Disk Diffusion Method

PRINCIPLE

This method is based on the principle that antibiotic-impregnated disk, placed

on agar previously inoculated with the test bacterium, pick-up moisture and the
antibiotic diffuse radially outward through the agar medium producing an antibiotic
concentration gradient. The concentration of the antibiotic at the edge of the disk
is high and gradually diminishes as the distance from the disk increases to a point
where it is no longer inhibitory for the organism, which then grows freely. A clear
zone or ring is formed around an antibiotic disk after incubation if the agent inhibits
bacterial growth.

MEDIA

The disk diffusion method is performed using Mueller-Hinton Agar (MHA), which
is the best medium for routine susceptibility tests because it has good reproducibility,
low in sulfonamide, trimethoprim, and tetracycline inhibitors, and gives satisfactory
growth of most bacterial pathogens.

The inoculum for the disk diffusion method is prepared using a suitable broth
such as tryptic soy broth. This medium is prepared according to manufacturer’s
instructions, dispensed in tubes at 4-5 ml and sterilized. Sterile 0.9% salt solution
may also be used.

Media are supplemented with 1-2% sodium chloride (NaCl) if intended for
marine organisms.

Preparation of agar medium

1 Prepare MHA from the dehydrated medium according to the manufacturer’s

instructions. Media should be prepared using distilled water or deionized
water.

2 Heat with frequent agitation and boil to dissolve the medium completely.
Sterilize by autoclaving at 121°C for 15 min.
3 Check the pH of each preparation after it is sterilized, which should be
between 7.2 and 7.4 at room temperature. This is done by macerating a small
amount of medium in a little distilled water or by allowing a little amount of
medium to gel around a pH meter electrode.

4 Cool the agar medium to 40-50°C. Pour the agar into sterile glass or plastic
petri dish on a flat surface to a uniform depth of 4 mm.

5 Allow to solidify.
 6 Prior to use, dry plates at
30-37°C in an incubator,
with lids partly ajar, for
not more than 30 minutes
or until excess surface
moisture has evaporated.
Media must be moist but
free of water droplets
on the surface. Presence
of water droplets may
result to swarming
bacterial growth, which
could give inaccurate
results. They are also
easily contaminated.

Storag e

1 If plates are not to be

immediately used,
they may be stored in
the refrigerator inside
airtight plastic bags
at 2-8°C for up to 4
weeks.

2 Unpoured media may be stored in airtight screw-capped bottles under the

conditions specified by the manufacturer.
 Control

Before use, check the ability of the agar to support the growth of control strains
(listed in the Introduction) by streaking bacterial cultures on the agar medium. It is
also advisable to check the ability of each batch of media to support the growth of a
representative member of the species to be tested.

INOCULUM

Preparation

1 From a pure bacterial

culture (not more than
48 hours, old except
for slow growing
organisms), take four
or five colonies with a
wire loop.

2 Transfer colonies to 5 ml of Trypticase soy broth or 0.9% saline.

Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
3 Incubate the broth at 30°C or at an optimum growth temperature until it
achieves or exceeds the turbidity of 0.5 MacFarland standard (prepared by
adding 0.5 ml of 0.048 M BaCl2 to 99.5 ml of 0.36 NH2SO4; commercially
available).

4 Compare the turbidity of the test

bacterial suspension with that of 0.5
MacFarland (vigorously shaken before
use) against a white background with
contrasting black line under adequate
light. Arrow points to tube with
correct turbidity.

5 Reduce turbidity by adding sterile saline or broth.

NOTE: Standardized inoculum has a concentration of 1-2 × 108 cfu/ml.

 Inoculation of plates

1 Dip a sterile cotton swab

into the standardized
bacterial suspension.

2 Remove excess inoculum

by lightly pressing the
swab against the tube
wall at a level above that
of the liquid.

3 Inoculate the agar by

streaking with the
swab containing the
inoculum.
4 Rotate the plate by 60° and repeat the rubbing procedure. Repeat two times.
This will ensure an even distribution of the inoculum.

5 Allow the surface of the medium to dry for 3-5 minutes but not longer than
15 minutes to allow for absorption of excess moisture.

ANTIMICROBIAL DISKS

Selection

The number of antimicrobial agents to be tested should be limited. To make the

test practical and relevant, include only one representative of each group of related
drugs; those indicated for veterinary use to control or prevent disease, and those that
can be useful for epidemiological or research purposes.

Use antibiotic disks purchased from a reputable manufacturer. The disk diameter
is approximately 6 mm. Disks should be properly stored in a tightly sealed container
with desiccant at 2-8°C. Expired disks should not be used.

Application

1 Using sterile forceps or disk

dispenser, place antibiotic disk on
the surface of the inoculated and
dried plate.
2 Immediately press it
down lightly with the
instrument to ensure
complete contact
between the disk and the
agar surface. Do not move
a disk once it has come
into contact with the
agar surface since some
diffusion of the drug
occurs instantaneously.

3 Position disks such that the

minimum center - center distance
is 24 mm and no closer than 10 to
15 mm from the edge of the petri
dish. A maximum of six disks may
be placed in a 9-cm petri dish and
12 disks on a 150 mm plate. Reduce
the number of disks applied per
plate if overlapping zones of
inhibition are encountered.

CONTROL PLATE

Include one plate inoculated with a control strain (Appendix 2.1) for every set of
plates and incubate together.

INCUBATION

1 Incubate plates in an inverted position at 30°C or at an optimum growth

temperature.
2 Observe for the zone of inhibition after 16 to 18 hours. Slow growing
organisms may require longer incubation period.

READING AND MEASUREMENT OF ZONES OF INHIBITION

Descr iption

1 The zone of inhibition (arrow) is

the point at which no growth is
visible to the unaided eye.

2 Record the presence of individual

colonies (arrow) within zones of
inhibition.
 3 Record occurrence of fuzzy zones
(arrow). In measuring the zone
diameter, the fuzzy portion of the
zone should be ignored as much
as possible. The zone limit is the
inner limit of the zone of normal
growth.

Reading

1 Read and record the diameter of

the zones of inhibition using a
ruler graduated to 0.5 mm.

2 Round up the zone measurement to the nearest millimeter.

Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
INTERPRETATION OF RESULTS

1 Compare the diameter of the zone of inhibition of the test isolates with those
in the chart of interpretative standard for veterinary pathogens (Appendix
2.2).

2 Report result as Resistant (R), Intermediate (I) or Susceptible (S).

Example
Disk used: Chloramphenicol, 30 µg (C-30)
Zone of inhibition: 16 mm
Result/ interpretation: Intermediate à based on the zone diameter
interpretative chart (Appendix 2.2)

3 Susceptibility test results using agents other than those listed in the chart
are interpreted on the basis of the presence or absence of a definite zone
of inhibition and is considered only as qualitative until such time as
interpretative zones have been established.

REJECTION CRITERIA

1 Do not read plates on which growth

of test bacteria have isolated
colonies or less than semi-confluent
growth (arrow).

Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
2 Do not read zones of inhibition
of two adjacent disks that
overlap (arrow) to the extent that
measurement of the zone diameter
cannot be made.

3 Do not read zones showing

distortion from circular (arrow).

4 Reject all data collected in a particular set if the zones of inhibition produced
on plate inoculated with a control strain are not within the tolerance limits
set.