Smear mounts and immersion oil

[Even]

I am playing around with some experiments found in "Illustrated guide to home biology experiments". I did my first gram staining of a smear mount I made with bacteria from the teeth gum line. I can clearly see both gram positive and negative bacteria and there are probably some epithelial cells too.

I would now like to use my oil immersion objective to get the highest magnification, but got a little confused: The book says that you should observe the smear mount without a cover slip. I can see from the "Common Beginners’ Mistakes" found from this site that all objectives are meant for using with a cover slip. And when using immersion oil you would most likely allways use a cover slip to avoid mixing the oil with the specimen. Am I wrong?

[zzffnn]

If your most powerful oil objective is marked as 170/- and not 170/0.17, then you don't need to use cover slip.

For those objectives marked with 0.17, you can skip cover slip at up to oil immersion objective NA 1.0 and dry objective NA 0.4.

If you dry your smear really well and fix smear to slide, using immersion oil without cover slip won't mix up specimens much.

Not all objectives require 0.17 mm cover. Some require no cover, some don't care, others require 1.0mm cover.

[kit1980]

There are oil objectives specifically designed to be used with smears without cover slips, and these are marked like "170/0" and also "NC". I think these are mostly for hospitals to do more observations faster, because coverslipping takes time.

[apochronaut]

Coverslip thickness has varied from manufacturer to manufacturer over the years but lately .17 has been a sort of standard for upright biological microscopes. Bausch & Lomb used .18, which is nice because a lot of water based covered samples can end up being too thick anyway. The extra .01 gives a tiny bit of leeway.

If you are using no cover slip, yes kit980 is correct, no cover objectives would be best but if all you have is a .17 corrected objective, using the thicker grade of immersion oil, will cause the oil to set up higher and provide closer to the .17 w.d. that the objective is corrected for.

[Tom Jones]

Even,

During my 40 years in clinical labs, I've looked at many, many thousands of hematology and gram stain smears with oil immersion and no cover slip. In fact, I've never seen anyone use a cover slip unless it is a permanently mounted slide. Just make sure they're dry first! While using a cover slip might be ideal in theory, you will find it's not worth the effort as the oil refractive index is generally very close to that of the cover slip. Even using non-oil lenses, a thin layer of oil, not thick enough to touch the objective lens, nicely replaces any mounting medium and cover slip combo. Dried smears need something to prevent diffraction around and through the dried material anyway, so using a cover slip will require some mounting medium under it, permanent or otherwise. Oil works here, too.

Even with initial low power scans with a non-oil lens, a thin layer of oil would be put over the specimen prior to viewing. That's done by putting a drop of immersion oil on the specimen, then wiping it across the slide with a glass rod to create the nice thin layer of oil. Think of it as a temporary cover slip made of immersion oil.

Tom

[billbillt]

Coverslip thickness has varied from manufacturer to manufacturer over the years but lately .17 has been a sort of standard for upright biological microscopes. Bausch & Lomb used .18, which is nice because a lot of water based covered samples can end up being too thick anyway. The extra .01 gives a tiny bit of leeway.

If you are using no cover slip, yes kit980 is correct, no cover objectives would be best but if all you have is a .17 corrected objective, using the thicker grade of immersion oil, will cause the oil to set up higher and provide closer to the .17 w.d. that the objective is corrected for.

It is wise to have something to measure the cover slip thickness if you are a perfectionist... I have found some variation with all brands of them.. Decent micrometers can be had from Ebay at a reasonable cost..

BillT

[apochronaut]

Coverslip thickness has varied from manufacturer to manufacturer over the years but lately .17 has been a sort of standard for upright biological microscopes. Bausch & Lomb used .18, which is nice because a lot of water based covered samples can end up being too thick anyway. The extra .01 gives a tiny bit of leeway.

If you are using no cover slip, yes kit980 is correct, no cover objectives would be best but if all you have is a .17 corrected objective, using the thicker grade of immersion oil, will cause the oil to set up higher and provide closer to the .17 w.d. that the objective is corrected for.

It is wise to have something to measure the cover slip thickness if you are a perfectionist... I have found some variation with all brands of them.. Decent micrometers can be had from Ebay at a reasonable cost..

BillT

My mistake in the text. I meant ; coverslip thickness requirement has varied over the years from microscope manufacturer to microscope manufacturer.

I wasn't referring to the cover slips themselves.