This study was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and Teaching Reform Research Project of China Pharmaceutical University (2019XJYB18).

1. Preparation of semisolid medium
<ol>
	<li>Traditional semisolid agar
	<ol>
		<li>Prepare the traditional semisolid agar according to the bacterial motility test medium recipe using the basic ingredients11. Dissolve 10 g of Tryptose, 15 g of NaCl, 4 g of agar in enough distilled water, adjust the pH to 7.2 &#177; 0.2, and make up the final volume to 1,000 mL.</li>
		<li>Autoclave the agar at 121 &#176;C for 20 min, and dispense it into 10 mL test tubes as a 3-cm high semisolid medium.</li>
	</ol>
	</li>
	<li>Traditional semisolid agar with TTC
	<ol>
		<li>After autoclaving the conventional semisolid medium, cool it to 50 &#176;C, add 5 mL of sterile 1% TTC solution to 100 mL of medium, mix, and dispense it into 10 mL test tubes to form a 3-cm-high semisolid medium.</li>
	</ol>
	</li>
</ol>
2. Bacterial strains
NOTE: Eighty strains were isolated from the aquatic environment and identified using an automated bacteria identification instrument (see the Table of Materials), including Escherichia coli, Pseudomonas aeruginosa, Salmonella spp., Vibrio spp., Klebsiella pneumoniae, and Aeromonas hydrophila (Table 1). Staphylococcus aureus (see the Table of Materials) was used as a negative nonmotile control; Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium (see the Table of Materials) were used as positive control strains.
<ol>
	<li>Identify test bacterial strains to be used for motility analysis.</li>
	<li>Include negative nonmotile controls and motile positive control strains.</li>
</ol>
3. TTC-enhanced bacterial motility observation
<ol>
	<li>Pick single colonies of test bacteria from agar plates and inoculate them into the above two semisolid media (steps 1.1.2 and 1.2.1) by puncture using inoculating needles.</li>
	<li>Culture the tubes at 37 &#176;C in the incubator for 24-48 h to observe the results.</li>
	<li>Observe the growth state: characterize the bacteria as nonmotile (-) if only the puncture line is red. Characterize the bacteria as motile (+) if the red color spreads lightly outward along the puncture line12.</li>
</ol>
4. Effect of different agar concentrations on bacterial motility
<ol>
	<li>Prepare semisolid media containing 0.3%, 0.5%, and 0.8% agar and inoculate them by puncture, as described above. Observe the results after 24-48 h of incubation.</li>
</ol>

<ol>
	<li>Jordan, E. O., Caldwell, M. E., Reiter, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+motility.">Bacterial motility.</a> Journal of Bacteriology. 27 (2), 165 (1934).</li><li>Lai, S. L., Hou, H., Jiang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+motility+and+its+role+during+initial+stage+of+pathogenesis.">Bacterial motility and its role during initial stage of pathogenesis.</a> Journal of Microbiology. 26 (5), 68-70 (2006).</li><li>Ding, S. S., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+flagella-dependent+motility+and+biofilm+in+bacteria+-+A+review.">Relationship between flagella-dependent motility and biofilm in bacteria - A review.</a> Acta Microbiologica Sinica. 49 (4), 417-422 (2009).</li><li>Zeng, J., Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+mechanism+of+bacterial+resistance+and+tolerance.">Recent advances in the mechanism of bacterial resistance and tolerance.</a> Chinese Journal of Antibiotics. 45 (2), 113-121 (2020).</li><li>Xu, M., Zhou, M. X., Zhu, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+the+mechanism+of+bacterial+flagellum+motility,+adhesion+and+immune+escape.">Progress in the mechanism of bacterial flagellum motility, adhesion and immune escape.</a> Chinese Journal of Veterinary Science. 37 (2), 369-375 (2017).</li><li>Leboffe, M. J., Pierce, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microbiology: laboratory theory and application. Third edition. , (2015).</li><li>Ball, R. J., Sellers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+motility+medium.">Improved motility medium.</a> Applied Microbiology. 14, 670-673 (1966).</li><li>An, S., Wu, J., Zhang, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Pseudomonas+aeruginosa+biofilm+dispersal+by+a+cyclic-di-GMP+phosphodiesterase+with+a+putative+hypoxia-sensing+domain.">Modulation of Pseudomonas aeruginosa biofilm dispersal by a cyclic-di-GMP phosphodiesterase with a putative hypoxia-sensing domain.</a> Applied and Environmental Microbiology. 76 (24), 8160-8173 (2010).</li><li>Chouhan, O. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+site-directed+mutagenesis+at+the+GGEEF+domain+of+the+biofilm+forming+GGEEF+protein+from+Vibrio+cholerae.">Effect of site-directed mutagenesis at the GGEEF domain of the biofilm forming GGEEF protein from Vibrio cholerae.</a> AMB Express. 6 (1), 2 (2016).</li><li>McLaughlin, 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=Simple+colorimetric+microplate+test+of+phage+lysis+in+Salmonella+enterica.">Simple colorimetric microplate test of phage lysis in Salmonella enterica.</a> Journal of Microbiological Methods. 69 (2), 394-398 (2007).</li><li>Difco Laboratories. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Difco+manual:+Dehydrated+culture+media+and+reagents+for+microbiology.">Difco manual: Dehydrated culture media and reagents for microbiology.</a> Difco Laboratories. , (1984).</li><li>Tittsler, R. P., Sandholzer, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+semi-solid+agar+for+the+detection+of+bacterial+motility.">The use of semi-solid agar for the detection of bacterial motility.</a> Journal of Bacteriology. 31 (6), 575 (1936).</li><li>Qian, Y., Tian, X. Y., Zhang, S. Y., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explore+the+influencing+factors+of+bacterial+motility.">Explore the influencing factors of bacterial motility.</a> Health Care Today. 6, 50-51 (2018).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamentous+Phytophthora+pathogens+deploy+effectors+to+interfere+with+bacterial+growth+and+motility.">Filamentous Phytophthora pathogens deploy effectors to interfere with bacterial growth and motility.</a> Frontiers in Microbiology. 11, 581511 (2020).</li><li>K&#252;hn, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+arrangement+of+several+flagellins+within+bacterial+flagella+improves+motility+in+different+environments.">Spatial arrangement of several flagellins within bacterial flagella improves motility in different environments.</a> Nature Communication. 9 (1), 5369 (2018).</li><li>Mitchell, A. J., Wimpenny, J. W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+agar+concentration+on+the+growth+and+morphology+of+submerged+colonies+of+motile+and+non-motile+bacteria.">The effects of agar concentration on the growth and morphology of submerged colonies of motile and non-motile bacteria.</a> Journal of Applied Microbiology. 83 (1), 76-84 (2010).</li><li>Xu, A., Zhang, M., Du, W., Wang, D., Ma, L. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+mechanism+for+how+sigma+factor+AlgT+and+transcriptional+regulator+AmrZ+inhibit+twitching+motility+in+Pseudomonas+aeruginosa.">A molecular mechanism for how sigma factor AlgT and transcriptional regulator AmrZ inhibit twitching motility in Pseudomonas aeruginosa.</a> Environmental Microbiology. 23 (2), 572-587 (2021).</li><li>Bartley, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attachment+and+invasion+of+Neisseria+meningitidis+to+host+cells+is+related+to+surface+hydrophobicity,+bacterial+cell+size+and+capsule.">Attachment and invasion of Neisseria meningitidis to host cells is related to surface hydrophobicity, bacterial cell size and capsule.</a> PLoS One. 8, 55798 (2013).</li></ol>

The authors have no conflicts of interest to disclose.

The detection of bacterial motility by the semisolid medium method is affected by many factors13,14. Bacterial growth conditions, such as oxygen (aerobic on agar surface, nonaerobic at the bottom of the tube with the semisolid medium), pH, and temperature, can affect the viability of bacterial flagella, which can lead to reduced motility or even loss of motility15. In addition, some mucus-type bacteria as their motility can be affected by the production of podoconjugates. 
The addition of TTC to the semisolid medium helps the observation of motility. The fermentative bacteria with motive power can grow in all directions along the puncture line after incubation. Hence, the medium around the puncture line becomes red. The nonfermentative bacteria with motive power have low oxygen content in the lower part of the medium. Hence, these bacteria grow poorly, so that only the upper layer of the medium is red. Bacteria without motive power can only grow on the inoculation line, and only the puncture line appears red. 
When detecting bacterial motility with TTC-semisolid medium, the longer the culture time, the more obvious are the results, especially with lower agar concentrations. If the result is difficult to interpret, the culture time should be extended appropriately. This may be related to the agar concentration of the semisolid medium, the number of bacteria, and their motility16. In addition, this method revealed that some strains of E. coli and P. aeruginosa could not grow around on conventional semisolid agar medium and showed only a faint red color on the surface of the medium on TTC semisolid agar medium. This may be due to the production of capsules by such strains, which affects their motility17. This phenomenon also occurs in Neisseria meningitidis18 because of its capsule. In conclusion, the detection of bacterial motility using a chromogenic semisolid medium containing TTC reduces the influence of bacterial factors on the test results and makes the results easier to observe with the naked eye. The advantage of a high detection rate makes this an effective method that can replace the traditional semisolid medium for detecting bacterial motility.

Bacterial motility plays a critical role in bacterial pathogenicity, biofilm formation, and drug resistance1. Bacterial motility is closely associated with pathogenicity and is necessary for bacterial colonization during early infection of host cells2. Biofilm formation is closely related to bacterial motility, where bacteria adhere to the surface of solid media through motility. Bacterial motility has long been considered to be positively correlated with biofilm formation. A high degree of bacterial drug resistance due to biofilm can lead to persistent infections that are a threat to human health3,4,5. Therefore, it is important to detect bacterial motility. The bacterial motility test is mainly used to examine the motility of different forms of bacteria in the living state, which can indirectly determine the presence or absence of flagella and, thus, has an important role in the identification of bacteria.
There are direct and indirect methods to detect bacterial motility6. As bacteria with flagella show motility, it is possible to detect whether bacteria are motile indirectly by detecting the presence or absence of flagella. For example, it is possible to detect motility indirectly by electron microscopy and flagellar staining to indicate that bacteria are motile. It is also possible to detect by direct methods, such as suspension drop and semisolid puncture methods. 
The semisolid puncture method commonly used in undergraduate microbiology laboratories to detect bacterial motility inoculates the bacteria into the puncture in the semisolid agar medium containing 0.4-0.8% agar, according to the direction of bacterial growth. If the bacteria grow along the puncture line to spread around, cloud-like (brush-like) growth traces appear, indicating the presence of flagella and, therefore, motility. If there are no puncture-line growth traces, the bacterium is neither flagellated nor motile. 
However, this method has its drawbacks: the bacteria are colorless and transparent, the flagellar activity is affected by the physiological characteristics of the living bacteria and other factors, and the concentration of agar and the small diameter of the test tube. Moreover, aerobic bacteria are only suitable for growth on the agar surface, affecting the observation of bacterial motility. Hence, to improve this experiment, 2,3,5-triphenyltetrazolium chloride (TTC) (colorless) was added to the medium to establish a more reliable and intuitive method of determining bacterial motility than the current direct-puncture method using intracellular dehydrogenases to catalyze the formation of a red product of TTC7,8,9,10.

<table><tbody><tr><td>Bacto Agar</td><td>Difco</td><td></td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt;</td><td>ATCC</td><td>ATCC25922</td><td>Positive control</td></tr><tr><td>&lt;em&gt;Pseudomonas aeruginosa&lt;/em&gt;</td><td>ATCC</td><td>ATCC27853</td><td>Positive control</td></tr><tr><td>&lt;em&gt;Salmonella typhimurium&lt;/em&gt;</td><td>ATCC</td><td>ATCC14028</td><td>Positive control</td></tr><tr><td>&lt;em&gt;Staphylococcus aureus&lt;/em&gt;</td><td>ATCC</td><td>ATCC25923</td><td>Negative nonmotile control</td></tr><tr><td>Tryptose</td><td>&amp;nbsp;OXOID</td><td></td><td></td></tr><tr><td>TTC</td><td>Sigma</td><td>298-96-4</td><td></td></tr><tr><td>VITEK 2 automated microbial identification system</td><td>Bio M&amp;eacute;rieux</td><td></td><td></td></tr></tbody></table>

visualizing bacterial motility based on a color reaction

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or dissemination of many pathogenic species. Therefore, it is important to detect bacterial motility. Bacterial growth conditions, such as oxygen, pH, and temperature, can affect bacterial growth and the expression of bacterial flagella. This can lead to reduced motility or even loss of motility, resulting in the inaccurate evaluation of bacterial motility. Based on the color reaction of 2,3,5-triphenyl tetrazolium chloride (TTC) by intracellular dehydrogenases of living bacteria, TTC was added to traditional semisolid agar for bacterial motility detection. The results showed that this TTC semisolid agar method for the detection of bacterial motility is simple, easy to operate, and does not involve large and expensive instruments. The results also showed that the highest motility was observed in semisolid medium prepared with 0.3% agar. Compared with the traditional semisolid medium, the results are easier to evaluate and more accurate.

Both standard strains and isolated strains were compared for motility detection, and the results are shown in Table 1. Due to the absence of flagella, Staphylococcus aureus and Klebsiella pneumoniae only grew along the inoculated line on both traditional and TTC semisolid media. In contrast, Pseudomonas aeruginosa, Escherichia coli, and Salmonella typhimurium showed growth in all directions around the inoculated line after culturing for 24 h on TTC semisolid medium. This was even more obvious after 48 h culture (Figure 1). Although the bacteria grew in all directions in the traditional semisolid medium, it was much more difficult to visualize than in TTC medium due to the small number of bacteria at the outer side of the inoculated line.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63706/63706fig01.jpg" /> 
Figure 1: Motility test results using TTC semisolid medium. Staphylococcus aureus on the left, Escherichia coli on the right. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63706/63706fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2" rowspan="2">Strain</td>
			<td colspan="2">Semisolid medium with 0.4% agar</td>
			<td colspan="2">Semisolid medium with 0.4% agar and 0.005% TTC</td>
		</tr>
		<tr>
			<td>24 h</td>
			<td>48 h</td>
			<td>24 h</td>
			<td>48 h</td>
		</tr>
		<tr>
			<td colspan="2">P. aeruginosa ATCC27853</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
		</tr>
		<tr>
			<td colspan="2">E. coli ATCC25922</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
		</tr>
		<tr>
			<td colspan="2">S. typhimurium ATCC14028</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
		</tr>
		<tr>
			<td colspan="2">S. aureus ATCC25923</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td colspan="2">E. coli (15)</td>
			<td>12</td>
			<td>14</td>
			<td>13</td>
			<td>14</td>
		</tr>
		<tr>
			<td colspan="2">Salmonella spp. (8)</td>
			<td>7</td>
			<td>8</td>
			<td>8</td>
			<td>8</td>
		</tr>
		<tr>
			<td colspan="2">A. hydrophila (20)</td>
			<td>18</td>
			<td>20</td>
			<td>20</td>
			<td>20</td>
		</tr>
		<tr>
			<td colspan="2">Vibrio spp. (8)</td>
			<td>7</td>
			<td>8</td>
			<td>8</td>
			<td>8</td>
		</tr>
		<tr>
			<td colspan="2">P. aeruginosa (24)</td>
			<td>18</td>
			<td>20</td>
			<td>22</td>
			<td>23</td>
		</tr>
		<tr>
			<td colspan="2">K. pneumoniae (5)</td>
			<td>-5</td>
			<td>-5</td>
			<td>-5</td>
			<td>-5</td>
		</tr>
		<tr>
			<td colspan="6">Numbers indicate the numbers of positive strains (+) and negative stains (-).</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of bacterial motility. 
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63706/63706fig02.jpg" /> 
Figure 2: Observation of Escherichia coli motility activity at different agar concentrations. Left to right: 0.3%, 0.5%, 0.8% agar. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63706/63706fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The influence of agar concentration on bacterial motility is shown in Figure 2. We found that the highest motility was observed in semisolid medium prepared with 0.3% agar. The medium color in the tube turned almost entirely red. In contrast, the area of red diffusion decreased, and the diffusion was prolonged with increasing agar concentration.

Watch this Scientific Journal Video about Visualizing Bacterial Motility Based on a Color Reaction at JoVE.com

Visualizing Bacterial Motility Based on a Color Reaction

Here, we present a protocol to detect bacterial motility based on a color reaction. Key advantages of this method are that it is easy to evaluate and more accurate, and does not require specialized equipment.

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or ...

visualizing-bacterial-motility-based-on-a-color-reaction

Nanyang Technological University

Research

JoVE Journal

Biology

3.8K Views.  China Pharmaceutical University. Here, we present a protocol to detect bacterial motility based on a color reaction. Key advantages of this method are that it is easy to evaluate and more accurate, and does not require specialized equipment.

Video: Visualizing Bacterial Motility Based on a Color Reaction

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of induced swarming motility, specific concentration&#160;of an inducer may not be able to report events occurring within the optimal concentration range to elicit the desired responses from a species. Semisolid plates containing multiple concentrations are commonly used to investigate the response within an inducer concentration range. However, separate semisolid plates increase variations in medium viscosity and moisture content within each plate due to nonuniform solidification time.
This paper describes a one-step method to simultaneously test surface swarming motility on a single gradient plate, where the isometrically arranged test wells allow the simultaneous acquisition of multiconcentration responses. In the present work, the surface swarming of Escherichia coli K12 and Pseudomonas aeruginosa PAO1 were evaluated in response to a concentration gradient of inducers such as resveratrol and arabinose. Periodically, the swarm morphologies were imaged using an imaging system to capture the entire surface swarming process.
The quantitative measurement of the swarm morphologies was acquired using ImageJ software, providing analyzable information of the swarm area. This paper presents a simple gradient swarm plate method that provides qualitative and quantitative information about the inducers&#39; effects on surface swarming, which can be extended to study the effects of other inducers on a broader range of motile bacterial species.

Here, we describe the use of inducer gradient plates to evaluate bacterial swarming motility while simultaneously obtaining multiple concentration responses.

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of ...

Biochemistry

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The bi-directional mitotic kinesin-5 Cin8 accumulates in clusters that split and merge during their motility. Accumulation in clusters also changes the velocity and directionality of Cin8. Here, a protocol for motility assays with purified Cin8-GFP and analysis of motile properties of single molecules and clusters of Cin8 is described.

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Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

Bioengineering

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 ...

Immunology and Infection

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Biophysical Characterization of Flagellar Motor Functions

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel in response to a variety of environmental stimuli and are among the triggers for surface colonization and infections. The precise mechanisms by which motors remodel and promote cellular adaptation likely depend on key motor attributes. The photomultiplier-based bead-tracking technique presented here enables accurate biophysical characterization of motor functions, including adaptations in motor speeds and switch-dynamics. This approach offers the advantage of real-time tracking and the ability to probe motor behavior over extended durations. The protocols discussed can be readily extended to study flagellar motors in a variety of bacterial species.

Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel ...

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, to elucidate the function and assembly of flagellar parts, and to examine and understand patterns of movement. Here we demonstrate a combination of three of these methodologies. Motility in soft agar is the oldest, offering a strong selection for isolating gain-of-function suppressor mutations in motility-impaired strains, where motility is restored through a second mutation. The cell-tethering technique, first employed to demonstrate the rotary nature of the flagellar motor, can be used to assess the impact of signaling effectors on the motor speed and its ability to switch rotational direction. The &#8220;border-crossing&#8221; assay is more recent, where swimming bacteria can be primed to transition into moving collectively as a swarm. In combination, these protocols represent a systematic and powerful approach to identifying components of the motility machinery, and to characterizing their role in different facets of swimming and swarming. They can be easily adapted to study motility in other bacterial species.

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Many bacteria use flagella-driven motility to navigate their environment and colonize favorable surroundings both individually and as a collective. Demonstrated here is the use of three established methods that exploit motility as a selection tool to identify components/pathways contributing to swimming and swarming motility.

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, ...

Bacterial Co-Incubation Assay: A Fluorescence Microscopy-Based Technique to Visualize Intraspecific Bacterial Competition at the Single-Cell Level

Source: Smith, S., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62851">Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging.</a> J. Vis. Exp. (2021).
This video demonstrates a fluorescence microscopy-based method to study bacterial competition at the single-cell level. This method provides insights into the structure and function of bacterial communities.

Test koinkubacji bakterii: technika oparta na mikroskopii fluorescencyjnej do wizualizacji wewnątrzgatunkowej konkurencji bakteryjnej na poziomie pojedynczej komórki

Visualizing Bacterial Motility Based on a Color Reaction

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A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Biophysical Characterization of Flagellar Motor Functions

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Test koinkubacji bakterii: technika oparta na mikroskopii fluorescencyjnej do wizualizacji wewnątrzgatunkowej konkurencji bakteryjnej na poziomie pojedynczej komórki