A. DISC DIFFUSION METHOD
FOR THE ANTIMICROBIAL SUSCEPTIBILITY TESTING
principle:
A standardized
inoculum of bacteria is swabbed onto the surface of a Mueller Hinton agar (MHA)
plate. Filter paper disc impregnated with antimicrobial agents are placed on
the agar. After overnight incubation, the diameter of the zone of inhibition is
measured around each disc. By referring to the tables in the CLSI disc
diffusion standard, a qualitative report of susceptible, intermediate or
resistant is obtained.
Quality control:
A. QC strains
1. Escherichia
coli
ATCC 25922 2. Staphylococcus aureus ATCC 25923
3. Enterococcus faecalis ATCC
29212
B.
Monitoring accuracy
1. Test QC strains by following routine
procedure, and record results. Record lot number and expiration date of discs
and agar.
2. Compare to expected results (CLSI QC
tables). Note any out of control result and document; proceed with corrective
action, if necessary.
3. Perform daily and weekly QC testing.
PROCEDURE:
- Brings agar plates and canisters of
discs to room temperature before use. Agar plates may be removed from
refrigerator and placed in a 35 C ambient air incubator with lids slightly
ajar to evaporate excess moisture. Do not leave in incubator for longer
than 30 min.
- Inoculum preparation
Using a loop or swab, transfer colonies as follows
- Direct colony suspension
method: - pick several colonies from a fresh (18 – 24 hr) nonselective
agar plate to broth or 0.9% NaCl.
- log phase method
a. Pick
four or five isolated colonies to 3.0 to 5.0 ml of broth.
b. Incubate
at 35 C for 2 to 8 hr until growth reaches the turbidity at or above that of a
0.5 McFarland standards.
- For either the log phase or
direct colony suspension method, vortex well and adjust turbidity
visually with sterile broth or 0.9% NaCl to match a 0.5% McFarland
standard.
- Inoculation of agar plates
- Within 15 minutes of
adjusting turbidity, dip a sterile cotton swab into the inoculum and
rotate against the wall of the tube above the liquid to remove excess
inoculum.
- Swab entire surface of agar
plate three times, rotating plates approximately 60˚ between streaking to
ensure even distribution. Avoid hitting the slides of the plate to avoid
aerosols. Finally, run swab around the edge of the agar to remove any
excess moisture.
- Allow inoculated plate to
stand for 3 to 15 min before applying discs.
- Application of discs
- Apply disc to agar surface
with dispenser or manually with a sterile forceps.
- Apply gentle pressure with
sterile forceps or needle to ensure complete contact of disc with agar.
- Do not place discs closer
than 24mm from center to center (no more than 12 discs on 150 mm plates
and 5 discs on 100 mm plates.
- Do not relocate a disc once
it has made contact with agar surface. Instead, place a new disc in
another location on the agar.
- Incubation
- Invert plates and incubate
within 15 min of disc application.
- Incubate for 16 to 18 at 35˚C
in an ambient air incubator.
- Reading plates
- Read plates only if lawn of
growth is confluent or nearly confluent.
- Hold inverted plate a few
inches above a black nonreflecting surface.
- Illuminate plate with
reflected light.
- Use a sliding caliper or
ruler held on the back of the plate to measure the diameter of inhibition
zone to nearest whole millimeter.
- When measuring zones for
sulfonamides, trimethoprim, or trimethoprim- sulfamethoxazole, disregard
light growth (20% less of lawn of growth) and measure edge of the more
obvious margin of the zone.
- Discrete colonies growing
within the inhibition zone may represent a mixed culture or resistant
variants; subculture single colonies from the primary culture plate,
re-identify, and retest for susceptibility. If the discrete colonies are
still apparent, measure the colony – free inner zone.
G. Interpretation and Reporting
Use criteria specified by the CLSI to interpret the
zone of inhibition for each
antimicrobial agents and report categorical result as either
susceptible(S), intermediate (I), or resistant (R).
Figure
1: Antibiotic Susceptibility test of a bacterial isolate.
precautions
The
following common sources of error should be investigated to verify that:
- Zone
diameters were measured and transcribed correctly;
- The
turbidity standard has not expired, is stored properly, meets performance requirements,
and was adequately mixed prior to use;
- All
materials used were within their expiration dates and stored at the proper
temperature;
- The
incubator is at proper temperature and atmosphere;
- Other
equipment used (e.g., pipettors) are functioning properly;
- Discs are
stored desiccated and at proper temperature;
- The control
strain has not changed and is not contaminated;
- Inoculum
suspensions were prepared and adjusted correctly; and inoculum for the
test was prepared from a plate incubated for the correct length of time
and in no case more than 24 hours old.
