Which Negative Stain is Best for Sensitive Large Molecular Complexes in EM?

In summary, the conversation discusses the preparation of a molecular complex for electron tomography. The integrity of the complex is sensitive to increasing ionic strength, so it must be prepared using low ionic strength solutions. The experiment must be conducted at room temperature due to a lack of a cold specimen holder. The question at hand is which negative stain to use for preparation, and the reason for this choice.
  • #1
i_a_n
83
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I have isolated a large molecular complex whose integrity is very sensitive to increasing ionic strength and thus had to be prepared for EM using low ionic strength solutions. And my goal is image this by electron tomography by collecting a dual tilt series of 120 images. Other experimental constraints are that I lack a cold specimen holder and thus must to this experiment at room temperature. From the possible negative stains that are available generally, which should I choose to prepare my specimen and why?
 
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  • #2
You forgot to change the last "your" to "my". Is this a homework question?
 
  • #3
Pythagorean said:
You forgot to change the last "your" to "my". Is this a homework question?
Yes. But can you help me somehow?
 
  • #4
No, but someone who would be able to help you would probably appreciate some effort in answering the question on your part.
 

FAQ: Which Negative Stain is Best for Sensitive Large Molecular Complexes in EM?

What is the purpose of using negative stains in microscopy?

Negative stains are used in microscopy to visualize the presence and morphology of structures that are too small or delicate to be stained with traditional dyes. This includes viruses, bacteria, and other microorganisms. Negative stains produce a dark background, allowing these structures to stand out and be easily observed.

What types of negative stains are commonly used in microscopy?

The most commonly used negative stains in microscopy are India ink, nigrosin, and uranyl acetate. India ink and nigrosin are both carbon-based dyes that produce a dark background, while uranyl acetate is a heavy metal stain that provides high contrast and detail.

How is a negative stain prepared and applied to a sample?

To prepare a negative stain, a small drop of the stain is placed on a clean microscope slide. A sample, such as a bacterial culture, is then added to the drop of stain and mixed gently. The mixture is allowed to air dry and is then observed under a microscope without any additional staining or fixing steps.

What are the advantages of using negative stains in microscopy?

Negative stains have several advantages over traditional staining methods. They are quick and easy to perform, do not require any special equipment, and do not interfere with the natural structure or morphology of the sample. Additionally, negative stains can be used to visualize samples that are difficult to stain with traditional methods, such as those with a high lipid content.

Are there any limitations or drawbacks to using negative stains in microscopy?

While negative stains have many benefits, they also have some limitations. They do not provide information about the chemical composition of the sample, and they can only be used on samples that are not heat-fixed. Negative stains also have lower sensitivity compared to traditional staining methods, meaning they may not be able to detect low levels of certain structures in a sample.

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