#alternative staining

Abstract

The efficacy of Clitoria ternatea L. flower (Pukingan) aqueous extract for use in staining cheek cells was studied to obtain non-toxic, environmentally friendly and low-cost dyes for use in staining. C. ternatea L. has a sparsely pubescent stem that sub-erect and woody at the base and may be up to 5 m long. The flower extract of C. ternatea L. have anthocyanins which are categorized as ternatins. A number of histological techniques have been identified to be used to provide a nuclear stain consist of natural phenolic compounds, structurally related to anthocyanins. The objective of this study is to determine the efficacy of aqueous extract of Pukingan on cheek cells considering several parameters. Dye extracts from C. ternatea L. were used to stain cheek cells using the existing standard staining procedures with little modification. One Way Analysis of Variance was used to analyze differences among the mean scores. A significant difference was determined using a post hoc analysis which is Tukey’s test using SPSS and the level of significance was set at 0.05. From the results of the descriptive, parameters for all sample populations have almost similar interpretations. The nucleus was stained satisfactorily, and the sharpness and contrast were excellent. The cytoplasm of the cheek cells was stained intensely, and the stained areas are homogeneous. The prepared extracts had affinity for the cell membrane and nucleus. Therefore, this study shows that dye extracts from C. ternatea L. could be used for cheek cell staining as alternative to Methylene blue stain.

Introduction

The complexity of tissue can be observed in the varied shapes, sizes, and arrangements of cells, and stains are advantageous in revealing these details and more (Nguyen, 2017). Dyes and stains are commonly used in biology to highlight cellular structures in different tissues. While microscopes allow for the magnification of minute anatomical features, dyes provide a clearer and more emphasized view of the specimens. Staining can also be used to highlight metabolic processes or differentiate between live and dead cells, and to enumerate cells to determine biomass in a given environment. Methylene blue is the preferred stain for animal cells, and is often used to view cheek cells in laboratory settings. However, it is toxic when ingested and causes skin and eye irritation (Chew Weng Cheng et al., 2014). Plant extracts are a potential source of natural dyes that produce varying color intensities from yellow to red to black, and are safe for users and the environment due to their herbal and organic nature. 

The Clitoria ternatea L., also known as butterfly pea or Pukingan, is a tropical legume that can grow vigorously as a trailing, scrambling, or climbing plant. Its stems are sub-erect and sparsely pubescent, and may be woody at the base, reaching up to 5 meters in length. The plant only roots at the tips of its stems, according to studies by Cook et al. (2005) and Staples (1992). The leaves are pinnate, with 5-7 elliptical leaflets that are about 3-5 centimeters in length. The flowers are typically solitary or paired, with a diameter of about 4 centimeters and can be either deep blue or pure white in color. 

Generally, this study aims to determine the efficacy of aqueous extract of Pukingan (Clitoria ternatea) aqueous extract on cheek cells. Specifically, this study aims to compare the mean staining scores of cheek cells stained with: (1) Stain 1, 5gram Pukingan flower/150ml distilled water subjected to 5minute boiling (2) Stain 2, 5gram Pukingan flower/150ml distilled water subjected to 10minute boiling (3) Stain 3, 10gram Pukingan flower/150ml distilled water subjected to 5minute boiling and (4) Stain 4, 5gram Pukingan flower/150ml distilled water subjected to 10minute boiling based on the following parameters: Nuclear staining; Nuclear Detail; Cytoplasmic staining and Uniformity of Stain.

Since many synthetic dyes pose a hazard to the health of students and those who prepare them, this study also aims to make use of a natural alternative to Methylene blue stain. Roots, Fruits, leaves and flowers from dye-producing plants can hypothetically provide an effective stain. An abundant number of potential plants with staining properties sprout throughout the Philippine setting, and one of them is Clitoria ternatea L. (Pukingan). The Fabaceae family in general has anthocyanins, which are water-soluble, vacuolar pigments capable of staining cellular structures. Therefore, Clitoria ternatea L. (Pukingan) presents a promising alternative as a non-toxic, accessible and cost-efficient organic chemical stain.