Southeast Asian Fisheries Development Center Aquaculture Department
SEAFDEC/AQD Institutional Repository http://repository.seafdec.org.ph
SEAFDEC/AQD-Government of Japan-Trust Fund (GOJ-TF) Laboratory Manuals
2004
Disk diffusion method
Tendencia, Eleonor
Aquaculture Department, Southeast Asian Fisheries Development Center
Tendencia, E. A. (2004). Disk diffusion method. In Laboratory manual of standardized methods
for antimicrobial sensitivity tests for bacteria isolated from aquatic animals and environment (pp.
13-29). Tigbauan, Iloilo, Philippines: Aquaculture Department, Southeast Asian Fisheries
Development Center.
http://hdl.handle.net/10862/1635
Downloaded from http://repository.seafdec.org.ph, SEAFDEC/AQD's Institutional Repository
13
CHAPTER 2
Disk Diffusion Method
Eleonor A. Tendencia
Aquaculture Department
Southeast Asian Fisheries Development Center
Philippines
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
14 CHAPTER 2. 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.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
15
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.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
16 CHAPTER 2. Disk Diffusion Method
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.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
17
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
18 CHAPTER 2. Disk Diffusion Method
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.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
19
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.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
20 CHAPTER 2. Disk Diffusion Method
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.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
21
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.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
22 CHAPTER 2. Disk Diffusion Method
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.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
23
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
24 CHAPTER 2. Disk Diffusion Method
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
25
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.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
26 CHAPTER 2. Disk Diffusion Method
REFERENCES
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.
Anonymous. 1986. Antimicrobial Susceptibility Testing: A System for Standardisation.
Becton Dickinson and Company, Hong Kong, 13 pp.
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
Prescott, L.M., J.P. Harley and D.A. Klein. 1993. Microbiology, Second Edition. Wm C
Brown Publishers, England, pp. 325-343.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
27
APPENDIX 2.1. Acceptable inhibitory zone diameter (mm) limit of control strains
recommended for use in the disk diffusion test of antimicrobial sensitivity testing of
bacteria isolated from animals.
Escherichia Staphylococcus Pseudomonas Streptococcus
Antimicrobial Disk
coli aureus aeruginosa pneumoniaea
Agent Content ATCC 25922 ATCC 25923 ATCC 27853 ATCC 49619
Amikacin 30 µg 19-26 20-26 18-26 -
Amoxicillin- 20/10µg 18-24 28-36 - -
Clavulanic acid b
Ampicillin 10µg 16-22 27-35 - 30-36
Cefazolin 30µg 21-27 29-35 - -
Cefoxitin 30µg 23-29 23-2 -
Cephalothin 30 µg 15-21 29-37 - 26-32
Chloramphenicol 30µg 21-27 19-26 - 26-32
Clindamycin 2 µg - 24-3 - 19-25
Erythromycin 15µg - 22-30 - 25-30
Gentamicin 10µg 19-26 19-27 16-21 -
Imipenem 10µg 26-32 - 20-28 -
Kanamycin 30µg 17-25 19-26 - -
Oxacillin 1µg - 18-24 - <12c
Penicillin 10 units - 26-37 - 24-30
Rifampin 5µg 8-10 26-34 - 25-30
Tetracycline 30µg 18-25 24-30 - 27-31
Ticarcillin 75µg 24-30 - 21-27 -
Ticarcillin- 75/10µg 24-30 29-37 20-28 -
Clavulanic acid
Spectinomycin 100 µg 21-25 13-17 10-14 -
Sulfisoxazole 250 µg or 300 µg 15-23 24-34 - -
Trimethoprim- 1.25/ 23-29 24-32 - 20-28
Sulfamethoxazoled 23.75 µg
Vancomycin 30µg- 17-21 - 20-27
* Adapted from M31-A2 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.
- no established range.
a applicable only using Mueller-Hinton Agar supplemented with 5% defibrinated sheep blood, incubated in 5% CO2.
b range for E. coli ATCC 35218 is 17-22 mm.
c best assessed using Staphylococcus auresus ATCC 25923 with acceptable zone diameter of 18-24 mm.
d very medium-dependent specially with enterococci.
NOTE: Reproduced with permission; from NCCLS publication M31-A2-Performance Standards for
Antimicrobial Disk and Dilution Susceptibility Test for Bacteria Isolated from Animals: Approved Standard-
Second Edition (ISBN 1-56238-461-9). Copies of the current edition may be obtained from NCCLS, 940 West
Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
28 CHAPTER 2. Disk Diffusion Method
APPENDIX 2.2. Zone diameter interpretative standard for veterinary pathogens.
Zone Diameter (mm)
Antimicrobial Agent Disk Content S I F R
Amikacin* 30µg >17 15-16 < 14
Gentamicin* 10µg > 15 13-14 < 12
Kanamycin* 30µg >18 14-17 < 13
Spectinomycin 100µg > 14 11-13 < 10
Amoxicillin-clavulanic acid*
Staphylococci 20/10µg > 20 - < 19
Other organisms 20/10µg >18 14-17 < 13
Ticarcillin-clavulanic acid*
Pseudomonas aeruginosa 75/10µg > 15 - < 14
Gram(-)enteric organisms 75/10µg >20 15-19 < 14
Ampicillin*
Enterobacteriaceae 10µg > 17 14-16 < 13
Staphylococci 10µg > 29 - < 28
Enterococci 10µg >17 - < 16
Streptococci (not S. pneumoniae) 10µg > 26 19-25 < 18
Oxacillin*
Staphylococci 1 µg > 13 11-12 < 10
Penicillin*
Staphylococci 10 units > 29 - < 28
Enterococci 10 units > 15 - < 14
S. pneumoniae 1µg oxacillin > 20 - -
Streptococci (not S. pneumoniae) 10 units > 28 20-27 < 19
Ticarcillin*
Pseudomonas aeruginosa 75µg > 15 - < 14
Gram (-) enteric organisms 75µg > 20 15-19 < 14
10 units/30
Penicillin-novobiocin > 18 15-17 < 14
µg
Imipenem* 10µg > 16 14-15 < 13
Cephalothin* 30µg > 18 15-17 < 14
Cefazolin* 30µg > 18 15-17 < 14
Ceftiofur 30µg > 21 18-20 < 17
Enrofloxacin (canine/feline) 5µg > 23 - 17-22 < 16
Enrofloxacin (chickens/turkeys) 5µg > 23 17-22 < 16
Enrofloxacin (bovine) 5µg > 21 17- 20 < 16
Difloxacin 10µg > 21 18- 20 < 17
Orbifloxacin 10µg > 28 - 18-22 < 17
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
29
APPENDIX 2.2. Continuation
Zone Diameter (mm)
Antimicrobial Agent Disk Content S I F R
Clindamycin 2µg > 21 15-20 < 14
Pirlimycin 2µg > 13 - < 12
Erythromycin*
Streptococci 15µg > 21 16-20 < 15
Organisms other than Streptococci 15µg > 23 14-22 < 13
Tilmicosin (Bovine) 15µg > 14 11-13 < 10
Tilmicosin (Swine) 15µg > 11 < 10
Chloramphenicol*
Streptococci (not S. pneumoniae) 30µg > 21 18-20 < 17
S. pneumoniae 30µg > 21 - < 20
Organisms other than Streptococci 30µg > 18 13-17 < 12
Florfenicol 30µg > 19 15- 18 < 14
Tiamulin 30µg >9 - <8
Trimethoprim-sulfamethoxazole*
Streptococcus pneumoniae 1.25/23.75µg > 19 16-18 < 15
Organisms other than S. pneumoniae 1.25/23.75µg > 16 11-15 < 10
Rifampin*
Streptococcus pneumoniae 5 > 19 17-18 < 16
Organisms other than Streptococci 5 > 20 17-19 < 16
Sulfisoxazole* 250 or 300 > 17 13- 16 < 12
Tetracycline*
Streptococci 30 > 23 19-22 < 18
Organisms other than Streptococci 30 > 19 15-18 < 14
Vancomycin*
Enterococci 30 > 17 15-16 < 14
Streptococci 30 > 17 - -
Other gram-positive organisms 30 > 12 10-11 <9
* human data taken from M100-S12 supplements to M2 and M7
S Susceptible
I Intermediate
R Resistant
F Flexible; should be considered susceptible if appropriate dosing modifications specified in the packaging insert are applied
NOTE: Reproduced with permission; from NCCLS publication M31-A2-Performance Standards for
Antimicrobial Disk and Dilution Susceptibility Test for Bacteria Isolated from Animals: Approved Standard-
Second Edition (ISBN 1-56238-461-9). Copies of the current edition may be obtained from NCCLS, 940 West
Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
