Agars used in Microbiological Media

There are many types of agar produced in many countries.

Agar

Agar is used to gel bacteriological broths (liquid media) to form slants, and to avoid sloushing so oxygen can't easily get to the bottom of the medim. Agar is obtained from seaweeds called kelps. There are many different grades of agar and most were developed for use in human food. The amont of agar needed to gel broths varies but 10 grams per liter is commonly used. For a harder drier agar, use 12 or 15 grams and for special purposes 20 or more grams is used. For top agar and motility assays use about 7 grams of agar per liter. For details on fractionation of agar, manufacture and special uses see page b041a.htm . This in blue is proposed new text. Move the black to b041a. add more to this blue text.

There are several kinds of agar which are long chain polymers made by certain marine alga. Commercial agar is often a mixture of molecules having some what differing chemical structures. Agar from different algae have different structures, and, therefore, have differing properties. While some marine bacteria can use agar as food and form pits when spread on a plate of agar, most bacteria can't damage agar.

Agar sold as "Bacteriological Agar" usually gels at 38-39C. "South American White" is mainly used in food manufacture and gels about 49C but is prefered at Indiana Biolab because it gels faster in hot classrooms and produces stronger gels. Some agars gel around 30C and are used when 40 or 45C would be harmful. For example, one often wants to mix bacteria or antiserum with melted agar and then pour this into individual wells of well plates, test tubes, or petri plants for special assays.

Since agar contains many different molecules and impurities, some companies fractionate agar and sell special products for use in foods and laboratories. Alginic acid is used in ice cream as a smoothener. In the presence of calcium ions alginic acid forms gels. Therefore, if you mix bacteria with alginic acid and drip the suspension into calcium ion solution, you obtain soft spheres containing trapped bacteria. You can then pass food thru the bed of spheres and food would diffuse into the spheres and the products will difusse out. If the soft sphere pack tightly, you can pass the flow upward in the "fluidized bed".

Agarose is another product obtained by fractionation of crude agar. Agarose is used to form special quality gels for electrophoresis of large molecules such as DNA. Ordinary agar and chemical gels have networks of polymer threads too dense to permit passage of the huge DNA molecules.

Noble Agar is obtained by cutting an agar gel into cubes and passing distilled water upward thru the tiny cubes to wash out salts and other small molecules which diffuse out of the agar.

Manufacture of Agar

Agar comes from huge alga plants known as kelps. These are harvested from the sea by floating devices or ships. The kelps are cooked with dilute acid and filtered, the acidic liquid is neutralized and cooled so that an agar gel is obtained. This is cut into blocks and frozen. When the blocks are thawed the agar is in shreds and the water drains away. In the old days, these dried shreds were sold as agar-agar and used in cooking. Professor ____ got the idea of using gelatin gelled media as a solid for bacteriological work, but it did not work very well because when incubated at moderate temperatures, the gelatin melted. Frau ____ working in his lab knew about agar-agar used in cooking and suggested he try using agar.

You can try this with with the agar in a petri plate or bottle and you will

Agar and other Gelling Agents

Notes on Choice of Ingredients

Scientists use high quality distilled water when doing exacting nutrition work, but tap water gives good results in the classroom. However, tapwater may give a cloudy preciptate in some instances. I use spring water, but tapwater is just as good. If it has lots of chlorine or junk, you may let it stand a few days before use--especially if you are growing animals in the water.

MgSO4.7 H2O is Epson Salt.

NaCl is table salt, but use normal salt not Morton's which contains insoluble aluminum salts which should be harmless but will give cloudy medium. I urge you to use only salt that dissolves to a clear solution. Afterall, aluminum is the active ingredient in underarm deodorants.

If you don't have all the minerals such as Mg, Ca, Mn, etc. stir soil into spring (or tap) water and let the mud settle a couple days and use the clear supernatant water.

If the chemical you have differs in water of hydrate. Solve a proportion to get the correct amount or just ignore the difference. You can't ignore the difference if you are preparing pH buffers by weight.

-------To be continued ------ in Dec or Jan


Revision #1 - 1999 FEB 5
Written by Harold Eddleman, Ph. D., President, Indiana Biolab, 14045 Huff St., Palmyra IN 47164
Please help by sending me an e-message containing your ideas for improvement. indbio@disknet.com

ubject: Re: Carageenan Date: 5 Feb 99 16:42:04 -0500 From: indbio@disknet.com To: Indbio

BacteriaStudyList - http://www.disknet.com/indiana_biolab/b.htm

bobwill@gsc.weblinq.com wrote: > > > Can I use solidified carageenan instead of agar-agar powder for > making solid media? This is commonly sold in our stores for > making gelatin. I also have knox powder available - but would > think that it is nutritionally quite different. Or is stiffness > of the final gel really all that matters? > Bob Williams > Stiffness of the gel, no inhibition of bacteria, and resistance to microbial digestion is all that matters. Carageenan from Irish Moss, Chondrus crispus, a marine red algae, and alginates from the California Coast, like agar from Japan are used as thickeners. That reference said no more. One would have to try Carageenan I guess. I have used sodium alginate as an adjuvant when injecting rabbits and I have seen it get fairly firm in Ca++ media. Sodium solutions of alginate are completely liquid. We use adjuvants with antigens to make the antigens more immunologenic. Therefore, carageenan and agar might make stronger gels when Ca++ is present. There is an expensive gum which can replace agar and produces very clear gels. I think it may be from a bacterium, but I have forgotten its name. After 25 years, it seems to be seldom used. I hope to cover all such alternatives on page b041.htm (URL below) but that is in the future. If you try carageenan, please send the results to this mail list. I can't find my copy of Merck Index which should have more information. Since I have never seen carageenan mentioned as a gel for microbiology, I assume it does not get thick. Perhaps a restaurant would know a source for food grade agar. More agar is used for cooking than for microbiological medium. Pectin makes jelly firm, but I have not tried it for gelling medium. -- Harold Eddleman Ph.D. Microbiologist. mailto:indbio@disknet.com Location: Palmyra IN USA; 36 kilometers west of Louisville, Kentucky http://www.disknet.com/indiana_biolab