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Norsk språkWhat is 100x Oil Objective and Oil Immersion?
Bacteria are very small of course, so it is necessary to view them at the highest magnification possible with the best resolution possible. In a light microscope this is about 1000x due to optical limitations. The Oil immersion technique is used in order to enhance resolution. This requires a special 100x objective. The 100x lens is immersed in a drop of oil placed on the slide in order to eliminate any air gaps and lossof light due to refraction (bending of the light) as the light passes from glass (slide) → air → glass (objective lens). Immersion oil has the same refractive index of glass. When used, light passes through glass → oil → glass without loss due to refraction.
When the oil immersion lens is in place, there are a few things to be aware of and very careful about-
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The working distance is VERY small.
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Depth of focus is also VERY small.
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Use only the fine focus, and focus slowly.
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Increase the light, as the lens opening is very small.
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Adjust the condenser and iris diaphragm as needed.
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Do not drag the 40x lens through the oil, if you need to go to lower power-4x or 10x, rotate the nosepiece in the opposite direction to avoid the 40x lens. Dragging the 40x lens through the oil will damage the lens!
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The immersion oil has the same refractive index as glass, so you may go back to low power (again, not 40x) and still see your specimen.
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Do not make any decisions or assessments about your bacterium until you are focused at 100x.
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If you see the cells clearly with the 4x, 10x, 40x, but don’t see it with 100x, your specimen may be upside down. Ask your instructor for help.
Viewing with oil immersion 100x objective lens-
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Place a stained slide on the stage in the clips.
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View the slide at low power (4x) and look for staining; for very small cells look for a pattern of regular tiny specks. Once in focus, move to the next objective (10X), focus with fine focus only. Move to 40x, fine focus only (if the 40x objective is not very clear—due to it being repeatedly dragged through the oil, skip it). Make sure that the area you want to see is dead center in the field of view.
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Rotate the objective aside so that you are between it and the 100x objective and let a small drop of oil fall onto the slide where your specimen is. Carefully rotate the 100x objective into place, immersing it into the drop of oil.
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While looking through the oculars, very carefully and slowly use the fine focus to bring the image into sharp focus. If you can’t get it into focus, or have lost what you are looking for you may go back to 4x or 10x and start over (do not remove the oil, it’s fine).
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When finished with your slides wipe the oil from the 100x objective with lens paper (DO NOT use anything else to clean the lens!). The lens retracts into the lens housing. Be sure to push up gently to remove oil that has moved up in the housing (this happens more when one is viewing a slide and over focuses and pushes the lens up into the housing). Then, use lens cleaner and lens paper to completely remove all oil from the lens. Keep the lens paper flat and use a circular motion. Do not wad up the lens paper. Finally, use lens paper to check all the other objectives and parts of the microscope—focus knobs, stage, drips onto the condenser, etc., and clean up any oil on them.
Why use immersion oil?
You’ll find that most high magnification objective lenses require immersion oil to reduce the impact of refraction.
Air, like any medium, refracts light to an extent. The air between your objective lens and your specimen is no exception – when the light hits the air, it bends and scatters.
At lower magnifications, the refraction isn’t particularly noticeable, and you can happily use your 4x, 10x and 40x objectives without any problems. But once you reach the higher magnifications, distortion becomes a real issue.
The solution? Replace that air with an oil with the same refractive index as the glass slide and coverslip. This reduces the amount of refraction, giving you a clearer image.
How to use immersion oil with objective?
First, make sure your lens is actually designed for oil immersion. Non-oil objectives are not properly sealed, and the oil will seep inside and damage them.
Once you’re sure you have an oil lens, here’s a general guide to using oil immersion microscopy with a standard compound microscope.
What you’ll need
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Oil objective lens
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Immersion oil
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Coverslips
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Glass slide
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Lens paper
Using the oil
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Prepare specimen on slide, make sure to use a coverslip.
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Put prepared slide on microscope working stage, using 4x, 10x objectives to find image first.
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Use 40x objectives and bring specimen into focus.
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Rotate the 40x lens out of the way, stopping halfway to the 100x (oil) objective.
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Place one small drop of immersion oil directly on your coverslip.
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Gently swing the 100x oil objective and position it over the slide. Then use the fine focus control to lower the objective lens until it just touches the oil. It’s often best to look at your slide from the side while you do this, so you get a clear view while you’re lowering the objective.
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Look down the eyepiece and bring your specimen into focus using the fine focus carefully. (Don’t use the coarse focus – you can easily ram the objective into the slide, damaging your lens.)
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Once you’ve finished inspecting your slide, roll the 100x objective out of position and remove the slide. Make absolutely sure that you don’t accidentally roll the 40x lens into the oily slide.
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Gently wipe the immersion oil off your oil objective lens by lens cleaning paper.
Huang Xin