Vegetable oils as clearing agents 2016 achievements in the life sciences

Achievements in the Life Sciences 10 (2016) 1–4

Contents lists available at ScienceDirect

Achievements in the Life Sciences
journal homepage: www.elsevier.com/locate/als

Vegetable Oils as Clearing Agents
I.M. Adeniyi ⁎,1, O.R. Adejoba, F.M. Akinlabi, O.J. Alao
Department of Forest Products Development and Utilization, Forestry Research Institute of Nigeria, P.M.B 5054, Forest Hill, Jericho Estate, Ibadan, Oyo-State, Nigeria

a r t i c l e

i n f o

Article history:
Received 14 October 2015
Received in revised form 5 April 2016
Accepted 9 May 2016
Available online 23 June 2016
Keywords:
Clearants
Dehydration
Sections
Oils
Xylene
Clove-oil

a b s t r a c t
Three types of vegetable oils namely, groundnut oil, coconut oil and palm kernel oil were investigated in this study for their clearing ability in wood histology. The vegetable oils were
compared with the conventional clearants like xylene and clove oil. Using a Riechet microtome

sliding machine, sections of wood that were about 20 μm were prepared and cleared in each of
the oils. Under a light microscope, the results showed that sections cleared in the vegetable oils
did not show any sign of distortion as details of wood features were distinct and clear after
processing in the vegetable oils. The study concluded that the vegetable oils could be used as
substitutes for xylene and clove oil. The respective photo micrographs are hereby presented
showing their efficacy after slide preparation. On the accounts of health hazards and cost of
conventional clearing agents, the study also recommended groundnut oil, coconut oil and
palm kernel oil as alternative clearants in wood histological processes.
© 2016 Far Eastern Federal University. Hosting by Elsevier B.V. This is an open access article
under the CC BY-NC-ND license ( />
Introduction
Transition step between dehydration and infiltration with embedding medium is often referred to as clearing or dealcoholization (Avwioro, 2002). It is a process that requires the use of solvents known as clearants or clearing agents that are
miscible with both absolute alcohol and the embedding medium. Alcohol is mostly used as a dehydrating agent before dealcoholization in a clearing agent, other dehydrating agents such as acetone and dioxane (diethylene dioxide) can also be used
(An et al., 2001). The most popular clearing agents are xylene, toluene, benzene, chloroform and cedar wood oil (Ochei, 2005),
though clove oil is mostly used in wood histology. These clearing agents had been substituted with vegetable oils and orange
oils at one time or the other on the account of occupational safety (Sermadi et al., 2014) or other reasons. The use of xylene
as a clearing agent in all cases might be discouraged on the account of its toxicity which ranges from acute neurotoxicity, cardiac
and kidney injury, cancer, blood dyscrasias, skin diseases, gastrointestinal disturbances, musculoskeletal system disorders,
fetotoxicity (Kandyala et al., 2010; Anderson et al., 1981), and tissue distortions as a result of long-term immersion of tissue in
xylene. However, in spite of its toxicity to laboratory personnel and the danger it poses to the environment, xylene has been
widely used as a clearant of choice. This might be due to the fact that xylene (when compared with other clearants) has lower
viscosity than immersion oil, vegetable oils and clove oil. This of course allows handling of fragile tissues in xylene to be easier
and convenient. Toluene is better at preserving tissue structure and is more tolerant of small amounts of water left behind in
the tissues than xylene, but it is less commonly used because it is more expensive and more toxic than xylene. Furthermore, chloroform not only acts slowly and has a severe health hazard, it may also lead to sectioning difficulties. Orange oil-based clearing

⁎ Corresponding author. Tel. +234 806 025504.
E-mail address: (I.M. Adeniyi).
Peer review under responsibility of Far Eastern Federal University.
1
Tel.: +234 806025504.

/>2078-1520/© 2016 Far Eastern Federal University. Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
( />

2

I.M. Adeniyi et al. / Achievements in the Life Sciences 10 (2016) 1–4

agents are excellent for preserving fine tissue structure, and can often be used in place of xylene with no alteration of protocol.
However, orange oils that are neither pure nor stable can break down to produce compounds that will interfere with staining procedures. Clove oil is an excellent clearing medium, but it has the power of extracting certain stains especially safranin stain, and so
it cannot be used in all cases. Coconut oil is a commonly used vegetable oil, available throughout the tropical world. It is nontoxic, heat stable, slow to oxidize and has highest resistance to rancidity (Fife, 2005), though the mixture of coconut oil and
clove oil as a clearing agent was ineffective (Rasmussen et al., 1992) because the resultant solution produced incomplete impregnation, leading to problems in the cutting sections. Furthermore, a study by Andre et al. (1994) stated that a mixture of peanut oil,
soyabean oil, coconut oil and cotton oil was a poor alternative for xylene. These earlier experiments suggested that mixtures of
clearants are not the best substitutes for widely used clearing agents. In addition, palm kernel oil (PKO), like coconut oil, is a commonly used vegetable oil that is available throughout the tropical world. It is less expensive, non-toxic and heat stable, and derived from oil palm. The fruit of oil palm yields two oils, palm oil and kernel oil, each exhibiting differences in composition,
properties and applications. Palm kernel oil is similar to coconut oil in composition and both are the only source of lauric oil available in the world market (Berger et al., 1991). The cake residue obtained after extracting oil is used in livestock feeds, while the
palm and kernel oils are used in soap production, vegetable oil and margarine (Ekpa, 1995). Beside these applications, the oils are
also used locally as body creams, cooking oils and medicinally as surface protectants for minor wounds. Apart from the identified
disadvantages of xylene, immersion oil and clove oil, they are also not cost effective, but expensive, whereas vegetable oils such as
coconut oil, palm kernel oil and groundnut oil are readily available even at affordable prices. It is therefore necessary to provide
less toxic and less expensive alternative s clearants in histology. Most of the past studies on alternative clearants based their findings on animal tissues such as skin, tendon, muscle, node etc.; however information on the efficacy of these substitute clearants
on wood specimens is limited. This present study therefore intend to investigate the efficacy of coconut oil, groundnut oil and
palm kernel oil on wood sections with a view to providing substitutes to toxic and expensive clearing agents.

Methodology
Five clearing agents namely clove oil, xylene, palm kernel oil, groundnut oil and coconut oil were used for this study. The
wood specimens were from a mature wood of Nauclea diderrichii. The choice of wood was informed based on its medium density
as opposed to the usual soft tissues used in the past studies (Sermadi et al., 2014). Wood microscopy was done in accordance
with ASTM D1413-61 (ASTM (American Society for Testing and Materials), 2007); wood sections of about 20 μm thick were produced in three planes namely cross sectional, tangential and radial sections using a Riechet sliding microtome. They were transferred into a dish containing methylated spirit using a soft brush. Sections were washed with distilled water and covered with
safranin for 2 min, and then they were dehydrated through a series of bath of increasing concentrations (30%, 50%, 75%, 90%
and 100%) of ethanol for about 15 min at each concentration (Kitin et al., 2000) with frequent changes of the respective solutions
of ethanol. After each section was carefully cut into four parts, each part was covered with a different clearant for 1 h, then placed

on microscope slides and fixed in a Canada balsam (Burger and Ritcher, 1991).

Results
All the clearing agents used in this study were colourless. Only the PKO (palm kernel oil) was dark. Each of the clearing agents
had a distinctive odour, except groundnut oil which was almost odourless. Xylene had an aromatic odour, while that of the clove
oil was a strong and slightly sweet odour that lingered for some time. Palm kernel oil (PKO) had a rancid smell, while coconut oil
possessed a sweet and pleasant odour. Groundnut oil was almost odourless. The wood features were distinct and clear in all the
clearing agents (Figs. 1–4), though each section cleared in respective clearing agent exhibited different nature of translucency as
shown by the micrographs. In the transverse section, the micrograph from coconut oil-cleared section was the least translucent of
all the micrographs (Fig. 1). The wood features were more distinct in sections cleared in clove oil than in any other section processed in other clearing agents. Perhaps safranin was moderately retained by wood cells when cleared in clove oil, although the
effect of the safranin stain was clearly visible in all the clearants at the transverse section (Fig. 1). It was also observed that
none of the wood sections was distorted after clearing in Groundnut oil, PKO and Coconut oil.

1

2

3

4

5

Fig. 1. Transverse sections (×100). 1: Xylene-cleared. 2: Groundnut oil-cleared. 3: PKO-cleared. 4: Clove oil-cleared. 5: Coconut oil-cleared.


I.M. Adeniyi et al. / Achievements in the Life Sciences 10 (2016) 1–4

6

7

10

9

8

3

Fig. 2. Tangential longitudinal sections (×100). 6: Xylene-cleared. 7: Groundnut oil-cleared. 8:PKO-cleared. 9: Clove oil-cleared. 10: Coconut oil-cleared.

12

11

13

15

14

Fig. 3. Radial longitudinal sections (×100). 11: Xylene-cleared. 12: Groundnut oil-cleared. 13:PKO-cleared. 14: Clove oil-cleared. 15: Coconut oil-cleared.

Discussion
This study aimed to examine the possibility of using PKO, groundnut oil and coconut oil in place of the conventional clearants
like xylene, toluene, benzene and clove oil in wood histology. The micrographs in Figs. 1–4 showed that PKO, groundnut oil and
coconut oil can replace the commonly used de-alcholization agents. The result obtained from coconut oil-cleared sections was in
accordance with an earlier study by Sermadi et al. (Rasmussen et al., 1992) that the oil was as effective as xylene and therefore it
is an efficient substitute of xylene, causing less shrinkage of tissues in the histopathological laboratory, and without losing the

quality of the histological details. PKO-cleared sections can also compete favourably well with those cleared in Clove oil or Xylene
(Figs. 1–4; nos. 3, 8, 13 and 18). Groundnut oil can also be employed in wood histology as sections processed in it presented distinct features. This is in agreement with the work of Esan et al. (2015) in which groundnut oil was stated as a suitable alternative
to xylene in histological tissue processing. The alternative oils used in this study are non-toxic and therefore do not pose a threat
to health or the environment during the clearing process.

Conclusion
This study has shown that palm kernel oil, cococnut oil and groundnut oil successfully cleared the wood sections processed in
the vegetable oils as did by xylene and clove oil. During handling, the oils were not hazardous, except for the mild rancid smell
from PKO that did not constitute any hindrance during clearing. No instance of feature distortion, inadequate translucency or
threat to human health was experienced during processing, therefore the use of these oils as alternatives to clove oil and xylene
will definitely reduce the cost slide preparation since the vegetable oils used in this study were far cheaper than the conventional
clearants.

16

17

18

19

20

Fig. 4. Radial longitudinal sections (×400). 16: Xylene-cleared. 17: Groundnut oil-cleared. 18:PKO-cleared. 19: Clove oil-cleared. 20: Coconut oil-cleared.


4

I.M. Adeniyi et al. / Achievements in the Life Sciences 10 (2016) 1–4

Recommendation
Other vegetable oils that are readily available locally should be experimented for clearing. Consequent to the findings in this
study, palm kernel oil, coconut oil and groundnut oil are therefore recommended for clearings in wood histology.
References
An, Y.H., Moreira, P.L., Kang, Q.K., Gruber, H.E., 2001. Handbook of Histology Methods and Cartilage. pp. 186–188.
Anderson, K., Fuxe, K., Nilsen, O.G., 1981. Production of discrete changes in dopamine and noradrenaline levels and turnover in various parts of the rat brain following
exposure to xylene. Toxicol. Appl. Pharmacol. 60, 535–548.
Andre, G.G., Wenger, J.B., Rebolloso, D., Arrington, J.B., Mehm, W.J., 1994. Evaluation of clearing and infiltration mixtures (CIMs) as xylene substitutes for tissue
processing. J. Histotechnol. 17, 137–142.
ASTM (American Society for Testing and Materials), 2007. Preparation of Decayed Wood for Microscopical Examination (ASTM D1413–61).
Avwioro, O.G., 2002. Histochemistry and Tissue Pathology. Claverianum Centre, Ibadan, pp. 31–140.
Berger, K.G., Andaner, W.T., Applewhite, T.H., 1991. The Lauric Oils Medium Chain Fatty Acids Source Champaign. American Oil Chemists Society, America.
Burger, L.M., Ritcher, H.G., 1991. Anatomia Da Madeira. Nobel Press, Brasil, pp. 97–105.
Ekpa, O.D., 1995. Bio-inorganic constituents and possible uses of the female influorescence of the oil palm. W. Afr. J. Biol. Appl. Chem. 40, 13–18.
Esan, E.O., Ajileye, A.B., Fasogbon, S.A., 2015. Groundnut oil as an alternative clearing agent to xylene in histological tissue processing. Adv. Lab. Med. Int. 5 (3), 24–31.
Fife, B., 2005. Coconut Cures: Preventing and Treating Common Health Problems with Coconut. Piccadilly Books, Ltd., USA: Colorado Springs, pp. 184–185.
Kandyala, R., Raghavendra, S.P., Rajasekharan, S.T., 2010. Xylene: an overview of its health hazards and preventive measures. J. Oral Maxillofac. Pathol. 14, 1–5.
Kitin, P., Funada, R., Sano, Y., Ohtani, J., 2000. Analysis by confocal microscopy of the structure of cambium in the hardwood Kalopanax pictus. Ann. Bot. 86, 1109–1117.
Ochei, J.K., 2005. Medical Laboratory Science Theory and Practical. pp. 220–400 (London).
Rasmussen, B., Hjort, K., Mellerup, I., Sether, G., Christensen, N., 1992. Vegetable oils instead of xylene in tissue processing. Acta Pathol. Microbiol. Immunol. Scand. 100,
827–831.
Sermadi, W., Prabhu, S., Acharya, S., Javali, S.B., 2014. Comparing the efficacy of coconut oil and xylene as a clearing agent in the histopathology laboratory. J. Oral
Maxillofac. Pathol. 18 (Suppl. S1), 49–53.