Agar Dilution Antimicrobial Susceptibility Testing
Antimicrobial Agents and Range of dilutions (mg/L):
Standard panel:
Penicillin G: 0.008 to 64.0
Tetracycline: 0.06 to 64.0
Spectinomycin: 128.0
Cefixime: 0.002 to 2.0
Ceftriaxone: 0.001 to 2.0
Ciprofloxacin: 0.001 to 16.0
Azithromycin: 0.008 to 16.0
Optional agent: Cefpodoxime: 0.008 to 8.0
Retest criteria:
If the MICs of strains are not determined at the highest concentration
of agent tested, the MIC
should be retested for susceptibility to a higher range of two-fold dilutions.
An endpoint must be
determined. See below for specific repeat testing criteria.
Preparation of Antibiotic-Containing Media
Difco GC medium base (Becton Dickinson, Cockeysville, MD) supplemented
with 1% IsoVitaleX (or an equivalent supplement) is used.
Prepare the required volume of GC base medium in single strength
according to the manufacturer's directions.
Autoclave the medium at 121 C for 15 min. Cool to 50 C in a waterbath.
Reconstitute the dehydrated IsoVitaleX with the provided diluent
according to the manufacturer's directions.
Add 10 ml of supplement per liter of base medium i.e., 1% (v/v);
mix thoroughly. This medium is GCS medium.
Dispense the required volume of medium into individual containers
for the addition of antimicrobial solutions. Maintain media at 50
C in a waterbath.
Prepare the working solutions and dilutions of antimicrobial agents
from the stock solutions or standard powder.
Note:It is important
that no longer than 1 hour elapse between the time that the stock
solution is thawed, the dilutions are prepared and added to the base
medium and the plates are poured.
Add the required volumes of the prepared working solutions and
dilutions of the antimicrobial agents to the respective bottles of
GCS medium, mix thoroughly and dispense into clearly labeled plates.
Thorough mixing of the antibiotics in the medium can be accomplished
by swirling the contents three times in a clockwise and counterclockwise
motion followed by inverting the bottle three times, minimizing bubble
formation.
Allow the plates to cool. Invert the plates and store them in sealed
plastic bags at 4 C to 8 C for no longer than two weeks prior to
use.
Agar-Dilution Susceptibility Test Procedure
Grow pure cultures of isolates to be tested on chocolate agar at
35 C to 36.5 C for 16 to 18 h in a carbon dioxide-enriched (5%) atmosphere.
Use pure cultures on chocolate agar to prepare the
inoculum. Do not use the first subculture from a frozen culture;
subculture these isolates again
before using to prepare the inoculum. Do not use isolates grown on
antibiotic-containing
media to prepare the inoculum. The complete set of control strains
provided by the CDC
should be included in each run. If two or more sets of plates (prepared
in the same batch) are
being inoculated on the same day, it is not necessary to inoculate
the complete set of control
strains on each set of plates. Two or three control strains may be
inoculated on each set of
plates, but all control strains should be included in each day's
test runs.
Use a cotton applicator or a bacteriologic loop to suspend isolated
colonies (or cells from less
dense areas of growth on the plate) in approximately 2 ml of Mueller-Hinton
(MH) broth. (The
exact volume of broth required will depend on the method for measuring
the turbidity of the
suspension. If a spectrophotometer is used, the volume of broth must
be sufficient to
completely cover the light path and will vary according to the type
of spectrophotometer).
Adjust the density of the suspension to contain 108 colony forming
units (CFU)/ml by
comparison with a 0.5 McFarland BaSO4 turbidity standard. (If a spectrophotometer
is used to
measure the optical density of the suspensions, set the wavelength
at 450 nm. Adjust the
turbidity to approximately 0.15. It may be necessary to dilute this
suspension further than
indicated in step #4. Determine the viable count for the suspension
and either adjust the initial
optical density to which the suspension is prepared or the dilution
to give a final viable count of
107 CFU/ml as indicated in step #4.)
Dilute this suspension 1:10 in MH (or equivalent) broth to give
107 CFU/ml.
Dispense an equal volume of each suspension into wells of a replicating
device, e.g., Steer's or
Cathra replicators. These replicating devices deliver 0.001 - 0.005
ml of the bacterial
suspension to the surface of the medium, i.e., 104 CFU.
Inoculate each plate of the set of antibiotic containing media plus
a plate of chocolate agar or GCS medium (as a control to determine
that all isolates grew). You may also wish to inoculated a GCS plate
between each set of antibiotic-containing medium to ensure against
carry-over of antimicrobial agents from one medium to another; these
plates also allow for monitoring for contamination of the inocula
during the inoculation process.
Note. The time elapsing between the preparation
of the strain suspensions and inoculation of the plates should not
exceed 1 h.
Allow the inoculated plates to air-dry at room temperature for
approximately 15 min. Invert the
plates and incubate at 35 C to 36.5 C in a CO2-enriched (5%) atmosphere
for 24 h.
Examine the plates for growth. Use a separate sheet to record the
results for each antibiotic
tested and record the growth for each isolate on each antibiotic
concentration tested. Record the
growth as good (+), poor (±), or no growth (-). By using this
scheme, the results can be
reviewed at a later date for transcription errors and, when isolates
grow on the highest
concentration tested, the degree of growth will indicate whether
the isolate is growing strongly
or is partially inhibited.
Agar Dilution Susceptibility Testing--CDC Reference Strains
of Neisseria gonorrhoeae
Abbreviations: ß-lac, ß-lactamase; Pen, penicillin; Tet, tetracycline;
Spc, spectinomycin; Cro, ceftriaxone; Cfx, cefixime; Cip, ciprofloxacin;
Azi, azithromycin;
Susc, susceptible to penicillin and tetracycline (MICs <2.0 µg/ml);
SpcR, spectinomycin-resistant (MIC >128.0 µg/ml); PP/TR, ß-lactamase-positive
strains with MICs ≥16.0 µg/ml of tetracycline; PPNG, penicillinase (ß-lactamase)-producing
N. gonorrhoeae; TetR, MIC <2.0 µg/ml of penicillin and MIC
≥2.0 µg/ml of
tetracycline; CipI, strain with MIC of 0.125-0.5 µg/ml of ciprofloxacin;
CipR, strain with MIC of ≥1.0 µg/ml of ciprofloxacin; Azi C,
isolates exhibiting critical
MICs (MIC ≥1.0 µg/ml) of azithromycin; CMRNG, ß-lactamase-negative
and MIC >2.0 µg/ml of penicillin and MIC ≥2.0 to 8.0 µg/ml
of tetracycline; Cfx DS,
isolates exhibiting decreased susceptibility (MIC ≥0.5 µg/ml)
to cefixime; ND, Not Determined.
aResistance phenotypes are composed of resistance
phenotypes for penicillin and tetracycline according to GISP definitions
supplemented
with specific
designations for spectinomycin, ciprofloxacin, azithromycin, and cefixime.
bATCC49226, NCCLS-recommended quality control strain
for N. gonorrhoeae; MIC ranges are those published by NCCLS.
cErythromycin range and modal values were calculated
from fewer results than range and modal values for the other antimicrobial
agents.
Isolates with Alert Value
MICs
Isolates with the following MICs require confirmation by retesting:
Ceftriaxone: MIC ≥ 0.25 µg/ml Cefixime: MIC ≥ 0.25 µg/ml
For ceftriaxone and cefixime, isolates should be tested for growth on
medium containing the antibiotics at concentrations ranging from two
dilutions below the initial MIC to sufficient concentrations above the
initial MIC to obtain an endpoint MIC.
Ciprofloxacin: MIC ≥ 1.0 µg/ml
For ciprofloxacin, isolates should be tested for growth on medium
containing two-fold dilutions of ciprofloxacin up to, and including, a
concentration of ciprofloxacin that will provide an endpoint MIC.
Azithromycin: MIC ≥ 1.0 µg/ml
For azithromycin, isolates should be tested for growth on medium containing azithromycin
at concentrations ranging from two dilutions below the initial MIC to sufficient
concentrations above the initial MIC to obtain an endpoint MIC.
Spectinomycin: MIC ≥ 128 µg/ml
For spectinomycin, isolates should be retested for growth on 128.0 µg/ml
of spectinomycin.
The GISP Coordinator and Manager in ESB, DSTDP, NCHSTP, CDC should be notified within one
working day by telephone or by e-mail of any isolate(s) identified to have a high MIC; the GISP Coordinator or Manager will then immediately communicate the results to the Sentinel Site and the pertinent local and/or state STD programs. Such isolates also should be shipped rapidly in
triplicate to CASPIR,
CDC; laboratory personnel should not wait until the request lists arrive from CDC to ship these isolates. A sheet marked “Alert Value Isolate Packing Form” that contains the original and the retest MIC values for each isolate should be included with the isolate shipment; a copy of this same sheet should also be sent separately to the GISP Coordinator.
Isolate Preservation: All isolates should be suspended in trypticase soy
broth containing 20% (v/v) glycerol and frozen at -70 C in duplicate at the
Regional Laboratories. When isolates are requested by CDC for further
characterization, triplicate copies of each requested isolate should be shipped
to CASPIR, CDC. A copy of each shipped isolate must be maintained at the
Regional Laboratory until notified by the GISP Coordinator or GISP Manager that
the isolates have been received. This will generally require maintenance of
isolates from the current year and the previous year.
