I found some papers (from ~1991) describing a technique using Acridine orange and crystal viole (AO-CV) to stain intracellular pathogens. The problem of this technique is that AO also detects lysosomes and at the end is difficult to discriminate what is all the green you see.
- So I would like to know if there is a special protocol with AO-CV to stain intracellular bacteria (without the lysosomes problem)?
- Or if AO-CV is not good to this purposes..
- If it's not, which technique is the best to visualize intracellular bacteria?
Look at this
https://histology.leeds.ac.uk/what-is-histology/histological_stains.php
If you looking for a specific bacteria it will be easier to choosing way of staining.
Hi Alireza,
Thank you for the link. But I didn't find anything for me there.
Yes, I want to stain Campylobacter jejuni invading Caco2 cells.
In my knowledge silver staining is very good method.
https://www.google.com/url?sa=t&source=web&rct=j&url=http://www.bioline.org.br/pdf%3Fmb09034&ved=2ahUKEwjG5Nub5ezdAhUQY1AKHZsIBW4QFjAEegQIBhAB&usg=AOvVaw2BXOGlbuozjFZPwjw8mbx6
Dear Sabrina Minatelli , you are aware that digested bacteria (states more/less from living whole bacteria to more/less totally digested bacteria remnants) entrapped into phagocytic vacuoles are stained perhaps too?
For sure you know about the article of FERNANDEZ et al,
Detection of Campylobacter jejuni Invasion of HEp-2 Cells by Acridine Orange-Crystal Violet Staining.
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 92 (4): 509-511, Jul./Aug. 1997
Citation/quote:
"In our study, invasion by C.jejuni was assessed by a double-stain technique
(AO-CV stain) that allows the optical observation (fluorescence microscopy) of internalized bacteria exclusively. This is an advantage of this method since it is difficult to diferentiate extra and intracellular bacteria by employing other methods. ....
"...using AO-CV stain (M Milliotis 1991 J Clin Microbiol 29: 830-831), ......... (cultured) coverslips were washed three times with PBS and, without fixing, stained with 0.01% acridine orange in Gey’s solution for 45 sec, rinsed with Hanks
balanced salt solution and counterstained with 0.05% crystal violet in 0.15 N NaCl for 45 sec, mounted on slides and sealed with colorless nail
polish and examined under epi-fluorescence microscopy at 400X magnification for screening and 1000X magnification for quantitative evaluation." ....
..."The results suggest that the acridine orange-crystal violet staining is a suitable technique to study in vitro the ability of C. jejuni to invade cell cultures, similarly to other bacteria as it was previously demonstrated for Arcobacter cryaerophylus
by H Fernández et al. (1995 Mem Inst Oswaldo Cruz 90 : 633-634)."
End of quote
cf:
https://pdfs.semanticscholar.org/e39e/055bdb132d2cd7fb1d93173d7b90f08849a3.pdf
NB: a good result might also depend on the quality (purity) of the AO-dye you will use as well as the pH of staining solution you apply.
Perhaps you might link to:
https://www.researchgate.net/post/What_is_the_role_of_pH_in_acridine_orange_staining, where you can find in answer #[003] a pdf with regard to ACRIDINES & PHENANTHRIDINES_Acridine orange [or link directly to: https://www.researchgate.net/profile/Wolfgang_MUSS/post/What_is_the_role_of_pH_in_acridine_orange_staining/attachment/59d6438fc49f478072eac138/AS%3A273810945708032%401442293024915/download/ACRIDINES+%26+PHENANTHRIDINES%2CAcridine+Orange%2Cex+Conns-10th+Ed%2C+p.32%2C253-254%2C257-258%2C2002%28collect.2015pdf%29.pdf]
Best wishes and good luck!
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining