I had used 3 different concentrations of plant extracts for different bacteria using ciproflaxacin as positive control. I had measure the zone of inhibition of extracts as well as control.
Determination of minimal inhibitory concentrations (MICs)
Microorganisms can be tested for their ability to produce visible growth on a series of agar plates (agar dilution), in tubes with broth (broth dilution), or in microplate wells of broth (broth microdilution) containing dilutions of an antimicrobial agent. Additionally, gradient MIC tests are also commercially available. MIC is defined as the lowest antibiotic concentration that prevents visible growth of bacteria. MIC methods are widely used in the comparative testing of new agents, or when a more accurate result is required for clinical management. As there are no CLSI (formerly NCCLS) (www.clsi.org) nor EUCAST (www.eucast.org) recommendations for the determination of MICs of P. larvae, MIC values of tetracycline and other antibiotics can be determined by the agar dilution method using MYPGP as basal medium (see section 3.1. for recipe) as described as follows:
1-Obtain antimicrobial powders directly from the manufacturer or from commercial sources.
The agent must be supplied with a stated potency (mg or International Units per g powder, or as percentage potency).
2-Store powders in sealed containers in the dark at 4 °C with a desiccant unless otherwise recommended by the manufacturer.
3-Prepare antibiotic stock solutions by using the following formula:
Weight of powder (mg) =
Volume of solvent (ml) X Concentration (µg/ml) / Potency of powder (µg /mg)
4-It is recommended that concentrations of stock solutions should be 1,000 µg/ml or greater.
In the case of tetracyclines, the tested concentrations can be achieved by using two stock solutions of 5,000 µg/ml and 1,000 µg tetracycline/ml in ethanol, stored at -20°C in darkness until used.
5-Prepare MYPGP agar flasks and maintain them at 45 °C until the antibiotic solutions are incorporated.
6-Pour 25 ml of culture medium onto each Petri dish of 90 mm in diameter to give a level depth of 4 mm ± 0.5 mm.
If using 150 mm diameter Petri dishes, 70 ml of culture medium should be dispensed.
7-Prepare plates with increasing concentrations of tetracycline i.e.: 0.03, 0.06, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 µg/ml. For the controls, MYPGP agar without antibiotic is used.
8-Allow the plates to set at RT before moving them.
Dry the plates in a sterile laminar flow cabinet so that no drops of moisture remain on the surface of the agar; do not over-dry plates.
9-Incubate each P. larvae strain to be tested on MYPGP agar for 48 h at 36°C to obtain mainly vegetative cells.
10-Adjust the bacterial suspension until the OD620 (density of a culture determined spectrophotometrically by measuring its optical density at 620 nm) is about 0.4.
11-Each bacterial suspension of each strain must be inoculated onto the surface of the culture medium by adding drops of 5 µl each by means of an automatic micropipette (usually 15-20 drops per plate).
It is possible to test different strains on the same plate. This procedure must be repeated at least twice for each strain and tetracycline concentration, and control plates without antibiotic must be used. It is strongly recommended to include control strains with known MICs in each batch.
12-Place the plates open into a sterile laminar flow cabinet until the drops are absorbed.
13-Incubate the plates in inverted position at 36°C ± 1 for 48 h.
14-After incubation, ensure that each tested strain has grown on the antibiotic-free plate control.
15-Read the MIC endpoint for each strain as the lowest concentration of antibiotic at which there is no visible growth.
The growth of one or two colonies or a fine film of growth should be disregarded.
Interpretation: for tetracyclines, P. larvae isolates should be considered as ”susceptible” when there MICs are
Hi Mukti,
For accurate MIC value I would recommend the broth micro-dilution using the 96-well micro-titre plate. So you are preparing serial dilutions of your plant extract and then inoculate the wells with the organisms after the incubation you check for the lowest concentration that inhibited a visible growth of the organism and that's should be you MIC value.
I recommend that you check out the book 'Antimicrobial Susceptibility Testing Protocols' which is edited by Richard Schwalbe, Lynn Steele-Moore and Avery C. Goodwin. It is published by CRC Press in 2007 and is a good place to start.
Determination of minimal inhibitory concentrations (MICs)
Microorganisms can be tested for their ability to produce visible growth on a series of agar plates (agar dilution), in tubes with broth (broth dilution), or in microplate wells of broth (broth microdilution) containing dilutions of an antimicrobial agent. Additionally, gradient MIC tests are also commercially available. MIC is defined as the lowest antibiotic concentration that prevents visible growth of bacteria. MIC methods are widely used in the comparative testing of new agents, or when a more accurate result is required for clinical management. As there are no CLSI (formerly NCCLS) (www.clsi.org) nor EUCAST (www.eucast.org) recommendations for the determination of MICs of P. larvae, MIC values of tetracycline and other antibiotics can be determined by the agar dilution method using MYPGP as basal medium (see section 3.1. for recipe) as described as follows:
1-Obtain antimicrobial powders directly from the manufacturer or from commercial sources.
The agent must be supplied with a stated potency (mg or International Units per g powder, or as percentage potency).
2-Store powders in sealed containers in the dark at 4 °C with a desiccant unless otherwise recommended by the manufacturer.
3-Prepare antibiotic stock solutions by using the following formula:
Weight of powder (mg) =
Volume of solvent (ml) X Concentration (µg/ml) / Potency of powder (µg /mg)
4-It is recommended that concentrations of stock solutions should be 1,000 µg/ml or greater.
In the case of tetracyclines, the tested concentrations can be achieved by using two stock solutions of 5,000 µg/ml and 1,000 µg tetracycline/ml in ethanol, stored at -20°C in darkness until used.
5-Prepare MYPGP agar flasks and maintain them at 45 °C until the antibiotic solutions are incorporated.
6-Pour 25 ml of culture medium onto each Petri dish of 90 mm in diameter to give a level depth of 4 mm ± 0.5 mm.
If using 150 mm diameter Petri dishes, 70 ml of culture medium should be dispensed.
7-Prepare plates with increasing concentrations of tetracycline i.e.: 0.03, 0.06, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 µg/ml. For the controls, MYPGP agar without antibiotic is used.
8-Allow the plates to set at RT before moving them.
Dry the plates in a sterile laminar flow cabinet so that no drops of moisture remain on the surface of the agar; do not over-dry plates.
9-Incubate each P. larvae strain to be tested on MYPGP agar for 48 h at 36°C to obtain mainly vegetative cells.
10-Adjust the bacterial suspension until the OD620 (density of a culture determined spectrophotometrically by measuring its optical density at 620 nm) is about 0.4.
11-Each bacterial suspension of each strain must be inoculated onto the surface of the culture medium by adding drops of 5 µl each by means of an automatic micropipette (usually 15-20 drops per plate).
It is possible to test different strains on the same plate. This procedure must be repeated at least twice for each strain and tetracycline concentration, and control plates without antibiotic must be used. It is strongly recommended to include control strains with known MICs in each batch.
12-Place the plates open into a sterile laminar flow cabinet until the drops are absorbed.
13-Incubate the plates in inverted position at 36°C ± 1 for 48 h.
14-After incubation, ensure that each tested strain has grown on the antibiotic-free plate control.
15-Read the MIC endpoint for each strain as the lowest concentration of antibiotic at which there is no visible growth.
The growth of one or two colonies or a fine film of growth should be disregarded.
Interpretation: for tetracyclines, P. larvae isolates should be considered as ”susceptible” when there MICs are
Hi Mukti,
To determine the MIC you have to perform microdilution method as other said.
For some known antibiotics, you can determine the MIC value by measuring the diameter of the inhibition zone. So this diameter is proportionnel to the MIC.
In you case the extract is not known, and depending on your disk concentration the diameter will be different. You have to do a correlation curve between your Disk concentration and your MIC. but to determine your MIC for each strain / species you have to do Microdilution method.
Or simply you can figure out your data only with the diameter of inhibition.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209125/
Thank you to all for giving best answer. I am really appreciate with these answers.
Thank you to all for giving best answer. I am really appreciate with these answers.
Thank you to all for giving best answer. I am really appreciate with these answers.
Thank you to all for giving best answer. I am really appreciate with these answers.
Dear Mukti
you can follow these steps to calculate antibiotic MIC:
1. preparation antibiotics serial dilution. the lowest and highest of your antibiotic concentration should be selected according to CLSI guideline for each selected antibiotics. you should select 2 less and more than CLSI break point for determination sensitive and resistance bacteria.
Note: it should be better if you examined antibiotic MIC for some of your strains that you can estimate your break point MIC.
2. Preparation of 0.5 Mac from your bacteria.
3. inoculate bacteria to muller hinton broth contain different concentration of antibiotics.
4. incubate at 37oC for over night.
5. the antibiotic concentration of the first completely clear test tube is your MIC.
Beat Regards
Hello Mr. Abdolaziz Rastegar Lari,
I was wondering if there is any method to correlate between zone of inhibition and MIC of antibiotic. Can I calculate the MIC from ZOI.
Best Regards
Hi Chetan, the ZOI is a measurement used to tell if the organism is susceptible or resistant. Using antibiotics E-strips give you an estimate of the level of MIC but broth micro-dilution assay is the most accurate and acceptable way in research to characterise the MIC value.
Hi, Ali A R Aldallal Sir,
I am not from biology background but I have done antimicrobial study of my samples during my PhD. I got values of Bacterial Count in terms (CFU/ml). How to convert these values in terms of MIC (50 or 90)? I plot regression line for (CFU/ml) Vs Conc. graph. Is this appropriate?
ciprofloxacin powder from himedia states that assay = 98%(HPLC). does this 98% refers to potency of the drug. how should i substitute this value in the following formula for antibiotic stock solution
W=C*V/P.
Can you help me?
Are there some excel template for calculate MIC 50 when I have OD for bacterial culture with tested compound?
@Durgadevi Harihara, 97% nthis refers fo the purity of the antibiotic.
Hello Mr. Abdolaziz Rastegar Lari
please how do i obtain MIC values from a range of values gotten from the anti cancer screening i have used the MTT assay for the screening.
Regards.
Hi. There are several ways to perform MICs. I use broth dilution. Remember to look at the manufacturer‘s info regarding antibiotic such as solubility, potency, storage temp etc.
Preparation of antibiotic stock solutions
• Choose a suitable range of antibiotic concentrations for the organisms to be tested.
• Prepare stock solutions using the formula 1000/Px V x C = W
where P = potency given by the manufacturer (μg/mg), V = volume required (mL), C = final concentration of solution (multiples of 1000) (mg/L), and W = weight of antibiotic in mg to be dissolved in volume V (mL).For example, 1000/x 20 x 10 = 204.08 mg
980
Powder (204.08 mg at a potency of 980 μg/mg) dissolved in 20 mL of solvent = 10,000 mg/L stock solution.
Method:
Grow the test strains in the chosen medium to the right A600 or McFarland std. Have antibiotic solutions and plates ready before the cultures reach the desired growth phase.
Thaw and weigh the antibiotics. Take a note of the purity at this stage, e.g. gentamicin, 577ug/mg solid. Dissolve the antibiotics (solvent depends on the compound), then dilute in the test medium to 2x the top concentration desired in the test, e.g. if highest desired concentration is 128ug/ml, dilute to 256ug/ml. Keep on ice until use.
Using the multipipettor, dispense 100ul of medium into all wells of a microtitre plate. Label the plate and lid.
Pipette 100ul of appropriate 2x antibiotic solutions into the wells in column 1 (far left of plate).
Using the multipipettor set at 100ul, mix the antibiotics into the wells in column 1 by mixing with pipette up and down in the wells ~6-8 times.
Withdraw 100ul from column 1 and add this to column 2. This makes column 2 a twofold dilution of column 1, e.g. for the example above this would be 64ug/ml. Mix up and down 6-8 times. Transfer 100ul to column 3. Repeat the procedure down to column 10 only. The same set of tips can be used for the entire dilution series.
Discard 100ul from column 10 rather than putting it in column 11.
Pour bacteria of the right A600 into a sterile petri dish. The bacteria may be diluted first depending on the desired inoculum. The appropriate inoculum size for standard MIC is 104 to 105 CFU/ml.
With the smaller multipipettor set to 5ul, dispense bacteria into wells in columns 11 to 1 in that order. Do not add bacteria to column 12 (sterility control and blank for the plate scanner).
Incubate the plates at 37oC or other desired temperature.
Streak the bacterial cultures on plates to check their purity.
When satisfactory growth is obtained (18-36 hours) scan the plates with an ELISA reader (e.g. Levy’s lab). Use column 12 as the blank (this means putting the plate in back-to-front).
MIC can be taken as the lowest concentration of drug that reduces, by more than 50% or 90% for MIC50 or MIC90 respectively.
Weight of powder(mg)=Volume of Solvent (ml) X Concentration (mg/ml)/Potency of powder(ug/mg)
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