IMMERSION OIL: COMPOSITION, CHARACTERISTICS, USES - BIOLOGY - 2024

The immersion oil is a viscous clear liquid having a refractive index high. For this reason it is widely used in microscopic observations, as it provides the property of concentrating light when it passes through the 100X objective of the microscope, increasing its resolving power.

This occurs because a viscous film is formed between the objective and the smear, preventing the light rays from scattering when they reach the air, thus concentrating the light beams towards the sample.

Dropper with oil immersion and microscope focused with 100X objective using oil immersion. Source: Pixabay.com

Immersion oil is used only with the 100X objective. That is why the 100X objective is also called an immersion objective. The rest are known as dry targets. This lens cannot be used without the immersion oil, as without it the visualization would not be satisfactory.

The main function of the immersion oil is to provide clearer, sharper and more defined images, allowing the observation of details that would not be possible with other lenses.

The first oil used for this purpose was anise oil, used together with the first immersion lens created by Giovanni Battista Amici. This only prevented chromatic aberration, but did not increase the numerical aperture of the lens system.

Then cedar oil was used, but it had many disadvantages that forced its disuse. Among them was its high acidity, which hurt short-term objectives.

Subsequently, other substances such as water and glycerin were used until synthetic immersion oils were created, which little by little were refined until they reached the current immersion oil, which is a complex compound of organic substances.

Composition of immersion oil

The immersion oil is made up of a mixture of organic compounds such as: terphenyl, hydrogenated terphenyl, natural hydrocarbons and polybutenes.

Immersion oil characteristics

Immersion oil is a light yellow liquid, slightly viscous in appearance, with a characteristic odor and a density between 0.92 to 0.99 g / cm ³.

Immersion oil is not soluble in water, and has a boiling point of 340 ° C. The refractive index a (n 20 ° C / D): ranges from 1.482 - 1.516. Meanwhile, the flash index is 110 ° C and the viscosity ranges from 100 to 120 mPa.s.

This product must be disposed of properly, as it is ecotoxic. In other words, it is harmful to the environment, mainly affecting aquatic animals. On the other hand, it is irritating to the skin and mucous membranes. If ingested it is nephrotoxic and cardiotoxic.

Immersion oil is stable at 15 to 25 ° C, the temperature at which it must be stored.

Immersion Oil Basics

When using higher magnification lenses, the intensity of the light should be increased. However, many of the light beams are lost because they are scattered upon reaching the air (space between the coverslip and the lens), and even some rays are totally reflected.

Immersion oil has a refractive index similar to glass. Therefore, when the oil comes into contact between the 100X lens and the coverslip, the light beams are concentrated.

The concentrated rays pass through the sample and sharpen the image, that is, increase the numerical aperture of the objective and correct for chromatic and spherical aberrations.

Uses or applications

The use of immersion oil is especially necessary when it is necessary to observe an image in a broader way, using the highest magnification objective (100X objective) to see details that are essential to establish a diagnosis.

Therefore, it is widely used in microscopic studies in various areas such as: histology, cytology, hematology and bacteriology. For example, it is useful for studying the characteristics of cells and tissues of a patient.

It is also used for the analysis of blood smears, where we want to detail the characteristics of hemoparasites inside and outside the erythrocytes, as well as in Gram preparations, to define the morphotintorial characteristics of the microorganisms.

It is generally used in fixed preparations, as it is not recommended in fresh preparations. This is because the surface tension of the oil causes the coverslip to move, causing the sample to shift during observation and prevent analysis.

On the other hand, immersion oil is not only useful in conventional light microscopy: it is also used in total internal reflection fluorescence (TIRFM) microscopes and in confocal fluorescence applications.

Steps to observe a preparation using immersion oil

To view a slide under a microscope, the slide should first be focused with the lowest-magnification objective, usually the 10X, with low light intensity.

Then it goes to the 40X objective, and the light path is slightly increased. Some analyzes can be performed at this magnification, but 100X magnification is required to see structural details.

Before moving to the 100X objective, a drop of immersion oil is placed on the coverslip, right where you want to observe, and then the microscope turret is moved to locate the 100x objective in the preparation.

The intensity of the light is regulated (increased) (by moving the condenser and opening the diaphragm). Then, with a few small movements of the micrometer screw (forward or backward) it should focus perfectly if the microscope uses parafocal objectives.

If you try to focus the preparation directly with the immersion lens, you may have trouble finding the focus. It is not an impossible action, but the difficulty increases significantly.

Care

To use the immersion oil, certain conditions must be taken into account.

When a colored preparation has been made, such as a smear stained with Giemsa or a Gram, wait for the smear to dry completely before placing the oil immersion. Otherwise, the oil together with the water will form micelles that will not allow the preparation to be seen.

On the other hand, after observing the batch of preparations, that is, at the end of the working day, the 100X objective should be carefully cleaned, using a lens paper with ethanol. If the lens is left dirty, the oil will dry on it and then it will be very difficult to remove it, damaging the field of view.

Likewise, it must be taken into account that the oil is flammable and must be kept away from heat sources (lighters). It is important to avoid heating it above 65 ° C.

Finally, oil is a toxic product. Therefore, direct contact with skin and mucous membranes should be avoided, where it can cause slight irritation. To avoid accidents, the use of gloves and safety glasses is recommended to handle it.

In the event of contact with the oil, the area should be washed with plenty of water. If the oil splashes into the eyes, it should be washed in the same way, keeping the eyes open. In case of accidental ingestion, it is important to ingest warm water and induce vomiting, in addition to going to the nearest doctor.

References

1. "Total Internal Reflection Fluorescence Microscope." Wikipedia, The Free Encyclopedia. 30 Dec 2018, 22:46 UTC. 14 May 2019, 01:54
2. IVD. Microscopy Oil immersion. Available at: Users / Team / Downloads.
3. Environmental Biology and Microbiology. Operation of the optical microscope. Available at: eumed.net/libros.
4. Sánchez Lera Rita María, Oliva García Ninfa Rosa. History of the microscope and its impact on Microbiology. Rev Hum Med, 2015; 15 (2): 355-372. Available in: scielo.
5. Herrero J. Practice No. 1: Fundamentals and use of the common compound optical microscope. Universitat d´Alacant Available at: rua.ua.es/dspace
6. Scharlau - MSDS. Immersion oil, for microscopy. 2001. Available at: insumos-labcentral.