General Research Topics => Microbiology => Topic started by: drain_master on August 06, 2006, 07:47:15 AM

Title: staining for wound surface bacteria
Post by: drain_master on August 06, 2006, 07:47:15 AM
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
Title: staining for wound surface bacteria
Post by: excalibur on August 07, 2006, 08:38:26 AM
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?
Title: BacLight stain for chronic wounds
Post by: drain_master on August 16, 2006, 09:13:06 AM
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)!
Title: staining for wound surface bacteria
Post by: excalibur on August 16, 2006, 02:11:50 PM
Well, Calcofluor is mainly used for fungus.

What fixative are you using?
Title: wound stain
Post by: drain_master on August 20, 2006, 08:13:30 AM
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.
Title: staining for wound surface bacteria
Post by: excalibur on August 20, 2006, 10:40:26 AM
Try a fixative that does not contain an aldehyde to reduce autofluorescents. Several companies are now selling them.

I suggest you take one sample, fix and process to paraffin, cut SERIAL sections and stain all with a Gram stain to get an idea of what you have.