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Wednesday, 14 December 2011

Stains in microscopy

This is probably the most technical and difficult section in this blog (and also the least illustrated). If you are not planning to cut thin sections and stain them, skip over this.  If you persevere, pay close attention to the safety messages.

Most of the things inside a cell are transparent, so it is hard to see any detail. A dye that attaches to one kind of cell part makes it show up more clearly, and we call that dye a stain.

There is a catch, because dyes often cause difficulties with authorities like parents, landlords (or landladies) and the like. These stains can easily stain baths, basins, carpets. people and pets—among other things. More importantly, while pale and colourless chemicals can also be dangerous, you are usually wise to assume that coloured chemicals are always dangerous. Any chemical which attaches to a biological molecule inside a cell (as stains do) is likely to cause damage in the cells that are attacked. Treat all biological stains as dangerous, to be on the safe side.

That means you need to discover the MSDS, the Material Safety Data Sheet. These are easy to find on the web by searching on <(name of chemical) MSDS>. Some MSDS sheets are hard to understand, but the ones at are reliable and clear. Try searching <methyl cellulose MSDS> for practice.

We are forever learning new things, and while methylene blue is regarded as safe now, that may change. Read the MSDS first, before buying or using any stain! You also need to understand that an MSDS will spell out all the risks: read the MSDS sheets for table sugar (sucrose to chemists), water and table salt (sodium chloride), and you will see how complete and obsessively thorough they are!

To get more information on the web, I suggest a search such as <microscopy stains safe>. Just be careful about what you believe!!

Here are some stains that I regard as fairly safe. Even so, you should handle and mix any stains out of doors in good weather. If the stain comes as a powder, think safety first. When you take the lid off, there is sometimes a puff of dust that you don't want to breathe. Try to choose a day when there is no breeze: if there is a light breeze, stand upwind of the bottle. Never mix stains in high winds. Use gloves, goggles and a face mask, or if possible, buy the stains as solutions.

In this outline, I indicate the uses for which each stain is most often used, but most stains will work on other tissues as well.

Basic fuchsin: used to stain nuclei. Dissolve 0.1 gram of the powder in 150 mL of distilled water and add 1 mL of 70% ethanol.

Eosin Y: used to stain muscle fibres, cytoplasm and collagen. Dissolve 1 gram in 100 mL of tap water.

Methylene blue: used to stain living organisms. Dissolve 1 gram in 100 mL of distilled water and add 0.5 gram sodium chloride. This stain can be obtained as a solution from some pet shops, but it will need to be diluted and may have nasty additives. Remember that this one will stain sinks, basins, baths and toilets—and skin!

Nigrosine: used to stain bacterial spores and capsules. Dissolve 1 gram in 20 mL of water.

You can also experiment with food colourings. Many of these are now accused of being dangerous, even when they are approved for adding to food. Treat them carefully, just in case.

Iodine: this is not the friendliest of materials, but it's the best stain for starch in plant materials. Add iodine crystals to a saturated solution of potassium iodide in water until it is saturated, filter and dilute to a pale golden brown. Check to see if you can buy 'tincture of iodine' from your pharmacist, but it will be expensive, and these days, in Australia at least, you will probably only be able to get it from a specialist pharmacist called a compounding chemist.

Malachite green: is not so safe but it is useful to stain plant cytoplasm. Dissolve 1 gram in 100 mL of tap water. This is available as an anti-fungal solution from aquarium shops, but that solution usually contains formalin, which is really dangerous. Read the label first!

When you come down to it, the staining of thin sections might be Too Much Trouble, so what else can you do?

One easy observation involves the large cells that are found in a layer called the epidermis on a piece of onion.  I touched on that a while back in A bit more about microscopes and hand lenses — and I may get back to it at some stage.

1 comment:

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