Applied Bioscience Consultants & Distributors

General product information

From: Applied Bioscience Consultants and Distributors, Mumbai
Title: From Agar to GELRITE, A paradigm Shift

GELRITE

Among the fundamental developments in Plant Tissue Culture and Microbiology, the use of solid substrate media for the support and growth of plants & microbes in propagation holds first place. The credit for the introduction of the solid culture technique in microbiology has been attributed to Robert Koch. Today, agar is utilized around the world in bacteriological culture media as perhaps the only gelling agent of choice due to its attractive features.

The natural gelling agent Agar-Agar is extracted from the seaweeds Gelidium and Gracilaria of Family Rhodophyceae. Gelidium is mostly found on the Atlantic coast of Southern Morocco, Spain, Portugal and France while Gracilaria grows on the coastline of Chile, South Korea, Japan and Argentina. Modern day production of agar involves an extraction of the seaweed liquor under high pressure. The gel is then pressed to eliminate free water to obtain a cake, which is then dried in large drying ovens. Impurities, debris, minerals and pigments are reduced to specified levels during manufacture. Agar finds wide applications in Scientific, Industrial, and Medical laboratories around the world as a culture medium for growing microorganisms i.e. bacteria and fungi and also forms a major component of Plant Tissue Culture media.

Chemically, Agar-Agar is a polysaccharide composed of units of galactose. It is a phycocolloid, insoluble in cold water but soluble in hot water and produces solutions that are viscous and fluid. This solution, when cooled, gels at 30 5 C for high grade agars and can be higher up to 40-45 C for low quality agars. Agar gels are thermo reversible gels that melt again at around 85 5 C and reform a gel on cooling down to 35 5 C.

Gel strength or firmness and gelling and melting temperatures are crucial parameters defining the quality of an agar. Other desired characteristics include clarity or the transparency of the gel, good diffusion characteristics, elasticity, and the absence of toxic substances inhibitory to growth of bacteria. Ideally gelling temperatures of around 35C; a melting temperature of around 85 C; a gel strength that must be over 800 gms/cm2 and an excellent quality agar has gel strength (measured in Nikkans) around 920 gms/cms2 at 20C; transparencies of approximately 5-10 NTU, divalent cations that must not be higher than 1.000 ppm, pH around neutrality define good quality agars. Optimal gelling temperature, clarity, presence of impurities are some of the most common problems associated with agars.

Thus, procurement of good quality agar is definitely a problem which plagues every Microbiology and Tissue culture laboratory and the search for both, good quality agars or its substitutes is unfortunately a never ending feature for every lab and a source of worry for every scientist.

Several polysaccharides of microbial origin, which have gelling properties have been investigated as gelling agents for commercial applications. These include xanthan gum, gellan gum, karrageenan, dextran, pullulan etc. (Kang et al 1982; Sanford et al 1984)

A substitute of agar, currently available in the markets is gellan gum based products under the brand name GELRITE and GELZAN CM. Gellan gum is a low-acyl microbial polysaccharide produced by the bacterium Pseudomonas elodea. It is a highly-purified, natural anionic heteropolysaccharide composed of glucose, glucuronic acid and rhamnose moieties.

Suggested structure of the repeating unit is a tetrasaccharide :
Lβ -D-Glcp-(1→ 4)-β -D-GlcpA-(1→ 4)-β -D-Glcp-(1→ 4)- δ-L-Rham-(1→ )

GELRITE is manufactured by a tightly-controlled fermentation process followed by a well-established purification process with stringent Quality Control measures in place so as to ensure consistent product quality and maintain batch-to-batch consistency. Therefore it contains very low levels of impurities and no contaminating matters like sulphur or phenolic compounds which are common contaminants found in agar and which adversely affect growth of sensitive organisms. Due to this reason, the growth supporting properties of Gelrite are better than those of agar. GELZAN CM gellan gum has demonstrated superiority over agar for cultivation of mesophilic Methanobacterium and Methanobrevibacter organisms. At the same time, it resists contamination by molds. Spreader colonies do not become too large. GELRITE itself is chemically inert to most biological growth media additives. Additives must be heated to just above GELRITE gel point before incorporation.

The main advantage of gels formed using these products is their remarkable optical clarity in comparison to those formed with agar making them excellent analytical tools. In Plant Tissue Culture media, this allows clear observation of root and tissue development. Thus, growth within the gel can be observed.

Stability of a product under the usual appropriate conditions of storage in a laboratory, determines its shelf life. GELRITE is unaffected by the unpredictable extremes of natural conditions which affect the basic properties of agar and have a profound effect on shelf life. GELRITE gels have essentially the same shelf life as agar gels. The gels have also been found to be resistant to enzymatic degradation.

Gels prepared with GELRITE are stable at high temperatures. In microbiological media, this supports incubation required by thermophilic microorganisms.

Several experiments have been conducted in order to elucidate physico-chemical properties of gellan gum. It was studied in aqueous solution and in the gel state by osmometry, viscometry, light scattering, polarimetry and NMR and by measurements of gel strength and cation-exchange selectivity. Measurements of optical rotation and NMR spectroscopy indicated that chain association and gelation were related phenomena. The results are interpreted as indicating that gelation occurs in two steps, namely, chain ordering and chain association.

Another very attractive feature of the gellan gums is that they can form rigid, brittle, agar-like gels at approximately half the use level of agar, in presence of soluble salts. In fact, GELZAN CM can be used at one-fifth the agar use level. In the presence of suitable soluble salts, GELRITE can be used to provide high gel strength at low GELRITE concentrations (normally at approximately half the concentration required for agar). It has been demonstrated that low concentrations of divalent cations are more important for gelation to take place rather than monovalent cations. A wide range of gel strengths are available simply by varying the concentration of Mg. Acceptable gel strengths can be obtained using GELZAN CM gellan gum at a lower level than agar. With greater gel strength, reduced preparation time for plates, drier media can be obtained. If gel strength and density is high then less space would be occupied by the polymer. This helps in enhancing liquid media ratio in the bottle.

Ease of Processing with GELRITE Media
GELRITE gellan gum disperses and hydrates easily in either hot or cold deionized water, forming viscous solutions in cold distilled waterAt high temperatures, the low viscosity of GELRITE solutions facilitates pipetting, pumping, and pouring; upon cooling, GELRITE solutions gel quickly and uniformly. It is able to withstand normal autoclaving conditions Gels prepared with GELRITE set faster than those made with agar. In microbiological applications this reduces plate preparation time. The gels are stable over a wide pH range. A wide range of media can be gelled. A wide range of gel textures can be produced

In conclusion, GELZAN CM gellan gum can not only replace agar in many routine media applications, but can also give a higher degree of cell growth in certain situations. GELRITE was used in concs of 0.2% for banana tissue cultures with good results. Plant species show that growth is indeed improved on media containing GELZAN CM gellan gum (Kulkarni et al 2006).

References:
1. K. S. Kang, G. Veeder, P. J. Mirrasoul, T. Kaneko and I. W. Cottrell. Applied and Environmental Microbiology, May 1982, Vol. 43, No. 5: 1086-1091. Agar-Like Polysaccharide Produced by a Pseudomonas Species: Production and Basic Properties.
2. P. A. Sandford, I. W. Cottrell, and D. J. Pettitt. Pure & Appi. C1-zem., Vol. 56, No. 7, pp. 879892, 1984. Microbial polysaccharides: new products and their commercial applications.
3. V. M. Kulkarni, P. Suprasanna and V. A. Bapat; Current science, vol. 90, no. 6, 25 March 2006. Plant regeneration through multiple shoot formation and somatic embryogenesis in a commercially important and endangered Indian banana cv. Rajeli

Note:
Both GELRITE and GELZAN CM are available ready stock, from Applied Bioscience India Pvt. Ltd.; Email : ; Tel : 022-2966 0575 ; Fax : 022- 2846 3990.