En ny metod för kvalitativ Multi-skala Analys av bakteriell Biofilmer på rådiga svampkolonier Använda Confocal och elektronmikroskopi

Immunology and Infection
 

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Miquel Guennoc, C., Rose, C., Guinnet, F., Miquel, I., Labbé, J., Deveau, A. A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy. J. Vis. Exp. (119), e54771, doi:10.3791/54771 (2017).

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Abstract

Bakteriella biofilmer bildar ofta på svamp ytor och kan vara inblandade i ett stort antal bakteriella svampinteraktionsprocesser, såsom metabolisk samarbete, konkurrens eller predation. Studiet av biofilmer är viktigt i många biologiska områden, inklusive miljövetenskap, livsmedelsproduktionen, och medicin. Emellertid har få studier fokuserat på sådana bakteriella biofilmer, delvis på grund av svårigheten att utreda dem. De flesta metoder för kvalitativ och kvantitativ biofilm analyser som beskrivs i litteraturen är endast lämpliga för biofilmer bildas på abiotiska ytor eller homogena och tunna biotiska ytor, såsom ett monoskikt av epitelceller.

Medan laserscanning konfokalmikroskopi (LSCM) används ofta för att analysera in situ och in vivo biofilmer, blir mycket utmanande denna teknik när den tillämpas på bakteriella biofilmer på svamphyfer, på grund av tjockleken och de tre dimensionerna hos hyfal netwOrks. För att övervinna denna brist, har vi utvecklat ett protokoll som kombinerar mikroskopi med en metod för att begränsa ansamling av hyfernas lager i svampkolonier. Med denna metod kunde vi undersöka utvecklingen av bakteriella biofilmer på svamphyfer vid flera skalor med både LSCM och svepelektronmikroskop (SEM). Denna rapport beskriver protokollet, inklusive mikroorganismer kulturer, bakteriella biofilm bildningsförhållanden, biofilm färgning och LSCM och SEM visualiseringar.

Materials

Name Company Catalog Number Comments
6 well Falcon Tissue Culture Plates Fisher Scientific 08-772-33 Used in 2.2 & 3.1
Congo Red Fisher Scientific C580-25 Used in 3.1.4.1
FUN 1 Cell Stain Thermo Fisher Scientific F7030 Used in 3.1.4.1
Wheat Germ Agglutinin, Alexa Fluor 633 Conjugate Thermo Fisher Scientific W21404 Used in 3.1.4.1
DAPI solution Thermo Fisher Scientific 62248 Used in 3.1.4.2
Propidium iodide Thermo Fisher Scientific P3566 Used in 3.1.4.3
FilmTracer SYPRO Ruby Biofilm Matrix protein Stain Thermo Fisher Scientific F10318 Used in 3.1.4.4
Fluoromount-G Slide Mounting Medium Fisher Scientific OB100-01 Used in 3.1.7
LSM780 Axio Observer Z1 Zeiss Used in 3.2.1
ZEN 2.1 lite black software  Zeiss Used in 3.2.1
High Vacuum Coater Leica EM ACE600 Leica Used in 4
GeminiSEM-FEG Zeiss Used in 4

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