1- Introduction

Dyes are chemical substances of chemical or synthetic origin, soluble in a medium used to impart a desired color to a non food material like paper, leather, wood,

textile and even cosmetics in a process known as dying (1). Dyes are also referred to as stains and can be used to add color to tissues and microbes to make them optically distinct (2).

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 (3).

Stains are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes. Stains may be used to define and examine bulk tissues (Highlighting, for instance, muscle fibers or connective tissues) cell populations, classifying different blood cells or organelles within individual cells (4).

Gram stain is named after its inventor, the Danish scientist Hans Christian Gram (1853–1938), who developed the technique in 1884 to discriminate between pneumococci and Klebsiella pneumoniaebacteria. Gram staining or the Gram's method is an empirical method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls (5).  

A counter stain is a stain with color contrasting to the principal stain, making the cells or structures more visible  when not completely visible with the principal stain. For instance crystal violet stain is applicable only to gram-positive bacteria in gram staining, while sufranin counter stains are applicable to all cells allowing the identification of Gram-negative bacteria as well as Gram-positive strains. Often these stains are called vital stains. These stains may be used in both living and fixed cells (6).  

Several studies have been carried out on the extraction of coloring component from different parts of plants i.e. leaves, roots, barks, trunks or fruits (7)as well as on the identification of chemical structure of the coloring component (8).

Henna, pomegranate, cabbage , bee roots, madder, turmeric, eucalyptus etc. are examples of some well-known natural dyes. The coloring components in most of the plants are usually flavonoids, anthraquinones and indigo.

Henna (Lawsonia inermis) is a plant, which grow wild in abandoned areas . It is widely known as cosmetic agent used to color hair, skin and nails . Henna has many traditional and commercial uses, the most common being as a dye for hair, skin and finger nails, as a dye and preservative for leather and cloth, and as an antifungal agent. Henna produces a red orange dye molecule, “lawsone”, which is known as hennotannic acid, that has an affinity for bonding with protein and this has been used to dye skin, fingernails, hair, leather, silk and wool (9).

Cabbage or headed cabbage (Brassica oleracea) is a leafy green or purple biennial plant, grown as an annual vegetable crop for its dense-leaved heads. Cabbage was most likely domesticated somewhere in Europe before 1000 BC. It is a good source of vitamin K, vitamin C and dietary fiberand it  prepared in many different ways for eating.

pomegranate  (Punica granatum )of Punicacea family is said to be originally found in Persia but also grows in all temperate countries. Pomegranate seeds, the edible portion of the fruit, are rich in sugars, vitamins, polysaccharides, polyphenols, and minerals (10) . They have low oil content and are rich in polyunsaturated (n-3) fatty acids. The pomegranate seed extract possesses high antioxidant activity (11).The coloring components of Pomegranate juice consists of different types of anthocyanins including pelargonidin-3-glucoside, cyaniding-3-glucoside, 5-diglucoside and delphinidin-3-glucoside. Granatonine is the chief coloring component in the pomegranate rind which is present in the alkaloid form N-methyl granatonine .

Beetroot is the taproot portion of the beet plant  usually known in North America as the beet, also red beet. It is one of several of the cultivated varieties of Beta vulgaris grown for their edible taproots and their leaves (called beet greens).Betanin, obtained from the roots, is used industrially as red food colorant, to improve the color and flavor of tomato paste, sauces, and jams among other applications( 12 ). Nowadays many natural roots were mentioned as a natural dye source in various scientific researches. Sivakumar et al. 2009( 13)  has been studied on the use of power ultrasound to improve the extraction of beetroot dye and application to the substrates such as leather and they reported that the beetroot is suitable for dying leather.

The increased awareness of health and environmental hazards associated with the manufacturing, processing and use of synthetic dyes that resulted to essential interest  of natural dyes in recent years. Therefore,  the current study aimed to fulfills the following objectives:

1.     To prepare hot aqueous  extraction  of  Henna, pomegranate ,cabbage and beet roots as  natural dyes .

2. Oxidation for all these extracts once with potassium alum, another with hydrogen peroxide and with potassium permanganate  .

3. Use of Henna , pomegranate, and beet roots solution as counter stain  

   of bacterial cell wall in Gram’s staining reactions.

4. Use of cabbage solution as principle stain of bacterial cell wall in

   Gram’s staining reactions.

2- Materials and Methods

2.1 Aqueous extraction of Henna

Henna dried leaves were grinded and a fifty gram of Henna powder has been soaked in 100 ml. of  boiled sterilized  water and was kept at a room temperature for 24 hours. The soaked powder was filtered by using Whatman no. 1 filter paper. The solution stored at 4°C in refrigerator until it will be used.

2.2 Aqueous extraction of cabbage

Fresh red cabbage has been chopped and then soaked in boiled sterilized water. The solid residues were filtered by textile mesh. Then, solution was filtered by using Whatman no. 1 filter paper. Filtration results were concentrated by a rotary evaporator at 50 degree and eventually stored at 4 degree before use.

2.3 Aqueous extraction of pomegranate

 The pomegranate seeds were squeezed into juice form. The solid residues were filtered by using Whatman no. 1 filter paper.The filtered solution  was used as  natural dyes in the staining process.

2.4 Aqueous extraction of Beet root

Fresh beet root has been chopped and then soaked in boiled sterilized water. The solid residues were filtered by textile mesh. Then, solution was filtered by using Whatman no. 1 filter paper. Filtration results were concentrated by a rotary evaporator at 50 degree and eventually stored at 4 degree before use.

2.5 Gram stain protocol

This technique is used to stain a slide to observe the bacterial present based on their gram stain reaction.

• “Heat-fix” the slide with the specimen by passing it over a heat source, such as a flame, several times using a clothes pin or forceps. The slide should be passed very quickly through the flame and not be heated excessively. Place slide on the staining tray.

• Flood the fixed smear with crystal violet solution  and allow to remain for 1 minute.

• Rinse off the crystal violet with distilled or tap water.

• Flood the slide with iodine solution , Allow to remain for one minute.

• Rinse off the iodine solution with distilled or tap water.

• Holding slide on a tilt with a clothes pin, flood the slide with decolorizer  for one to five seconds.

• Rinse off the decolorizer with distilled or tap water.

• Flood the slide with safranin . Allow to remain for 30 seconds.

• Rinse off the safranin with distilled or tap water.

• Dry the slide on bibulous paper or absorbent paper and place in an upright position.

Microscopically examine the slide for bacterial organisms under a 100X objective. Observe several fields on the slide for bacterial organisms. Describe the gram reaction of any organisms seen. Gram-positive bacteria stain deep violet to blue and gram-negative bacteria stain pink to red. If your slide is all one color (either pink or blue), then the slide may either have been over or under decolorized. Unless it is a gram stain of a pure culture, there normally should be material staining both colors somewhere on the slide.

3- References

1- Green CL (1995). Nutritional Colourant and Dyestuff: A review of Production Markets and Development Potential. Published by the Food and Agricultural Organization of the United Nations, Rome, Italy, pp. 1-34.

2- Ragaswami G, Bagyaraj DJ (1993). Agricultural Microbiology, 2nd edn., Prentice-Hall of India Private Limited, New Delhi, India, pp. 18-29.

3- Bhuyan R, Saikia CN (2004). Isolation of colour components from native dye bearing plants in North Eastern India. Am. J. Pathol., 164(5): 873-877.

4- Penney, D.P., Powers, J.M., Frank, M., Willis, C., and Churukian, C. (2002). Analysis and testing of biological stains – the Biological Stain Commission procedures. Biotechnic and Histochemistry 77: 237–275.

5-M.T. Madigan, , J. Martinko and J. Parker (2004). Brock Biology of Microorganisms, 10th Edition, Lippincott Williams & Wilkins.

6- Horobin, R.W., and Kiernan, J.A. (2002) Conn's Biological Stains. 10th ed. Oxford: BIOS.

7-Bhuyan, R. and Saikia, C. N. (2005). “Isolation of colour components from native dye-bearing plants in northeastern India”. India Bioresour. Technol. 96, 363-372.

8- Guinot, P., Gargadennec, A., Valette, G., Fruchier, A. and Andary, C. (2008). “Primary flavonoids in marigold dye: extraction, structure, and involvement in dyeing process”. Phytochemical Analysis19, 46–51.

9- Singh, Y.V. Kumar, S. and Singh, M. (2005). Agro History, Uses, Ecology. And Distribution of Henna (Lawsonia Inermis) Henna Cultivation, Improvement and trade Central and Arid zone Jodpur, India. Pp 11 – 12.

10. Melgarejo P, Artés F. (2000). Total lipid content and fatty acid composition of oilseed from lesser known sweet pomegranate clones.  J Sci Food Agric.;80(10):1452–1454.

11. Singh R.P, Murthy K.N.C, Jayaprakasha G.K. (2002 ) Studies on the antioxidant activity of pomegranate (Punica granatum) peel and seed extracts using in vitro models. J Agric Food Chem.;50(1):81–86.

12- Grubben, G.J.H. & Denton, O.A. (2004) Plant Resources of Tropical Africa 2. Vegetables. PROTA Foundation, Wageningen; Backhuys, Leiden; CTA, Wageningen.

13- Sivakumar, V.; Lakshmi, A.J.; Vijayeeswarri, J.; Swaminathan, G. (2009). Ultrasound Assisted Enhancement in Natural Dye Extraction from Beetroot for Industrial Applications and Natural Dyeing of Leather. Ultrasonics Sonochemistry 16: 782-789.