Fish disease expert and leader of the Regional Fish Disease Project Southeast Asian Fisheries Development Center, Aquaculture Department Tigbauan 5021, Iloilo, Philippines. The data collected from the tests are essential in the selection of the most effective antimicrobial agents against pathogenic bacteria. Two important methods for the in vitro determination of the susceptibility of microorganisms to antimicrobials are the disc agar diffusion test, which uses antibiotic-impregnated discs with an agar medium, and the dilution techniques, in which the test microorganism is exposed to increasing concentrations of an antibiotic in broth or agar.
It also provides an accurate determination of the susceptibility of an organism to a measured amount of test antibiotics. However, only a few antimicrobials have veterinary-specific interpretive criteria; therefore, human interpretation criteria are used for the majority of chemotherapists. The minimal inhibitory concentration (MIC) using agar dilution is one of the appropriate in vitro tests widely used in various laboratories (McDonald et al., 1995).
Basically, a series of plates are prepared with an agar medium to which different concentrations of the antimicrobial agents are added. The selection of the control strain should be based on the type of bacteria being examined in a series of experiments.
Bacterial Isolation, Identification, and Storage
Disk Diffusion Method
The concentration of the antibiotic at the edge of the disc is high and gradually decreases with increasing distance from the disc to the point where it is no longer inhibitory to the organism, which then develops freely. Before use, test the ability of the agar to support the growth of the control strains (listed in the introduction) by plating the bacterial cultures on the agar medium. It is also recommended to check the ability of each batch of medium to support the growth of a representative representative of the species to be tested.
4 Compare the turbidity of the test bacterial suspension with that of 0.5 MacFarland (shaken vigorously before use) against a white background with contrasting black line under sufficient light. 2 Remove excess inoculum by gently pressing the swab against the tube wall at a level above that of the liquid. 5 Allow the surface of the medium to dry for 3-5 minutes, but no longer than 15 minutes, so that excess moisture can be absorbed.
1 Using sterile forceps or disc dispenser, place an antibiotic disc on the surface of the inoculated and dried plate. Do not move a disk once it has contacted the agar surface, as some diffusion of the drug occurs instantaneously. 3 Place discs so that the minimum center-to-center distance is 24 mm and no closer than 10 to 15 mm from the edge of the petri dish.
Reduce the number of discs per plate if overlapping inhibition zones are encountered. 1 The zone of inhibition (arrow) is the point where growth is not visible to the naked eye. When measuring the diameter of the zone, the soft part of the zone should be neglected as much as possible.
1 Read and record the diameter of the inhibition zones using a ruler with a 0.5 mm scale. 1 Compare the diameter of the zone of inhibition of the test isolates with that in the standard interpretation scheme for veterinary pathogens (Appendix 2.2). 3 Susceptibility test results using means other than those listed in the table are interpreted based on the presence or absence of a particular zone of inhibition and are considered qualitative only until the interpretive zones are established.
2 Do not read the braking areas of two adjacent discs that overlap (arrow) to the extent that the diameter of the area cannot be measured. 4 Reject all data collected in a particular group if the zones of inhibition produced on the plate inoculated with a control strain are not within the specified tolerance limits.
Lila Ruangpan
Minimal Inhibitory Concentration (MIC) Test and Determination of
The agar dilution technique is used to qualitatively measure the in vitro activity of an antimicrobial agent against the test bacteria. If the organism under study is sensitive to the incorporated antibiotic, no bacterial growth is expected in agar plates containing larger amounts of the drugs. 1 Remove the antimicrobial from the freezer and allow it to reach room temperature before opening to prevent water condensation.
NOTE: Standard units of antimicrobial activity may vary widely based on the actual weight of the powder or may vary within a product batch. 1 Label each blank sterile plate to identify the antimicrobial agent and its concentrations. 2 Place the label on the top of the underside of the Petri dish to ensure that the plate is inserted at the correct point on the basal stand A of the multiple inoculation apparatus.
5 Pipette 1 ml of appropriate dilutions of the test antimicrobial agent (previously prepared, page 32) into the labeled plate (prepared in number 1). 6 Pipette 9 ml of MHA (keep warm at 48-50°C), add to the plate with appropriate dilution of the test antimicrobial agent and mix thoroughly. In the absence of a multi-dispenser (pictured right), sterile test tubes of the same size can be used to hold the diluted standardized inoculum.
Inoculate the plates with 1-3 µl of the inoculum if using an automatic multidispenser and 10 µl if done manually. Inoculate a second plate with final control agar to ensure that there is no contamination or carryover of the antimicrobial agent during inoculation. 2 When manually inoculating, it is only important to include a plate without drug or control at the beginning of the inoculation series.
Place the plate on the basal stand A of the multiple inoculation apparatus in such a way that the label on the plate faces the front. 4 Adjust the apparatus to ensure that the surface of each multiple inoculator rod will properly touch the surface of the medium in agar plate. 3 Read and record the MIC at the lowest concentration of antimicrobial that completely inhibits growth of the organism as observed with the naked eye.
6 Repeat the test if two or more colonies persist at concentrations of the agent beyond a clear endpoint; that is, if there is no growth at lower concentration, but there is growth at higher concentrations. However, if the "observed" MIC values for the tested plates are different, e.g. used two-fold dilutions to test OTC and the "observed" MIC value for OTC in the first plate was 6.25 µg/ml and the second plate was 3.125 µg/ml, the "true" MIC for OTC would be between 6.25 µg/ml and 3.125 µg/ml.
Determination of Antimicrobial Resistant Bacteria
Appendix 3.4. Preparation of dilutions of antimicrobial agents for use in the agar dilution method of minimal inhibitory concentration (MIC) test
- Safety in Laboratory Practice
Laboratory Manual of Standardized Methods for Antimicrobial Susceptibility Tests for Bacteria Isolated from Aquatic Animals and the Environment. Example: MIC values of bacterial strains against OTC, OA, S, SD, CP and TM in different provinces of Thailand in 2002.
