General Research Topics > Microbiology

staining for wound surface bacteria

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Hi everyone!
i am trying to use confocal microscopy to look for bacterial biofilms on surface of human chronic wound. Attempted to use invitrogen BacLight stain (Syto9/PI) but unsuccessfully so far b/c of autofluorescence being very similar to stain itself (used 488 laser). Would be extremely grateful for any suggestion ? eliminate background autofluorescence somehow ? use different stain ?used different filters

Baclight is probably a great product if you were just staining cultures or smears. However, you say you are staining chronic wounds. Chronic wounds will contain live and dead bacteria as well as mostly dead tissue, of which the cells have lysed and could also pick up staining. Necrotic tissue is notorious for this.

Are there RBCs and WBCs present? Are you staining a tissue sample of the wound or a swabbing? Were the wounds non-healing and wet or healing and dry? What fixative are you using? How thick are the sections? Have you performed a Gram stain on any sections?

Hi Excalibur,
thanks for the prompt reply. My samples are fresh wound biopsies 1x1 cm and approx 0.5-1 mm thick. Some have RBC, WBC on them. I would like to use confocal microscope to do optical sections rather than do the parafin sections. I am hoping to perhaps see the biofilms by Baclight (for bacterial DNA) + a lectin or Calcofluor stain (for biofilm polysaccharide matrix). My concern is non-specific stainings. Would be grateful for your view (or anyone else's)!

Well, Calcofluor is mainly used for fungus.

What fixative are you using?

I aim to stain the tissue biopsy 1st BEFORE fixing with paraformaldehyde 3.7%. If i fix before staining, the live/dead stain won't work so well as all bacteria will have died.


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