International Journal of Scientific & Engineering Research, Volume 5, Issue 2, February-2014 1586

ISSN 2229-5518

Application of aqueous plant extracts as

Biological stains.

Korade Deepali1, Sonawane Lalita2,Mahale Deepika3

Abstract— Aqueous extracts from local dye yielding plants: Henna leaves, Madder stem and Flowers of Hibiscus, Bougainvillea, Fire flame bush and Madder were used for histological, fungal and Paramecium staining. The acidic cytoplasmic natural stains of Rose and Bougainvillea showed best results for fungal and plant tissue staining, whereas Rose, Hibiscus and Henna (instead of Eosin) were foremost for animal histological staining in combination with haematoxyline.

Index Terms— Aqueous plant extracts, Bougainvillea, Biological stains, Henna, Hibiscus, Madder.

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1 INTRODUCTION

TAINS are generally used to add color to animal tissues, plant tissues, microbes and spores to make them optically distinct and technique is known as staining. Most stains in current use are chemically synthesized from cheap petroleum sources, shows superior fastness properties, are widely availa- ble at an economical price and produce wide variety of color [1]. However, they cause skin allergies and other harms to human body on exposure and produce toxic waste, also re- duce soil fertility. The use of non-allergic, non-toxic, and eco- friendly stains has become a matter of significant importance due to the increased environmental awareness in order to
avoid some hazardous synthetic ones.
Despite the biotechnological advance in medical science to- day, biological stains are vital in laboratory diagnosis and dif- ferent staining methods remains an important simple diagnos- tic tool in diagnostic and research laboratories [2]. Extracts obtained from natural sources such as animal and vegetable sources, plants, insects and soil hold promise as a potential source of cheaper stains. Over 2000 dyes are synthesized from various parts of more than 500 dye-yielding plant species, of which only about 150 have been commercially exploited [1]. In present study, Six local dye yielding plants were chosen to obtain aqueous extracts and their applications in fungal, par- amecium staining and histological staining were explored.

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1. Corresponding Author- Dr. Korade Deepali, Department of Biotechnology, K.T.H.M. College, Nashik, Maharashtra, India (deepsk_19@yahoo.co.in)

2,3 Post graduated students of Department of Biotechnology, K.T.H.M. Col-

lege, Nashik, Maharashtra, India

2 METHODOLOGY

2.1 AQUEOUS EXTRACTS PREPARATION

Six local plants: Henna: Lowsonia inarmis; Hibiscus: Hibiscus rosa-sinensis, Madder: Rubia tictorium L., Fire flame bush: Butea monosperma, Rose: Rosa indica Bougainvillea: Bougainvillea gala- bra chosen in this study based on their availability. Aqueous (aq.) extracts of dried powered of Henna leaves, Madder stems and flowers of Hibiscus, Fire Flame bush and Rose were pre- pared by Soxhlet extraction [3]. Staining solutions/ natural stains were prepared using dried powder in water (50mg/ml).

2.2 FUNGAL STAINING

Lactophenol-in-cotton blue is standard stain (acidic pH 3.6) for fungal specimens. A drop of 70% alcohol was placed on a mi- croscope slide and specimen of cottony white fungus was im- mersed in the drop of alcohol. One/two drops of the Lacto- phenol-in-cotton blue before the alcohol dries out [1]. pH of the stains was 6-6.5 that was lowered to 4 -5 then used as stains replacing Lactophenol-in-cotton blue. Slides were ob- served under microscopes and compared.

2.3 PARAMECIUM STAINING

Paramecia were cultured by Hay culture method using pond water as initial inoculum. One drop of paramecium rich cul- ture was placed on clean glass slide and stained using either Methylene blue dye or stains on different slide, observed un- der the microscope.

2.4 HISTOLOGICAL STAINING

Angiospermic tissue staining: Fresh stem of Hibiscus were collected and thin sections of stem tissue were stained using Saffranin and all stains differently. Stained slides were ob- served under simple light microscope (4x), their staining in- tensity were observed and compared [4].

Animal tissue staining: Permanent slides of animal tissue sections were obtained by Paraffin embedding method. For staining, slides were dewaxed by dipping in the xylene fol- lowed by hydration by a series of alcohols (Absolute to 90% to

70% to 50% to 30% alcohol) then dipped into distilled water for 3 minutes at each step [5]. After rinsing into water the slides were dipped into the Haematoxyline jar for 5 to 10

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minutes then only rinse with distilled water followed by aq. extract (each separately) for 15 to 20 minutes. Dehydration of the slides was carried out by passing the sections through 30,
50, 70, and 95% alcohol series for few minutes in each strength. Slides stained with only Haematoxyline were further stained by Eosin dye by first dehydration of the slides and then im-
mersion of slide in eosin jar for 3 to 5 minutes. Then wash the excess Stain in 95% alcohol. (which stain cytoplasm of the tis- sue.) After clearing the slides, the sections were mounted with help of DPX and observation under the microscope [6], [7], [8], [9].

3 RESULTS AND DISCUSSION

The ability to stain specific tissue structures is determined by the pH values of stain. According to staining theory, acidic structures are stained by basic dyes while basic structures are stained by acidic dyes [3]. The stains treated with acid and base were reported to improve staining potentials for moulds [10]. Hence, in this study, it was necessary to low the pH val- ues of natural stain using dilute HCL. The result of stained white cottony fungus using the natural stains and lactophenol- in-cotton blue is shown in Fig.1. The fungus stained with Bou- gainvillea and Rose natural stains found to be as efficient as Lactophenol-in-cotton blue in staining. These results are con- comitant with work done in 2010 by Briade and his colleagues using extracts of four Nigerian local plants for staining bacte- ria and moulds [10]. Hibiscus and Butea monosperma stains shown good staining compare to Madder and Henna extracts. Application methanolic extracts of Hibiscus as biological stain for staining some fungal species was found to be successfully explored by Ihuma et al. (2012) [1]. Madder stain was show clump formation it interfere with the staining and Henna stain shows low capacity to stain the fungal specimen than other ones. Hence both were shows poor staining may be because of their composition of pigments.

Lactophenol–in-cotton blue, Henna,

Hibiscus Rose

Bougainvillea

Fig.1 Specimens of White cottony fungus were stained with different aq. plant extracts

The potential natural extracts as a staining agent for living cell i.e. Paramecium was investigated in this study. Paramecium staining is useful for study of external morphology, different organelles, behavior and response to various stimuli. It is also used for observe the process of food cycle of paramecium. Figure 2 shows the comparative photographs of stained Para- mecium using natural stains and Methylene blue dye. All natural stains stained cytoplasm, cytoplasmic components or cell organelles of paramecium very effectively. Cytoplasm of the cell is usually stained with Acidic stains, while the basic stains usually stain the nucleus of the cell [6]. From this obser- vation it can be estimated that the natural stains were acidic in nature and can replace methylene blue like synthetic stains for paramecium staining. To our knowledge this is the first at- tempt of paramecium staining using aq. plant extracts as stains.

Unstained Methylene blue

Henna Hibiscus

Rose Madder

Madder Fire flame bush

Bougainvillea Fire flame bush

Fig.2 Paramecium: unstained/ stained with various stains

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The results of histological staining of angiospermic tissue are presented in Fig 3. Henna and Fire flame bush stains were imparted grayish green colour to the sclerenchyma and paren- chyma stem tissue. These findings are similar to the work done by Jan et al. (2011) using henna dried leaves extract as angiospermic tissue stain [4]. Bougainvillea natural stain also showed effective staining same as saffranin synthetic dye. Vascular bundles, xylem cells were stained effectively, while cortex and medulla were stained less effectively. Xylem is the water conducting tissue cells therefore; they may be effectively stained with aqueous natural stains. Rose extract showed good staining, whereas as madder and Hibiscus extract showed poor staining because they were form clumping with the fluid of stem tissue. Hence, they were not suitable for staining the stem tissue of Hibiscus plant. Any previous re- ports on plants like Fire flame bush and Bougainvillea for plant tissue staining have not been found.

Saffranin Henna

Madder Fire flame bush

Bougainvellea Hibiscus

Rose

Fig.3: Cross sections of angiospermic stem tissues were stained with different stains

and insect parts used in histological staining as natural dyes are Haematoxylon campechiaumn, from which haematoxyline is obtained and Dactylopius cacti, from which carmine stain is obtained. The haematoxyline in combination with Eosin which is synthetic dye is used for the demonstration of general tissue structures such as muscle fiber, connective tissue etc. [11]. Haematoxyline is a basic dye that stains acidic components of the cell. Eosin is an acidic dye that stains the basic cytoplasmic components of the cells. A counter stain is a stain with colour contrasting to the principal stain, making the stained structure more easily visible [11]. It is the application to the original stain, usually nuclear stains. Some counter stains which are acidic may lighten or remove the nuclear stains. Nuclei of the cells take the haematoxyline dye and appear dark violet or blue in color, cytoplasm of some epithelial cells; erythrocytes take eosin dye and stain pink [7].
The dyeing of tissues is dependent on binding forces or link to the tissue; or they will simply be rinsed out of the tissue when the section is washed in another reagent [8]. Ionic bond- ing involves electrostatic attraction between oppositely charged ions is the most important form of bonding in histo- logical staining. Selectivity of staining depends on a sufficient- ly low stain concentration, on the time of action on the solvent, its aqueous or alcoholic nature and its pH [6]. Eosin is an alco- holic stain and Haematoxyline is aqueous stain extracted from logwood.

According to the results obtained natural stains from Rose, Hibiscus, Henna and Bougainvellea stained cytoplasmic com- ponents of the tissue showed better histological staining same as the combination of Haematoxyline with counter stain Eosin. Madder stain and Fire flame bush stain showed poor histolog- ical staining (Fig. 4). Histomorphological studies of testis tis- sue using Curcuma Longa extracts as stains has been reported by Bossey et al. (2012) [6]. Hence, in present study, the aque- ous extracts can be called as Cytoplasmic stain which can be used as a counter stain with Haematoxyline instead Eosin.

(A) (B)

(C) (D)

Plant and insect parts have found place in histological staining due to their coloring and dying effects. For instance, plants

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(E) (F)

(G) (H)

(I)

Fig 4 : Histological staining of Animal tissue by various stains

(A)Section of the gills of bioworm stained with Eosin only, (B) Section of the Gills of bioworm stained with Haematoxyline only.(C) Section of the gills of bioworm stained by Haematox- yline with Eosin stain (D)Section of the gills of bioworm stained by Haematoxyline with Rose stain(E) Section of the pancreas tissue of Albino rat stained by Haematoxyline with Hibiscus stain (F) Section of the Pancreas tissue of Albino rat stained by Haematoxyline with Henna stain (G)Section of the gills portion of Bioworm stained by Haematoxyline with Fire flame bush stain (H) Section of the of gills of Bioworm stained by Haematoxyline with Madder stain (I)Section of the Kidney tissue of Albino rat Stained with Haematoxyline with Bou- gainvillea dye.

4 CONCLUSION

Six local plants were selected and their aqueous extracts used as eco-friendly and cost effective biological stains. Acidic cy- toplasmic natural stains have affinity with basic components of tissue/ fungal species due to which Rose and Bougainvillea extracts stained plant tissue well, may replace saffranin whereas Rose, Hibiscus and Henna worked best in combina- tion with haematoxyline for animal tissues staining instead of Eosin.

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