The author would like to thank Li Zhang, Xian-Zhi Li, Aaron Hinz, and Clayton Hall for editorial help with this manuscript. This assay was initially developed in the lab of George O&#8217;Toole, Geisel School of Medicine at Dartmouth. Research in Dr. Mah&#8217;s lab is supported by grants from the Natural Sciences and Engineering Research Council of Canada and Cystic Fibrosis Canada.

1. MBC-B
<ol>
	<li>Growing a biofilm (adapted from O&#39;Toole9).
	<ol>
		<li>Grow a culture of the wild-type strain of interest and mutant strain for 16 hr in a rich medium at 37 &#176;C.</li>
		<li>Dilute the saturated overnight cultures 1:100 into fresh medium for antibiotic resistance assays. A standard medium for P. aeruginosa is M63 minimal medium supplemented with magnesium sulfate and arginine (see Table 1). This medium stimulates the formation of a more robust biofilm.</li>
		<li>Add 100 &#956;l of the dilution per well in a 96-well microtiter dish (see Table 1). Since these assays are typically performed in triplicate for each strain, there should be 24 wells of each strain.</li>
		<li>Incubate the microtiter plate for 24 hr at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Exposing the preformed biofilm to a concentration gradient of antibiotic
	<ol>
		<li>Prepare a 10x&#160;dilution series of antibiotic for 7 wells. Example: for the antibiotic tobramycin, the final concentrations in the wells are 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, and 0.006 mg/ml 5-8 . From a stock of 25 mg/ml, prepare 10x&#160;dilutions of 4, 2, 1, 0.5, 0.25, 0.125, and 0.06 mg/ml. Leave on ice.</li>
		<li>Remove the spent supernatant (containing planktonic cells) using a multichannel pipette (see Table 1).</li>
		<li>Add 90 &#956;l M63 (Mg/Arg) to all of the wells.</li>
		<li>Add 10 &#956;l of each 10x&#160;antibiotic concentration in order to achieve the desired final concentrations. Add 10 &#956;l water (no antibiotic control) to the final well for each replicate and strain.</li>
		<li>Incubate the microtiter plate for 24 hr at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Refreshing the media and allowing for the detachment of live cells.
	<ol>
		<li>Remove the spent supernatant (containing planktonic cells) using a multichannel pipette.</li>
		<li>Add 115 &#956;l M63 (Mg/Arg) to all of the wells.</li>
		<li>Incubate the microtiter plate for 24 hr at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Assay for live cells.
	<ol>
		<li>Label two LB agar plates per 96-well microtiter plate.</li>
		<li>Sterilize the multiprong device (see Table 1) by dipping the prongs in 100% ethanol and passing the prongs across the open flame of a Bunsen burner. Repeat. Let the prongs cool slightly. Using the multiprong device, transfer ~3 &#956;l (amount that is typically retained on the tips of the prongs) of planktonic culture from each well of the microtiter&#160;plate to the surface of an LB agar plate.</li>
		<li>Incubate the LB agar plates for 16 hr at 37 &#176;C.</li>
		<li>Determine minimal bactericidal concentration of the antibiotic by identifying, by eye, the cut-off for bacterial growth (Figure 1).</li>
	</ol>
	</li>
</ol>
2. MBC-P&#160;
<ol>
	<li>Preparing bacterial strains
	<ol>
		<li>Grow a culture of the wild-type strain of interest and mutant strain for 16 hr in a rich medium at 37 &#176;C.</li>
		<li>Dilute the saturated overnight cultures 1:100 into fresh medium for antibiotic resistance assays. A standard medium for P. aeruginosa is M63 minimal medium supplemented with magnesium sulfate and arginine (see Table 1).</li>
		<li>Add 90 &#956;l of the dilution per well in a 96-well microtiter dish (see Table 1). Since these assays are typically performed in triplicate for each strain, there should be 24 wells of each strain.</li>
	</ol>
	</li>
	<li>Exposing planktonic cells to a concentration gradient of antibiotic
	<ol>
		<li>Prepare 10x&#160;dilution series of antibiotic for 7 wells. Example: for the antibiotic tobramycin, the final concentrations in the wells are 0.032, 0.016, 0.008, 0.004, 0.002, 0.001, and 0.0005 mg/ml. From a stock of 25 mg/ml, prepare dilutions of 0.32, 0.16, 0.08, 0.04, 0.02, 0.01, and 0.005 mg/ml. Leave on ice.</li>
		<li>Add 10 &#956;l of each 10x&#160;antibiotic concentration in order to achieve the desired final concentrations. Add 10 &#956;l water (no antibiotic control) to the final well for each replicate and strain.</li>
		<li>Incubate the microtiter plate for 24 hr at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Assay for live cells.
	<ol>
		<li>Label two LB agar plates per 96-well microtiter plate.</li>
		<li>Sterilize the multiprong device (see Table 1) by dipping the prongs in 100% ethanol and passing the prongs across the open flame of a Bunsen burner. Repeat. Let the prongs cool slightly. Using the multiprong device, transfer ~3 &#956;l (amount that is typically retained on the tips of the prongs) of planktonic culture from each well of the microtiter plate to the surface of an LB agar plate.</li>
		<li>Incubate the LB agar plates for 16 hr&#160;at 37 &#176;C.</li>
		<li>Determine minimal bactericidal concentration of the antibiotic by identifying, by eye, the cut off for bacterial growth (Figure 2).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Hoiby, N., Bjarnsholt, T., Givskov, M., Molin, S., Ciofu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+of+bacterial+biofilms.">Antibiotic resistance of bacterial biofilms.</a> Int. J. Antimicrob. Agents. 35 (4), 322-332 (2010).</li><li>Mah, T. F., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+biofilm+resistance+to+antimicrobial+agents.">Mechanisms of biofilm resistance to antimicrobial agents.</a> Trends Microbiol. 9 (1), 34-39 (2001).</li><li>Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm-specific+antibiotic+resistance.">Biofilm-specific antibiotic resistance.</a> Future Microbiol. 7 (9), 1061-1072 (2012).</li><li>Mah, T. F., Pitts, B., Pellock, B., Walker, G. C., Stewart, P. S., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+basis+for+Pseudomonas+aeruginosa+biofilm+antibiotic+resistance.">A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.</a> Nature. 426 (6964), 306-310 (2003).</li><li>Zhang, L., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Involvement+of+a+Novel+Efflux+System+in+Biofilm-Specific+Resistance+to+Antibiotics.">The Involvement of a Novel Efflux System in Biofilm-Specific Resistance to Antibiotics.</a> J. Bacteriol. 190 (13), 4447-4452 (2008).</li><li>Zhang, L., Hinz, A. J., Nadeau, J. P., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+tssC1+Links+Type+VI+Secretion+and+Biofilm-specific+Antibiotic+Resistance.">Pseudomonas aeruginosa tssC1 Links Type VI Secretion and Biofilm-specific Antibiotic Resistance.</a> J. Bacteriol. 193 (19), 5510-5513 (2011).</li><li>Beaudoin, T., Zhang, L., Hinz, A. J., Parr, C. J., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Biofilm-Specific+Antibiotic+Resistance+Gene,+ndvB,+is+Important+for+Expression+of+Ethanol+Oxidation+Genes+in+Pseudomonas+aeruginosa+Biofilms.">The Biofilm-Specific Antibiotic Resistance Gene, ndvB, is Important for Expression of Ethanol Oxidation Genes in Pseudomonas aeruginosa Biofilms.</a> J. Bacteriol. 194 (12), 3128-3136 (2012).</li><li>O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtiter+Dish+Biofilm+Formation+Assay.">Microtiter Dish Biofilm Formation Assay.</a> J. Vis. Exp. (47), e2437 (2011).</li><li>Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidrug+tolerance+of+biofilms+and+persister+cells.">Multidrug tolerance of biofilms and persister cells.</a> Curr. Top. Microbiol. Immunol. 322, 107-131 (2008).</li><li>Merritt, J. H., Kadouri, D. E., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+and+Analyzing+Static+Biofilms.">Growing and Analyzing Static Biofilms.</a> Curr. Protoc. Microbiol. 1, 1B.1.1-1B.1.17 (2005).</li><li>Ramey, B. E., Parsek, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+1.+Growing+and+analyzing+biofilms+in+fermenters.">Chapter 1. Growing and analyzing biofilms in fermenters.</a> Curr. Protoc. Microbiol. 1, 1B.3.1-1B.3.14 (2005).</li></ol>

The author declares that she has no competing financial interests.

Antibiotic resistance in planktonic cells is defined as an increase in the minimum inhibitory concentration (MIC) of an antibiotic due to a permanent change in the cells (e.g. a mutation). The mechanisms of biofilm-specific resistance or tolerance that have been identified to date are the result of the expression of wild type genes within biofilms. Thus, the classical definition of resistance does not apply to biofilms. However, another set of definitions has been presented: resistance mechanisms prevent the antibiotic from accessing its target, while tolerance mechanisms shut down the targets of antibiotics10. Biofilm-specific antibiotic resistance mechanisms encompass both of these definitions. The term &#8220;resistance&#34; has been used throughout this protocol, but further experimentation must be carried out in order to determine if a gene is contributing to resistance or tolerance.
The MBC-P and MBC-B assays may be adapted for any biofilm-forming bacterial strain and any bactericidal antimicrobial agent. The two important criteria for adapting this assay to other bacteria and other antimicrobial agents are determining conditions that lead to biofilm formation and determining the appropriate range of concentrations for the antimicrobial agent using the wild type strain of interest. Proper biofilm-formation conditions can be determined using a JoVE biofilm formation protocol published by George O&#39;Toole9. In order to determine an appropriate concentration range for the MBC-B assay, a good rule of thumb is to use 25 x&#160;MIC (minimal inhibitory concentration11) as the middle concentration of the range and adjust accordingly. For the MBC-P assay, use 1/10 of the values identified for the MBC-B assay. The goal is to define a range of concentrations that will allow for a clear difference between the MBC-B of the wild-type strain and a sensitive mutant strain. Thus, the top end of the range should be close to the MBC-B of the wild-type strain. We repeat this experiment at least three times, giving a minimum of 9 data points per strain.
The most critical step of these assays is to avoid contamination. To prevent contamination between strains, an empty column should be left between strains. Another critical step is to allow enough time for the metal prongs on the multiprong device to cool enough. This can be tested by flaming the prongs, timing cooling period and quickly touching the prongs with one&#39;s&#160; hands.
If detachment of the cells from the biofilm after exposure to antibiotics is an issue, the MBC-B method can be modified to include a sonication step. Skip step 1.3.3 (24 hr incubation after addition of fresh media). Instead, cover the wells of the 96-well microtiter plate after adding 115 &#956;l&#160;of fresh media (step 1.3.2) with sterile adhesive plate seal and sonicate plate to dislodge biofilm cells from the walls of the wells. Use a water bath sonicator. Specific conditions (time, intensity) for the efficient removal of biofilm cells from the plastic must be determine beforehand.
A variation of the MBC-B was used to screen a library of 4,000 transposon-insertion mutants of P. aeruginosa for biofilm-specific antibiotic resistance mutants. A concentration of tobramycin (50 &#956;g/ml) was chosen that would not kill wild type biofilm cells but would allow for the identification of mutants that were 3-fold more sensitive than the wild type biofilm. Instead of inoculating columns of wells with one specific strain of bacteria, each well was inoculated with a different mutant. The mutants were left to form a biofilm, exposed to 50 &#956;g/ml&#160;tobramycin, left to recover from tobramycin exposure and then assayed for viability. Mutants that did not survive 50 &#956;g/ml&#160;tobramycin exposure were retested under the same screening conditions. The MBC-P and MBC-B protocols were utilized to confirm the tobramycin-sensitive phenotypes of the mutants.
There are other methods to assay antibiotic resistance in biofilms, i.e. colony biofilms and biofilms grown in bioreactors12,13. These methods are considerably more laborious and limited in the number of strains that can easily be assayed. Thus, an advantage that the MBC-B has over these other methods is that it is high-throughput, allowing the researcher to conduct screens or to assay several strains, as well as several different antibiotic concentrations at one time.
<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>1x M63</td>
			<td></td>
			<td></td>
			<td>Prepare as a 5x M63 stock by dissolving 15 g KH2PO4, 35 g K2HPO4 and 10 g (NH4)2SO4 in 1 L of water. This stock does not need to be autoclaved and can be stored at room temperature.&#160; Dilute 5x stock 1:5, autoclave, cool, then add the desired components.</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Fisher</td>
			<td>P288-500</td>
			<td></td>
		</tr>
		<tr>
			<td>(NH4)2SO4</td>
			<td>Sigma</td>
			<td>A5132</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Fisher</td>
			<td>M63-500</td>
			<td>Add to 1 mM final concentration.&#160; Prepare as a 1 M stock in water and autoclave.</td>
		</tr>
		<tr>
			<td>Tobramycin</td>
			<td>Sigma</td>
			<td></td>
			<td>Prepare 50 mg/ml stock. Aliquot and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Arginine</td>
			<td>Sigma</td>
			<td>A5131</td>
			<td>Add to 0.4% final concentration.&#160; Prepare as a 20% stock in water and filter sterilize.&#160; This alternative carbon/energy source can replace glucose and casamino acids</td>
		</tr>
		<tr>
			<td>96-well microtiter plates</td>
			<td>Corning</td>
			<td>3595</td>
			<td>Sterile, flat-bottom, low evaporation</td>
		</tr>
		<tr>
			<td>Tranferpette (multichannel pipette)</td>
			<td>BrandTech</td>
			<td>2703610</td>
			<td>8-channel, 20-200 &#956;l</td>
		</tr>
		<tr>
			<td>Multiprong device</td>
			<td>Dan-Kar</td>
			<td>MC48</td>
			<td>48 prongs fit into &#189; of a 96-well microtiter plate</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;

Antibiotic resistance assays were initially developed to assay resistance of planktonic (free-swimming) cultures of bacteria. Since many bacterial infections involve biofilms (surface-attached cells), we were interested in developing a method to assay biofilm-specific antibiotic resistance. However, most antibiotic resistance assays are poorly suited for measuring the resistance of biofilms. For example, determining the minimal inhibitory concentration (MIC) is the gold standard for determining antibiotic resistance of planktonic bacterial cultures 1. This assay entails mixing a diluted planktonic culture with a dilution series of antibiotic.&#160; The concentration of antibiotic that inhibits the visible growth of the planktonic cells is the MIC. Since this assay relies on inhibition of growth, by definition, it cannot work with biofilm cultures, which requires examining the antibiotic sensitivity of cells in a pregrown biofilm. Instead of measuring growth inhibition, the MBC-B assay described here determines the concentration of antibiotic that kills cells already existing in a biofilm. Thus, this assay aims to mimic antibiotic treatments of established biofilm infections, and provide a more relevant view of the bacterial antibiotic resistance in vivo.
Since biofilms are generally more antibiotic resistant than planktonic cultures 2-4 , it was necessary to devise a method that directly relates the antibiotic resistance of a biofilm to that of a planktonic culture. Thus another goal of this method is to be able to directly compare the level of antibiotic resistance between planktonic and biofilm cells. The MBC-P and MBC-B assays described here make this possible because cells are cultured under similar conditions. We have utilized this method to study several genes that are important for biofilm-specific antibiotic resistance 5-8 .

<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td style="width:160px;">
			1x&#160;M63
			</td>
			<td style="width:160px;">
			
			</td>
			<td style="width:160px;">
			
			</td>
			<td style="width:160px;">
			Prepare as a 5x&#160;M63 stock by dissolving 15g KH2PO4, 35g K2HPO4 and 10g (NH4)2SO4 in 1 L of water. This stock does not need to be autoclaved and can be stored at room temperature.&#160; Dilute 5x&#160;stock 1:5, autoclave, cool, then add the desired components.
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			KH2PO4
			</td>
			<td style="width:160px;">
			Fisher
			</td>
			<td style="width:160px;">
			P285-500
			</td>
			<td style="width:160px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			K2HPO4
			</td>
			<td style="width:160px;">
			Fisher
			</td>
			<td style="width:160px;">
			P288-500
			</td>
			<td style="width:160px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			(NH4)2SO4
			</td>
			<td style="width:160px;">
			Sigma
			</td>
			<td style="width:160px;">
			A5132
			</td>
			<td style="width:160px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			Magnesium sulfate
			</td>
			<td style="width:160px;">
			Fisher
			</td>
			<td style="width:160px;">
			M63-500
			</td>
			<td style="width:160px;">
			Add to 1 mM final concentration.&#160; Prepare as a 1 M stock in water and autoclave.
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			Tobramycin
			</td>
			<td style="width:160px;">
			Sigma
			</td>
			<td style="width:160px;">
			
			</td>
			<td style="width:160px;">
			Prepare 50 mg/m&#160;stock. Aliquot and store at -20&#176;C.
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			Arginine
			</td>
			<td style="width:160px;">
			Sigma
			</td>
			<td style="width:160px;">
			A5131
			</td>
			<td style="width:160px;">
			Add to 0.4% final concentration.&#160; Prepare as a 20% stock in water and filter sterilize.&#160; This alternative carbon/energy source can replace glucose and casamino acids
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			96-well microtiter plates
			</td>
			<td style="width:160px;">
			Corning
			</td>
			<td style="width:160px;">
			3595
			</td>
			<td style="width:160px;">
			Sterile, flat-bottom, low evaporation
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			Tranferpette (multichannel pipette)
			</td>
			<td style="width:160px;">
			BrandTech
			</td>
			<td style="width:160px;">
			2703610
			</td>
			<td style="width:160px;">
			8-channel, 20-200 &#956;l
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			Multiprong device
			</td>
			<td style="width:160px;">
			Dan-Kar
			</td>
			<td style="width:160px;">
			MC48
			</td>
			<td style="width:160px;">
			48 prongs fit into &#189; of a 96-well microtiter plate
			</td>
		</tr>
	</tbody>
</table>

establishing the minimal bactericidal concentration of an antimicrobial agent for planktonic cells (mbc-p) and biofilm cells (mbc-b)

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro. Bacteria are inoculated into the wells of a 96-well microtiter plate. In the case of the planktonic assay, serial dilutions of the antimicrobial agent of choice are added to the bacterial suspensions. In the biofilm assay, once inoculated, the bacteria are left to form a biofilm over a set period of time. Unattached cells are removed from the wells, the media is replenished and serial dilutions of the antimicrobial agent of choice are added. After exposure to the antimicrobial agent, the planktonic cells are assayed for growth. For the biofilm assay, the media is refreshed with fresh media lacking the antimicrobial agent and the biofilm cells are left to recover. Biofilm cell viability is assayed after the recovery period. The MBC-P for the antimicrobial agent is defined as the lowest concentration of drug that kills the cells in the planktonic culture.&#160; In contrast, the MBC-B for a strain is determined by exposing preformed biofilms to increasing concentrations of antimicrobial agent for 24 hr. The MBC-B is defined as the lowest concentration of antimicrobial agent that kills the cells in the biofilm.

MBC-P and MBC-B assays were carried out, comparing the sensitivity of PA14 wild type with PA14 &#8710;ndvB. Tobramycin was used as the antibiotic. Results corresponding to step 1.4.4 (Figure 1) and step 2.3.4 (Figure 2) are presented. PA14 and &#8710;ndvB were inoculated into the MBC-P and MBC-B assays in triplicate. After completing steps 1.0-1.4 of the MBC-B protocol and steps 2.0-2.3 of the MBC-P protocol, the viable cells were plated onto an LB agar plate. Concentrations of tobramycin (&#956;g/ml) are indicated to the left of the cells. The MBC-B for PA14 is 100 &#956;g/ml&#160;and the MBC-B for &#8710;ndvB is 12.5 &#956;g/ml. The MBC-P for both PA14 and the &#8710;ndvB mutant is 8 &#956;g/ml.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50854/50854fig1highres.jpg" src="/files/ftp_upload/50854/50854fig1.jpg" /> 
Figure 1.&#160; Representative results from an MBC-B assay with PA14 wild type vs. PA14 &#8710;ndvB and tobramycin. Values refer to the final concentration of tobramycin (&#956;g/ml).
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50854/50854fig2highres.jpg" src="/files/ftp_upload/50854/50854fig2.jpg" /> 
Figure 2.&#160; Representative results from an MBC-P assay with PA14 wild type vs. PA14 &#8710;ndvB and tobramycin. Values refer to the final concentration of tobramycin (&#956;g/ml).

Watch this Scientific Journal Video about Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B at JoVE.com

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro using a 96-well microtiter plate. Planktonic or biofilm bacteria are exposed to serial dilutions of the antimicrobial agent of choice. Viability is assayed by growth on agar plates.

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells (MBC-P) and Biofilm Cells (MBC-B)

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro. Bacteria ...

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Universidad San Sebastian

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JoVE Journal

Immunology and Infection

19.9K Views.  University of Ottawa. This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro using a 96-well microtiter plate. Planktonic or biofilm bacteria are exposed to serial dilutions of the antimicrobial agent of choice. Viability is assayed by growth on agar plates.

Video: Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key players in health and disease, acting as phagocytes during immune defense and mediating trophic, maintenance, and repair functions. Although it has been possible to study some of the molecular processes involved in human macrophage function, it has proved difficult to apply genetic engineering techniques to primary human macrophages. This has significantly hampered our ability to interrogate the complex genetic pathways involved in macrophage biology and to generate models for specific disease states. An off-the-shelf source of human macrophages that is amenable to the vast arsenal of genetic manipulation techniques would, therefore, provide a valuable tool in this field. We present an optimized protocol that allows for the generation of macrophages from human induced pluripotent stem cells (iPSCs) in vitro. These iPSC-derived macrophages (iPSC-DMs) express human macrophage cell surface markers, including CD45, 25F9, CD163, and CD169, and our live-cell imaging functional assay demonstrates that they exhibit robust phagocytic activity. Cultured iPSC-DMs can be activated to different&#160;macrophage states that display altered gene expression and phagocytic activity by the addition of LPS and IFNg, IL4, or IL10. Thus, this system provides a platform to generate human macrophages carrying genetic alterations that model specific human disease and a source of cells for drug screening or cell therapy to treat these diseases.

This protocol describes the robust generation of macrophages from human induced pluripotent stem cells, and methods for their subsequent characterization. Cell surface marker expression, gene expression, and functional assays are used to assess the phenotype and function of these iPSC-derived macrophages.

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key ...

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to animal infections in vivo. The existing in vitro tests typically overestimate antimicrobial efficacy as the presence of host tissue as a diffusion barrier is not accounted for. To overcome this bottleneck, we have developed an ex vivo porcine corneal model of bacterial keratitis using Pseudomonas aeruginosa as a prototypic organism. This article describes the preparation of the porcine cornea and protocol for establishment of the infection. Bespoke glass molds enable straightforward setup of the cornea for infection studies. The model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Establishment of infection is verified as an increase in the number of colony forming units assessed via viable plate counts. The results demonstrate that infection can be established in a highly reproducible fashion in the ex vivo corneas using the method described here. The model can be extended in the future to mimic keratitis caused by microorganisms other than P. aeruginosa. The ultimate aim of the model is to investigate the effect of antimicrobial chemotherapy on the progress of bacterial infection in a scenario more representative of in vivo infections. In so doing, the model described here will reduce the use of animals for testing, improve success rates in clinical trials and ultimately enable rapid translation of novel antimicrobials to the clinic.

This article describes a step-by-step protocol to set up an ex vivo porcine model of bacterial keratitis. Pseudomonas aeruginosa is used as a prototypic organism. This innovative model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue.

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to ...