According to H. J. Croxx, when microscopists first began to use stains in the sixties and seventies, the demand for dyes for this purpose was naturally too small to justify a special source of supply. They therefore had to make use of textile dyes, which were then very crude and were not constant in their composition. Plant-based dyes such as wood, indigo, saffron, and madder were raised commercially and were important trade goods in the economies of Asia and Europe. Across Asia and Africa, patterned fabrics were produced using resist dyeing techniques to control the absorption of color in piece-dyed cloth. Dyes from the New World such as cochineal and logwood were brought to Europe by the Spanish treasure fleets, and the dyestuffs of Europe were carried by colonists to America.

According to Bhuyan and Saikia, After a number of years, however, the demand for biological stains grew which led to the discovery of man-made synthetic dyes which were made from coal tar late in the 19th century ended the large-scale market for natural dyes. Most dyes in current use are chemically synthesized. Besides been expensive, they are also hazardous to human health. Some dye components are carcinogenic or at least strongly allergenic resulting in their withdrawal as their hazard becomes recognized.

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One of the most important and widely used staining technique is Gram staining. This was introduced by Christian Gram in 1884. In this process, the fixed smear is subjected to the following staining solution in the following order: Crystal Violet (initial stain), Iodine solution, alcohol (decolorizing agent), and Safranin (counterstain). Bacteria stained by the Gram staining method fall into two groups. One group is the Gram-positive bacteria which retain the Crystal Violet and thus, appear deep violet. The Gram-negative bacteria is another group which lose the Crystal Violet and is counterstained by Safranin. These bacteria, therefore, appear red in color. The most plausible explanation for this phenomenon is associated with the structure and composition of the cell wall. The cell walls of Gram-negative bacteria are generally thin.

Only three natural dyes are still used by botanical technicians: brazilin from brazil wood, hematoxylin from logwood, and hematein from Caesalpinaceae. The researchers saw the need to broaden these sources of natural dyes by investigating the effectiveness of Basella rubra fruit extract for bacterial staining.

Significance of the Study

Synthetic stains may contain highly toxic materials such as iodine that may be detrimental to health. The suggested stain is cheaper than conventional stains and is all-natural which helps in conserving the environment such as reducing water contamination brought about by improper disposal of used and expired chemical stains. An all-natural also means that it is non-toxic.

Review of Related Literature

Basella rubra is known as Malabar Spinach, Malabar Nightshade, Indian Spinach or Climbing Spinach. It is popularly known in the Philippines as Alugbati. It is a perennial vine found in the tropics and it is widely used as a leaf vegetable (Dictionary of Pilipino Vegetables).

The fruit of this herbaceous vine is fleshy, stalk less, ovoid or nearly spherical, 5 to 6 millimeters long, and purple when mature. The fruit is also said to yield mucilage and iron. When crushed, it produces a reddish-violet juice.

According to studies, the fruit extract contains anthocyanin, which makes it a good natural food colorant due to its stability. Studies showed that the anthocyanin extracted from Basella rubra fruits produced a stain comparable with synthetic stains like Crystal Violet and Safranin, and, therefore, can be used as an alternative microbiological stain such as Gram staining (Philippine Alternative Medicine).

According to Francis (1992), anthocyanins are glycosylates from anthocyanidins, the nucleus of which is the structure of a 4'-hidroxiflavilum ion. All anthocyanins are composed of two or three parts: the basic structure, which is aglycone (anthocyanidin), sugar and frequently an acyl group. According to Hidrazina (1982), anthocyanins are responsible for the blue, red, violet and purple coloration in most species of plant kingdom. According to Mazza (1995), anthocyanins predominantly exist in their non-colored forms in neutral to slightly acidic pH.

According to Brouillard (1982), anthocyanins are more stable in acidic than in neutral or alkaline solutions. One of the main characteristics of anthocyanins is the change in solution coloration in response to the pH of the environment. The color and stability of an anthocyanin in solution is highly dependent on pH. Anthocyanins are most stable and most highly colored at low pH values and gradually lose color as the pH increases, becoming almost colorless between pH 4.0 and 5.0. This color loss is reversible and the red hue will return upon acidification; which makes it suitable as a counterstain in Gram staining.

Natural stains are best known for its distinct property: permanence of coloration. Most of the stains used nowadays are synthetic and are made up of chemical compounds made from a substance found in coal tar. Yet, natural stains are more superior to synthetic stains. They preserve the specimen for a long time while the synthetic ones easily fade away. Permanence of coloration is important especially for preparations that require considerable handling over a period of time. Only three natural dyes are still used by bio-technicians: brazilin from brazil wood, hematoxylin from logwood and hematein from caesalpinacae.

According to Kjell et.al (2004), colors of anthocyanin in freshly made samples at pH 1.1 to 10.5 in experiments performed at a fixed temperature of 25ºC showed color differences in varying pH. These pigments had light pinkish at the lowest pH values. By stepwise pH increase until 7.3, the color gradually changes toward more bluish tones.

According to Mazza and Minati (1993), Musa acuminata bract showed colourless structure at higher pH 10.5. At these pHs, the flavylium cation hydrated to yield the colorless carbinol. Bathochromic shifts were observed for colorants at pH 1.1, 3, 4.1 and highest bathochromic shifts were observed at 6, 6.6. Increasing anthocyanin concentration, increase absorbance and the bathochromic shift occurs. Musa acuminata bract anthocyanin was stable at these pHs.

Biological stains make possible the viewing of microscopic plant and animal tissues under microscopes. When stained, the specimens are viewed clearer and they become more defined. Berries of Basella rubra (alugbati) were crushed using mortar and pestle. The crude extract obtained was filtered and used as a substitute for crystal violet as primary stain and for safranin as counterstain in the Gram staining of Bacillus subtilis and Escherichia coli. (http://www.investigatoryprojectexample.com/science/basella-rubra-biological- stain.html)

According to Ozela et al., The stability of anthocyanin in the extract of spinach vine fruit (Basella rubra L.) was studied in relation to degradative factors such as light, temperature and pH acting alone or in combination. In this work, the possible use of spinach vine fruit as a source of natural pigments for use in food coloring was evaluated. Extraction of the pigment was carried out with 99.9% methanol at pH 2.0. The stability of the anthocyanin extract was estimated. From these values, reaction velocity constants (k) as well as the half-life time (tl;2) were calculated at pH 4.0, 5.0, and 6.0 in the presence and absence of light both at 40 and 60°C.

Results indicate that, independent of pH values, spinach vine extract suffered an interference of light in its anthocyanin degradation kinetics with the mean tl;2being greater in samples place in darkness (654.5 + 66.6 h) compared to exposed to light (280 + 60.62 h). In the presence of light, degradation of the anthocyanin pigment increased with increased temperature and had an average half life time of 280 + 60.62 h, 6.88 + 0.76 h and 2.42 + 0.31 h at room temperature (25 + 1°C), 40 and 60°C, respectively. Spinach vine extract was more stable at pH 5.0 and 6.0 than at pH 4.0 both in the presence and absence of light. This characteristic differs from other anthocyanins. This property could facilitate its application as a natural food colorant.

According to Chen J. et al., Anthocyanin dye of the acai fruit (E. oleracea) and the dyes from cochineal (D. coccus) and chlorophyll extract from alfalfa (M. sativa) resulted in the best capability for posterior hyaloid and ILM staining in human cadaveric eyes and may be a useful tool for vitreoretinal surgery. Anthocyanins are water-soluble flavonoid pigments that are found in many plants. They have been studied and used for thousands of years and are known to contribute to flower colors across the visible spectrum; the only color yet to be recorded is green. One study identified 43 different types of anthocyanins in four wild forms of garden iris, and concluded, “There was no particular relationship between the type of pigment present, and the flower color,” This suggests that plant colors (and changes) are the result of structural changes the anthocyanins undergo. However, in laboratory conditions, anthocyanins can also act as pH indicators. Indicators change colors because of structural changes the compound undergoes in solutions of differing acidity.

A major function of anthocyanins is to provide color to most flowers and fruits. The colors can help attract pollinating animals to flowers and animals that will help disperse seeds. Anthocyanins have a positive charge on the molecule which enables it to absorb light and thus have color. Anthocyanin pigments are responsible for the red & purple colors of many fruits, vegetables, cereal grains, and flowers. They are water soluble, vacuolar pigments that may appear red, purple, or blue depending on the pH. At pH7, they are purple; at >pH7 they appear greenish yellow; at <pH7 they are color pink. Anthocyanins seem to be tissue-specifically biosynthesized, resulting in formation of a color pattern.