Disk diffusion method

The Government of Japan Trust Fund through SEAFDEC Aquaculture Department provided financial support for the research project and publication of this manual. We would like to express our sincere thanks to Dr. Yasuo Inui and Dr. Kazuya Nagasawa, the former and current Fish Disease Experts and Leaders of the Regional Fish Disease Project, for their encouragement and critical suggestions; and the staff of the Fish Health Section, SEAFDEC Aquaculture Department, for their kind cooperation, especially to Dr. Celia R. Lavilla-Pitogo for carefully reading the manuscript and Dr. Edgar C. Amar for serving as the over-all coordinator in the preparation of drafts. Thanks also to Dr. Hisatsugu Wakabayashi, Emeritus Professor of Fish Pathology Laboratory at the University of Tokyo, for reviewing the manuscript.

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

Storag e
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
2 Transfer colonies to 5 ml of Trypticase soy broth or 0.9% saline.
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.
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 BaCl 2 to 99.5 ml of 0.36 NH 2 SO 4 ; 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.   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.

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.  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.  (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