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

In summary, there is difficulty in staining dormant or lag phase bacteria with Nucleic acid dye like SYTO9/SYBR Green Ⅰ. Possible reasons for this include the complexity of DNA 3D structure, high amount of DNA binding protein, low material transport efficiency in bacteria, or other factors. There is research that supports this, and it mentions membrane permeability as a contributing factor. Further information may be available from @Ygggdrasil. Attempts to increase membrane permeability have shown little effect on staining efficiency for inactive bacteria. Actively dividing bacteria have higher levels of DNA, which may contribute to the difficulty in staining them. It is also possible that the density of DNA and its binding protein in dormant bacteria affects the
  • #1
littledog
6
0
TL;DR Summary
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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  • #3
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.
 
  • #4
Actively dividing bacteria are replicating their DNA and could have 2x or more DNA/cell than dormant bacteria that are not replicating.
 
  • #5
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？
 
  • #6
Have you asked the dye manufacturer?
Do other DNA dyes have the same effect?
 
  • #7
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.
 

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

The main reason for the difficulty in staining dormant bacteria with Nucleic acid dyes is that these dyes primarily target actively dividing cells. Dormant bacteria are in a state of low metabolic activity and do not have actively dividing cells, making it challenging for the dyes to bind to their nucleic acids.

2. Can dormant bacteria be stained with Nucleic acid dyes at all?

Yes, it is possible to stain dormant bacteria with Nucleic acid dyes, but it requires specialized techniques and longer staining times. These techniques include using higher concentrations of dyes, prolonged incubation periods, and the addition of chemical enhancers to improve dye penetration into the bacteria.

3. How do Nucleic acid dyes work to stain bacteria?

Nucleic acid dyes, such as DAPI and SYBR Green, work by binding to the DNA or RNA present in the bacterial cells. They have a high affinity for these nucleic acids and emit fluorescence when excited by a specific wavelength of light. This fluorescence allows for the visualization and detection of bacteria under a microscope.

4. Are there alternative methods to stain dormant bacteria?

Yes, there are alternative methods to stain dormant bacteria, such as using cell membrane dyes or metabolic dyes. These dyes target different components of the bacterial cell, such as the cell membrane or metabolic activity, and can provide valuable information about the viability and activity of dormant bacteria.

5. Why is it important to be able to stain dormant bacteria?

Staining dormant bacteria is crucial for studying their behavior and understanding their role in various biological processes. It also allows for the identification and detection of these bacteria in environmental samples or clinical specimens, which can aid in the diagnosis and treatment of bacterial infections.

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