Name: Forfatter i søkelyset: Fremme forskning innen mikrobiell autoaggregering ved bruk av avbildningsstrømningscytometri
Uploaded: 2023-09-29T14:00:00
Description: Read this Scientific Article Protocol about Author Spotlight: Advancing Research in Microbial Auto Aggregation Using Imaging Flow Cytometry at JoVE.com

Dette arbeidet ble støttet av Israeli Science Foundation (Grant 119/16) og IMoh-stipend (3-15656) til IKG. R.S. støttet av Kreitman-stipendet.

.ast-filen, med malen for Lacticaseibacillus rhamnosus GG (LGG) som eksempel, er gitt i Supplementary Coding File 1.
1. Forberedelse av media
<ol><li>Tilbered Lactobacilli MRS-buljong i henhold til produsentens instruksjoner (55 g i 1 L avionisert vann) og Lactobacilli MRS-agarplater med 1.5 % (vekt/volum) agar (se materialtabell). Etter autoklavsterilisering er mediet klart til bruk direkte eller kan lagres ved 4 °C.</li>
	<li>Til...

Kvantitativ analyse av mikrobiell autoaggregering ved bruk av avbildningsstrømningscytometri

<ol>
	<li>Trunk, T., Khalil, H. S., Leo, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+autoaggregation.">Bacterial autoaggregation.</a> AIMS Microbiology. 4 (1), 140-164 (2018).</li><li>Ishikawa, M., Nakatani, H., Hori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AtaA,+a+new+member+of+the+trimeric+autotransporter+adhesins+from+Acinetobacter+sp.+Tol+5+mediating+high+adhesiveness+to+various+abiotic+surfaces.">AtaA, a new member of the trimeric autotransporter adhesins from Acinetobacter sp. Tol 5 mediating high adhesiveness to various abiotic surfaces.</a> PLoS One. 7, e48830 (2012).</li><li>Inoue, T., 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=Molecular+characterization+of+low-molecular-weight+component+protein,+Flp,+in+Actinobacillus+actinomycetemcomitans+fimbriae.">Molecular characterization of low-molecular-weight component protein, Flp, in Actinobacillus actinomycetemcomitans fimbriae.</a> Medical Microbiology and Immunology. 42 (4), 253-258 (1998).</li><li>Boddey, J. A., Flegg, C. P., Day, C. J., Beacham, I. R., Peak, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-regulated+microcolony+formation+by+Burkholderia+pseudomallei+requires+pilA+and+enhances+association+with+cultured+human+cells.">Temperature-regulated microcolony formation by Burkholderia pseudomallei requires pilA and enhances association with cultured human cells.</a> Infection and Immunity. 74 (9), 5374-5381 (2006).</li><li>Suissa, R., 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=Context-dependent+differences+in+the+functional+responses+of+Lactobacillaceae+strains+to+fermentable+sugars.">Context-dependent differences in the functional responses of Lactobacillaceae strains to fermentable sugars.</a> Frontiers in Microbiology. 13, 949932 (2022).</li><li>Isenring, J., Geirnaert, A., Lacroix, C., Stevens, M. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bistable+auto-aggregation+phenotype+in+Lactiplantibacillus+plantarum+emerges+after+cultivation+in+in+vitro+colonic+microbiota.">Bistable auto-aggregation phenotype in Lactiplantibacillus plantarum emerges after cultivation in in vitro colonic microbiota.</a> BMC Microbiology. 21, 268 (2021).</li><li>Kos, B., 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=Adhesion+and+aggregation+ability+of+probiotic+strain+Lactobacillus+acidophilus+M92.">Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92.</a> Journal of Applied Microbiology. 94 (6), 981-987 (2003).</li><li>Zommiti, M., 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=In+vitro+assessment+of+the+probiotic+properties+and+bacteriocinogenic+potential+of+Pediococcus+pentosaceus+MZF16+isolated+from+artisanal+tunisian+meat+&amp;#34;Dried+Ossban&amp;#34;.">In vitro assessment of the probiotic properties and bacteriocinogenic potential of Pediococcus pentosaceus MZF16 isolated from artisanal tunisian meat &amp;#34;Dried Ossban&amp;#34;.</a> Frontiers in Microbiology. 9, 2607 (2018).</li><li>Suissa, R., 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=Metabolic+rewiring+of+the+probiotic+bacterium+rhamnosus&lt;/em&gt;+GG+contributes+to+cell-wall+remodeling+and+antimicrobials+production.">Metabolic rewiring of the probiotic bacterium rhamnosus&lt;/em&gt; GG contributes to cell-wall remodeling and antimicrobials production.</a> bioRxiv. , (2023).</li><li>Connell, J. L., Kim, J., Shear, J. B., Bard, A. J., Whiteley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+quorum+sensing+in+3D-printed+bacterial+aggregates+using+scanning+electrochemical+microscopy.">Real-time monitoring of quorum sensing in 3D-printed bacterial aggregates using scanning electrochemical microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (51), 18255-18260 (2014).</li><li>Misawa, N., Blaser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+characterization+of+autoagglutination+activity+by+Campylobacter+jejuni.">Detection and characterization of autoagglutination activity by Campylobacter jejuni.</a> Infection and Immunity. 68 (11), 6168-6175 (2000).</li><li>Sherlock, O., Schembri, M. A., Reisner, A., Klemm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+roles+for+the+AIDA+adhesin+from+diarrheagenic+Escherichia+coli:+cell+aggregation+and+biofilm+formation.">Novel roles for the AIDA adhesin from diarrheagenic Escherichia coli: cell aggregation and biofilm formation.</a> Journal of Bacteriology. 186 (23), 8058-8065 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+flow+cytometry.">Imaging flow cytometry.</a> Nature Reviews Methods Primers. 2, 87 (2022).</li><li>Wang, Y., 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=Metabolism+characteristics+of+lactic+acid+bacteria+and+the+expanding+applications+in+food+industry.">Metabolism characteristics of lactic acid bacteria and the expanding applications in food industry.</a> Frontiers in Bioengineering and Biotechnology. 9, 612285 (2021).</li><li>Turroni, F., 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=Molecular+dialogue+between+the+human+gut+microbiota+and+the+host:+a+Lactobacillus+and+Bifidobacterium+perspective.">Molecular dialogue between the human gut microbiota and the host: a Lactobacillus and Bifidobacterium perspective.</a> Cellular and Molecular Life Sciences. 71, 183-203 (2014).</li><li>Zuba-Surma, E. K., Kucia, M., Abdel-Latif, A., Lillard, J. W., Ratajczak, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ImageStream+System:+a+key+step+to+a+new+era+in+imaging.">The ImageStream System: a key step to a new era in imaging.</a> Folia Histochemica et Cytobiologica. 45, 279-290 (2007).</li><li>Dashkova, V., Malashenkov, D., Poulton, N., Vorobjev, I., Barteneva, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+flow+cytometry+for+phytoplankton+analysis.">Imaging flow cytometry for phytoplankton analysis.</a> Methods. 112, 188-200 (2017).</li><li>Maan, H., Gilhar, O., Porat, Z., Kolodkin-Gal, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+subtilis+colonization+of+arabidopsis+thaliana+roots+induces+multiple+biosynthetic+clusters+for+antibiotic+production.">Bacillus subtilis colonization of arabidopsis thaliana roots induces multiple biosynthetic clusters for antibiotic production.</a> Frontiers in Cellular and Infection Microbiology. 11, 722778 (2021).</li><li>Maan, H., 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=Imaging+flow+cytometry+reveals+a+dual+role+for+exopolysaccharides+in+biofilms:+To+promote+self-adhesion+while+repelling+non-self-community+members.">Imaging flow cytometry reveals a dual role for exopolysaccharides in biofilms: To promote self-adhesion while repelling non-self-community members.</a> Computational and Structural Biotechnology Journal. 20, 15-25 (2022).</li><li>Konieczny, M., Rhein, P., Czaczyk, K., Bialas, W., Juzwa, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+flow+cytometry+to+study+biofilm-associated+microbial+aggregates.">Imaging flow cytometry to study biofilm-associated microbial aggregates.</a> Molecules. 26 (23), 7096 (2021).</li><li>Niederdorfer, R., Peter, H., Battin, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attached+biofilms+and+suspended+aggregates+are+distinct+microbial+lifestyles+emanating+from+differing+hydraulics.">Attached biofilms and suspended aggregates are distinct microbial lifestyles emanating from differing hydraulics.</a> Nature Microbiology. 1, 16178 (2016).</li></ol>

Flowcytometri er en mye brukt metode for å kvantifisere fluorescensintensiteter i eukaryote celler, men det gir kanskje ikke nøyaktige målinger for bakterieceller på grunn av deres større størrelse eller små aggregater. Disse faktorene kan ha betydelig innvirkning på den nøyaktige kvantifiseringen av autoaggregering og det basale nivået av aggregatdannelse under forskjellige forhold. For å løse dette ble avbildningsflowcytometri (IFC) brukt for å få en bedre oppløsning av hvordan karbohydrater påvirker ag...

Bakteriell autoaggregering regnes som et primært trinn i biofilmdannelse. I denne prosessen (noen ganger også kalt autoagglutinasjon eller flokkulering) danner bakterier av samme type flercellede klumper som til slutt legger seg i bunnen av kulturrør eller fester seg til målvevet eller overflaten1.
Autoaggregering er et mye observert fenomen og har så langt blitt vist i gramnegative patogener som det opportunistiske patogenet Acinetobacter baumannii2, tannpatogenet Aggregobacter actinomycetemcomitans3 og det nye patogenet Burkholderia pseudomallei4. Autoaggregering har også blitt beskrevet i flere probiotiske grampositive stammer 5,6,7,8. I Lactobacillus (L.) acidophilus ble autoaggregering delvis mediert av S-lagsproteiner og korrelert med en adhesjon til xylan7. På samme måte fant vi en sammenheng mellom glukoseavhengig autoaggregering og induksjon av limegenskapene (bedømt ved adhesjon til mucin) til de probiotiske artene Lacticaseibacillus rhamnosus GG, Lacticaseibacillus casei, L. acidophilus, Lacticaseibacillus paracasei og Lactiplantibacillus plantarum5. Den økte autoaggregeringen reflekterte mest sannsynlig endringer i ekspresjonen av cellulære adhesiner som respons på glukose og dets katabolitter9. Mens de molekylære mekanismene for autoaggregering gjenstår å bestemme, har det vist seg at denne prosessen endrer fenotypen til de aggregerte bakteriene og gir dem økt toleranse for miljøstressfaktorer1, samt økt følsomhet for quorum sensing molekyler10.
Flere tilnærminger har blitt brukt for å måle autoaggregering; En eksperimentell tilnærming er å la kulturer stå statisk i smale kulturrør i en gitt tid. Kontrollkulturer forblir grumsete, mens autoaggregeringskulturer vil legge seg i bunnen av røret. En mer kvantitativ tilnærming måler autoaggregering ved sedimentering eller sedimenteringsanalyse11.
Flowcytometri har også blitt i økende grad brukt de siste årene for å undersøke bakteriell autoaggregering. Denne metoden er egnet for å analysere partikler mellom ca. 0,5 og 1000 μm i størrelse. Enkeltbakterien eller dannede aggregater suspenderes i væske, mates inn i en bekk og kan påvises en etter en11. Opptak av spredt lys fremover gjør det mulig å måle den relative størrelsen på cellen eller aggregatet. Den er relativt rask og grei, men kan ikke oppdage flere parametere, for eksempel aggregatstørrelse eller gjennomsnittlig antall celler i aggregater. Derfor kan denne tilnærmingen kompletteres mikroskopisk, slik at flere parametere kan kontrolleres12. Tradisjonell mikroskopi er imidlertid tidkrevende og begrenser dermed antall testede prøver og analysens statistiske styrke. Generelt gir avbildning av flowcytometri flere funksjoner sammenlignet med tradisjonell flowcytometri, for eksempel samtidig analyse av cellemorfologi og fenotype, gjennomføring av bildebasert analyse, oppdagelse av sjeldne hendelser og validering av flowcytometridata13. Disse fordelene forbedrer mulighetene til flowcytometri og letter en mer detaljert undersøkelse av cellepopulasjoner.
Denne studien gir en verdifull protokoll for avbildning av flowcytometri for å overvåke autoaggregering i melkesyrebakterier (LAB). Disse grampositive stavene er fakultative anaerober og tilhører LAB-gruppen. Disse effektive glukosefermentorene genererer melkesyre som deres viktigste sluttprodukt av karbohydratmetabolismen14. Disse bakteriene er gunstige kjernemedlemmer av mikrobiomet og finnes naturlig i mage-tarmkanalen (GIT) hos mennesker og dyr, så vel som i urogenitalkanalen til hunner15. Derfor er den nøyaktige karakteriseringen av deres autoaggregeringsegenskaper av høy bioteknologisk og klinisk interesse.
Våre tidligere funn indikerte at det basale nivået av autoaggregering varierer mellom ulike probiotiske stammer. Denne heterogeniteten påvirkes av de forskjellige karbohydratene som brukes som karbonkilde5. For å overvinne denne grunnleggende egenskapen til probiotiske bakterier, ble effekten av karbohydrater fra kostholdet overvåket på autoaggregering på enkeltcellenivå ved hjelp av IFC. Denne IFC-baserte tilnærmingen kombinerer kraften og hastigheten til tradisjonelle flowcytometre med mikroskopets oppløsning. Derfor tillater den høyhastighets komplekse morfometriske målinger på en fenotypisk definert måte16,17. Denne tilnærmingen kan utvides til andre probiotiske og patogene bakterier, kombinert med fluorescerende reportere for å overvåke genuttrykk, og fluorescerende merkede stammer for å overvåke tilstedeværelsen og overfloden av spesifikke bakteriearter i heterogene aggregater.

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			<td>14 mL culture tubes</td>
			<td>Falcon</td>
			<td>352051</td>
			<td></td>
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		<tr>
			<td>15 mL centrifuge tube</td>
			<td>Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>Baeton,Dickinson and Company</td>
			<td>214010</td>
			<td></td>
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			<td>Bacto Typtic Soy Broth</td>
			<td>Baeton,Dickinson and Company</td>
			<td>211825</td>
			<td></td>
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		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>G7021-1KG</td>
			<td></td>
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		<tr>
			<td>D-(+)-Raffinose pentahydrate</td>
			<td>Sigma</td>
			<td>83400-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Lactobacilli MRS broth</td>
			<td>Baeton,Dickinson and Company</td>
			<td>288130</td>
			<td></td>
		</tr>
		<tr>
			<td>EASY-LOCK MICROPR. 1.5 mL (Eppendorf)</td>
			<td>FL medical</td>
			<td>23053</td>
			<td></td>
		</tr>
		<tr>
			<td>IDEAS Software</td>
			<td>Amnis/EMD Millipore</td>
			<td>N/A</td>
			<td>&#160;Details available at: https://www.merckmillipore.com/INTL/en/20150212_144049?ReferrerURL=https%3A%2F%2Fwww.google.com%2F&#38;bd=1</td>
		</tr>
		<tr>
			<td>ImageStream X Mark II</td>
			<td>Amnis/EMD Millipore</td>
			<td>N/A</td>
			<td>&#160;Details available at: https://www.merckmillipore.com/INTL/en/20150121_205948?ReferrerURL=https%3A%2F%2Fwww.google.com%2F</td>
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			<td>MOPS, 3-(N-morpholino)propanesulfonic acid</td>
			<td>Fisher bioreagents</td>
			<td>BP308-500</td>
			<td></td>
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			<td>Potassium phosphate dibasic</td>
			<td>Fisher Scientific, 174.18 g/mol</td>
			<td>BP363-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma, 136.09 g/mol</td>
			<td>P0662-500G</td>
			<td></td>
		</tr>
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imaging flow cytometry to study microbial autoaggregation

Gunstige og probiotiske bakterier spiller viktige roller i vertene sine, og gir ulike helsemessige fordeler, inkludert immunitet mot smittsomme sykdommer. Lactobacillaceae-familien består av grampositive bakterier med bekreftede probiotiske egenskaper. Denne studien bruker Lactobacillaceae-arter som en modell for å demonstrere effektiviteten av encellet høykapasitetsanalyse for å studere cellulær aggregering. Fokuset er på å analysere responsen til disse gunstige artene på enkle karbohydrater fra kostholdet.
Studien viser hvordan Imaging Flow Cytometry (IFC) kan overvinne de grunnleggende forskjellene i sammensetningen av probiotiske bakterier i nærvær og fravær av karbohydrater. IFC kombinerer kraften og hastigheten til konvensjonell flowcytometri med den romlige oppløsningen til mikroskopi, og muliggjør høyhastighets komplekse morfometriske målinger på en fenotypisk definert måte på tvers av et bibliotek med fordelaktige bakteriestammer og tilstander. Denne protokollen gir innsikt i autoaggregering av Lactobacillaceae-arter og kaster lys over deres respons på karbohydrater i kosten, og bidrar til å forstå mekanismene bak de gunstige effektene av disse probiotiske bakteriene.

Bacterial autoaggregation is considered a primary step in biofilm formation. In this process (sometimes also called autoagglutination or flocculation), bacteria of the same type form multicellular clumps that eventually settle at the bottom of culture tubes or attach to their target tissue or surface1.
Autoaggregation is a widely observed phenomenon and has been shown so far in Gram-negative pathogens such as the opportunistic pathogen Acinetobacter baumannii2, the dental pathogen Aggregobacter actinomycetemcomitans3, and the emerging pathogen Burkholderia pseudomallei4. Autoaggregation has also been described in several probiotic Gram-positive strains5,6,7,8. In Lactobacillus (L.) acidophilus, autoaggregation was partially mediated by S-layer proteins and correlated with an adhesion to xylan7. Similarly, we found a correlation between glucose-dependent autoaggregation and induction of the adhesive properties (as judged by adhesion to mucin) of the probiotic species Lacticaseibacillus rhamnosus GG, Lacticaseibacillus casei, L. acidophilus, Lacticaseibacillus paracasei, and Lactiplantibacillus plantarum5. The increased autoaggregation most likely reflected changes in the expression of cellular adhesins in response to glucose and its catabolites9. While the molecular mechanisms of autoaggregation remain to be determined, it has been shown that this process alters the phenotype of the aggregated bacteria and grants them enhanced tolerance to environmental stressors1, as well as increased sensitivity to quorum sensing molecules10.
Several approaches have been used to measure autoaggregation; one experimental approach is to let cultures stand statically in narrow culture tubes for a given time. Control cultures remain turbid, whereas autoaggregation cultures will settle at the bottom of the tube. A more quantitative approach measures autoaggregation by sedimentation or settling assay11.
Flow cytometry has also been increasingly employed in recent years to investigate bacterial autoaggregation. This method is appropriate for analyzing particles between approximately 0.5 and 1000 &#181;m in size. The single bacterium or formed aggregates are suspended in fluid, fed into a stream, and can be detected one by one11. Recording forward scattered light allows measuring the relative size of the cell or aggregate. It is relatively fast and straightforward but cannot detect several parameters, such as aggregate size or the average number of cells in aggregates. Therefore, this approach can be complemented microscopically, allowing more parameters to be checked12. However, traditional microscopy is time-consuming and thereby limits the number of tested samples and the statistical power of the analysis. In general, imaging flow cytometry provides several features compared to traditional flow cytometry, such as simultaneous analysis of cell morphology and phenotype, conducting image-based analysis, detecting rare events, and validating flow cytometry data13. These advantages enhance the capabilities of flow cytometry and facilitate a more detailed examination of cell populations.
This study provides a valuable protocol for imaging flow cytometry to monitor autoaggregation in lactic acid bacteria (LAB). These Gram-positive rods are facultative anaerobes and belong to the LAB group. These efficient glucose fermenters generate lactic acid as their main end-product of carbohydrate metabolism14. These bacteria are beneficial core members of the microbiome and are naturally found in the gastrointestinal tract (GIT) of humans and animals, as well as in the urogenital tract of females15. Therefore, the exact characterization of their autoaggregation properties is of high biotechnological and clinical interest.
Our previous findings indicated that the basal level of autoaggregation differs between different probiotic strains. This heterogeneity is affected by the different carbohydrates used as a carbon source5. To overcome this fundamental property of probiotic bacteria, the effects of carbohydrates from the diet were monitored on autoaggregation on the single-cell level using IFC. This IFC-based approach combines the power and speed of traditional flow cytometers with the microscope&#39;s resolution. Therefore, it allows high-rate complex morphometric measurements in a phenotypically defined way16,17. This approach can be extended to other probiotic and pathogenic bacteria, combined with fluorescent reporters to monitor gene expression, and fluorescently labeled strains to monitor the presence and abundance of specific bacterial species in heterogeneous aggregates.

Forfatter i søkelyset: Fremme forskning innen mikrobiell autoaggregering ved bruk av avbildningsstrømningscytometri

Imaging Flow Cytometry for å studere mikrobiell autoaggregering

This study utilized Lactobacillus species as a model to demonstrate the effectiveness of imaging flow cytometry analysis in studying microbial autoaggregation. The focus is on analyzing the response of autoaggregation of Lactobacillus species to simple carbohydrates from the diet. The most used methods for measuring autoaggregation are spectral photometry, which measures microbial suspensions, turbidity, or optical density, and traditional microscopy techniques, such as bright field, phase contrast, and fluorescent microscopy.When talking about hight-throughput imaging, one major experimental challenge is single event segmentation. In this protocol, it is a single aggregate. Imaging flow cytometry addresses this challenge elegantly and give researchers a powerful tool for studying autoaggregation.

Resultatene viser at denne metoden enkelt kan måle forskjellene i autoaggregering som respons på kostholdssukker i LAB-bakterier. Ved å skille individer fra aggregater, tillater metoden å beregne prosentandelen av befolkningen av aggregeringshendelsene av alle hendelser som svar på fermenterbart eller ikke-fermenterbart sukker fra kostholdet. I tillegg var det mulig å måle om det er forskjeller i gjennomsnittsstørrelsen på aggregatpopulasjonen mellom behandlingene.
De representative b...

Read this Scientific Article Protocol about Author Spotlight: Advancing Research in Microbial Auto Aggregation Using Imaging Flow Cytometry at JoVE.com

Denne protokollen beskriver en kvantitativ tilnærming for å måle mikrobiell autoaggregering ved hjelp av avbildningsflowcytometri.

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The ...

Denne studien brukte Lactobacillus-arter som en modell for å demonstrere effektiviteten av avbildningsflowcytometrianalyse for å studere mikrobiell autoaggregering. Fokuset er på å analysere responsen til autoaggregering av Lactobacillus-arter på enkle karbohydrater fra kostholdet. De mest brukte metodene for å måle autoaggregering er spektral fotometri, som måler mikrobielle suspensjoner, turbiditet eller optisk tetthet, og tradisjonelle mikroskopiteknikker, som lysfelt, fasekontrast og fluorescerende mikroskopi.Når vi snakker om avbildning med høy gjennomstrømning, er en stor eksperimentell utfordring segmentering av enkelthendelser. I denne protokollen er det et enkelt aggregat. Imaging flow cytometry løser denne utfordringen elegant og gir forskere et kraftig verktøy for å studere autoaggregering.

imaging-flow-cytometry-to-study-microbial-autoaggregation

Research

JoVE Journal

Biology

Imaging Flow Cytometry to Study Microbial Autoaggregation

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The Lactobacillaceae family consists of Gram-positive bacteria with confirmed probiotic properties. This study utilizes Lactobacillaceae species as a model to demonstrate the effectiveness of single-cell high throughput analysis in studying cellular aggregation. The focus is on analyzing the response of these beneficial species to simple carbohydrates from the diet.
The study showcases how Imaging Flow Cytometry (IFC) can overcome the fundamental differences in the assembly of probiotic bacteria in the presence and absence of carbohydrates. IFC combines the power and speed of conventional flow cytometry with the spatial resolution of microscopy, enabling high-rate complex morphometric measurements in a phenotypically defined manner across a library of beneficial bacterial strains and conditions. This protocol provides insights into the autoaggregation of Lactobacillaceae species and sheds light on their response to dietary carbohydrates, contributing to understanding the mechanisms behind the beneficial effects of these probiotic bacteria.

This protocol describes a quantitative approach to measure microbial autoaggregation using imaging flow cytometry.

Lactobacillaceae Sample Preparation for Flow Cytometric Analysis

Start by inoculating five milliliters of lactobacilli MRS broth with a single colony of the lactobacilli strain, incubated at 37 degrees Celsius under static conditions overnight. Dilute the cell culture in a one-to-100 ratio in three milliliters of 50%supplemented tryptic soy broth, vortex the culture, and incubate it. Then invert the tube to mix the cells in the medium before transferring 200 to 300 microliters of the sample into a 1.5 milliliter micro centrifuge tube.

Slekten Lactobacillaceae Prøveforberedelse for flowcytometrisk analyse

Flow Cytometric Data Acquisition of Microbial Aggregates

To begin the flow cytometric analysis, first set the 785-nanometer laser to a power of five milliwatts for dark field measurements. Use a 60X lens with a numerical aperture of 0.9. Then select the high-sensitivity setting and set the speed to low.To ensure calibration bead stability, press the Play button in the fluidics box and examine the bead images, which should appear sharp and clear. Generate a new scatterplot in the workspace box by selecting New Scatterplot. Then select the area in the Brightfield channel that corresponds to the SSC channel intensity and exclude the calibration beads that are running concurrently within the sample.Next, press the Load button and position a tube containing the Lactobacilli sample in the holder. In the Acquisition Settings box, enter the sample name and specify the location for data storage. Set the number of events to be collected, typically within the range of 10, 000 to 20, 000 events.Now, begin the acquisition process by clicking the Record button located in the Acquisition box. The process will halt after reaching the specified volume of events. Press the Return button to unload the tube and remove it from the holder as prompted in the popup window.After repeating the process for all samples, save the template to maintain data acquisition uniformity. For data analysis, to load the raw file into the analysis software, click on File and open the rif file. In the opened window, select Use Acquisition Analysis and then click OK.Next, to create a histogram of the gradient root mean square, click the New Histogram button and choose the population from the acquisition to analyze.Under the X Axis feature, select Gradient RMS of the Brightfield channel. Next, place a gate to exclude non-focused events by clicking the Create Line Region button in the analysis area. Create a scatterplot of the area versus the aspect ratio by clicking the New Scatterplot button in the analysis area.Select the focused population in the New Scatterplot window. Then select the area of the Brightfield channel for the X-Axis feature. Choose the aspect ratio for the Y-Axis feature.Draw a gate on the scatterplot using the Create Rectangle button based on the area value for the aggregate events population. Similarly, draw another gate for the singles and small aggregates population. Save your data analysis as a template by clicking the File tab, choosing Save As Template ast, and then open the next sample file under the same template to create the data analysis file.To measure the aggregate size distribution, start by plotting a histogram of the area of the aggregation events population. Save the data analysis by clicking on the file and selecting the Save As Template option. After saving, open the next sample file under the same template for the data analysis file.Single cells, small aggregates, large aggregates, and chains were observed in LGG and L.paracasei but it was not possible to distinguish between chains and larger aggregates. Significant variations in the aggregation characteristics of different LAB strains were observed in response to fermentable or non-fermentable sugars.