Minimal Inhibitory Concentration Test
Minimal Inhibitory Concentration Test
Minimal Inhibitory Concentration Test
To cite this document : Ruangpan, L. (2004). Chapter 3. Minimal inhibitory concentration (MIC) test
and determination of antimicrobial resistant bacteria. In Laboratory manual of standardized
methods for antimicrobial sensitivity tests for bacteria isolated from aquatic animals and
environment (pp.31-55). Tigbauan, Iloilo, Philippines: Aquaculture Department, Southeast Asian
Fisheries Development Center.
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CHAPTER 3
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Lila Ruangpan
Chanthaburi Coastal Fisheries Research and Development Center
Department of Fisheries
Ministry of Agriculture and Cooperatives
Thailand
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
32 CHAPTER 3. Minimal Inhibitory Concentration (MIC) Test and Determination of Antimicrobial Resistant Bacteria
PRINCIPLE
The agar dilution technique is used to measure qualitatively the in vitro activity
of an antimicrobial agent against the test bacteria. In this method, graded amounts
of antibiotics are incorporated in agar plates and inoculated in spots with the
organisms under study. If the organism under study is susceptible to the incorporated
antibiotic, no bacterial growth is expected in agar plates with higher amounts of
the drugs. Bacterial growth is observed as the antibiotic concentration in the agar
plate diminishes. Inhibition of growth at the minimum or lowest concentration of
antibiotic is regarded as the end point.
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MEDIA
Refer to Appendix 3.1 for the different culture media, solvents, antimicrobial
agents, control strains and apparatus needed for the minimal inhibitory concentration
(MIC) test.
The inoculum is prepared using a suitable broth such as heart infusion broth (HIB).
This medium is prepared according to the manufacturer’s instructions, dispensed in
tubes at 3 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 species.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
33
2 Weigh appropriate amount of the powdered antimicrobial agent.
NOTE: The standard units of activity of the antimicrobial agents may differ widely by the actual weight of
the powder or may differ within a drug production lot. Each laboratory must standardize the antimicrobial
agent solutions whenever they are being used. See Appendix 3.2 for the formula used to determine the amount
of powder needed for a standard solution.
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b. Some drugs must be dissolved in solvents other than water. In such cases, it is necessary to:
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
34 CHAPTER 3. Minimal Inhibitory Concentration (MIC) Test and Determination of Antimicrobial Resistant Bacteria
4 Dispense the stock solution into sterile diluent using two-fold dilution
technique.
NOTE: As an example, a range of 0.012-100 µg/ml two-fold dilutions may be used as test concentrations
for different antibiotics. For Sulfadimethoxine and Sulfadiazine, 400 µg/ml may be used as the highest test
concentration (see Appendix 3.4).
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5 Set aside.
1 Label each empty sterile plate in order to identify the antimicrobial agent and
their concentrations.
2 Place the label on the upper portion of the bottom side of the petri dish to
ensure that the plate is inserted at the correct point of the basal stand A of the
multiple inoculating apparatus.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
35
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
36 CHAPTER 3. Minimal Inhibitory Concentration (MIC) Test and Determination of Antimicrobial Resistant Bacteria
6 Pipette 9 ml of MHA
(keep warm at 48-
50°C), add into the
plate with appropriate
dilution of the test
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Prepare control agar plates by pipetting 10 ml of MHA into a sterile petri dish. Do
not add any antimicrobial agent. There should at least be 2 control plates.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
37
INOCULUM
Preparation
2 Shake vigorously in a
water bath at 30°C 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). The inoculum
may also be standardized
based on optical density
[OD625 of 0.08-0.1 (1cm
light path)] using a
spectrophotometer. This is
usually achieved after 18-
24 hours.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
38 CHAPTER 3. Minimal Inhibitory Concentration (MIC) Test and Determination of Antimicrobial Resistant Bacteria
NOTE: The final concentration of the bacterial suspension is approximately 105 cfu/ml well.
5 Store at room temperature for not more than 2 hours before testing.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
39
Inoculation volume
Inoculation sequence
Inocula tion
1 Place a completely dried agar plate on the basal stand A of the multiple
inoculating apparatus. Insert the plate on the basal stand A of the multiple
inoculating apparatus in such a way that the label on the plate is facing the
front direction.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
40 CHAPTER 3. Minimal Inhibitory Concentration (MIC) Test and Determination of Antimicrobial Resistant Bacteria
front direction.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
41
CONTROL PLATE
INCUBATION
1 Let the inoculated agar plates remain at room temperature until the moisture
in the inoculum spot is absorbed by the agar or until all spots are dry.
NOTE: The incubation time is extremely important to obtain reliable end points when reading the results.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
42 CHAPTER 3. Minimal Inhibitory Concentration (MIC) Test and Determination of Antimicrobial Resistant Bacteria
3 Read and record the MIC at the lowest concentration of antimicrobial agent
that completely inhibits growth of the organism as detected by the naked
eye.
Example:
a) No growth
in plates
with 250
µg/ml OTC,
this means
that OTC at
250 µg/ml
completely inhibited all strains.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
43
4 The MIC is the lowest concentration of antimicrobial agent that completely
inhibits colony formation.
7 When reporting the MIC values of antimicrobial agents that will prevent
the growth of bacterial strains, include important information such as the
locality and date the samples were isolated or collected.
9 Compare the MIC breakpoint of the test isolates with those in the chart
(Appendix 3.5).
NOTE: The “true” MIC value is the lowest test concentration that completely inhibits the growth of
organisms in both replicate plates. However, if the “observed” MIC values of the tested plates are different,
for example, two-fold dilutions were used to test OTC and the “observed” MIC value of OTC in the first
plate was 6.25 µg/ml, and the second plate was 3.125 µg/ml, the “ true” MIC of OTC would be between 6.25
µg/ml and 3.125 µg/ml.
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44 CHAPTER 3. Minimal Inhibitory Concentration (MIC) Test and Determination of Antimicrobial Resistant Bacteria
After the MIC values are read, further interpretation of the inhibitory concentration
at 50% (IC50) and 90% (IC90) of the bacterial strains are carried out.
Basically, the IC50 or IC90 is used to determine the dosage regimen of antimicrobial
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agent for in vivo treatment against the pathogen in the field to know the effectiveness
of each drug. The formula used to determine IC50 and IC90 values are presented in
Appendix 3.6.
When the IC50 and IC90 values could not be determined from any value of the MIC
series results, these must be determined from the average value between the nearest
percent that is lower and higher than the IC50 and IC90 values (see Appendix 3.6).
In case a number of bacterial strains have been tested using MIC standardized
method, the MIC values obtained from the study can be used to determine the
breakpoint of each agent and interpretation of results for drug resistance.
1 A large number of bacterial strains (at least 300 strains) should be tested for
each antimicrobial agent using MIC standardized method.
2 Arrange the MIC values of each bacterial strain against an agent and fill into
the assigned table (see example in Appendix 3.7).
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
45
3 Analyze the results by using a program in WHONET to evaluate the breakpoint
of each antimicrobial agent. To access the program: go to www.who.int, click
drug resistance on the right side of the web page. On the left side of the web
page is the table of contents, click # 2 How to obtain WHONET 5 through the
internet. This will give you instructions on how to download the software.
REFERENCES
Ericsson, H.M. and J.C. Sherris. 1971. Antibiotic sensitivity testing. Report of an
international study. Acta Pathol. Microbiol. Scand. B, 217 (suppl.): 1-90.
Jones, R.N., A.L. Barry, T.L. Gavan and J.A. Washington. 1985. Susceptibility tests:
Microdilution and macrodilution. In: Lennette, E.M., Balows, A., Hausler, W.J.
and Shadomy, H.J. (eds.), Manual of Clinical Microbiology. Fourth Edition.
American Society for Microbiology, Washington, DC, USA, pp. 972-977.
McDonald, C.L., W.E. Maher and R.J. Fass. 1995. Revised interpretation of oxacillin
MICs for Staphylococcus epidermidis based on MecA detection. Antimicrob.
Agents Chemother., 39: 982-984.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
46 CHAPTER 3. Minimal Inhibitory Concentration (MIC) Test and Determination of Antimicrobial Resistant Bacteria
Thornsberry, C., T.L. Gaven and E.H. Gerlach. 1977. New developments in
antimicrobial agent susceptibility testing. Cumitech, 6: 1-13.
Watts, J.L. and S.C. Nickerson. 1986. Evaluation of a rapid inoculum standardization
system of antibiotic susceptibility testing of bovine mammary gland isolates. Vet.
Microbiol., 2: 269-276.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
47
APPENDIX 3.1. List of culture apparatus, culture media, solvents, control strains,
and antimicrobial agents needed for the minimal inhibitory concentration (MIC)
test.
Apparatus:
Culture media:
Solvents:
1. Distilled water
2. 0.1 N sodium hydroxide (NaOH)
3. Absolute ethanol
4. Dimethyl sulfoxide
5. N-N-Dimethyl formamide
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48 CHAPTER 3. Minimal Inhibitory Concentration (MIC) Test and Determination of Antimicrobial Resistant Bacteria
Antimicrobial agents:
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1. Chloramphenicol (CP)
2. Erythromycin (E)
3. Furazolidone (FD)
4. Oxolinic acid (OA)
5. Oxytetracycline (OTC)
6. Norfloxacin (NFX)
7. Prefloxacin (PFX)
8. Trimethoprim (TM)
9. Sulfadiazine (SD)
10. Sulfadimethoxine (S)
NOTE: Each laboratory should consider which agents to routinely test or report, in accordance with the
needs of each institute’s study plan or of the farmers in particular aqua culture areas. The test reports should
help to minimize the resistant strains due to misuse or overuse of the agents. At present, except for CP and
FD, the listed antibiotics are FDA approved agents for use in food animal production (NCCLS, 1998).
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
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APPENDIX 3.2. Preparation of antimicrobial agent stock solution.
Example:
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Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
50 CHAPTER 3. Minimal Inhibitory Concentration (MIC) Test and Determination of Antimicrobial Resistant Bacteria
APPENDIX 3.3. List of solvents and diluents needed for the preparation of stock
solutions of antimicrobial agents.
NOTE: For antimicrobial agents which must be dissolved in solvents other than water, it is necessary to
follow this guide:
a. Use only enough solvent to dissolve the powdered antimicrobial agent (in our case, 1 ml of
solvent was used).
c. If sterile solutions are desired, they are to be filtered through a membrane filter.
d. Use antimicrobial solution immediately after preparing and discard unused portion at the end of
the day.
e. Stock solution of antimicrobial agents can be stored at – 70°C or colder for 6 months without
significant loss of activity.
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Appendix 3.4. Preparation of dilutions of antimicrobial agents for use in the agar
dilution method of minimal inhibitory concentration (MIC) test.
Final Conc. At 1:
Concentration Volume Intermediate
Step Source Distilledwater 10 dilution in Agar
(µg/ml) (ml) Conc. (µg/ml)
(µg/ml)
1 1000 Stock 10 10 1000 100
2 1000 Step 1 5 5 500 50
3 500 Step 2 5 5 250 25
4 250 Step 3 5 5 125 12.5
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NOTE:
b. For S and SD, stock solutions were prepared at 4,000 µg/ml and the concentrations were made
by the same method as shown in the scheme.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
52 CHAPTER 3. Minimal Inhibitory Concentration (MIC) Test and Determination of Antimicrobial Resistant Bacteria
* based on 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).
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53
APPENDIX 3.6. Determination of inhibitory concentrations at 50% and 90% (IC50
and IC90 values).
Formula:
50 × MIC value of the next % lower than 50%
IC50 = A+B
2 : when A = the next % lower than 50%
Example: How to find IC50 and IC90 of OTC from the following MIC values.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
54 CHAPTER 3. Minimal Inhibitory Concentration (MIC) Test and Determination of Antimicrobial Resistant Bacteria
Example: The MIC values of the bacterial strains against OTC, OA, S, SD, CP and TM
in various provinces of Thailand in 2002.
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APPENDIX 3.8. Notes on laboratory practice (adapted from NCCLS, 2002)
results.
The following are common sources of error and should be checked whenever
the results of MIC are outside the accuracy control limits:
a. Reader error in observation of the apparent bacterial growth on each
concentration;
b. Contamination or other changes in the control plates;
c. Inoculum adjusted too heavy or too light;
d. Failure to thoroughly mix the antimicrobial dilution tube;
e. Variability in the performance of antibiotic media. Each new lot should be
checked before use;
f. Loss of drug potency during handling or storage in the laboratory; and
g. Mistake in labeling antimicrobial concentration and/or bacterial code.
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment