Keywords: tryptone,peptone,agar,peptides,amino acids,sugars,carbohydrates,yeast extract,digests,tryptic digest,viruses,phages,bacteriophages,bacteria,yeast,fungi,microbiology,bile salts, If this page gets too long, let 41a be Agar page.

Ingredients used in Microbiological Media

The Manufacture and Purpose of These Ingredients

put a list of jumps to agar, petontes etc

All formulas refer to one liter. Thus "10 grams of agar" means 10 grams per liter.

The early microbiologists used the foods available for homes and restaurants as foods for their bacteria. They supplemented these with blood, fruit juices and other mateials found in the environment where they found the bacteria, yeasts, and fungi they wished to study. When viruses were discovered, living cells became "media".

Any microbiological medium and environment must provide everything the species under cultivation requires. These are oxygen (or other electron acceptor), water, nitrogen source, carbon source, energy source, minerals, vitamins, and trace biochemicals. Many bacteria can use glucose as energy and carbon sources. Some bacteria can use light as energy source and others can oxidize sulfur as energy source. As nitrogen source, most bacteria require protein, peptides, or amino acids, but many can use ammonia, nitrates or nitrogen molecules. Bacteria capable of fermentation can oxidize one molecule and use another as electron acceptor; thus, they are not able to obtain as much energy as would be available were the energy oxidized completely to carbon dioxide and water.

Some organisms can make almost everything them need, but others require a long list of vitamins and required factors. Some metabolize their food down to carbon dioxide and water, others have very limited in disimilatory metabolism and leave their food practically untouched. Bacteria used in food processing typically are able to use only a few of the components of the food they used to process. Knowledge of what a bacterium needs helps you design a medium for growing it.

Water

All bacteria require some amount of water. Some can live in evaporating salt water or moist pastes, but completely dry foods such as a bag of sugar are free from bacterial attack. Dried meat or fruit are free from attack by most bacteria, but those foods may be attacked when the humidity increases. However, attack of damp dried foods is usually by fungi rather than bacteria.

While scientists use distilled water in vitamin studies, in instances where hard water might affect pH of the medium, or in situations where the water might contain interferring substances, many bacteriologists prefer tap water for making medium to save money or supply trace minerals.

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Water is the main ingredient in any medium. I usually prefer tap water because it contains some minerals and is cheap. Advanced experiments may require distilled water when you are testing exactly which minerals are reguired by an organism. Since most commercial media contain calcium and other ingredients found in tapwater, it is seldom neccessary to buy distilled water. When growing protozoa or animal cells, the chlorine in tap water may kill the animals. If so, you can let the tapwater age open to the air so the chlorine leaves, or do as many professionals do, use water from a well and call it spring water as bottled water companies do. I do use distilled water to prepare all my reagent solutions such as phosphate solutions. Otherwise a precipitate LINK/CHEM may result. As you experiment and read you will develop your own ideas of when you will use distilled water.

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

While a few bacteria from the ocean can

Carbon Sources

Glucose is the ideal carbon source for many bacteria. Many can make everything they need from glucose and a nitrogen source. Some bacteria can't use sugars and need carbon dioxide or carbonate or bicarbonate. Bacteria found in the cow's rumen do better on organic acids because glucose is rare in that environment and they have developed mechanisms to fit the environment.

Energy Sources

Most bacteria, yeasts, and fungi do best when glucose is provided as the primary energy source because they may not be able to digest other carbohydrates. All bacteria found on and in animals and most of those found on plants and soils do well on glucose as the sole carbon and energy source. However, bacteria which use light or oxidation of minerals for their energy source may do better in relatively organic-free media. In water we find many little studied bacteria which do better in media containing only 0.5 gram of peptone and 0.5 gram of starch.

Nitrogen Sources

Bacteria require nitrogen to make proteins and nucleic acids. Only a few genera of bacteria can use free molecular nitrogen from the air. Others require fixed nitrogen in the organic or inorganic form. Some can use nitrate or nitrite salts, but most require amino acids, peptides, peptones, or proteins.

Peptones

Peptones are the most widely used source of nitrogen in microbial media. Some are made by cooking milk or meat products in acid, but most are made by incubating milk or meat with trypsin, pepsin, or other proteolytic enzymes to digest the protein to a mixture of amino acids, peptides, and polypeptides. Many microbes, called proteolytic, can digest proteins, but most can't. The choice of peptone is sometimes of importance. For example, for a positive hydrogen sulfide test, the medium must contain sulfur-containing amino acids.

Table of Equivalent Peptones

Source Common Use DIFCO BBL GIBCO Sheffield OXOID
Casein acid
hydrolysis
  Casamino Acids Acidicase Peptone 5   L41
Same extra
soluble
  Paptamin   Peptone 20    
Casein acid
hydrolysis
  Pepticase   Peptone 50    
Lactalbumin
enzymic digest
  Lactaltone Lactalysate Peptone
60
  Lactalbumin
hydrolsate
Meat
enzymic digest
  Proteose
Peptone
Thiotone Peptone 100   Peptone P
Soymeal
enzymic digest
  Soytone Phytone Peptone 110   Soya Peptone
Casein
trypsin digest
  Tryptone
Casitone
Trypticase Peptone
140
  Tryptone
Animal
casein
polypeptone
  Proteose Peptone #2
Proteose Peptone#3
Tryptose
Poly-
peptone
Peptone
180
  Tryplose
Gelatin pan-
creatic digest
  Peptone Gelysate Peptone
190
  Peptone
Yeast casein
polypeptone
  Bitone Biosate Peptone
220
   

Tryptone is a tryptic digest of casein. Casein is a complex of proteins found in milk. Trypsin is an important digestive enzyme produced by the pancreas. It cleaves proteins into shorter pieces called peptones. Tryptic digests of dried milk are called tryptones. Tryptones are the best choice for bacteria media because they are used by most bacteria from animals and supply nitrogen, energy, and carbon. Tryptone water (tryptone + water) will support the growth of many species of bacteria. Tryptones are not pure substances. They are a mixture of left over trypsin, salts, and vitamins, amino acids, peptides, peptones (longer than peptides), and polypeptides (longer than peptones). Tryptones are used in foods for flavoring and nutrition, in electroplating for smooth plating, and in media for microbes.

Not all tryptones are identical. Many manufactuers make several grades of tryptones. Some dissolve in water to give a totally transparent yellowish solution free of any turbity or sediment. These expensive tryptones are often used in diagnostic media when super clarity is needed. Some tryptones are much cheaper and form quite turbid, darker colored solutions with some sediment and often support better growth of microbes. These are commonly used in manufacteuring antibiotics and other drugs.

Phytone is a tryptic digest of soybean proteins. Some bacteria isolated from plants may grow a little better on phytones than on tryptones.

Peptones are enzymic digests of other proteins often meat scraps.

Minerals

Oxygen

Nitrates as honorary oxygen.

Vitamins

Yeast Extract

Yeast extract is used in most media for microbes from plants and animals. Some of these microbes require vitamins and other growth factors from their plant or animal hosts and yeast extract is rich in vitamins, minerals, and digested nucleic acids. However, yeast extract is very hydroscopic and is difficult to keep dry in the classroom and is sometimes difficult for students to obtain. See YIB on page b030 for a method for making your own yeast extract.

Commercial preparation of yeast extract begins with a tanker load of waste yeast from a brewery. The yeast is dumped to huge stainless steel tanks and it is incubated about 50C. At that temperature, health of the cell is ruined and the internal partions degrade and enzymes begin at the cell from within (autolysis = self lysis). This process of self-lysis occurs in all cells at death. After a day or so, stirring stops, and the liquid is separated from the solids (yeast cell walls and bits of hops and grain). The wonderfully flavored yeast extract is then used for icecream syrup, candy, breakfast foods, gravy to add flavor and vitamins, sugars, and nucleic acids and other nutrients. A tiny part of the production is used in bacterial medium to replace some or all of the meat extract. Yeast extract is high in B-complex vitamins.

It is easy to make. If neccesary you can grow your own yeast, but you can usually buy compressed yeast at any bakery. I usually comes in one pound foil wrapped bricks.

Yeast Autolysate is often added to conventional media to supply growth factors for fastidious microbes.

Yeast Infusion Broth is prepared by

YIB is excellent for cultivation of lactic acid bacteria. For even better growth of the bacteria, do not remove the spent baker's yeast. Filter and use egg only if needed for clarity.

Agar and other Gelling Agents

now list each source carbon sources and list the ingrediens under it.

ammonium salsts, nitrates, proteinsk, digests of milk, plant proteins,

In addition, certain aspects stresses of culture might require additonal substance not needed for basic surviva

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 #2 - 1999 January 2
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