
Why perform AST?
 Determination of the sensitivity of a
certain microorganism to a drug whose
concentrations is within achievable serum
level
 Used to determine the etiologic agent of
disease
 AST results are often used to dictate
specific management for patients
Why is AST important?
1. It indicates which antibiotics are most
likely to cure an infection.
2. It reduces the empirical prescription of
“broad-spectrum” antibiotics.
3. It avoids unnecessary prescription of
antibiotics thus reducing health care costs.
Properties of an Ideal Antimicrobial Agent
1. It should be with the most activity against
the pathogen
2. With the least toxicity to the host
3. With the appropriate pharmacological
effect
4. It should be economical
2 Actions of Microbial Control Agents
1. Alteration of Membrane Permeability
 Damage to the lipids or proteins of
the plasma membrane by
antimicrobial agents causes
cellular contents to leak into the
surrounding medium and
interferes growth.
2. Damage to Proteins and Nucleic Acids
 DNA and RNA are the carriers of
the cell’s genetic information
 Damage to these nucleic acids is
frequently lethal to the cell
Factors Affecting Effectiveness of
Antimicrobial Treatments
1. Number of Microbes
 The more microbes there are to begin
with, the longer it takes to eliminate
the entire population
 Drugs > Microbes = false resistant
 Drugs < Microbes = false sensitive
2.
 The presence of organic matter often
inhibits the action of chemical
antimicrobials
 Fats and proteins are specially
protective, and a medium rich in these
substances protects microbes, which
will then have a higher survival rate
 Use the right culture media
3. Time of Exposure
 Chemical antimicrobials often require
extended exposure to affect more
resistant microbes or endospore
 16-24 Hours
4. Microbial Characteristics
 MEC-A Gene
Mueller – Hinton Agar (MHA)
 The standard medium for non-
fastidious bacteria (General Purpose
Culture Media)
 Has reduced concentration of
sulfonamide, trimethoprim, and
tetracycline inhibitors
 Composed of beef infusion, nucleic
acids, vitamins, casein hydrosylate
(casein neutralizes fatty acids), and
agar.
Methods of AST
1. Dilution Method
 A test tube dilution method
 Time consuming (only 1 antibiotic every
tube)
 2 types
1. Microdilution: 0.05 – 0.1mL total
broth
2. Macrodilution: 1.0mL total broth
2. Agar Overlay Disk Diffusion Method
 Procedure:
o Pour a thin layer of agar on a petri
dish, allow to solidify, inoculate
organisms, place the disks
o Pour another layer of agar on top of
the disks, incubate overnight, note for
growth indication on top layer of agar

3. Growth Disk Elution Method
 Elution = mga nilutaw
 Antibiotics are incorporated in the
medium
 Inoculate organism by shaking
 Incubate
 Note for elution of organisms which
indicates sensitivity
4. Disk Agar Diffusion Method (Kirby-Bauer
Method)
 The most commonly used AST method
 Procedure:
 After standardization (0.5% MacFarland),
inoculate culture on Muller-Hinton Agar
(standard concentration: approx. 1-2 x 10 8
CFU/mL)
 Stand for 2-3 minutes
 Place disks on MH agar with spaces in
between, incubate
 Note for zone of inhibition (ZOI)
around disks which means organism is
sensitive to the antibiotic
 Turbidity Standard
o 0.5% MacFarland – (barium-
sulfate suspension)
o 99.5mL of 1% H2SO4 (Sulfuric
Acid) and 0.5mL of 1.175% barium
chloride
 During Streaking and Placing of
Disks
o The plate must be turned 60°
between each streaking
(overlapping)
o Distance of disks: no closer than
24mm from disk centers
o Distance from edge of plate: no
closer than 10 to 15mm
o Plates are inverted and incubated
at 35°C for 16-18 hours not more
than 24 hours
 Reading of Plates and Interpretation
of Results
o The “lawn of growth” must be
confluent or almost confluent; if
not, repeat
o The plates are examined two to
three inches above a black, non-
reflective surface
o The diameter of each inhibition
zone is measured using caliper or
ruler; read from the back of the
plate
o Interpret zone measurement using
CLSI
 Factors Affecting the K-B Technique
o pH of the media (7.2 – 7.6)
o thickness of the agar (10-15mL; 4-
6mm)
o growth rate of microorganism (16-
24 hours)
o amount of inoculum (increase =
false resistance; decrease = false
sensitivity)
o suitability of the medium to be
used (BAP = H. influenzae; usually
MH)
o diffusability of antibiotic disks
(check expiration date)
o discrepancy in inoculum (check
technique and apparatus)
Automated Procedures
• Use broth
• Place antibiotic disks
• Inoculate and incubate
• Check for turbidity which is measure
spectrophotometrically
Minimum Inhibitory Concentration (MIC)
• A measure of a microorganism’s
susceptibility or resistance to an antibiotic
• The lowest concentration of antibiotic that
inhibits in vitro bacterial growth
• As the MIC increases, the zone diameter
decreases
Interpretations of Susceptibility Testing

 Susceptible
o Indicates that the antimicrobial
agent in question may be an
appropriate choice for treating the
infection caused by the organism
 Intermediate
o The potential utility of the
antimicrobial agent in body sites
where it may be concentrated or if
high concentrations of the drug are
used
o Possible effectiveness of the
antimicrobial agent against the
isolate, but possibly less so than
against a susceptible isolate
o Use as an interpretive safety
margin to prevent relatively small
changes in test results from
leading to major swings in
interpretive category
 Resistant
o Indicates that the antimicrobial
agent in question may not be an
appropriate choice for treatment,
either because the organism is
NOT inhibited with serum-
achievable levels of drug