CHAPTER 2
Disk Diffusion Method
Eleonor A. Tendencia
Aquaculture Department
Southeast Asian Fisheries Development Center
Philippines
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 1210C 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 40-500C. 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
30-370C 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.
Storage
1. If plates are not to be
immediately used, they may be stored in the refrigerator inside airtight plastic bags
at 2-80C 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.
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.
3. Incubate the broth at 300C 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 600 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.
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 2-80C. 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 300C 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
Description
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.
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 -> 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.
1. Do not read plates on which growth of test bacteria have isolated colonies or less than semi-confluent growth (arrow).
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.
Alderman, D.J. and P. Smith. 2001. Development of draft protocols of standard reference methods for antimicrobial agent susceptibility testing of bacteria associated with fish diseases. Aquaculture, 196: 211- 243.
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Bailey, W.R. and E.G. Scott. 1966. Diagnostic Microbiology, Second Edition. Toppan Company Ltd., Japan, pp. 257- 270.
Finegold, S.M. and W.J. Martin. 1982. Bailey and Scott's Diagnostic Microbiology, Sixth Edition. The CV Mosby Company, London, pp. 532- 557.
NCCLS. 2002. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Approved Standard- Second Edition. NCCLS document M31-A2 (ISBN 1-56238-461-9). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898,USA
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