Name: visualizing bacterial motility based on a color reaction
Uploaded: 2022-02-15T15:00:00
Description: Watch this Scientific Journal Video about Visualizing Bacterial Motility Based on a Color Reaction at JoVE.com

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-...

<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 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 ...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bacto Agar</td>
			<td>Difco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli</td>
			<td>ATCC</td>
			<td>ATCC25922</td>
			<td>Positive control</td>
		</tr>
		<tr>
			<td>Pseudomonas aeruginosa</td>
			<td>ATCC</td>
			<td>ATCC27853</td>
			<td>Positive control</td>
		</tr>
		<tr>
			<td>Salmonella typhimurium</td>
			<td>ATCC</td>
			<td>ATCC14028</td>
			<td>Positive control</td>
		</tr>
		<tr>
			<td>Staphylococcus aureus</td>
			<td>ATCC</td>
			<td>ATCC25923</td>
			<td>Negative nonmotile control</td>
		</tr>
		<tr>
			<td>Tryptose</td>
			<td>&#160;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&#233;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 m...

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 ...

Detection of bacterial motility is important because motility is crucial for pathogenicity. The protocol described here is a reliable method for the detection of bacterial motility, because it is based on the color reaction of TTC with intracellular dehydrogenases of living bacteria. The TTC semi-solid agar method for detecting bacterial motility is simple, easy to operate and does not involve large and expensive instruments.The method can be used by clinical microbiologists or researchers to investigate pathogenic bacteria. Demonstrating the procedure will be Xiyi Zhuang, a senior experimentalist from my laboratory. To begin, prepare the traditional semi-solid medium by adding 10 grams of Tryptose, 15 grams of sodium chloride and four grams of agar in an adequate volume of distilled water.Adjust the pH to 7.2 and make up the final volume to 1, 000 milliliters. Autoclave the medium at 121 degrees Celsius for 20 minutes and dispense the medium into 10 milliliter test tubes to a height of three centimeters. To prepare the TTC medium, cool the autoclaved traditional medium to 50 degrees Celsius then add five milliliters of sterile 1%TTC solution to 100 milliliters of medium.Mix and dispense the medium into 10 milliliter test tubes to a height of three centimeters. Pick single colonies of bacteria from agar plates and inoculate them into both the semi-solid media by puncture using inoculating needles. Remember to include non-modal strains as negative controls and modal strains as positive controls.To observe the effect of agar concentration on bacterial motility, prepare semi-solid media containing different agar concentrations and inoculate them by puncture. Incubate the tubes at 37 degrees Celsius and observe the results after 24 to 48 hours of incubation. If only the puncture line is red, characterize the bacteria as non-modal.If the red color spreads outward along the puncture line, characterize the bacteria as modal. Bacterial motility was compared for various strains on traditional and TTC semi-solid media and summarized in this table. After culturing for 24 hours on TTC semi-solid medium, Staphylococcus aureus showed growth along the inoculated line.Whereas, Escherichia coli showed growth in all directions around the inoculated line. Highest motility for Escherichia coli was observed in TTC semi-solid medium prepared with 0.3%agar in which the medium turned almost entirely red. In contrast, the area of red diffusion decreased in 0.5%agar and the diffusion was prolonged with increasing agar concentration to 0.8%It is important to adjust the pH to 7.2 and cool the autoclaved medium to 50 degrees Celsius before inoculation using the needles.Bacteria should be inoculated by puncture into the semi-solid medium containing TTC. It is a direct method for detecting bacterial motility. It is also possible to detect motility indirectly by electronic microscope and

Research

JoVE Journal

Biology

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Visualizing RNA Localization in Xenopus Oocytes

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Gastrointestinal Motility Monitor (GIMM)

Gastrointestinal Motility Monitor GIMM

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

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 ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. ...