Vegetable Oils as Clearing Agents

Abstract

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.

Keywords

Clearants ; Dehydration ; Sections ; Oils ; Xylene ; Clove-oil

Introduction

Transition step between dehydration and infiltration with embedding medium is often referred to as clearing or de-alcoholization (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 de-alcoholization 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 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 non-toxic, 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 (Fig. 1 , Fig. 2 , Fig. 3  ;  Fig. 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. 1. Transverse sections (× 100). 1: Xylene-cleared. 2: Groundnut oil-cleared. 3: PKO-cleared. 4: Clove oil-cleared. 5: Coconut oil-cleared.

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

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

Fig. 4. Radial longitudinal sections (× 400). 16: Xylene-cleared. 17: Groundnut oil-cleared. 18:PKO-cleared. 19: Clove oil-cleared. 20: 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 Fig. 1 , Fig. 2 , Fig. 3  ;  Fig. 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 (Fig. 1 , Fig. 2 , Fig. 3  ;  Fig. 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.

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.

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