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1. Antibiotic Minimum Inhibitory Concentration (MIC) Assay in 96-well Plates
<ol>
	<li>Set up overnight cultures of the strains to be tested in LB (Luria-Bertani) with and without NTBC (2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione). 
	NOTE: This protocol is described using the representative level of 300 &#181;M NTBC.
	<ol>
		<li>Add 300 &#181;M NTBC to 2 ml LB. Add an equivalent volume of vehicle (Dimethyl sulfoxide, DMSO) to 2 ml LB for the no NTBC condition.</li>
		<li>Using sterile toothpicks, inoculate tubes with one isolated colony of bacteria. There will be one culture with NTBC and one culture without NTBC for each strain. Incubate overnight at 37 &#176;C with aeration using a tissue culture rotator in an air incubator.</li>
	</ol>
	</li>
	<li>The following day, make LB + NTBC and LB + DMSO master solutions for setting up the MIC assay. Add NTBC at a concentration of 600 &#181;M as this will be diluted two-fold when inoculum is added, yielding a final concentration of 300 &#181;M.
	<ol>
		<li>To test one antibiotic for one strain, add 600 &#181;M NTBC to 2 ml LB and mix to make the NTBC master solution. Add an equivalent volume of vehicle (DMSO) to 2 ml LB and mix to make the no NTBC master solution. Use these solutions for creating antibiotic stock solutions as well as for setting up the dilution series in 96-well plates. 
		NOTE: The master solution formulations will yield extra solutions to account for pipetting errors. The master solutions can be scaled up or down as required depending on the number of antibiotics and strains tested.</li>
	</ol>
	</li>
	<li>Prepare the antibiotic solutions in the LB + NTBC or LB + DMSO master solutions. 
	NOTE: The antibiotic concentration in these solutions should be double the final desired concentration. Enough solution should be made to transfer 100 &#181;l to four wells in a 96-well plate.
	<ol>
		<li>Prepare gentamicin +/- NTBC stock solution at 64 &#181;g/ml. To make this solution, add 0.288 &#181;l of gentamicin stock (100 mg/ml) to 450 &#181;l LB + NTBC or LB + DMSO master solution. 
		&#8203;NOTE: The maximum concentration of gentamicin for Pseudomonas aeruginosa PAO1 is 32 &#181;g/ml.</li>
		<li>Make the kanamycin +/- NTBC stock solution at 256 &#181;g/ml. To make this solution, add 3.84 &#181;l of kanamycin stock (30 mg/ml) to 450 &#181;l LB + NTBC or LB + DMSO master solutions. 
		NOTE: The maximum concentration of kanamycin for P. aeruginosa PAO1 is 128 &#181;g/ml.</li>
		<li>Prepare tobramycin +/- NTBC stock solution at 8 &#181;g/ml. To make this solution, add 0.36 &#181;l of tobramycin stock (10 mg/ml) to 450 &#181;l LB + NTBC or LB + DMSO master solutions. 
		NOTE: For P. aeruginosa PAO1, the maximum concentration of tobramycin is 4 &#181;g/ml. 
		NOTE: The antibiotics and concentrations can be adjusted for the bacteria to be tested.</li>
	</ol>
	</li>
	<li>Add 100 &#181;l of each 2x antibiotic solution to four wells in a 96-well plate. Place these solutions in row A. For example, gentamicin should be placed in A1 through A4, kanamycin should be placed in A5 through A8, and tobramycin should be placed in A9 through A12. See Figure 1A for a diagram of a 96-well plate setup. 
	NOTE: Multiple antibiotics can be tested in one plate, but only one strain should be tested per plate to eliminate the potential for cross-contamination from other strains.</li>
	<li>Add 50 &#181;l of the LB + NTBC or LB + DMSO master solution to rows B through H of the 96-well plate. Ensure that one plate is LB + NTBC and one plate is LB + DMSO. See Figure 1A.
	<ol>
		<li>Use LB + NTBC for the antibiotics in LB + NTBC. Use LB + DMSO for the antibiotics in LB + DMSO.</li>
	</ol>
	</li>
	<li>Using a micro pipettor perform two-fold serial dilutions of the antibiotics by transferring 50 &#181;l of the solution from row A to row B. Mix the solution, change the pipet tips, and transfer 50 &#181;l of the solution from row B to row C. Repeat for the remaining rows. After diluting row G, remove 50 &#181;l of the solution from that row and discard. Use row H as a no-antibiotic control for bacterial growth. See Figure 1B. 
	NOTE: Each well in rows A through G now contains 50 &#181;l of antibiotic in LB + NTBC or LB + DMSO at 2X the final desired concentration. Row H contains LB + NTBC or LB + DMSO with no antibiotics.</li>
	<li>Measure the OD600 of the overnight cultures. Wash all cultures before taking OD600 readings to eliminate pyomelanin present in the media.
	<ol>
		<li>Wash the cultures by centrifuging 1 ml of culture in a microcentrifuge at 16,000 x g for 2 min. Remove the supernatant with a micro pipettor and resuspend the cell pellet in 1 ml LB.</li>
	</ol>
	</li>
	<li>Dilute the overnight cultures to 2.75x105 CFU/ml in LB. 
	NOTE: Assume that one OD600 unit is the equivalent of 1x109 CFU/ml for P. aeruginosa. OD to CFU/ml conversions may be different in other bacteria.</li>
	<li>Add 50 &#181;l of the diluted bacterial culture to the appropriate well. 
	NOTE: Cultures grown in NTBC should be added to the wells containing NTBC and cultures grown in DMSO should be added to the wells containing DMSO. See Figure 1B.
	<ol>
		<li>Add bacteria to three wells for each strain and antibiotic concentration. Add 50 &#181;l of LB to the fourth well to act as a control for bacterial contamination. See Figure 1B.</li>
		<li>Use a multi-channel micro pipettor to inoculate the wells. Ensure that pipet tips are near the bottom of the wells when adding inoculum to prevent contamination of neighboring wells. 
		NOTE: Adding bacterial culture to the wells will dilute the antibiotic and NTBC concentrations twofold.</li>
	</ol>
	</li>
	<li>Cover the 96-well plates with parafilm and incubate for approximately 24 hr at 37 &#176;C. Incubate 96-well plates statically, in an air incubator.</li>
	<li>Examine the plates for bacterial growth in the wells. The MIC is the lowest concentration of antibiotic in which no bacterial growth is seen for all three replicates of each strain.
	<ol>
		<li>Visually examine the plate for growth or read using a plate reader set to OD600.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)</td>
			<td>Sigma-Aldrich</td>
			<td>SML0269-50mg</td>
			<td>Also called nitisinone. Soluble in DMSO.</td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Sigma-Aldrich</td>
			<td>216763-100ML</td>
			<td>30 wt. % in H2O. Stabilized.</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gold Bio</td>
			<td>G-400-100</td>
			<td>Soluble in H2O. Filter sterilize.</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td>Soluble in H2O. Filter sterilize.</td>
		</tr>
		<tr>
			<td>Tobramycin</td>
			<td>Sigma-Aldrich</td>
			<td>T4014-100MG</td>
			<td>Soluble in H2O. Filter sterilize.</td>
		</tr>
	</tbody>
</table>

a microtiter plate method to assess the minimum inhibitory concentration of an antibiotic

Source: Ketelboeter, L. M., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/53105">Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress.</a> J. Vis. Exp. (2015).
This video demonstrates a microtiter plate-based method for determining the Minimum Inhibitory Concentration (MIC) of test antibiotics against bacteria. Upon adding a suspension of the bacterial pathogen Pseudomonas aeruginosa to a microtiter plate containing serial dilutions of a test antibiotic, and an inhibitor of the production of pyomelanin by the bacteria, the optical density of the wells is measured to compute the minimum inhibitory concentration of the antibiotic, and the impact of the pyomelanin production inhibitor the antibiotic sensitivity of the bacteria.

<img alt="Figure 1" src="/files/ftp_upload/22004/22004_Figure1.jpg" /> 
Figure 1: Schematic of antibiotic MIC assay 96-well plate setup. (A) 100 &#181;l of 2x antibiotics of the highest starting concentration are in row A. Rows B through H are filled with 50 &#181;l of either LB + NTBC or LB + DMSO without antibiotics. (B) Two-fold serial dilutions are performed in rows A through G, resulting in 50 &#181;l of diluted...

A Microtiter Plate Method to Assess the Minimum Inhibitory Concentration of an Antibiotic

1. Antibiotic Minimum Inhibitory Concentration (MIC) Assay in 96-well Plates

	Set up overnight cultures of the strains to be tested in LB (Luria-Bertani) ...

Take microtiter plates containing a serially diluted aminoglycoside antibiotic. One of the plates contains NTBC, a synthetic drug.
Introduce Pseudomonas aeruginosa, a gram-negative bacteria, and incubate.
The bacteria produce pyomelanin &#8212; a protective extracellular pigment &#8212; in the NTBC-untreated wells, while the drug prevents the production of pyomelanin.
The cationic antibiotic binds to anionic lipopolysaccharides, or LPS, on the bacterial outer membrane, displacing the cationic bridges and destabilizing the membrane.
Upon entering the bacteria, the antibiotic binds to the aminoacyl site, or A site, in the 30S ribosomal subunit.
The bound antibiotic prevents the aminoacyl-tRNA binding and ribosomal movement across mRNA, interfering with protein synthesis and inhibiting bacterial growth.
Post-incubation, measure the culture&#39;s optical density to assess bacterial growth.
The lowest antibiotic concentration that prevents bacterial growth is the minimum inhibitory concentration or MIC.
Similar MIC values for NTBC-treated and NTBC-untreated wells indicate no impact of NTBC on the antibiotic sensitivity of bacteria.

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435 Views. Source: Ketelboeter, L. M., et al. Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress. J. Vis. Exp. (2015). This video demonstrates a microtiter plate-based method for determining the Minimum Inhibitory Concentration (MIC) of test antibiotics against bacteria. Upon adding a suspension of the bacterial pathogen Pseudomonas aeruginosa to a microtiter plate containing serial dilutions of a test antibiotic, and an inhibitor...

Video: A Microtiter Plate Method to Assess the Minimum Inhibitory Concentration of an Antibiotic - Concept

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail treatment. This Gram-negative bacterium causes gonorrhea exclusively in humans. During infection, it is able to form aggregates and/or biofilms. The minimum inhibitory concentration (MIC) test is used for to determine susceptibility to antibiotics and to define appropriate treatment. However, the mechanism of the eradication in vivo and its relationship to laboratory results are not known. A method that examines how GC aggregation affects antibiotic susceptibility and shows the relationship between aggregate size and antibiotic susceptibility was developed. When GC aggregate, they are more resistant to antibiotic killing, with bacteria in the center surviving ceftriaxone treatment better than those in the periphery. The data indicate that N. gonorrhoeae aggregation can reduce its susceptibility to ceftriaxone, which is not reflected using the standard agar plate-based MIC methods. The method used in this study will allow researchers to test bacterial susceptibility under clinically relevant conditions.

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

A simple ATP-measuring assay and live/dead staining method were used to quantify and visualize Neisseria gonorrhoeae survival after treatment with ceftriaxone. This protocol can be extended to examine the antimicrobial effects of any antibiotic and can be used to define the minimal inhibitory concentration of antibiotics in bacterial biofilms.

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail ...

JoVE Journal

Immunology and Infection

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

A Microtiter Plate Method to Assess the Minimum Inhibitory Concentration of an Antibiotic

Transcript

A Microtiter Plate Method to Assess the Minimum Inhibitory Concentration of an Antibiotic

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung