Kumkuma the Social and religious marking of India is used as stain to improve the contrast of Pectinolytic actinomycetes VIT S2

 

Suneetha V*, Praveen Kumar G

School of Biosciences and Technology, VIT University, Vellore

 

ABSTRACT:

Background: Kumkuma is a red colour powder which is mainly used for the religious purpose and it was made from saffron flowers of Crocus sativus L with mild use of turmeric. The utilization of kumkuma by Hindu religious people is immense and it is applied at the forehead where people believe it as so divine and religious marking.

Objective: The kumkuma samples ,the natural stain  were used to stain the Pectinolytic actinomycetes VIT S2for contrast and it is easily available and economically viable.

Methods: The kumkuma samples were collected from local market, Vellore. The samples were used for the staining of Pectinolytic actinomycetes.

Result: The Kumkuma (K1) and Kumkum (K2). The Kumkuma (K1) shows better result compared to that of Kumkum. The results revealed that kumkuma (K1) shows positive result compared to that of kumkum (K2).  This may be due to the presence of  more Ca (OH)₂   in kumkuma (K1) is basic (cationic) which retains on the  cell layer present in all gram positive Actinobacteria. The positive ions which are present in kumkuma attracted to the negatively charged bacterial cell and retain the stain.

Conclusion: This is the foremost study to report on staining of pectinolytic strain using kumkuma (K1) and kumkum (K2).  The turmeric has beneficial effects present in samples were less harmful compared with chemical dyes. But, the saffron present in kumkuma shows various pharmacological properties. The study supports with positive result that kumkuma (K1) is better than kumkum (K2) and the use of kumkuma (K1) is safer than other chemical dyes which can be used for the staining of actinomycetes in near future.

 

 

 

 

 

INTRODUCTION:

Actinomycetes are group of bacteria which are enormously present and have significant characteristic of degrading the biomass in nature¹. These are filamentous group of organisms which represents as an extensive group of soil population².

 

Streptomyces represents most of the actinomycete group and have been widely reported for their application in various fields ³.  Stains or dyes are charged molecules which are mainly adhere to the cell structure and provide them color. Pectinases are the group of catalysts that breakdown pectic substances in plant tissues⁴. They were used for different application in various industrial sectors like food and drug delivery studies^5,6,7.  Pectinase are cocktail catalysts or enzymes which degrade the pectin substrate present in plant tissues⁸. Staining also utilized to increase the cell visibility and contrast between the gram-positive and gram-negative cells⁹. Stains are normally utilized in microscopy techniques to make the microorganisms to differentiate from their respective (light or dark) backgrounds. Simple or monochrome staining is a technique which involves the usage of single dye to provide basic cell structures and arrangement .The colour of basic dyes is in the presence of positive ion^10-14. Kumkum is a red colour powder which is mainly used for the religious purpose and it was made from dried turmeric (Curcuma longa) powder with slaked lime Ca(OH)₂ which helps in the colour conversion of yellow into bright red colour with addition of water. The chemical analysis showed that Crocus sativus L. contains 150 components¹⁵. Saffron consists of a water-soluble carotenoid which has main pigment named alpha-crocin¹⁶. It also reported that α-crocin have high antioxidant, anticancer properties and other pharmacological properties¹⁷. Earlier reports showed that it can be used as histological stain¹⁸. The utilization of kumkuma by Hindu religious people is immense and it is applied at the forehead where people believe it as so divine¹⁹. Murein is a special type of peptidoglycan present in all Gram-positive bacteria. The peptidoglycans are the polymer sheets composed of disaccharides that are cross linked with peptide chains. N-acetylmuramic acid deserves a main component of murein. Curcumin main component of turmeric which exhibits vast range of pharmacological activities including anti-inflammatory, anti-cancer and antioxidant properties. Recently, natural resources utilization had been efficiently increased¹⁹. This is the first study to report on the  staining of Pectinolytic actinomycetes by using kumkuma (K1) and kumkum (K2) samples.

 

Table 1: Different names of kumkuma in different origins of India

 

MATERIALS AND METHODS:

Manufacturing of kumkuma

 

Fig no 1: Steps involved in manufacture of kumkuma

 

Inoculum preparation:

The potential Pectinolytic actinomycete was screened from fruit industrial dump sites. The 1µL of inoculum was taken in a CSPYME broth and kept in incubation at 30 °C for 120 hrs. The fresh inoculum was taken for further studies.  

 

Slide culture technique for actinomycetes:

The CSPYME plates were prepared and autoclaved at 121°C at 15 psi for 15 min. The plates were taken to the laminar air flow chamber. The agar blocks (8mm×8mm) were cut with the help of sterile blade and placed it on a clean sterile grease free slide. The inoculums were stabbed with the help of stabbing needle at the centre of four sides of the agar blocks. The cover slip was placed above the agar block and it was incubated at 30° C for 120 hrs. Then, the slide was used for the further staining procedures.

 

Kumkuma samples:

The kumkuma samples were collected from local market, Vellore by using sterile containers. The samples were of different colour and designated as Kumkuma (K1) and Kumkum (K2) were diluted with dis.H₂O.  

 

Stain preparation:

The stains were prepared in different concentration of 0.5%, 1% and 1.5%   and observed the chromophore and auxochrome properties and used for this study . The concentration of stain 1% was optimized and used for further studies.

 

Staining procedure:

 

Fig no 2: shows steps involved in staining of pectinolytic actinomycete

 

Microscopic examination

The slides were stained with samples and it was viewed under light microscope and Scanning electron microscope.

 

RESULTS AND DISCUSSIONS:

 

Fig no 3: The staining of Pectinolytic actinomycete VIT S2 with kumkuma (K1)

 

Fig no 4: Shows chain morphology with Kumkuma (K1)( 6 days old culture)

 

Fig no 5: The staining of Pectinolytic actinomycete VITS2 with kumkum (K2)

 

Fig no 6: The  chain morphology of Pectinolytic actinomycete VIT S2with Kumkum (K2)

 

Fig no 7 The scanning electron microscopic image of pectinolytic actinomycetes VIT S2

 

RESULTS AND DISCUSSION:

The main objective of this study is to stain the Pectinolytic actinomycetes with the help of kumkuma (K1) and kumkum (K2) in alternate to chemical dyes. The slide culture technique was followed for the preparation of culture and it was used for further studies. The staining was carried out by using both kumkuma (K1) and kumkum (K2). The novel protocol was designed for the staining of actinomycetes. The stained slides were observed with light microscope. The staining of Pectinolytic actinomycete by using kumkuma (K1) was shown in (Fig 3). The chain morphology of Pectinolytic actinomycetes clearly stained by kumkuma (K1) (Fig 4). The staining and chain morphology of Pectinolytic actinomycete by using kumkum was shown in (Fig 5) (Fig 6). The results reveal that the Ca(OH)₂ which is present in kumkuma (K1) is basic (cationic) which retains on the peptidoglycan layer present in all gram positive bacteria. The bacterium becomes slightly negative when it reaches pH 7. The positive ions which are present in kumkuma (K1) attracted to the negatively charged bacterial cell of gram positive bacteria and it retains the stain. The chain morphology also clearly seen in isolate stained with kumkuma (K1). Whereas, the kumkum (K2) cant able to stain the actinomycete and spore morphology was also not clearly visible on observation. 

 

In addition to that, the isolate was characterized by using scanning electron microscopy (SEM) (Fig. 7) which shows the shape, spore and spore chain morphology of Pectinolytic actinomycetes.

 

CONCLUSION:

The study ends with positive result that kumkuma (K1) is better than kumkum (K2) and the use of kumkuma (K1) is more beneficial with pharmacological properties than other chemical dyes. Thus, Kumkuma can be promising natural stain which can be used for the staining of actinomycetes in near future and we can able to see viability for few hours compare to chemical stains.

 

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Accepted on 21.03.2017           © RJPT All right reserved