The most powerful lens of the light microscope is the 100x oil immersion objective. Because light is refracted every time it passes through a medium with a different refractive index, (air to glass or vice versa) the quality of the image is reduced with each passage. Thus, by reducing the number of such passages to a minimum, the clarity, brilliance and resolving power is preserved. You can see the difference between 400x and 1000x in the image to the left.
Immersion oil has been formulated so that it has a refractive index identical to that of glass. (It is written on the label of the immersion oil container as n D 25 : record it in your notebook.) Thus there is no refraction of light when it passes from glass to oil and vice versa. You can see the effect of this by removing the glass dropper rod from the oil, and re-immersing it. What happens to the image of the glass rod? How do you explain this observation?
Thus, two changes in refractive index can be eliminated by placing a drop of immersion oil on the specimen, and immersing the 100x oil immersion objective directly into the drop. You should be struck by the clarity that results.
Illustrate the four stage process of using the oil immersion lens:
Three important rules attend the use of this lens:
- Never use an oil immersion lens without the oil.
- Never get oil on any other lens.
- Clean up all oil when finished.
1. Focus at low power on a region of a smeared and stained specimen which is well-spread and stained (not too thin, nor too thick).
Focus only with fine focus. Hopefully, the specimen will come into focus easily. Do not change focus dramatically. If you still have trouble, move the slide slightly left and right, looking for movement in the visual field, and focus on the object which moved.
5. With more than one specimen on a slide, do not alter focusing, rather, place a drop of oil on the second specimen, and slide the slide laterally until it is in place.
6. Never go back to the 10x or 40x objectives after you have applied oil to the specimen since oil can ruin the lower power objectives. [The 4x objective can be used because it is high enough to be above the oil.]
7. Clean up!: When you have finished for the day, wipe the 100x oil immersion objective carefully with lens paper to remove all oil. Wipe oil from the slide thoroughly with a Kimwipe. Cleanse stage should any oil have spilled on it. Recap the immersion oil container securely, replace in drawer.