Sorghum Bicolor – The Staining Effect Of Sorghum Bicolor On Human Tissues

Sorghum Bicolor  – The Staining Effect Of Sorghum Bicolor On Human Tissues

Sorghum Bicolor  – Tissues and their constituent cells are usually transparent and colourless and different structures cannot be easily distinguished from each other when examined with the conventional light microscope. To make the required optical differentiation of tissue constituents possible, the practice of staining is found necessary. Stains are substances which colour tissues in order to aid optical differentiation of tissue components. The process by which stains are applied to sections is known as staining.

The mechanisms of staining of tissues are histochemical in nature. Histochemistry is the application of chemical substances on to a tissue in order to bring about a visible end product of reaction in varying degree of colours. Tissues are made up of organic and inorganic compounds that are capable of undergoing chemical reactions in-vitro to form new products. Advantage is made of this fact and such chemical reactions are made to terminate in an opaque or coloured end products. (Avwioro, 2002).

A dye is defined as an organic compound consisting of a benzene ring to which has been or attached both a chromophore and an auxochrome, which confers the property of imparting long-lasting colour.   Although the great majority of dyes in use today are synthetic, there are still a few natural dyes in use. (Culling, 1974).

Natural dyes are stains which are derived from organic substances or extracted from plants, insects, and other creatures. (Avwioro, 2002). Among the natural dyes that are of histopathological importance are: haematoxylin, carmine and orcein. Haematoxylin is extracted from the log wood, Haematoxylon campechianum, carmine is obtained from the female insect Dactylopius cacti while orcein is extracted from lichens.

This work is aimed at detecting a new natural dye which can provide an alternative to Haematoxylin and Eosin staining technique for the demonstration of general tissue structures. It will also provide a cheaper approach to staining.

STAINING.

The mechanisms of staining of tissues are histochemical in nature. Histochemistry is the application of chemical substances on to a tissue in order to bring about a visible end product of reaction in varying degrees of colours (Avwioro, 2002).

The various types of staining are:

Regressive staining: This is a type of staining in which the stains, when applied to tissue, over stain the tissue. These tissues require differentiation i.e removal of excess stain in order to bring about either a varying degree of staining or staining of specific structures. (culling, 1974).

Progressive staining: It is a type of staining technique which stains tissue in order to reveal relevant structures without the use of a differentiation.

Negative staining: this is the staining of the background. The object to be demonstrated is not stained. Therefore, the unstained object gives a contrast against the stained background (Avwioro, 200).

Direct staining: This is the staining of tissue with simple solutions of dye in water or alcohol. It does not involve the use of mordants.

Indirect staining: this is dye assisted staining. It is used when there is no direct union between dye and tissue. It involves the addition of certain elements to solution of the dye to bring about combination of dye to tissue. Where a mordant is used, a tissue mordant-dye complex is formed.

Metachromatic staining: certain dyes have more than one absorption band in the visible spectrum. They react with acid mucopolysacchari-des to produce a colour, which is different from the colour of a weak solution of the dye and that of the surrounding tissue (Carleton, 1974).

Vital staining: This is the staining of living cells or living tissues. In vital staining, the nucleus is not stained because a living nucleus does not permit entry of stains. However, the cytoplasm can be stained either as a result of phagocytic activity of the cell concerned or by simple absorption of dyes into the cytoplasm where staining of specific structures takes place (Avwioro,2002).

Intra vital staining (in vivo): This is the staining of structures in a living whole body. Appropriate dilution of the dye is made and injected into the body. Staining takes place within the living body from where specimen is taken and examined either as a wet preparation or processed, sections cut and counterstained.

Supra vital staining (in vitro): This is the staining of living cells  outside the whole body. This method is particularly used for the demonstration of nerve endings by the Ehlich’s methylene blue technique. Supravital staining is also of value in differentiating between living and non-living spermatozoa in cases of infertility (Carleton, 1979).

PHYSICAL THEORIES OF STAINING.

Adsorption: This was the property by which a large body attracted to itself minute particles from a surrounding medium, and was a phenomenon well known to chemists. This theory was developed by Bayliss, (1900) and he called it an electrical theory of staining (culling, 1974).

Absorption:  certain tissue components such as those with mineral salts attracted dye molecule and caused them to infiltrate the tissue and thereby effected staining (Avwioro, 2002).

Solubility: Some dyes are more attracted to and more soluble in substances which they stain than in the solvent in which they are dissolved. Examples of this method of staining were fat stains which were effective simply because the stain was more soluble in the fat than in the 70% alcohol or other solvent in which it may be dissolved.(culling, 1974).

 Osmotic pressure:  This is the movement of dye molecules from the region of higher concentration to a region of lower concentration until this was sufficient to cause staining.

Capillary Attraction: staining can also occur due to movement of dye molecules in tiny vessels in tissue. This movement is sometimes due to negative intracapillary pressure. It is also affected by ionic strength, concentration of dye, temperature and type of tissue (Avwioro, 2002).

CHEMICAL THEORIES OF STAINING

Tissues were found to be composed of substances which were acidic, basic or amphoteric in reaction. This is in relation to the iso-electric points of the dye molecules. This means a cell constituent of pH 5 is basic to a dye which stains at pH less than 5. Tissue components such as parts of cytoplasm which are basic in reaction are stained by acid dyes while tissue components such as of the nucleus which are acidic in reaction are stained by basic dyes (culling, 1974). Some dyes stain both basic and acidic structures. These are called amphoteric dyes. Amphoteric dyes were found to contain both acidic and basic radicals. A third staining reaction was found in amphoteric dyes as a result of the staining effect exhibited by the product of reaction between the acidic and basic dye components (Avwioro, 2002).  Whether a dye is basic or acidic in reaction depends on the nature of the auxochrome because it is the auxochrome that carried the basic and acidic radicals (Avwioro, 2002).

CLASSIFICATION OF DYES.

Dyes that were used for staining were classified in many ways based on different criteria like mode of action on tissue, source, type of solvent required, purpose of use, PH etc. not only was the classification of dyes a complex problem but their names also created difficulties (Carleton,1974). The classification which is of great interest to this work is that based on the source of dye and the PH of the dye in solution.

CLASSIFICATION BASED ON SOURCE OF DYE.

Natural dyes: Natural dyes were derived from organic substances, plants, insects and other creatures. Below is a list of some common natural dyes:

i)       Haematoxylin: This dye was extracted from the logwood of the Mexican tree Haematoxylon campechianum by waldeyer (1864) using either as the extraction agent. It has poor staining properties and is normally used in conjunction with a mordant. (Baker et al 1998).

ii)     Orcein: Orcein is a vegetable dye extracted from certain lichens by the action of ammonia and air. Engle et al, (1952) showed that orcein could be fractionated by chromatograpy into at least four components. Two of these fractions stained elastic tissue selectively only when acid alcohol is used as solvent. However, in aqueous media, these two fractions stained many other structures in addition to the elastin. These structures were found to be effectively stained between the PH range of 3-8 than at the extremes of the PH scale (1-2 and 9-14). Orcein has a violet to demonstrate elastic fibre.

iii)   Litmus: this was also obtained from lichens which were treated with lime ash or soda, in addition to exposure to ammonia and air. Although litmus was a poor dye, it was used widely as an indicator (Baker at al, 1998).

iv)   Saffron: this natural pigment extracted from crocus sativus was not widely in histology, but it was incorporated into connective tissue stain (Avwioro,2002).

v)     Carmine:  This was a natural dye obtained from the female insect Dactylopius cacti and it is used for the staining of glycogen and neural cells when combined with picric acid.

Synthetic dyes: Synthetic dyes are chemically refined dyes derived from coal tar (Avwioro, 2002). They were produced by combining certain chemicals which resulted in a product and imparted colour on a tissue. Synthetic dyes are sometimes referred to as “coal-tar dyes” since they were sometimes manufactured from substances which were in the past only obtained from coal to all these compounds were derivatives of the hydrocarbon benzene (C6H6), which consists of six carbon atoms at the corners of a regulate hexagon with   hydrogen atom attached to each carbon atom. Simple benzene compound was found to have absorption bands in the ultraviolet range of the spectrum. Certain substances known as chromophores were said to be capable of moving the absorption band of benzene into the visible portion of the spectrum, this therefore produced visible colour. Benzene compounds containing chromophores were known as chromogens. Koopman et al (2001) developed a stain permitting automated qualification myocellular lipid deposits in skeletal muscle sections together with mmunolocalisation, other myocellular constituents by fluorescence microscopy. Lipid droplets were detected in skeletal muscle by oil red O. conventional oil red O was modified to diminish background staining, preventing crystallization of oil red O and optimizing lipid retention in cryostat sections. He concluded that oil red O stained lipid droplets in skeletal muscle sections together with multiple staining of other immunodetectable proteins present in skeletal muscle by quantitative fluorescence microscopy. A new silver stain was developed by Sinha et al (2001), which was said to be specially developed for staining large gels (25cmx20cm) from the Hoefer 150-DECT system for matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis of proteins. He concluded that the gels were stained in a new apparatus that reduced gel handling to a minimum, thus also reducing the contamination with keratins to a minimum. The development times in potassium carbonate were very long, thus improving batch-to batch reproducibility. Only the surface of the protein was stained and the silver could be oxidized. Thereafter, MALDITOF can be performed with protein loads as little as 100 micrograms per gel.

CLASSIFICATION BASED ON pH.

The nature of the auxochrome ordinarily determined whether the resulting dye was acidic or basic in character. (Culling, 1974).

Acid Dye:The staining components of an acid dye were acidic in reaction. They possesses a colourless base such as sodium. Acid dyes reacted with basic components of the tissue such as cytoplasm. Such includes eosin and acid fuchsin (culling 1974).

Basic Dye: The coloring substances in basic dye was in the acidic components. Basic dyes react with acidic components of the tissues such as the nuclei.  Included in this group was methylene blue and basic fuchsin (Avwioro, 2002).

Amphoteric Dyes: Amphoteric dyes contain both basic and acid dyes.

They are said to combine the properties of both acid and basic dyes to give a triple staining effect. (weigwert, 1904). It involves staining due to product of interaction between basic and acid dyes. The component dyes of amphoteric dyes were found to be soluble in aqueous solutions separately but when combined, they were soluble in alcohol and rarely in aqueous solutions. This group of dyes includes Romamowsky dyes: Leishman, Giemsa and Wright stains. (Carleton 1979).

Neutral Dyes: Neutral dyes exhibit neither acidic nor basic properties. They do not stain by salt linkages, their staining characteristic depends essentially on physical properties.(Avwioro, 2002). Example of neutral dyes is Sudan black B which stains lipids by selective solubility (Carleton, 1979).

FACTORS AFFECTING STAINING

MORDANT

Mordants are hydroxides of salts of divalent and trivalent metals, which form salt bridges between tissues and dye. (Avwioro, 2002). Without mordant, staining was found to be impossible in certain dyes where there was no direct union between dye molecules and tissue. The combination of the dyes and the mordant forms a compound, which is sometimes called a lake, and is capable of attaching itself firmly to the tissue (culling, 1974).

The complex formed is not soluble in ordinary aqueous or alcoholic solution. Mordants are applied to effect staining reaction in three ways:

1. Pre- Mordanting: the mordant was applied on the tissue before application of the stain.
2. Metachrome staining: the mordant was added in conjunction with the stain.
3. Post- mordanting: this mordant was applied  after the application of the stain. (Avwioro, 2002). The term mordant is only strictly applicable to salts and hydroxides of divalent and trivalent metals and is not used to indicate any substance to improve or aided staining in some other manner.(culling, 1974).

TRAPPING AGENTS

Trapping agents hold dyes in combination with tissue and bacteria. (Carleton,1899). Examples are tannic acid and iodine. Laveran (1899) showed that a blood smear stained with the methlyene blue-eosin selectively retained the methylene blue in chromatin after treatment with tannic acid. The findings of Gram (1884) that certain bacteria stained with gentian violet and that iodine resisted alcoholic discoloration was also due to the trapping action of iodine on the bacteria- dye complex. It was believed that the iodine did not alter the capacity of the dye to react with bacteria but tended to hold the dye and thus prevented it from escaping from the tissue during differenciation.(Culling,1974).

FIXATION

Fixation has a marked effect on the subsequent staining  of tissue. It generally facilitates the dye, assisting the interaction of tissue and dyes.(Carleton, 1979). Chromatin probably splits into DNA and protein by fixation, allowing the DNA to be stained by a basic dye. Mercuric chloride, formaldehyde and ethyl alcohol act in this way. Proteins are also more easily stained after fixation, formaldehyde and mercuric chloride favoured basic dyes while trichloroacetic acid, picric acid and chromium compounds facilitated the action of acidic dyes. After fixation with acetic acid or ethyl alcohol, tissues take up both basic and acidic dyes easily. Certain fixatives also act as a mordant.(Carleton,1979).

SORGHUM BICOLOR

Sorghum bicolor is an annual,summer, coarse and erect plant with much variability in growth characteristics, culms solid or sometimes with spaces in pith, 0.6-5m tall, depending on variety and growing condition, 5 to over 30mm in diameter, either dry at maturity or with sweet insipid juice.

            Leaves of sorghum bicolor are broad and corn– like but with shorter and wider blades that are glabrous and waxy with overlapping margins. (Bukantis,1980). Though sorghum is used largely for forage in the US, it is very important in the world’s human diet with over 300 million people depended on it. (Bukantis, 1980). The leaves are also of some local medical importance. For example, it is widely used among West Africans to make local antibiotic syrup. (Reed, 1976). It is also employed as a colouring flavour in cooking some species of white cowpea (Ricaud et al, 1981).

Sorghum are high on the priority list of energy crops. The genus sorghum includes grain sorghums noted for their ability to grow in dry climates and to manufacture starch effectively. Sweet sorghums are noted for their high yields of directly fermentable stalk sugars.  Sorghum is prone to various  pests, including birds and some parts of Africa parasitic Withchweed (Stiga).

Sorghum contains hydrocyanic acid and the alkaloid hordenine. Varieties differ considerably in HCN poisonings. Danger is light when grain is nearly mature. Young plants and suckers are dangerous, particularly when suffering from drought.

Sorghums are antiabortive, cyanogenetic, demulcent, diuretic, emollient, introicant and poison. Sorghum is a folk remedy for cancer, epilepsy, flux and stomachache (Duke, 1982). The root is used for malaria in southern Rhodesia, the seed has been used for breast disease and diarrhea, the stem for tubercular swellings. The stomachic seeds are considered  beneficial in fluxes (Perry, 1980).

According to Morton (1981), Curacao natives drink the leaf decoction for  measles, grinding the seeds with those of the calabash tree (Cresentia) for lung ailments.

Sorghum is usually grown as a field crop. In Africa, there are two basic types, white sorghum which is sweeter and used as a grain crop and red sorghum, which is  less tasty to eat, but is not as badly attacked by birds and makes good beer. Sorghum is also planted for cattle fodder and other purposes.

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This article was extracted from a Project Research Work/Material Topic “THE STAINING EFFECT OF SORGHUM BICOLOR LEAVES ON HUMAN TISSUES”

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