Why is it difficult to stain dormant bacteria with Nucleic acid dyes?

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SUMMARY

The difficulty in staining dormant bacteria with nucleic acid dyes such as SYTO9 and SYBR Green I is primarily attributed to factors like membrane permeability and the complex 3D structure of DNA. Research indicates that dormant bacteria have significantly less DNA per cell compared to actively dividing bacteria, which affects staining efficiency. Additionally, attempts to enhance membrane permeability have shown minimal impact on the staining of inactive bacteria. The discussion references a relevant article from FEMSEC that supports these findings.

PREREQUISITES
  • Understanding of nucleic acid dyes, specifically SYTO9 and SYBR Green I
  • Knowledge of bacterial growth phases, particularly lag and active division phases
  • Familiarity with membrane permeability concepts in microbiology
  • Basic comprehension of DNA structure and its interaction with binding proteins
NEXT STEPS
  • Research methods to enhance membrane permeability in bacteria
  • Investigate alternative DNA dyes from Thermo Fisher and their staining efficacy
  • Explore the impact of DNA structure density on dye affinity
  • Review literature on staining techniques for dormant bacteria
USEFUL FOR

This discussion is beneficial for microbiologists, researchers in bacterial genetics, and laboratory technicians involved in staining techniques and bacterial viability assessments.

littledog
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TL;DR
The correlation between bacterial physiological activity and Nucleic acid staining (SYTO) efficiency?
I found that it's difficult to stain dormant bacteria or bacteria in lag phase with Nucleic acid dye like SYTO9/SYBR Green Ⅰ, does anyone know why?
DNA 3D structure too complex? DNA binding protein too much? Low material transport efficiency in bacteria? Or anyother factors?
Is there any research about this?
 
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jim mcnamara said:
This article agrees with your assessment of less staining for inactive bacteria.
https://academic.oup.com/femsec/article/29/4/319/526255
The authors mention membrane permeability.
@Ygggdrasil may have more information.
I have try some methods to increase membrane permeability, the result shows that few effect on staining efficiency to inactive bacteria.
 
Actively dividing bacteria are replicating their DNA and could have 2x or more DNA/cell than dormant bacteria that are not replicating.
 
Ygggdrasil said:
Actively dividing bacteria are replicating their DNA and could have 2x or more DNA/cell than dormant bacteria that are not replicating.
But as what I observe in my experiment，there are a lot of bacteria in dividing phase couldn't be stained by SYTO13，and what I wonder is whether the DNA structure or I should say the density of DNA and its binding protein in dormant bacteria greatly effect the affinity between DNA and nucleic dye？
 
Have you asked the dye manufacturer?
Do other DNA dyes have the same effect?
 
BillTre said:
Have you asked the dye manufacturer?
Do other DNA dyes have the same effect?
YES，I almost buy all the DNA dyes of Thermo Fisher.
 

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