ABSTRACT
Monstaining is a special kind of staining procedure that highlights single cells. Most stains used in histopathology are synthetic, toxic and costly for use hence this study was carried out at the histopathology laboratory of Imo state university to research more on the probability of replacing routine toxic and costly stains for natural, non toxic, less costly and effective stains that can highlight observably single cellular structures when viewed microscopically. Twelve sections were used for this study (6 liver and 6 kidney sections), obtained from the federal medical center Owerri of which was divided into four groups of liver and kidney sections respectively. This division was according to stains to be used on both sections either with haematoxylin and eosin or prepared adjusted PH extracts of zobo and turmeric viz: The group 1 (3 liver sections) were stained with haematoxylin and eosin, group 2 (3liver sections) stained with zobo and turmeric plant extracts, group 3 (3kidney sections) stained with haematoxylin and eosin stains and group 4 (3kidney sections) stained with zobo and turmeric plant extracts. After analysis it was concluded that zobo and turmeric were not very good stains in highlighting single cell structures when compared to routinely used haematoxylin and eosin stains.
CHAPTER ONE
- INTRODUCTION
A stain is a dye consisting of a colored ion (a chromophore) and a counter ion to balance the charge. Attachment of the chromophore part of the dye complex to a cellular component represents the staining reaction. Monostaining or Monochrome staining is the use of a single colour stain to colour tissues. It is mainly used on bacterial cells (Gurr, 2010). Simple staining implies the use of only a single stain, which is usually sufficient to reveal the morphological features of most microbial cells, including relative size, shape, and characteristic arrangements for groups of cells. It is possible to see structural details by staining with a single dye if the sections are sufficiently thin. To obtain additional information, various specialized differential staining procedures have been developed. Examples of monostains used includes Heidenhain’s iron-hematoxylin which is a classical method of this type, Eriochrome cyanine R an anionic hydroxytriarylmethane dye that has various uses as an industrial colorant, a reagent in analytical chemistry and a biological stain, Hyman and Poulding’s method advantage which taken of the existence of red and blue dye-iron(III ) complexes with affinities for different components of animal tissues or an epoxy or polyester resin, Alkaline toluidine blue and so on (Baker, 2002; Bettinger and Zimmermann, 2001)
Pathology may be used broadly to refer to the study of disease in general. Pathology addresses four components of disease: cause (etiology), mechanisms of development (pathogenesis), structural alterations of cells (morphologic changes), and the consequences of changes (clinical manifestations)(Stanley, 2010). Pathology deals with the diagnosis of disease based on the macroscopic, microscopic, biochemical, immunologic and molecular examination of organs and tissues. Pathology is a scientific study of disease (Bezabeh et al., 2004). Tissue damage can occur as a result of an adverse stimulus which disrupts the normal homeostasis of affected tissues. Among other causes, this can be due to physical, chemical, infectious, biological, nutritional or immunological factors. Tissue damage can be reversible or irreversible. Depending on the extent of injury, the tissue response may be adaptive and where possible, homeostasis is restored (Ronni,2011). Some changes occur in pathological tissues. Tissue swelling may occur due to tissue hypoxia, loosening of intercellular attachments, mitochondrial changes dilation of the endoplasmic reticulum, tissue damage causes inadequate metabolism of fat (Hayes, 2008). Histopathology is the microscopic study abnormal tissues of the body it can also be called Tissue pathology (Proctor, 2015). Structure of these abnormal tissues gives a lot information about their function but the structures cannot be easily differentiated under the microscope unless by the use of stains. For normal or diseased tissues of humans and other vertebrate animals, a routinely used staining method for use on paraffin or frozen (cryostat) sections 4-7 μm thick is expected to provide intense blue, purple or black coloration that is largely confined to chromatin in the nuclei of cells, together with a contrasting and paler color such as pink or yellow in the surrounding cytoplasm and in extracellular structures (notably collagen fibers). Staining can be done either manually or in an automatic stainer. Conventional histological staining methods have been established for decades, some for more than a century (Dettmeyer, 2011). Heamatoxylin and eosin are routine stains used on most pathological tissues.
Hematoxylin and eosin (H&E) stains have been used for at least a century and are still essential for recognizing various tissue types and the morphologic changes that form the basis of contemporary cancer diagnosis. The stain has been unchanged for many years because it works well with a variety of fixatives and displays a broad range of cytoplasmic, nuclear, and extracellular matrix features. Hematoxylin has a deep blue-purple color and stains nucleic acids by a complex, incompletely understood reaction. Eosin is pink and stains proteins nonspecifically. In a typical tissue, nuclei are stained blue, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining. Hematoxylin and Eosin (H&E) staining is used routinely in histopathology laboratories as it provides the pathologist or researcher a very detailed view of the tissue. It achieves this by clearly staining cell structures including the cytoplasm, nucleus, and organelles and extra-cellular components. (Fischer et al., 2008; Anderson, 2016). Stains other than heamatoxylin and eosin are referred to as special stains. When a specific component of tissue e.g. fibrous tissue, elastic tissue, nuclear material is to be stained, certain special stains are used which specifically stain that component tissue and there are thousands of such stains. Before tissue can be stained and viewed, it must be prepared so that a very thin section, only one cell thick, can be cut and placed onto a microscope slide. This involves fixing the tissue (so it does not decay) then hardening and supporting it so that it can be cut to the very thin sections needed (typically 2–7 µm). There are two main techniques used for this, referred to as frozen sections and paraffin-embedded sections. Frozen sections are used when answers are needed fast (Talukder, 2007; Anderson, 2016). Simple staining implies the use of only a single stain (Monostain), which is usually sufficient to reveal the morphological features of most microbial cells, including relative size, shape, and characteristic arrangements for groups of cells. To obtain additional information, various specialized differential staining procedures have been developed. Stains that react differently with different cell types are known as differential stains, and they have an important role in the identification of taxonomic groups (Gurr, 2010).
Tumeric (Curcuma longa), belonging to the family Zingiberaceae has creeping tuberous rhizomes that are harvested for food. It is distributed throughout tropical and subtropical regions of the world, such as Asia, Africa and the Americas. The rhizomes are harvested and processed before they are ground to yield the yellow powder known as turmeric. The yellow colored compound fond in turmeric is called Curcumin. The curcuminoids are phenols and hence are strong antioxidants. curcuminoids are non-toxic ( Alshuler, 2011; Roy, 2013). It has been in use for centuries as a dye and as a component of curry powder, and more recently as a pH indicator; therefore, it is a common commercially used substance. (Avwioro et al., 2007). When used as a counterstain for haematoxylin, the staining reaction was similar to eosin except for its yellow colour also the ability of a dye to stain specific tissue structures was determined by certain factors, one of which was the acidity of the stain. Acidic structures would be stained by basic dyes while basic structures would be stained by acidic dyes. The Curcuma longa dye distinctly stains the seminiferous epithelium and interstitium yellow. Curcuma longa provided a good counter stain for Haematoxylin, taking up the acidic staining characteristics with Haematoxylin staining the basic staining characteristics. Turmeric can be used as a counterstain after hematoxylin, its staining ability was also good and comparable to that of eosin dye with a special affinity for collagen and muscle fibers (Avwioro et al., 2007; Bassey et al., 2012; Kumar et al., 2014).
Hibiscus (Hibiscus sabdariffa) is a bushy annual plant. Parts of the flower are used to make a popular drink in Egypt called Karkade. Various parts of the plant are also used to make jams, spices, soups, and sauces. The flowers are used to make medicine. It is native to tropical Africa, but today grows throughout many tropical areas. This annual herb produces elegant red flowers. The major producing countries are Jamaica and Mexico. The hibiscus has had a lengthy history of use in Africa and neighboring tropical countries. Its fragrant flowers have been used in sachets and perfumes. In areas of northern Nigeria, this plant has been used to treat constipation (kluwa, 2016). The Roselle extract mordanted with potassium alum can be used to counter stain with eosin. A section of testis showed a better nuclear and cytoplasmic differentiation compared to that given by the haematoxylin and eosin method according to research (Egbujo et al., 2008). Hibiscus Sabdariffa solution can be used as a cytoplasmic stain instead of eosin in the H&E method to stain formalin fixed paraffin embedded skin tissue sections (Raheem et al., 2015).
Development of new histological staining method remains justified especially if the new stain is harmless, easy to use, cheap, commercially favorable, and gives the desired practical results. Many developing countries can no longer afford the ever increasing cost of synthetic dyes, The use of cheaper, naturally occurring dyes from plants is being viewed as an alternative to synthetic dyes. There has been a rising need for efficient, accurate and less complex staining procedures (Avwioro et al.,2007; Raheem et al., 2015; Hanni et al., 2016).
1.1 JUSTIFICATION
The results of recent study suggested that most stains used in staining pathological tissues are allergic, toxic and costly (Bassey et al., 2012). Therefore a need arises to discover alternative stains which would defeat this deficiency. Natural dyes such as turmeric and hibiscus are non toxic cheap and environment friendly thus they could serve as an alternative to synthetic and toxic stains.
1.2 AIM
- To highlight single cells of pathological tissues using extracts from turmeric and hibiscus.
1.3 OBJECTIVES
- To evaluate the individual staining effect of turmeric and hibiscus.
- To compare the staining effect of haematoxylin and eosin to turmeric and hibiscus extracts.
- To investigate if turmeric and hibiscus are suitable alternatives for routine stains in the histopathology laboratories.
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