이 연구는 장쑤 고등 교육 기관의 우선 학업 프로그램 개발(PAPD) 및 중국 약학 대학의 교육 개혁 연구 프로젝트(2019XJYB18)의 지원을 받았습니다.

1. 반고체 배지의 제조
<ol><li>전통적인 반고체 한천
	<ol><li>기본 성분11을 사용하여 박테리아 운동성 시험 배지 레시피에 따라 전통적인 반고체 한천을 준비한다. 충분한 증류수에 트립토오스 10g, NaCl 15g, 한천 4g을 녹이고 pH를 7.2 ± 0.2로 조정하고 최종 부피를 1,000mL로 구성합니다.</li>
		<li>한천을 121°C에서 20분 동안 오토클레이브하고 3cm 높이의 반고체 배지로 10mL 시험관에 분주합니다.</li>
	</ol></li>
	<li>TTC를 사용한 전통적인 반고체 한천
	<ol><li>종래의 반고체 배지를 오토클레이빙한 후 50°C로 냉각하고 100mL의 배지에 멸균된 1% TTC 용액 5mL를 넣고 혼합한 후 10mL 시험관에 분주하여 3cm 높이의 반고체 배지를 형성합니다.</li>
	</ol></li>
</ol>2. 박테리아 균주
참고: 대장균, 녹농균, 살모넬라종, 비브리오종, 폐렴간균 및 에어로모나스 하이드로필라를 포함한 80개의 균주를 수생 환경에서 분리하고 자동 박테리아 식별 기기(재료 표 참조)를 사용하여 식별했습니다(표 1). 황색포도상구균(Staphylococcus aureus)(재료 표 참조)은 음성 비운동성 대조군으로 사용되었습니다. 대장균(Escherichia coli), 녹농균(Pseudomonas aeruginosa) 및 살모넬라 티피뮤리움(Salmonella typhimurium)(재료 표 참조)을 양성 대조군 균주로 사용했습니다.
<ol><li>운동성 분석에 사용할 테스트 박테리아 균주를 식별합니다.</li>
	<li>음성 비운동성 대조군 및 운동성 양성 대조군 균주를 포함합니다.</li>
</ol>3. TTC 강화 박테리아 운동성 관찰
<ol><li>한천 플레이트에서 테스트 박테리아의 단일 콜로니를 선택하고 접종 바늘을 사용하여 구멍을 뚫어 위의 두 반고체 배지(단계 1.1.2 및 1.2.1)에 접종합니다.</li>
	<li>튜브를 37°C에서 24-48시간 동안 배양하여 결과를 관찰하였다.</li>
	<li>성장 상태 관찰: 펑크 라인만 빨간색인 경우 박테리아를 운동성이 없는(-) 특성화합니다. 붉은 색이 천공선12를 따라 바깥쪽으로 가볍게 퍼지면 박테리아를 운동성(+)으로 특성화합니다.</li>
</ol>4. 다양한 한천 농도가 박테리아 운동성에 미치는 영향
<ol><li>0.3%, 0.5%, 0.8% 한천을 함유하는 반고체 배지를 준비하고, 상기와 같이 천자에 의해 접종한다. 배양 24-48 시간 후에 결과를 관찰하십시오.</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>

저자는 공개할 이해 상충이 없습니다.

반고체 배지 방법에 의한 박테리아 운동성의 검출은 많은 요인에 의해 영향을 받는다13,14. 산소(한천 표면의 호기성, 반고체 배지가 있는 튜브 바닥의 비호기성), pH 및 온도와 같은 박테리아 성장 조건은 박테리아 편모의 생존력에 영향을 미칠 수 있으며, 이는 운동성 감소 또는 운동성 상실로 이어질 수 있다15. 또한, 일부 점액형 박테리아는 이들의 운동성이 podoconjugates의 생성에 의해 영향을 받을 수 있다.
반고체 배지에 TTC를 첨가하면 운동성 관찰에 도움이 됩니다. 원동력을 가진 발효 박테리아는 배양 후 펑크 라인을 따라 모든 방향으로 자랄 수 있습니다. 따라서 펑크 라인 주변의 매체가 빨간색으로 변합니다. 원동력을 가진 비발효성 박테리아는 배지 하부의 산소 함량이 낮습니다. 따라서 이러한 박테리아는 잘 자라지 않아 배지의 상층만 빨간색입니다. 원동력이 없는 세균은 접종선에서만 자랄 수 있고, 천자선만 빨갛게 보입니다. 
TTC-반고체 배지로 박테리아 운동성을 검출할 때 배양 시간이 길수록 특히 한천 농도가 낮을수록 결과가 더 명확해집니다. 결과를 해석하기 어려운 경우 배양 시간을 적절하게 연장해야 합니다. 이것은 반고체 배지의 한천 농도, 박테리아의 수 및 운동성과 관련이 있을 수 있다16. 또한, 이 방법은 대장 균과 녹농균의 일부 균 주가 기존의 반고체 한천 배지에서 자랄 수 없었고 TTC 반고체 한천 배지에서 배지 표면에 희미한 붉은 색만 나타났음을 밝혀냈습니다. 이것은 운동성에 영향을 미치는 그러한 균주에 의한 캡슐의 생산 때문일 수 있습니다17. 이 현상은 캡슐 때문에 Neisseria meningitidis18 에서도 발생합니다. 결론적으로, TTC를 함유한 발색 반고체 배지를 이용한 박테리아 운동성 검출은 박테리아 요인이 검사 결과에 미치는 영향을 줄이고 결과를 육안으로 더 쉽게 관찰할 수 있도록 합니다. 높은 검출률의 장점은 박테리아 운동성을 검출하기 위한 기존의 반고체 배지를 대체할 수 있는 효과적인 방법입니다.

박테리아 운동성은 박테리아 병원성, 생물막 형성 및 약물 내성에 중요한 역할을 합니다1. 박테리아 운동성은 병원성과 밀접한 관련이 있으며, 숙주 세포의 초기 감염 동안 박테리아 집락화에 필요하다2. 생물막 형성은 박테리아 운동성과 밀접한 관련이 있으며, 박테리아는 운동성을 통해 고체 배지의 표면에 부착됩니다. 박테리아의 운동성은 오랫동안 생물막 형성과 양의 상관관계가 있는 것으로 여겨져 왔습니다. 생물막으로 인한 높은 수준의 박테리아 약물 내성은 인체 건강에 위협이되는 지속적인 감염을 유발할 수 있습니다 3,4,5. 따라서 박테리아의 운동성을 감지하는 것이 중요합니다. 박테리아 운동성 검사는 주로 살아있는 상태에서 다양한 형태의 박테리아의 운동성을 검사하는 데 사용되며, 이는 편모의 유무를 간접적으로 결정할 수 있으므로 박테리아 식별에 중요한 역할을 합니다.
박테리아의 운동성을 감지하는 직간접적인 방법이 있다6. 편모를 가진 박테리아는 운동성을 나타내기 때문에 편모의 유무를 감지하여 박테리아가 운동성인지 여부를 간접적으로 검출할 수 있습니다. 예를 들어, 전자 현미경 및 편모 염색에 의해 간접적으로 운동성을 감지하여 박테리아가 운동성이 있음을 나타낼 수 있습니다. 서스펜션 드롭 및 반고체 펑크 방법과 같은 직접적인 방법으로 감지하는 것도 가능합니다. 
세균 운동성을 감지하기 위해 학부 미생물학 실험실에서 일반적으로 사용되는 반고체 천자 방법은 세균 성장 방향에 따라 0.4-0.8% 한천을 함유한 반고체 한천 배지에 박테리아를 천자 내로 접종합니다. 박테리아가 펑크 라인을 따라 자라서 주변으로 퍼지면 구름 모양 (브러시 같은) 성장 흔적이 나타나 편모의 존재와 운동성을 나타냅니다. 펑크 라인 성장 흔적이 없으면 박테리아는 편모도 운동성도 없습니다. 
그러나이 방법은 박테리아가 무색이고 투명하며 편모 활성은 살아있는 박테리아 및 기타 요인의 생리적 특성과 한천의 농도 및 시험관의 작은 직경에 의해 영향을받는 단점이 있습니다. 또한, 호기성 박테리아는 한천 표면에서의 성장에만 적합하여 박테리아 운동성 관찰에 영향을 미칩니다. 따라서이 실험을 개선하기 위해 2,3,5- 트리 페닐 테트라 졸륨 클로라이드 (TTC) (무색)를 배지에 첨가하여 TTC 7,8,9,10의 적색 생성물 형성을 촉매하기 위해 세포 내 탈수소 효소를 사용하는 현재의 직접 천자 방법보다 박테리아 운동성을 결정하는 더 신뢰할 수 있고 직관적 인 방법을 확립했습니다.

<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

박테리아 운동성은 박테리아 병원성, 생물막 형성 및 약물 내성에 매우 중요합니다. 박테리아 운동성은 많은 병원성 종의 침입 및/또는 보급에 매우 중요합니다. 따라서 박테리아의 운동성을 감지하는 것이 중요합니다. 산소, pH 및 온도와 같은 박테리아 성장 조건은 박테리아 성장과 박테리아 편모의 발현에 영향을 줄 수 있습니다. 이것은 운동성을 감소시키거나 심지어 운동성을 상실하여 박테리아 운동성에 대한 부정확한 평가를 초래할 수 있습니다. 살아있는 박테리아의 세포 내 탈수소효소에 의한 2,3,5-트리페닐 테트라졸륨 클로라이드(TTC)의 색상 반응을 기반으로 박테리아 운동성 검출을 위해 전통적인 반고체 한천에 TTC를 첨가했습니다. 그 결과 박테리아 운동성 검출을 위한 이 TTC 반고체 한천 방법이 간단하고 작동하기 쉬우며 크고 값비싼 기구가 필요하지 않다는 것을 보여주었습니다. 그 결과, 0.3% 한천으로 제조된 반고체 배지에서 가장 높은 운동성이 관찰되는 것으로 나타났다. 기존의 반고체 매체와 비교하여 결과를 평가하기 쉽고 정확합니다.

운동성 검출을 위해 표준 균주와 분리된 균주 모두 비교하였으며, 그 결과를 표 1에 나타내었다. 편모가 없기 때문에 황색포도상구균과 폐렴간균은 전통적인 배지와 TTC 반고체 배지 모두에서 접종된 라인을 따라서만 성장했습니다. 대조적으로, 녹농균(Pseudomonas aeruginosa), 대장균(Escherichia coli) 및 살모넬라 티피뮤리움(Salmonella typhimurium)은 TTC 반고체 배지 상에서 24시간 동안 배양한 후 접종된 라인 주위의 모든 방향에서 성장을 보였다. 이는 48시간 배양 후에 더욱 분명해졌습니다(그림 1). 박테리아는 전통적인 반고체 배지에서 모든 방향으로 성장했지만 접종 라인의 바깥쪽에 박테리아 수가 적기 때문에 TTC 배지보다 시각화하기가 훨씬 더 어려웠습니다.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63706/63706fig01.jpg"> 그림 1: TTC 반고체 배지를 사용한 운동성 테스트 결과. 왼쪽은 황색포도상구균, 오른쪽은 대장균입니다. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63706/63706fig01large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2" rowspan="2">거르다</td>
			<td colspan="2">0.4% 한천을 함유한 반고체 배지</td>
			<td colspan="2">0.4% 한천 및 0.005% TTC를 갖는 반고체 배지</td>
		</tr><tr><td>24 시간</td>
			<td>48시간</td>
			<td>24 시간</td>
			<td>48시간</td>
		</tr><tr><td colspan="2">녹농균(P. aeruginosa) ATCC27853 시리즈</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
		</tr><tr><td colspan="2">대장균 ATCC25922 시리즈</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
		</tr><tr><td colspan="2">S. 티피 뮤리움 ATCC14028 시리즈</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
		</tr><tr><td colspan="2">S. 아우레우스 ATCC25923 시리즈</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr><tr><td colspan="2">대장균 (15)</td>
			<td>12</td>
			<td>14</td>
			<td>13</td>
			<td>14</td>
		</tr><tr><td colspan="2">살모넬라 종 (8)</td>
			<td>7</td>
			<td>8</td>
			<td>8</td>
			<td>8</td>
		</tr><tr><td colspan="2">A. 친수성 (20)</td>
			<td>18</td>
			<td>20</td>
			<td>20</td>
			<td>20</td>
		</tr><tr><td colspan="2">비브리오 종 (8)</td>
			<td>7</td>
			<td>8</td>
			<td>8</td>
			<td>8</td>
		</tr><tr><td colspan="2">녹농균 (24)</td>
			<td>18</td>
			<td>20</td>
			<td>22</td>
			<td>23</td>
		</tr><tr><td colspan="2">K. 폐렴 (5)</td>
			<td>-5</td>
			<td>-5</td>
			<td>-5</td>
			<td>-5</td>
		</tr><tr><td colspan="6">숫자는 양성 균주(+)와 음성 얼룩(-)의 수를 나타냅니다.</td>
		</tr></tbody></table>표 1: 박테리아 운동성 비교. 
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63706/63706fig02.jpg"> 그림 2: 다양한 한천 농도에서 대장균 운동성 활성 관찰. 왼쪽에서 오른쪽으로 : 0.3 %, 0.5 %, 0.8 % 한천. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63706/63706fig02large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>
박테리아 운동성에 대한 한천 농도의 영향은 그림 2에 나와 있습니다. 우리는 0.3% 한천으로 제조된 반고체 배지에서 가장 높은 운동성이 관찰되었음을 발견했습니다. 튜브의 중간 색상은 거의 완전히 빨간색으로 변했습니다. 대조적으로, 적색 확산 면적은 감소하고, 한천 농도가 증가함에 따라 확산이 연장되었다.

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

여기에서는 색상 반응을 기반으로 박테리아 운동성을 감지하는 프로토콜을 제시합니다. 이 방법의 주요 장점은 평가하기 쉽고 정확하며 특수 장비가 필요하지 않다는 것입니다.

색상 반응을 기반으로 한 박테리아 운동성 시각화

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

Universidad San Sebastian

Research

JoVE Journal

Biology

3.8K 조회수.  China Pharmaceutical University. 여기에서는 색상 반응을 기반으로 박테리아 운동성을 감지하는 프로토콜을 제시합니다. 이 방법의 주요 장점은 평가하기 쉽고 정확하며 특수 장비가 필요하지 않다는 것입니다.

비디오: 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.

안정적인 농도 기울기에 박테리아 Chemotaxis을 Quantifying위한 Microfluidic 장치

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

연구

생물학

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.

유도기 그라디언트 플레이트의 박테리아 표면 떼 운동성 정량화

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

생화학

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.

S. cerevisiae 세포로부터 정제된 양방향 Kinesin-5 Cin8의 단일 분자 및 클러스터의 이동성

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of ...

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.

미생물 행동을 연구하기 위한 제어된 동적 화학 적 경관 생성

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

생체공학

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.

형광 항생제 프로브를 사용한 세균 저항성 시각화

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

면역학 및 감염병학

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.

단세포 종 간 세균성 상호 작용의 운동 시각화

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.

세균 표면 운동성 분석의 준비, 이미징 및 정량화

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.

편모 모터 기능의 생물 물리학 적 특성

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.

에슈리치아 대장균에서 모토렐라 구동 운동성 조사

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.bdigitaluss.remotexs.co/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.