On-line version ISSN 0717-9502
Int. J. Morphol. vol.25 no.2 Temuco June 2007
Int J. MorphoL, 25(2):335-336,2007.
Studies on the Effects of Fixatives on the Staining Ability of Morinda lucida Extracts on Tissue Sections
Estudios en los Efectos de Fijadores en la Capacidad de Tinción de los Extractos de Morinda lucida en Secciones de Tejidos
*O. G. Avwioro; **I. O. Imosemi; ***T. Oduola; ****O. J. Adisa & *****A. A. Oladele
*Department of Histopafhology, School of Medical Laboratory Science, Obafemi Awolowo University Teaching Hospital, Ile-Ife, Nigeria.
SUMMARY: The objective of the study was to determine the best fixative for tissue sections, which were to be stained with the dye extracts oí Morinda lucida, a stain for collagen fibres and muscle fibres. The effects of 10% formalin saline, Carnoy's fluid, Zenker's fluid, Helly's fluid and Bouin's fluid on the staining ability of the dye extracted from Morinda lucida on tissue sections were studied. Human tissues were fixed in the afore mentioned fixatives and the sections stained with acidified alcoholic solution of Morinda lucida extract. No visible difference in the staining reactions was observed when the stained slides from the different fixatives were compared. The colour, staining time and staining intensity were the same in all the sections studied.
KEYWORDS: Formalin saline; Carnoy's fluid; Zenker's fluid; Helly's fluid and Bouin's fluid.
RESUMEN: El objetivo del estudio fue determinar el mejor fijador para secciones de tejidos, los cuales fueron teñidos con el extracto de Morinda lucida, una tinción para fibras colágenas y musculares. Fueron estudiados los efectos de formalina salina al 10%, líquido de Carnoy, líquido de Zenker, líquido de Helly y líquido de Bouin en la capacidad de tinción de los extractos de Morinda lucida sobre los tejidos. Fueron fijados tejidos humanos en los fijadores antes mencionados y las secciones teñidas con solución alcohólica acidificada de extracto de Morinda lucida. No se observaron diferencias visibles en las tinciones cuando fueron comparadas con los diferentes fijadores. El color, tiempo de tinción e intensidad de tinción fueron iguales en todas las secciones estudiadas.
PALABRAS CLAVE: Formalina salina; Líquido de Carnoy; Líquido de Zenker; Líquido de Helly; Líquido de Bouin.
Description of Morinda lucida. Morinda lucida has previously been described (Avwioro et ah, 2005) as a tree which is 9 to 18m high bearing a dense crown of slender crooked branches and which is 20 to 30 cm in diameter. Morinda lucida is abundant in Northern and Southern Nigeria and Fernando po and over the Congo basin. (Keay, 1989). The wood is moderately coarse, open grained with a tendency to spiral, though not warping nor altering, fairly hard and of medium weight (Taylor, 1960).
Histological and other uses of Morinda lucida. An alternative cheaper and bio-friendly natural dye had previously been extracted from Morinda lucida for the staining of collagen fibres and muscle fibres (Avwioro, 2005). The staining component was described as an anthraquinone, which stained best at an acid pH. Morida lucida has also been used as a grand medicament of West African traditional medicine, valued for its antipyretic and antimalaria properties, and in the treatment of ulcers, leprosy and gonorrhoea (Durodola, 1974). Extracts of the leaves and stem have been recommended in the treatment of hypertension and cerebral complication showing distinct diuretic and tranquillizing effects (Debray et al., 1974).
Effects of fixatives on staining reactions. The choice of a fixative for tissues sometimes influences subsequent histochemical reactions (Culling, 1974). This is because fixatives are chemicals and they react with tissue structures to produce new compounds, which are capable of withstanding autolysis and putrefaction. Generally, fixatives, which contain acetic acid or whose pH is less than 4.4 do not favour the staining of cytoplasm (Avwioro, 2002). Formalin a major constituent of 10% formol saline supports many staining techniques, Carnoy's fluid, which contains alcohol and acetic acid, is a good fixative for chromosome studies. Zenker's fluid is good for the trichrome methods for collagen fibres while Helly's fluid is said to be a good fixative for micro-anatomical and cytological studies (Avwioro, 2002).
MATERIAL AND METHOD
Preparation of M. lucida extract and staining solutions.
Fresh roots of M. lucida were collected from the forest at Ile-Ife, Nigeria and identified at Botany Department Obafemi Awolowo University He Ife, Nigeria. They were rinsed several times in distilled water and drained. The roots were cut into tiny bits of about 1 mm in diameter and dried in an open air oven at 60°C for 96 hours. They were milled until they became fine powder. About 3 kg of the powdered plant material was extracted with 2 litres 70% ethanol under soxhlet for 72 hours until completion. The extract was filtered and concentrated in vacuo at 50°C and finally dried in a descicator. A very fine powder was obtained. 2g of the very fine powder was dissolved in 100ml of 1 % glacial acetic acid in 70% alcohol. The mixture was boiled in a water bath for 5 minutes in order to dissolve the solute completely.
Fixation, Preparation of sections and Staining of Sections
3 mm thick human tissues were obtained from intestine, skin, kidney, heart, liver and spleen at post mortem. They were fixed in 10% formalin saline, Carnoy's fluid, Zenker's fluid, Helly's fluid and Bouin's fluid for 24 hours and processed for paraffin wax embedding by dehydrating through 70%o, 90%>, 95%> and two changes of absolute ethanol for 1 hour 30 minutes each. Clearing was achieved through changes of xylene twice for two hours each, infiltrating through two changes of paraffin wax at 70°C and embedded in paraffin wax. Sections were cut at 4^im with the rotary microtome. (Sakura fine tech, Netherlands), attached to slides and dried at 65°C for 45 minutes. Sections were dewaxed in xylene, hydrated through graded solutions of alcohol and stained in 2g of the powder in 100ml of 1% glacial acetic acid in 70%o alcohol for 15 minutes. Sections were finally differentiated in 1% acid alcohol for 10 seconds, rinsed in water for 10 minutes, dehydrated through ascending grades of alcohol, cleared and mounted in a DPX synthetic mountant.
There was no significant difference in the staining reactions of the various tissue sections fixed in 10% formol saline, Carnoy's fluid, Zenker's fluid Helly's fluid and Bouin's fluid when they were stained with acidified alcoholic extracts of Morinda lucida. There was also no significant difference in the staining time, staining intensity and the ability to withstand the bleaching effect of the dehydrating alcohols when compared with those of the tissues fixed in 10%> formol saline.
The staining ability of some dyes has been said to be influenced by some fixatives, staining been faster and more intense after some fixatives than others (Culling). This is obviously due to the interaction, first between the tissue and the fixative to produce new products and second between the new products and the dye. The influence of fixatives has not been observed in staining with the dye extracts of M. lucida. Several different staining reactions are also not affected by the nature of fixatives used. For instance, the most important and the most used histological technique, haematoxylin and eosin technique is not affected by the nature of the fixative. The experiment was performed on several post mortem tissues from intestine, skin, kidney, heart, liver and spleen to enable a reasonable number of tissue structures from different parts of the body to be investigated, because no single tissue is a representative of all the tissues of the body.
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Dr. Godwin O. Avwioro PhD