Saturday, October 27, 2012

Agar for Plant Tissue culture








 Agar :

Agar is a galactose polymer (or agarose) obtained from the cell walls of some species of red algae or *seaweed (Sphaerococcus euchema) and species of **Gelidium and ***Gracilaria, chiefly from eastern Asia, Chile and California. It is also known as Kanten, Agar-Agar, or Agal-Agal (Ceylon Agar).
-----------------------------------------------------------------------------------------------------------------------
*Seaweed : Seaweed is a loose colloquial term encompassing macroscopic, multicellular, benthic marine algae. The term includes some members of the red, brown and green algae. Seaweeds can also be classified by use (as food, medicine, fertilizer, industrial, etc.).

 
Taxonomy : A seaweed may belong to one of several groups of multicellular algae: the red algae, green algae, and brown algae. As these three groups are not thought to have a common multicellular ancestor, the seaweeds are a polyphyletic group. In addition, some tuft-forming bluegreen algae (Cyanobacteria) are sometimes considered as seaweeds — "seaweed" is a colloquial term and lacks a formal definition.
 
Structure :Seaweeds' appearance somewhat resembles non-arboreal terrestrial plants.
  • thallus: the algal body
    • lamina: a flattened structure that is somewhat leaf-like
      • sorus: spore cluster
      • on Fucus, air bladders: float-assist organ (on blade)
      • on kelp, floats: float-assist organ (between lamina and stipe)
    • stipe: a stem-like structure, may be absent
    • holdfast: specialized basal structure providing attachment to a surface, often a rock or another alga.
    • haptera: finger-like extensions of holdfast anchoring to benthic substrate
 
The stipe and blade are collectively known as the frond.

Ecology :Two specific environmental requirements dominate seaweed ecology. These are the presence of seawater (or at least brackish water) and the presence of light sufficient to drive photosynthesis. Another common requirement is a firm attachment point. As a result, seaweeds most commonly inhabit the littoral zone and within that zone more frequently on rocky shores than on sand or shingle. Seaweeds occupy a wide range of ecological niches. The highest elevation is only wetted by the tops of sea spray, the lowest is several meters deep. In some areas, littoral seaweeds can extend several miles out to sea. The limiting factor in such cases is sunlight availability. The deepest living seaweeds are some species of red algae.

 

A number of species such as Sargassum have adapted to a fully planktonic niche and are free-floating, depending on gas-filled sacs to maintain an acceptable depth.
Others have adapted to live in tidal rock pools. In this habitat seaweeds must withstand rapidly changing temperature and salinity and even occasional drying.

Use: Seaweed has a variety of purposes, for which it is farmed or foraged from the wild.

 
At the beginning of 2011, Indonesia produced 3 million tonnes of seaweed and surpassed the Philippines as the world's largest seaweed producer. By 2012 the production will hit 10 million tonnes.

Food :Seaweeds are consumed by coastal people, particularly in East Asia, e.g., Brunei, Japan, China, Korea, Taiwan, Singapore, Thailand, Cambodia, and Vietnam, but also in South Africa etc.

Seaweeds are also harvested or cultivated for the extraction of alginate, agar and carrageenan, gelatinous substances collectively known as hydrocolloids or phycocolloids. Hydrocolloids have attained commercial significance as food additives. The food industry exploits their gelling, water-retention, emulsifying and other physical properties. Agar is used in foods such as confectionery, meat and poultry products, desserts and beverages and moulded foods. Carrageenan is used in salad dressings and sauces, dietetic foods, and as a preservative in meat and fish products, dairy items and baked goods.
 
Herbalism :Alginates are used in wound dressings, and production of dental moulds. In microbiology research, agar - a plant-based goo similar to gelatin and made from seaweed - is extensively used as culture medium. Carrageenans, alginates and agaroses (the latter are prepared from agar by purification), together with other lesser-known macroalgal polysaccharides, also have several important biological activities or applications in biomedicine.
 
Seaweed is a source of iodine, necessary for thyroid function and to prevent goitre. However, an excess of iodine is suspected in the heightened cancer risk in Japanese who consume a lot of the plant, and even bigger risks in post-menopausal women.

Seaweeds may have curative properties for tuberculosis, arthritis, colds and influenza, worm infestations and even tumors. In Japan, seaweed eaten as nori is known as a remedy for radiation poisoning.

 
Seaweed extract is used in some diet pills. Other seaweed pills exploit the same effect as gastric banding, expanding in the stomach to make the body feel more full.

Other uses : Other seaweeds may be used as fertilizer, compost for landscaping, or a means of combating beach erosion through burial in beach dunes. Seaweed is currently under consideration as a potential source of bioethanol. Seaweed is an ingredient in toothpaste, cosmetics and paints.

Alginates enjoy many of the same uses as carrageenan, and are used in industrial products such as paper coatings, adhesives, dyes, gels, explosives and in processes such as paper sizing, textile printing, hydro-mulching and drilling.

Health risks : Rotting seaweed is a potent source of hydrogen sulfide, a highly toxic gas, and has been implicated in some incidents of apparent hydrogen-sulphide poisoning. It can cause vomiting and diarrhoea.

-------------------------------------------------------------------------------------------------------------
** Gelidium : Gelidium is a genus of thalloid alga comprising 124 species. Its members are known by a number of common names. Specimens can reach around 2 to 40 cm in size. Branching is irregular, or occurs in rows on either side of the main stem. Gelidium produces tetraspores. Many of the algae in this genus are used to make agar. Chaetangium is a synonym.



red algae




Chemically, agar is a polymer made up of subunits of the sugar galactose; it is a component of the algae's cell walls. Dissolved in hot water and cooled, agar becomes gelatinous; its chief use is as a culture medium for microbiological work. Other uses are as a laxative, a vegetarian gelatin substitute — a thickener for soups, in jellies, ice cream and Japanese desserts such as anmitsu, as a clarifying agent in brewing, and for paper sizing fabrics.

Species
----------------------------------------------------------------------------------------------------------------------------------

Gelidium affine                                            Gelidium allanii
Gelidium amamiense                                   Gelidium amansii
Gelidium ambiguum                                    Gelidium americanum                         Gelidium anthonini                                      Gelidium applanatum
Gelidium arborescens                                  Gelidium arenarium
Gelidium asperum                                       Gelidium australe
Gelidium bernabei                                       Gelidium bipectinatum
Gelidium canariense                                    Gelidium cantabricum
Gelidium capense                                        Gelidium caulacantheum
Gelidium ceramoides                                  Gelidium chilense
Gelidium coarctatum                                  Gelidium concinnum
Gelidium congestum                                  Gelidium corneum
Gelidium coronadense                                Gelidium coulteri
Gelidium crinale                                         Gelidium crispum
Gelidium deciduum                                    Gelidium decompositum
Gelidium delicatulum                                 Gelidium divaricatum
Gelidium elegans                                       Gelidium elminense
Gelidium fasciculatum                               Gelidium filicinum
Gelidium flaccidum                                   Gelidium floridanum
Gelidium foliaceum                                   Gelidium foliosum
Gelidium galapagense                                Gelidium hancockii
Gelidium heterocladum                              Gelidium hommersandii
Gelidium howei                                          Gelidium hypnosum
Gelidium inagakii                                       Gelidium inflexum
Gelidium intertextum                                  Gelidium isabelae
Gelidium japonicum                                   Gelidium johnstonii
Gelidium kintaroi                                        Gelidium latiusculum
Gelidium lingulatum                                   Gelidium linoides
Gelidium longipes                                       Gelidium macnabbianum
Gelidium madagascariense                          Gelidium maggsiae
Gelidium masudae                                       Gelidium microdentatum
Gelidium microdon                                      Gelidium microdonticum
Gelidium microphyllum                              Gelidium microphysa
Gelidium micropterum                                Gelidium minusculum
 Gelidium multifidum                                  Gelidium musciforme
Gelidium nova-granatense                           Gelidium nudifrons
Gelidium omanense                                     Gelidium pacificum
Gelidium planiusculum                                Gelidium pluma
Gelidium pristoides                                      Gelidium profundum
Gelidium pseudointricatum                          Gelidium pteridifolium
Gelidium pulchellum                                    Gelidium pulchrum
Gelidium pulvinatum                                    Gelidium purpurascens
Gelidium pusillum                                        Gelidium reediae
Gelidium refugiensis                                     Gelidium regulare
Gelidium reptans                                           Gelidium rex
Gelidium rigens                                             Gelidium robustum
Gelidium samoense                                       Gelidium sclerophyllum
Gelidium secundatum                                    Gelidium semipinnatum
Gelidium serrulatum                                      Gelidium sinicola
Gelidium spathulatum                                    Gelidium spinosum
Gelidium subfastigiatum                                Gelidium tenue
 Gelidium tsengii                                            Gelidium umbricola
Gelidium usmanghanii                                   Gelidium vagum
Gelidium venetum                                          Gelidium venturianum
Gelidium versicolor                                        Gelidium vietnamense
Gelidium vittatum                                           Gelidium yamadae
Gelidium zollingeri
-------------------------------------------------------------------------------------------------------------
*** Gracilaria : Gracilaria is a genus of red algae (Rhodophyta) notable for its economic importance as an agarophyte, as well as its use as a food for humans and various species of shellfish. Various 

  
 

species within the genus are cultivated among Asia, South America, Africa and Oceania.


 

Distribution

Gracilaria bursa-pastoris (S.G.Gremlin) Silva and Gracilaria multipartita (Clemente) Harvey have long been established in southern England and northwestern France, but confusion between Gracilaria gracilis (Stackhouse) 

gracilaria

 

Steentoft, L.Irvine & Farnham and Gracilariopsis longissima (S.G.Gmelin) Steentoft, L. Irvine & Farnham, (as Gracilaria verrucosa (Hudson) Papenfuss or Gracilaria confervoides (L.) Greville), has prevented recognition of the northern boundaries.

 


 

Use : Gracilaria is used as a food in Japanese, Hawaiian, and Filipino cuisine. In Japanese cuisine, it is called ogonori or ogo. In the Philippines, it is called gulaman or guraman.





------------------------------------------------------------------------------------------------------------
The nutrient broth includes complex mixture of compounds that support metabloic activity of bacteria and provide nutrients for growth and cell division.

Agar or agar-agar is a gelatinous substance derived by boiling a polysaccharide in red algae, where it accumulates in the cell walls of agarophyte and serves as the primary structural support for the algae's cell walls. Agar is a mixture of two components: the linear polysaccharide agarose, and a heterogeneous mixture of smaller molecules called agaropectin.


Throughout history into modern times, agar has been chiefly used as an ingredient in desserts throughout Asia and also as a solid substrate to contain culture medium for microbiological work. Agar (agar-agar) can be used as a laxative, an appetite suppressant, vegetarian gelatin substitute, a thickener for soups, in fruit preserves, ice cream, and other desserts, as a clarifying agent in brewing, and for sizing paper and fabrics.

The gelling agent is an unbranched polysaccharide obtained from the cell walls of some species of red algae, primarily from the genera Gelidium and Gracilaria, or seaweed (Sphaerococcus euchema). For commercial purposes, it is derived primarily from Gelidium amansii. In chemical terms, agar is a polymer made up of subunits of the sugar galactose.

Background

Agar consists of a mixture of agarose and agaropectin. Agarose, the predominant component of agar, is a linear polymer, made up of the repeating monomeric unit of agarobiose. Agarobiose is a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose. Agaropectin is a heterogeneous mixture of smaller molecules that occur in lesser amounts.

Agar exhibits hysteresis, melting at 85 °C (358 K, 185 °F) and solidifying from 32-40 °C (305-313 K, 90-104 °F). This property lends a suitable balance between easy melting and good gel stability at relatively high temperatures. Since many scientific applications require incubation at temperatures close to human body temperature (37 °C), agar is more appropriate than other solidifying agents that melt at this temperature, such as gelatin.

The word "agar" comes from agar-agar, the Malay name for red algae (Gigartina, Gracilaria) from which the jelly is produced. It is also known as kanten, China grass, Japanese isinglass, Ceylon moss or Jaffna moss. Gracilaria lichenoides is specifically referred to as agal-agal or Ceylon agar.

Uses

Microbiology

Agar is used throughout the world to provide a solid surface containing medium for the growth of bacteria and fungi. Microbial growth does not destroy the gel structure because most microorganisms are unable to digest agar. Agar is typically sold commercially as a powder that can be mixed with water and prepared similarly to gelatin before use as a growth medium. Other ingredients are added to the agar to meet the nutritional needs of the microbes. Many specific formulations are available, because some microbes prefer certain environmental conditions over others.

Motility assays

As a gel, an agarose medium is porous and therefore can be used to measure microorganism motility and mobility. The gel's porosity is directly related to the concentration of agarose in the medium, so various levels of effective viscosity (from the cell's "point of view") can be selected, depending on the experimental objectives.

A common identification assay involves culturing a sample of the organism deep within a block of nutrient agar. Cells will attempt to grow within the gel structure. Motile species will be able to migrate, albeit slowly, throughout the gel and infiltration rates can then be visualized, whereas non-motile species will show growth only along the now-empty path introduced by the invasive initial sample deposition.

Another setup commonly used for measuring chemotaxis and chemokinesis utilizes the under-agarose cell migration assay, whereby a layer of agarose gel is placed between a cell population and a chemoattractant. As a concentration gradient develops from the diffusion of the chemoattractant into the gel, various cell populations requiring different stimulation levels to migrate can then be visualized over time using microphotography as they tunnel upward through the gel against gravity along the gradient.

Plant biology

Research grade agar is used extensively in plant biology as it is supplemented with a nutrient and vitamin mixture that allows for seedling germination in Petri dishes under sterile conditions (given that the seeds are sterilized as well). Nutrient and vitamin supplementation for Arabidopsis thaliana is standard across most experimental conditions. Murashige & Skoog (MS) nutrient mix and Gamborg's B5 vitamin mix in general are used. A 1.0% agar/0.44% MS+vitamin dH2O solution is suitable for growth media between normal growth temps.

The solidification of the agar within any growth media (GM) is pH-dependent, with an optimal range between 5.4-5.7. Usually, the application of KOH is needed to increase the pH to this range. A general guideline is about 600 µl 0.1M KOH per 250 ml GM. This entire mixture can be sterilized using the liquid cycle of an autoclave.
plants growing in agar dish
This medium nicely lends itself to the application of specific concentrations of phytohormones etc. to induce specific growth patterns in that one can easily prepare a solution containing the desired amount of hormone, add it to the known volume of GM, and autoclave to both sterilize and evaporate off any solvent that may have been used to dissolve the often-polar hormones. This hormone/GM solution can be spread across the surface of Petri dishes sown with germinated and/or etiolated seedlings.

Experiments with the moss Physcomitrella patens, however, have shown that choice of the gelling agent — agar or Gelrite - does influence phytohormone sensitivity of the plant cell culture.

Molecular biology

Agar is a heterogeneous mixture of two classes of polysaccharide: agaropectin and agarose. Although both polysaccharide classes share the same galactose-based backbone, agaropectin is heavily modified with acidic side-groups, such as sulfate and pyruvate.

The neutral charge and lower degree of chemical complexity of agarose make it less likely to interact with biomolecules, and, therefore, agarose has become the preferred matrix for work with proteins and nucleic acids. Gels made from purified agarose have a relatively large pore size, making them useful for separation of large molecules, such as proteins and protein complexes >200 kilodaltons, as well as DNA fragments >100 basepairs. Agarose has been used widely for immunodiffusion and immunoelectrophoresis, as the agarose fibers functions as an anchor for immunocomplexes. Agarose is used generally as the medium for analytical scale electrophoretic separation in agarose gel electrophoresis and for column-based preparative scale separation as in gel filtration chromatography and affinity chromatography.

Culinary

Agar-agar is a natural vegetable gelatin counterpart. White and semi-translucent, it is sold in packages as washed and dried strips or in powdered form. It can be used to make jellies, puddings, and custards. For making jelly, it is boiled in water until the solids dissolve. Sweetener, flavouring, colouring, fruit or vegetables are then added and the liquid is poured into molds to be served as desserts and vegetable aspics, or incorporated with other desserts, such as a jelly layer in a cake.

Agar-agar is approximately 80% fiber, so it can serve as an intestinal regulator.

In Philippine cuisine, it is used to make the jelly bars in the various gulaman refreshments or desserts such as sago gulaman (aka gulaman at sago), buko pandan, agar flan, halo-halo, the various Filipino fruit salads, black gulaman, and red gulaman. One use of agar in Japanese cuisine is anmitsu, a dessert made of small cubes of agar jelly and served in a bowl with various fruits or other ingredients. It is also the main ingredient in Mizuyōkan, another popular Japanese food. In Vietnamese cuisine, jellies made of flavored layers of agar agar, called thạch, are a popular dessert, and are often made in ornate molds for special occasions. In Indian cuisine, agar agar is known as "China grass" and is used for making desserts. In Burmese cuisine, a sweet jelly known as kyauk kyaw  is made from agar. In Russia, it is used in addition or as a replacement to pectin in jams and marmalades, as a substitute to gelatin for its superior gelling properties, and as a strengthening ingredient in souffles and custards. Agar-agar may also be used as the gelling agent in gel clarification, a culinary technique used to clarify stocks, sauces, and other liquids.

Agar  of various qualities

Agar and the solidifying agents in solid growth media should be well chosen, with certain criteria to consider.

Agar is available in a range of qualities dictated by the target application. For example, Fluka brand agar  is highly purified and ideal when high transparency and brightness is needed, as in nutritional studies (Vitamin Assay Media) and sensitivity testing procedures, or when high purity and good diffusion of substances is essential. For identification and differentiation  using a purified or even highly purified agar is necessary. However, when isolating a single colony in most cases a standard quality will suffice. Typical solid media has an agar concentration of 1.0 - 1.5%, to accommodate the requirements of different applications and growth habits of the target microorganisms.

Agar Products -  Agar qualities for Microbiology 

Agar, highly purified
Agar, purified
Agar, standard quality
Agar, fibers
Select Agar
Bacteriological agar
Noble Agar

Ingredients for agar

Here are the ingredients for Nutrient Broth
proportions are for 1000ml
--------------------------------------------------------------------------------------------------------------
Peptone 10gram
  Beef/Yeast Extract  3gram
  Sodium Chloride    5gram
  Distilled Water      
1000ml
Ph 7-7.2


If you want to make agar just add
30gram agar-agar to the above fluid.
This makes Nutrient Agar.

--------------------------------------------------------------------------------------------------------------













No comments:

Post a Comment