Evaluation of Some Essential Oils as Clearing Agents on the Histology of Some Selected Organs of Adult Wistar Rats
THE OBJECTIVES OF THE STUDY
The objectives of the research are to study:
- The effectiveness of essential oils namely citrodora oil, citrus oil, and neem oil as clearing agents in tissue processing.
- The effect of these oils on the structures of some tissues of adult Wistar
- The effects of these oils on the staining qualities of brain, liver, kidney, intestine and testes of adult Wistar rats.
- The clearing ability of these essential oils with themselves and the standard
CHAPTER TWO
LITERATURE REVIEW
INTRODUCTION
Before the microscopic examination of tissues, the tissues must undergo preparatory treatment entailing fixation, dehydration, clearing, infiltration and embedding. This preparatory treatment is known as tissue processing (Bancroft and Gamble 2008). Tissue processing is all the necessary treatments given to tissues from procurement and sacrificing of the animal, excising, fixation and labeling of the tissue. It also include the various stages of histological section preparations; dehydration, clearing, infiltration and embedding to the production of sections for microscopy (Bancroft and Stevens, 1990). This process has been designed to remove all extractable water from the tissue, replacing it with a support medium which provides sufficient rigidity and a suitable consistency for sectioning of the tissues without damage or distortions (Kieranan, 2010).
Paraffin wax is used universally to infiltrate and embed tissues in the final step prior to microtomy. Paraffin wax does not mix with water due to their hydrophobic nature; therefore water present in tissues has to be removed by dehydration using alcohols in ascending grades (Baker, 1966). Following dehydration, the tissues are saturated with alcohols which also are not miscible with paraffin wax. Prior to infiltration and embedding, the alcohols in the tissues have to be removed and replaced by certain chemical agents, a process called clearing. Clearing agents are both miscible with alcohols and paraffin wax. These clearing agents usually leave the tissues translucent after the clearing process. Impregnation is the complete removal of clearing reagents by media that is miscible with both the dehydrating agents and the impregnation. Embedding is the orientation of tissue in melted paraffin which when solidified provides a firm medium for keeping intact all parts of the tissue when sections are cut (Baker, 1966).
According to Kieranan (2010), there are many clearing agents sometimes called ―de- alcoholization agents‖. Most clearing agents are hydrocarbons with refractive indices similar to proteins. Some are aromatic derivatives of benzene with different levels of toxicity. Others are natural essential oils such as cedar wood oil and anicine (Hans et al., 1995). Common examples of clearing agents include xylene, toluene, chloroform, acetone, kerosene, diaxane, benzene etc.
CHAPTER THREE
MATERIALS AND METHODS
REAGENTS AND MATERIALS
The reagents and materials for this study include xylene, ethanol, neutral buffered formalin, chloroform, paraffin wax, egg albumen, Oven, Rotary microtome made in England Search Tech Instrument, Duplex Shandon Procesor serial number 02030, glass slides and microscope. The reagents were manufactured by Mayer and Baker Limited Dagenham England with product number 28304. They were purchased from Cardinal Scientific Supply by Zaria Hotel, Kwangila Zaria.
PLANT MATERIALS FOR THE OIL.
Orange (Citrus Sinensis) peels of ripened fruits of Citrus (sweet orange) plant, leaves of Eucalyptus (Eucalyptus Citrodora. Hook) plants and the seed of Neem trees were used in this study. These plant materials were all sourced within Zaria metropolis.
CHAPTER FOUR
RESULTS
RESULT OF PHYTOCHEMICAL SCREENING OF THE OILS
The phytochemical screening showed positive result for carbohydrate in citrus oil, citrodora oil, neem oil and xylene and also showed positive result for glycosides, steroid and tripenes in citrus and citrodora. Absence of anthraquinones, anthracene, saponins, flavonoid, tannins and alkaloid were observed (Table 4.1).
CHAPTER FIVE
DISCUSSION
Color of the oils
The colors observed were similar to what has been widely documented. Gbenou, (2013) reported that perfectly pure, essential oils can be colorless; some are brown, blue, or green or may have a pale yellow tint. Most of the work on colors of essential oils reported has to do with mixing of essential oils based on color for acupuncture and aromatherapy (Chiazzari 1998; Mary 2005).
CHAPTER SIX
SUMMARY, CONCLUSION AND RECOMMENDATIONS
SUMMARY
The present study have demonstrated that in tissue processing, conventional clearing and dewaxing agents (aromatic and aliphatic hydrocarbon) may be substituted by non- hazardous compounds with no loss of quality. The results from the present study indicated that essential oil namely citrus oil, citrodora oil and neem oil can clear and dewax tissue during processing and staining in their undiluted form. These essential oils may be viable alternative clearing agents to xylene especially the citrodora oil. With the exception of the neem oil, the citrus oil and citrodora oil has similar physical properties such as low specific gravity and density, viscosity, flash point and refractive index to that of xylene, placing them in the same clearing group. Citrus oil and citrodora oil are naturally extracted from plant with no or little known toxicity; however, citrodora oil seemed to be a better clearing agent than citrus oil and neem oil. Compared with xylene, citrus oil and citrodora oil have a sweet smell unlike xylene that have a pungent offensive smell. Citrus oil, citrodora oil and xylene had lower flash point values, meaning they are more flammable than neem oil which had a very high flash point. Microscopic examination showed no significant changes in section and staining quality between the sections cleared with citrodora oil and xylene at I and 2 hours respectively. Artifacts observed normally in tissue cleared using xylene were the same as that using citrodora oil. The sections of citrodora oil and xylene stained with all the special stains demonstrated the respective structures well.
There was no change of volume of the clearing agents used as against the standard two changes of xylene. This implies that these essential oils citrus, citrodora and neem will require less frequent changing than xylene, thereby reducing the quantity of clearing agent used. It was also observed that the better mean clearing time was 1hour with citrodora oil. This is a gain in time when compared to standard xylene clearing time of 2hours two changes each (4hours). The cost of 2.5litres of citrus oil, citrodora oil was cheaper when compared to that of neem oil and xylene is about. The high cost of this essential oil is species dependent in terms of oil yield; however, it is still better than xylene when the safety is considered.
CONCLUSION
This present study concludes that citrus oil, citrodora oil and neem oil can clear and dewax Wistar rat tissues during tissue processing and staining. There was significant difference in some of the tissues between citrus oil and neem oil when compared to xylene especially at 15 and 30 minutes respectively. There was no significant difference between citrodora oil and xylene when used as clearing agent for Wistar rat tissues at 1 and 2 hours respectively. Citrodora oil is a better clearing agent than citrus oil while neem oil is the poorest of clearing oil used in this present study.
Although the long time stability of these oil in clearing tissues still remain to be clarified, it can be said that by substituting the clearing and dewaxing agent using citrodora oil, and modifying the mounting procedure, xylene or similar organic solvent may be removed completely from the repertoire of reagents used in histology laboratories without losing valuable information.
RECOMMENDATIONS
The following were recommendations:
- Studies using advance techniques such as immunohistochemistry, Molecular and enzymatic study, Flurescence and electron microscopy techniques of the effects of these essential oils in tissue
- The study on the long time effects of this oil on tissues
- The long time mounting effect of these oils using different mounting
- Study on the mixture of these oils with other clearing
LIMITATIONS
Although, the data obtained from the present study suggest that a comparable section quality and staining result can be achieved with citrodora oil and xylene, the criteria for assessment were somewhat subjective. In the present study, the major focus was on the general appearance of the tissue. For it is usually difficult to give a clear definition of the ideal appearance of a tissue section by using only bright field microscope.
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