The Potential use of Beta Vulgaris as a stain to demonstrate Schistosoma Haematobium in bladder biopsy

The Potential use of Beta Vulgaris as a stain to demonstrate Schistosoma Haematobium in Bladder Biopsy

CHAPTER ONE

Staining In Histopathology

Colour is one of the elements of nature that has made human living more aesthetic and fascinating in the world. (Bassey et al., 2011). Staining is a techniques or a method that has been employed in microscopy which enable visualization and identification of structures of tissues and micro-organism. The process of staining enables certain cell or structure to be readily identified and studied because of the contrasting colours (s) they take on base on their chemical and physical properties. (Adio , 2015).

The physical and chemical makeup of cellular component of a tissue allows for uptake of different shade resulting in a contrast. Stains have been used to enhance accurate description of microscopic structures of tissues which is necessary for histopathologic analysis (Egbujo et al., 2008). The use of different stains has enabled the preferential staining of certain cellular component such as the nucleus, cytoplasm, and mitochondria.  Most stains can be used on fixed or non living cell while only a few can be used on either living or non living cells. The process of staining which involves staining living cells is called vital staining.

In many cases, stains are affected by heat and may become reactive enough to bind with underlying materials. A mordant which is a chemical compound is required by some dyes to form an insoluble coloured precipitate.  This enables the stain to remain on the stained structure after excess of the dye have been washed away.  The use of accelerators helps increased the speed, intensity and specificity of staining while the conversion of inactive to active compound is achieved through the use of oxidants. (Awvioro, 2002).

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There are various methods of staining many of which are  example of  simple staining which employs the use of only one stain or dye, counter staining in which a stain makes a cell more visible when not completely visible with the primary stain. (Hafiz et al., 2012).

Beta vulgaris (beetroot) 

Beta vulgaris is an herbaceous biennial or rarely perennial plant with leafy stems. It grows with a height of 1 – 2m, with heart shaped leaves. It has dense spike flowers, which are small measuring 3 – 5mm in diameter, green or tinged reddish with five (5) petals. The fruit is a cluster of hard nut lets.

Beta vulgaris belongs to the family Amaranthaceae. The root vegetable beetroot or garden beet is the most popularly known species. Other varieties include the leaf vegetable chord, the sugar beet and mangelwurzel.

The roots are commonly deep red- purple in colour, but less common varieties include golden – yellow and red and white stripped roots. (Zeldes, 2011).

Blood turnip was once a common name for beetroot cultivars for gardens. Beta vulgaris contains a variety of pigment called betalian which gives it its characterstic red colour.( Robinson and Tevor ,1963).

Figure 1.Chemical Structure of Betanin.

https://en.m.wikipedia.org

The composition of different betalian pigment can vary resulting in the different strains of colours in addition to the familiar deep red. ( Hamilton,2005). Some varieties of the betalians include betanin, isobetanin, probetanin and neobetanin while the red to violet ones are collectively known as betacyanin. Indicaxanthin and vulgaxanthin are pigment also found in beets.

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Beeturia is referred to the red colouratim of urine found in individual who are not able to break down betacyanin from ingested beetroot (Eastwood and Nyhlin, 1995).

The pigments which are quite unstable and will leak when cut, heated or when in contact with air or sunlight are contained in cell vacuoles.

 Roman and Jewish Literature sources suggest that in the 1st century BC, the domestic beet was represented in the Mediterranean basin primarily by leafy forms like chard and spinach beet. (Hopf and Zohary , 2000).

Urinary Bladder

The urinary bladder is a hollow elastic organ that functions as the body´s urine storage tank. Urine enters the bladder via the ureters and exists via urethra. There is no exact measurement for volume but it’s around 500ml – 1000ml.

Anatomy of the Urinary bladder

The urinary bladder is roughly spherical in shape, although its shape and size vary among individuals and depends greatly upon the volume of urine that it contains. It is located in the pelvic cavity anterior to the rectum and superior to the reproductive organs of the pelvis.

Figure 2: Anatomy of the Urinary Bladder

(Moore and Agur, 2007)

Histology of the Urinary Bladder

The urinary bladder is lined with transitional epithelium. It does not produce mucus. (Chin et al., 2007). The internal lining of the bladder wall is termed the urothelium  and  lamina  propria and this layer is thought to regulate some aspects of overall bladder physiology in response to stimuli such as stretch during filling. (Moro et al., 2011)

Figure 3: Histology of the Urinary Bladder

(Mescher, 2010)

Tissue Parasite

A parasite is an organism that lives on or in a host and gets its food from or at expense of its host. Parasites can cause disease in humans. The parasites benefits at the expense of the host ( Bartel,  2011). Unlike predators, parasites typically do not kill their host, are generally much smaller than their host and will often live in or on their host for an extended period. Parasites show a high degree of specialization and reproduce at a faster rate than their host. (Getz, 2011). Parasites are classified base on their interactions with their hosts and on their life cycles. An obligate parasite is totally dependent on the host to complete its life cycle while a facultative parasite is not. Parasites inhabit various tissue of man and some require those tissues for the completion of their life cycle. If the parasites enter the body, the immune system is a major defense against parasitic invasions. The immune system is made up of different families of molecules. These include serum proteins and pattern recognition receptors. Pattern recognition receptors are intracellular and cellular receptors that activate dendrite cells, which in turn activate the adaptive immune systems lymphocytes. Lymphocytes such as the T cells and antibody producing B cells with variable receptor that precognitive parasites. (Maizels ,2009).

Schistosoma haematobium

Figure 4: Schistosoma haematobium

https://en.m.wikipedia.org

Schistosoma haematobium is an important digenetic trematode and is found in Africa and the Middle East. It is a major agent of schistosomiasis more specifically; it is associated with urinary schistosomiasis.

Adults are found in the Venus plexus around  the urinary bladder and the released eggs travel to be wall of the urinary bladder causing  haematuria and fibrosis of the bladder. The bladder becomes calcified, and there is increased pressure on ureters  and kidneys otherwise  known’s as hydronephrosis. Inflammation of the genitals due to Schistosoma haematobium may contribute to the propagation of Human Immune Deficiency Virus (HIV) (Leutscher et al., 2005).  There is a relationship between Schistosoma haematobium  infection and development of squamous cell carcinoma of the bladder (Khurana et al., 2005).  The ova are initially deposited in the muscularis propria which leads to ulceration of the overlaying tissue. Infections are characterized by pronounce acute inflammatory, squamous metaplasia, blood and reactive epithelial changes.Granulomatous and multinucleated giant cells may be seen. The main cause of  Schistosomaisis is the dumping of human waste into supplies. Hygienic disposal of waste would be sufficient to eliminate disease (Black, 2005)

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Mordant

A mordant is a substance used to set dyes on tissue sections by forming a coordination complex with the dye which then attaches to the tissue( IUPAC, 1997). Stains such as heamatoxylin require an intermediate substance such as a mordant to satisfactory combine with a tissue. This process is called in direct staining. Mordant are used in staining to intensive stained cells or tissue preparations.

Mordant was derived from the French present participle mordre “to bite” because in those days bite into the fiber to avoid washing off. The coordination complex of a dye and on ion is colloidal and can either be acidic or alkaline. Mordant is a polyvalent metal ion ( Llewellyn, 2005).

Mordant include tannic acid, alum, urine, chrome alum, sodium chloride and certain salt of aluminum, chromium, copper, iron, iodine, potassium, sodium and ion. Iodine which is referred to as a mordant in gram stains is bests a trapping agent.

In histological staining, the dye and mordants are either used together; meta-mordant is added to the dye bath itself example Haematoxylin and potassium alum in Elich heamatoxylin or pre modanting (on chrome) in which the mordant may be used first before applying the stain examples iron alum used prior to Heidentaris haematoxylin or post mordanting (after – chrome) in which the dyed material is treated with a mordant.

Mordants are affected by certain factors which include;

The action of the  mordant on the substrate; pre mordanting and post mordanting will limit the potential for damage if the mordant and dye  methods are horsh, the stability of the mordant or dye mordant can be added without fear of losing the dye. Some most dyes require the use of mordants, mordants tend to have a mark, effect on the final colour. Each dye can have different reaction to each mordant; example cochineal carlet used cochineal along with a thin mordant to create a brilliant orange hued red (Phipps ,2010).

Accentuators 

Accentuators are chemical substances that increase the staining prior of a dye but do not form lakes with the dye and are not essential with the chemical union of the dye with tissue. Accetuators fall into three (3) groups PH control, salt and phenol potassium hydroxides in Loeffler’s methylene blue and phenol in control thiamine and carbon fuctisin acts as accentuators increasing intensity and selectivity of the stain.

 Justification of the Study

Several stains have been used in histopathology to demonstrate the general tissue structures or specific organelles in cells. Most of these stains which include Eosin, orange G are synthetically derived. The chemicals use to produce these dyes are often highly toxic, carcinogenic, expensive and hazardous to humans and the environment. Therefore it has become necessary to create healthier, easier and quality stain for use in the demonstration of tissue structures and over the years, natural dyes derived from plant and animals such as heamatoxylin, carmine have proved to be effective. Cultivation and processing of the root crop (Beta vulgaris) will help create employment for farmers thus boosting the country’s economy. Demonstration of Schistosoma haematobium in bladder biopsy which is a major health challenge will help in its diagnosis. Therefore the potential of Beta Vulgaris extract as a staining agent for demonstrating tissue structure will be investigated in the study.

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Significance of the Study

The study will be carried out in an effort to find an alternative which will improve the quality of diagnosis by enhancing visibility of general tissue structure and clear demonstration of the individual cell. Demonstration of the parasite in the bladder biopsy may help in the diagnosis of various diseases affecting such organs

Aims and Objectives

The general aim of the study is to produce a cheaper, healthier and a quality demonstration of general tissue structure using Beta Vulgaris extracts and modification in demonstrating the present of parasite in urinary bladder biopsy

The objectives include;

  1. To find out the potential use of Beta vulgaris as a stain to demonstrate schistosoma haematobium in bladder biopsy
  2. To find out the effect of pH on the staining property of Beta vulgaris
  • To find out the effect of concentration on the staining property of Beta vulgaris
  1. To find out the effect of time on the staining property of Beta vulgaris
  2. To find out the effect of temperature on the staining property of Beta vulgaris
  3. To find out the effect of accentuator on the staining property of Beta vulgaris

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