Immersion Oil Microscopy

	IMMERSION OIL MICROSCOPY David B. Fankhauser, Ph.D. Professor of Biology and Chemistry U.C. Clermont College Batavia OH 45103
Applying oil to the slide	This page has been accessed times since 11 July 2001. 24 February 1986, rvsd 8 July 94, 7 July 95, 28 June 96, 3 July '97, 8 July 98, 29 June 00, 25 June 01	100x objective has been rotated into the oil

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 teh 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_D²⁵ : 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 reimmersing 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:

1) Focus very carefully with the 40x objective over the stained specimen on the slide.
(Once focused, do not alter focus for the next three steps!)
2) Rotate turret half way so that the 40x and 100x objectives straddle specimen.
3) Apply a small drop of oil directly on the slide over the specimen.
4) Rotate 100x objective into the immersion oil .

Three important rules attend the use of this lens:

1. Never use an oil immersion lens without the oil.
2. Never get oil on any other lens.
3. Clean up all oil when finished.

PROTOCOL:

	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).
	2. Rotate turret to 40x objective, locate desired portion of specimen in the center of the field. Refocus very carefully so that the specimen is focused as sharply as possible. (Do not alter focus for the following steps )
	3. CRITICAL STEP: Partially rotate turret so that 40x and 100x objectives straddle the specimen. Place a small drop of oil on the slide in the center of the lighted area. (Take care not to dribble on the stage.) Note the small drop of oil directly over the area of the specimen to be examined.
	4. Rotate turret so that the 100x oil immersion objective touches the oil and clicks into place. 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.

IMMERSION OIL MICROSCOPY