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Journal of the Anatomical Society of India

Cost Effective, Qualitative Immersion Oil for Microscopy

Author(s): Victor R, Jayalakshamma, Rajangam Ssayee

Vol. 54, No. 2 (2005-07 - 2005-12)

Victor R#, Jayalakshamma*, Rajangam Ssayee*
# Cytogenetic laboratory, Lal Bagh Nursing Home, Jayanagar, Bangalore
* Division of Human Genetics Department of Anatomy, St. John's Medical College, Bangalore

Abstract: An attempt has been made to identify a cost effective, but qualitative immersion oil for microscopy and photomicroscopy. Pure Castor oil (Oleum Ricini) and refined sunflower oil have been tried along with commercially available immersion oil on GTG banded chromosome preparations. Metaphase spreads were analysed, photographed and the results were compared. Applicaton of castor oil has been observed to be better and it could be recommended as alternative immersion oil.

Key words: chromosome preparation, immersion oil, castor oil.


plays an important role. In the absence of immersion oil, flat images with little contrast may be observed. Immersion oil, available commercially, contains Benzyl benzoate. There is no proof that Benzyl benzoate could affect the image. Cedarwood oil and paraffin oils are also in use as immersion oil for microscopy.

GTG banded chromosome preparations could be analysed with or without coverslips with immersion oil. While analysing the chromosomal preparations without the coverlips, consequent removal of the oil with Xylene, tend to fade the chromosomal spreads. Normally, GTG banded slides need not be coverslipped, as the slides may have to be destained for rebanding. Also it has been observed that there is not much difference between the coverslipped and not coverslipped slides in the obtained images.

An attempt has been made to identify immersion oil, which will suit the chromosomal preparations and also any other histopathological preparations with following objectives:

  1. The oil should be cost effective when compared with the commercially available immersion oil for microscopy and photomicroscopy.
  2. It should not have any adverse effect on the preparation after its removal with Xylene.
  3. It should give a better image
  4. It should not damage the oil immersion objective with its frequent use.

Materials and Methods:

Castor oil is clear colorless viscid oil and has excellent keeping quality. It has varied use, mostly, as a purgative and lubricant. Commercially it is the chief raw material for the production of resins / fibres and in the cosmetic preparations. Thus, arose the idea of its possible use as an alternative to the usual immesion oil. In Division of Human Genetics, patients are referred for karyotyping and counseling. Hence, along with the commercially available immersion oil, castor oil also was put to use.

Lymphocyte cultures set up by the method of Arakaki and Sparkes (1963) harvested slides prepared and GTG banded by the method of Seabright. (1971).

Three sets of banded slides with coverslips and without coverslips were kept separately for the Analyzing, Photomicrography with 25 ASA black and white film and Computer print outs with KOCOM CCD camera

The following immersion oils were used:

  1. Pure castor oil (Oleum Ricini) Commercially available oil was filtered thorugh a gauze cloth and stored in a bottle.
  2. Refined Sunflower oil
  3. Commercially available immersion oil

Each set of slides observed with oil objective X 100 in Leitz Laborlux microscope with green filter using oils 1, 2, 3 respectively. Five spreads were drawn and photographed. After observations, the slides without coverslips were kept in individual coplin jars containing xylol overnight to remove the oil. Slides were kept overnight to see whether long time exposure to xylene affects the image / stain Next day, slides were removed, dried, observed and photographed and computer print out.

Results and Discussion:

There is not much difference between the slides with and without coverslips; neither with the castor oil or with immersion oil (Figure I)

The Merck index describes castor oil as Lioinis oil / oil of palma Christi / Tangantangen oil / Neoloid. Fixed oil is obtained by cold pressing of the seeds of Ricinus communis. L. Euphor biaceae, Triglyceride of fatty acids, Fatty acid composition is approximately: Ricinoleic 87%, Oleic 7%, Linoleic 3%, Palmitic 2%, Stearic 1% and dihydroxy stearic trace amounts (Merck Index 1989).

Table 1:

Sl.No Immersion oil Observation Photomicrography Computer print out
1. Castor oil (Oleum Ricini) Images are clear. No strain to the eye V Good V Good
2. Refined Sunflower oil Images are very clear. Strain to the eye Fair Fair
3. Commercially available immersion oil Images are clear. No strain to the eye Good Good

GTG-Metaphase spreads

Fig. 1: GTG-Metaphase spreads (a) Castor oil, (b) Immersion oil, (c) Castor oil without cover slip, (d) Immersion oil without cover slip

In Microscopy, while using oil immersion objective the immersion oil used must have proper refractive index and density. Lens affects the rays because of its material. The speed of light traveling through the lens is reduced as compared to its speed in the air. The ratio of the speed of light in air to that in lens medium is called refractive index. Refraction is dependent not only on the angle at which the ray strikes the surface of the lens but also on the refractive index of the medium. Light entering with dense medium bends towards the normal, while it bends away the normal while entering a less dense medium.

The commercially available immersion oil has a refractive index 1.515 with a density of 1.303. Pure castor oil (Oleum ricini) has a refractive index of 1.477 to 1.481 with a density of 0.953 to 0.964. Cedar wood oil, which is also used as immersion oil, has a refractive index of 1.495 to 1.510 (British Pharmacopeia 1963). The mounting medium normally used are also have the same refractive index. DPX Mountant 1.5240 and Canada balsam 1.5250.

While comparing the commercially available immersion oil and pure castor oil (Oleum ricini) castor oil has a lower value. But the performance is better as observed.

The major advantage in using castor oil (Oleum ricini) is that, after its use, the observed slides could be put in xylene for removal of oil even overnight and the chromosome spreads were intact and no fading was observed. A good quality of xylol must be used (AR grade)

Also, it is observed that, there is not much difference between the banded slides with or without converslips, either in observing, photomicrographs or computer printouts. Hence, coverslipping is not necessary for normal cases, for abnormal cases the slides could be mounted. Even mounted slides tend to fade after sometime.

Now, with the advantage of computer printouts images could be stored in floppies. Hence, coverslips are not necessary. If a slide has to be destained for rebanding after observing in oil, the slide can be put in xylol for removal of oil dried and destained with methanol / acetic acid fixative (3:1) ratio.

It may be concluded that pure castor oil (Oleum ricini) could be cost effective qualitative immersion oil for microscopy, photomicrography and computer printouts for karyotyping, and for any histopathological analysis.


  1. Arakaki D T and Sparkes RS: Micro technique for culturing leukocytes from whole blood. Cytogenetics 1963; 2 : 57.
  2. British Pharmacopeia, The pharmaceutical press, 17 Bloomsbury Square London WC1, UK, 1963; pp 144.
  3. The Merck Index. An encyclopedia of chemicals, drugs and biologicals. 11th Edn; Merck & Co. Inc, Rahway New Jersey, USA, 1989;pp 1137 and 1901.
  4. Seabright M. A rapid banding technique for human chromosomes. Lancet 1971; 971.
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