This chapter reviews the literature related to the plant extracts that will be used in this study, which will provide basis for the interpretation of the data that shall be gathered later.
Staining is a technique utilized in microscopy to enhance structures of organisms. It is considered as biochemical technique to emphasize images under a microscope, it will aid by adding a specific substance to a specimen to measure and to view it clearly. A stain or a pigment is used to dye certain cells, tissues, or cell contents and even microorganisms.
Stains are used to feature structures while looking at a microscope (Adiyemo, et. al, 2017).
Staining technique have originated from the second half of last century, pigments which are called stains have been used to amplify precise descriptions of the microscopic structures of organisms (Penney et. al., 2002).
Stain is any colored organic compound usually called dye, used to stain or give tincture to tissues, cells, cell components or microorganisms.
Stains and dyes are frequently used to highlight structures for viewing often with the aid of different microscope (Hafiz, et.al, 2012).
Moreover, even if specimens can be noticed or viewed by using microscope in the laboratory, it should be fixed or stained first, to extend its visibility and morphological features and even preserve them for further study (Prescot, et. al, 2009). This way, specimens viewed under a microscope will be clearly and properly seen and examined (Estandarte, 2012).
Stains can either be natural or synthetic. Synthetic stains became popular and are widely used nowadays, because of its lasting color pay-off and wide range of color choices.
Synthetic stains like Giemsas, Aceto-orcein and Aceto-carmine have been utilized to stain chromosomes during meiotic and mitotic cell division. However, most synthetic stains that are chemically combined used in most laboratory activities are high-priced, carcinogenic, and have a toxic effect to humans and animal health and can even be a threat to the environment (Elgubbi & Asayh, 2017)
On the contrary, natural stains are acquired from organisms such as fruits, minerals, animals, and other natural resources. Thats why natural stains are harmless and safe for the environment. More so, these natural stains are found to be inexpensive, and easy to acquire in the laboratory for plant histology (Cheng, et. al., 2014).
In this peak of technological expansion and advancement, many people would rather use natural and organic substances if given a chance to pick over commercial and chemically synthesized substances (Bondoc, 2018). Since there is an increasing demand in the use of natural resources in our ecosystem, to maintain environmental stability worldwide, plants which can produce stains and staining technique is highly recommended for use in microbiological and other laboratories.
Furthermore, the use of these natural stains in the laboratories will able to facilitate the practice of protective measures for students, proper treatment and disposal of these stains (Adeyemo, et. al., 2017). Natural herbal dyes used in staining specimens such as microorganisms are found out to be extracted from the different parts of a plant, such as the fruits, leaves, flower, stem, stem bark or fruit skins (Akinloye, et.al., 2010).
Specimens such as cells needs to be stained not just to enhance visualization of cellular components but also to highlight metabolic processes (Bruckner, 2017). Cell cycle one of the basic and common topics discussed in laboratories, is the sequence of events that take place in a cell, this will cycle will eventually lead to cell division and duplication of the cells DNA.
Cell division has two processes: mitosis and meiosis. Meiosis is a cell division that produces reproductive cells, specifically on fungal spores, and reproductive cells. Whereas, mitosis is a cell division that allows cells to reproduce daughter cells that are the exact copies of themselves (Vidyasagar, 2018). Organisms genetic make-up resides in the individuals DNA chromosomes or molecules. In plants, the roots continue to grow as they search for nutrients and food. Since cells which are rapidly growing and actively dividing mitotically can be located at the root tip region of a plants, this is the area which is ideal for viewing mitosis. In laboratories, the most commonly used specimens for viewing mitotic chromosomes are, onion, wheat, alfalfa and barley. Since onions have larger chromosomes compared to other plants, the chromosomes are easier to see in a microscope.
Onion (Allium cepa L.) is a multicellular, herbaceous biennial vegetable crop in the family Alliaceae and is planted for its edible bulb. It is grown and widespread throughout the world, and is a native to Southwestern Asia, and are mostly found in temperate areas. Onion is considered to be a multicellular plant organism (Nedungadi, et. al., 2013). In fact, onion is one of the fifteen (15) most commonly grown vegetable worldwide (Jahromi & Amirizadeh, 2015). This vegetable crop is popularly grown for its pungent bulbs which are used by almost every household as one of its seasoning in different dishes. It has been estimated that onion is one of the most consumed vegetable on the planet, with its average consumption per year of roughly 11kg (Shultz, 2010).
Since Philippines is known to be a tropical country, onion is able to grow well. In fact, many farmers all over the archipelago have shifted from growing rice to onion farming, because it is a highly versatile, high value crop. In addition, onion is a source of different active biological compounds, like phenolic acids, thiosufinates and flavonoids. Studies have shown also that onion is one of the worlds healthiest vegetable, it provides a great deal of health benefits, which includes heart health, anti- inflammatory benefits, blood-sugar regulator and has antioxidant effects (Kuete, 2017).
Human cells contains forty six (46) chromosomes, whereas, onion possesses only eight (8) chromosomes. The tip of the onion is an actively growing part of the plant, so, many cells will be able to have various phase of mitosis. Onion root tips can be prepared and squashed so that it will be able to be flattened on a slide, this way chromosomes can be visualized easily by using specific stains.
Alugbati (Basella alba) is a cool season vegetable which is wildly cultivated. Its stems are color purple or green. It has a succulent branch, which has a twining herbaceous vine that can reach several meters in length. The leaves of this plant has a unique shape, its either ovate or has a heart shape, the leaves are also fleshy, that is 5 to 12cm long. Fruits of alugbati are fleshy, and dont have stalk, tha is ovoid or spherical in shape and is 5-6 mm long, and is purple when it matures. (Adhikari, et. al., 2012).
It is a vital green leafy vegetable which can be found abundantly in tropical Africa, Caribbean, tropical South America, Malaysia and Philippines, this is somehow used as an alternative for true spinach (i.e.Spinacea oleracea L.) and has its great ethnomedicinal importance (Deshmukh & Gaikwad, 2014).
Izonfuo, et al., (2006), in their study, Acid-base indicator properties of dyes from local plants: dyes from Basella alba (Indian spinach) and Hibiscus sabdariffa (Zobo), determined that the acid base indicator properties of extracts acquired from the ripened fruits of Basella alba are photo chemically unstable and can be utilized as an indicator. They reported that stains obtained from the fruit pulp of the plant can be used as an alternative for crystal violet otherwise known as safranin in Gram staining and as a biological stain for plant organelles or nuclei.
Coleus blumei Benth., most commonly known as Mayana is described as an ornamental plant due to its assorted varieties of foliage, this plant is highly and broadly cultivated and collected in the Philippines (Medina & Cardenas, 2017). It can be grown all year round in the country. It has acer rubrum leaves which are dotched or colored with mostly purple. Some leaves are of color yellow, green, red, pink and sometimes even blue (Rodriguez, 2007). Mayana is a a fleshy, branched and erect annual herb, that is about one meter high. Stems of the plant are purplish and are 4-angled. Leaves are numerously smeared or colored that is about 5 to 10 centimeters long and is more or less hairy (Stuart, 2015)
Furthermore, mayana contains anthocyanin, a pigment that produce orange, pink, red, purple and blue that has a great potential for use as alternative stains.
Cris (2008), in his study on Using Mayana (Colleus scutellariodes) as Biological Stain, showed that mayana leaves extract can be partially used as stain for a plant. He also concluded that cell components, such as nucleus and cell wall of the onion cell can be seen under a microscope.
Sweet Potato (Ipomea batatas) is an annual plant grown through vegetative propagation. The stem is cylindrical and its length depends on the growth habit of the plant and on the availability of resources. The commercial part of the sweet potato are its roots. Leaves of this plant are simple, which are arranged spirally and alternatively on the stem. Its colors can be yellowish-green, green or it can be purple (Mohanraj & Sivasankar, 2014).
Sweet potato leaves is a popular known worldwide and is consumed by people, especially in Southeast Asia (Nagai, et.al., 2011). The leaves of this plant is an exceptional source of carotenes and chlorophyll (Dinu, et. al., 2018). Sweet potato leaves contains protein and carbohydrates (Bell et al., 2000). It is an excellent source of ascorbic acid and vitamin A and also contains anthocyanins and phenolic compounds (Zannou, 2017).
Carotenoids (from yellow to deep orange) or flavaonoids, are some of the sources of natural colorants that can be extracted from sweet potato, specifically anthocyanin that gives a purple pigment. Therefore, sweet potato contains anthocyanin that is an essential source of pigments (Tan, 2015).