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Vegetable oils as clearing agents 2016 achievements in the life sciences Vegetable oils as clearing agents 2016 achievements in the life sciences Vegetable oils as clearing agents 2016 achievements in the life sciences Vegetable oils as clearing agents 2016 achievements in the life sciences Vegetable oils as clearing agents 2016 achievements in the life sciences Vegetable oils as clearing agents 2016 achievements in the life sciences Vegetable oils as clearing agents 2016 achievements in the life sciences Vegetable oils as clearing agents 2016 achievements in the life sciences 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 April 2016 Accepted 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 (http://creativecommons.org/licenses/by-nc-nd/4.0/) 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: micifad@gmail.com (I.M Adeniyi) Peer review under responsibility of Far Eastern Federal University Tel.: +234 806025504 http://dx.doi.org/10.1016/j.als.2016.05.001 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 (http://creativecommons.org/licenses/by-nc-nd/4.0/) 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 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 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 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 Fig 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 10 Fig 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 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 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 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 (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