Published TCIMAIL newest issue No.196
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Biofilm Tissue-Clearing Reagents
Products
Advantages
- Can make biofilm transparent by simply adding iCBiofilm.
- Suitable for various kinds of biofilm.
- Cleared biofilm can be observed by light-sheet fluorescent microscopy (LSFM) or confocal laser-scanning microscopy (CLSM).
Example of observation after using iCBiofilm-H1
Figure. Images of methicillin-resistant Staphylococcus aureus (MRSA) observed microscopically after incubated overnight at 37°C,
fixed at room temperature for 30 minutes, stained with FM®1-43 at 4°C overnight, washed, and dipped in PBS or iCBiofilm-H1.
(FMⓇ is a trademark of Thermo Fisher Scientific Inc.)
Application: Observation of biofilm treated with iCBiofilm-H1 or iCBiofilm-H2
iCBioflim-H1 (Product No. 4031) is applicable to clearing many kinds of biofilms. If the transparency with iCBiofilm-H1 is insufficient, iCBiofilm-H2 (Product No. T4032) can be used instead. Since iCBiofilm-H2 is prepared at high component concentration, iCBiofilm-H2 is available as a stock solution. Before addition to biofilm, iCBiofilm-H2 has to be diluted with deionized water to the appropriate concentration. (Undiluted iCBiofilm-H2 may work well.)
[Direction for Use]
- Prepare a biofilm in a glass bottomed culture dish (35mm diam.).
- After removing planktonic cells and the culture medium from the dish, add 1% glutaraldehyde solution diluted with PBS to fix the biofilm at room temperature for 30 minutes. To preserve immunogenicity for immunofluorescence applications, the use of 4% paraformaldehyde in PBS is recommended.
- Remove the glutaraldehyde solution and wash three times with PBS or distilled water.
- Stain the biofilm with appropriate fluorescence reagents.
- Remove staining reagents and add iCBiofilm solution.
- Observe the biofilm by optical microscopy.
Application: Live-Cell-Imaging of Biofilms using iCBiofilm-D
[Directions for Use]
- Prepare live-cell-imaging medium: Dissolve [1] Brain Heart Infusion (BHI) powder to twice the normal culture concentration and [2] glucose powder to a final concentration of 2% in deionized water and filter sterilize. Then mix with an equal volume of iCBiofilm-D (Product No. I1275) to make BHI medium containing 1% glucose with 50% iCBiofilm-D.
- Add MitoTracker® Deep Red to the live-cell-imaging medium and culture Methicillin‐Resistant Staphylococcus Aureus (MRSA) in a glass bottom dish.
- Observe the biofilm via microscopy to obtain time lapse images.
(MitoTrackerⓇ is a registered trademark of Thermo Fisher Scientific Inc.)
Video of Live-Cell-Imaging of Bioflims using iCBiofilm-D
Note:
If clear images cannot be obtained or biofilm formation is prevented when first trying 100% iCBiofilm-D, try diluting iCBiofilm-D with culture medium at a ratio of 1:1 according to the above application method. If iCBiofilm-D must be used at the original concentration, but with culture medium, prepare live-cell-imaging medium using iCBiofilm-D in place of water.
This products are commercialized under instruction of Assoc. Prof. Shinya Sugimoto.
Figures and videos were provided by Assoc. Prof. Shinya Sugimoto.
![Clearing Reagent iCBiofilm-D [for Biofilm]](/_ui/responsive/theme-tci/images/missing_product_srp.jpg "Clearing Reagent iCBiofilm-D [for Biofilm]")
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