Media for Bacteria Stock Cultures

This list of simple media is all you need to maintain a culture collection.

At the peak, in 1985, my collection of microbial cultures had 2000 strains of 250 species. These are the only media I used for stock cultures. ** marks the media I use for many species, * marks media I use for a few species. I needed the other media only for rare species. Notice the abbreviation listed before each medium. I mark that abbreviation on each tube of the medium and use it on all my web pages. Notice I use spring water (well water or tap water; not distilled water). There is seldom any reason to purchase distilled water. I regret that most of these media use professional ingredients. The high school student can try to improvise see B041 for descriptions of tryptone, yeast extract, etc and ideas for substitutes. Besides being used for stock cultures, many of these media are excellent for classroom experiments. TGY is an excellent medium; most students and teachers can get by using it alone. If you have questions contact me at the e-address below. I autoclave all these media for 15 minutes at 15 pounds pressure.

AM * Acetobacter Medium

Autoclave the liver in the water for 15 minutes 15 pounds. Remove the liver particles by filtering thru a handkerchief. To save work, you may leave the liver particles in the medium. Then add the other ingredients. Use for Acetobacter, Gluconobacter and, perhaps, other fastidious species. To avoid settling of CaCO3, stir while filling tubes. Use as slants.

B6 Medium for Thiobacillus

To be completed, I rarely used it.

CS Cottonseed medium

Gives very high counts of fungi, and Streptomyces. Since the pH will be about 4.5, it selects fungi. If you do not have cottonseed, grinding peanut or bean to a powder. You try using peanut butter but it has added oil and cottonseed meal has the oil removed. Cottonseed or corn steep liquor are often used in commercial media for growing bacteria and fungi to produce antibiotics because they are cheap and often produce high yields of antibiotics.

DLM * Deep Liver Medium

For clear medium, filter through a handkerchief after cooking but before adding the other ingredients. Most bacteria do better when the particles are not removed. Excellent for protelytic anaerobes, lactic acid bacteria and many others.

E E. coli Stock Culture Medium

Use as stabs to maintain E. coli cultures at room temperature. Supports many other species. Notice the glucose is used at a low level. That helps reduce acid and other harmful products.

HAL * Halobacterium Medium

PART A

PART B

Autoclave Part A and Part B separately. Place 1.5 mL of part B in culture tube. Autoclave and hold near boiling point of water so the agar does not gel. Add 3.5 mL of very hot Part A, cap, mix, and cool as slants. For extreme halophiles. Contaminants probably not able to grow in this high salt. Try this medium when you are trying to isolate bacteria from salt lakes that have salt crystallizing out, such as on the Bonneville Salt Flats or some of the nearly dry lakes in CA and NV.

LP ** Liver Particles

Add water to the ground liver to make a total volume of 1000 mL. Simmer for 30 minutes, and add the other ingredients and add more water, if needed, to make final volume of 1000 mL. Fill tubes 7 to 10 cm deep and autoclave. Excellent for protelytic anaerobes and lactic acid bacteria.

M Mg++ Casitone

Or use 0.01 M phosphate buffer (pH 7.6) in place of the water. Used for many Myxobacteria. Used as slants.

MPSS Modified Peptone Succinate Salts

The reason for using the free acid is to lower the pH, but if you plan to use HCl to adjust the pH then you can use any succinate salt. Adjust pH to 7.0 before adding the agar. Transfer Spirillum volutans weekly and hold at 30 deg C. Spirillum volutans is very difficult to keep alive; transfer twice per week until you are sure it will survive longer periods between transfers. Aquaspirillum do not require this special medium and can be held on TGY, nutrient agar, etc. for 30 days at room temperature. Use as deeps (deep stabs).

R Rumen Bacteria Medium

This medium supports a variety of rumen bacteria. Since all important bacteria of the rumen are strict anaerobes, the headspace of the tube must be filled with carbon dioxide. The medium must be colorless. If the resazurin turns pink oxygen is present, and strict anaerobes will not grow and they may be killed by the oxygen. Autoclave 15 minutes at 15 pounds. Omitting any of the above sugars limits the growth of some species. Usually used as deeps.

Mineral #1 = 6 g K2HPO4 per liter of spring water.

Mineral # 2:

NA Nutrient Agar

Supports the growth of many common bacteria from the intestines and many other species. Nutrient agar is the most widely used medium in highschools, but not in my laboratory. TGY supports the growth of many more species. If your bacterium requires NaCl just add NaCl to TGY. Nutrient Agar is specified in many texts because it has been used since the 1880's when nothing better was available. Nutrient agar is a very low quality medium.

NB Nutrient Broth

Nutrient Broth is Nutrient Agar without the agar. In my laboratory, I use NB only when I have a special reason not to use a tryptone broth.

NFM * Nitrogen Free Medium

    Mo stock solution = 0.1 mg Mo/ml
    Fe stock solution  = 1.0 mg Fe/ml
Combine the ingredients, heat to dissolve, dispense into tubes, autoclave. Sucrose or other sugars may be used, but growth of Azotobacter species varies on other sugars. This medium is mainly use for culturing Azotobacter species as slants. .

PDA ** Potato Dextrose Agar

Autoclave 200 grams of sliced potato in 1000 mL of tap water. Filter through a hankerchief. Make potato salad for the family out of the potato residue. To the turbid filtrate add 20 grams of glucose and 10 grams of agar. Add water to make 1 liter total volume. Usually used as slants. PDA slants give good growth of most fungi, Agrobacterium, Nocardia, and many others. This is a cheap simple medium which I highly recommend for student projects. Omit the agar for a useful liquid medium.

PYE Pate's Yeast Extract

For Cytophaga and many other genera of similar nutritional needs. These species survive longer on PYE than on TGY. Many bacteria isolated from streams grow better on very dilute media. On dilute media, these water bacteria may outgrow the common intestinal bacteria found in fresh and marine waters. Notice this medium contains no carbohydrates which intestinal organisms would convert to acids which might retard growth of some species. Instead this medium contains a small amount of acetate (vinegar) which many common bacteria can't use.

RG Rabbit Dung Agar

Place 4 pellets of rabbit dung in tube and add enough 1% agar so that when slanted some pellets extend above the agar but are held in place by the agar. Autoclave and slant. Use antibiotic-free rabbit dung when available. Inoculate directly onto the pellets. This medium supports excellent growth of Myxobacteria. This medium is also good for many fungi; especially Coprinus and others which grow on manure. For such fungi, you may use other types of dung such as deer, horse, cow, elephant, and other herbivores, but not dung of carnivores. If you have no agar, just autoclave dung pellets after adding a little water.

RZB * Rhizobium Medium

Make soil extract by autoclaving 400 grams of rich garden soil in 1 or 2 liters of spring water for one hour at 15 pounds pressure. Allow to cool slowly to avoid foaming out of container. Allow to stand a few days (need not be sterile) and decant (pour off) the clear liquid. If you wish you may autoclave again for 15 minues at fifteen pounds and allow to stand for a few weeks to obtain super clear soil extract.
    Use RZB as slants. It gives good growth of Rhizobium species which are found in the nodules on the roots of legumes. Rhizobium species grow well on mannitol which is a rare sugar alcohol.

SG Silica Gel

Many of my friends have had good success preserving cultures on silica gel, I have not tried it and can't give details. I only mention this method as some readers may want to look into this method. This is not medium for growing organisms. A drop or so of a liquid culture is added to the dry gel beads and they sometimes remain viable for years, but I have had problems (no living cells).

S Soil

This method has been used by many scientists to preserve cultures in dry form. None of my cultures survived. I assume my cultures were not well sporulated. I also had problems with mold contamination while waiting for the tubes to dry out. Even when I used paper towels over the loose screw caps.

SPA ** Spizizen Potato Agar - A special medium for producing Bacillus spores.

Autoclave 200 grams sliced potato in 1 liter of tap or spring water for 15 minutes at 15 pounds pressure. Filter through a hankerchief. To this potato water add 10 grams agar, 5 mg manganese sulfate, and 1 ml of 1.0 Molar magnesium sulfate. Heat to dissolve the agar. add water to make volume of 1 liter. Dispense as slants. Autoclve 15 minutes at 15 pounds pressure. This medium produces heavy crops of spores for all common species of Bacillus. However, certain Bacillus species such as Bacillus popilliae, pathogen of Japanese Beetle, require complex medium and will not sporulate on this medium. Bacillus produces spores when it is starving to death. This medium contains no glucose and the cells seem to think they are facing starvation. If you have no manganese sulfate try using soil extract to replace the spring water.

SSW * Synthetic Seawater Medium

This medium is excellent for growing most bacteria from the oceans. The glycerol helps bioluminescent bacteria glow as brightly as they are capable. This medium gives bright glow from Vibrio (Photobacterium) phosphoreum provided oxygen is present. Add 10 grams of agar if slants are desired. I maintain all my marine bacteria on slants.

There are simpler ways to make medium. If you live near the ocean and can get full strength seawater (not water from a river), you can use that water and add yeast extract, glycerol and peptone. Or you can get dry seasalt from a pet store and use that for the ocean water. If you don't have peptone, you could use meat flavoring such as bouillon cube or meat broth. You could try some yeast if you do not have yeast extract. I have not tried these substitutes.

WR * Water on Rocks

It is not necessary to autoclave this unless you are making stock cultures. Actually, steaming on 3 successive days may be better as autoclaving 15 minutes @ 15 pounds pressure will melt the sulfur into little balls, but that is not serious as the Thiobacillus can still use it. Adding a chip of limestone or marble to each tube will neutralize the sulfuric acid produced by the bacteria as they oxidize the sulfur. My Thiobacillus survived a year on this medium at room temperature.

TGY ** Tryptone Glucose Yeast

This is called Plate Count Agar by many people. It supports more species of bacteria than any other medium. If in doubt try TGY. Bacterial pigments which are pale on some media are usually much brighter on TGY in air at room temperature. Used as slants and stabs. I often add 1 gram of powdered CaCO3 to counteract the acid generated by many bacteria from the glucose. With the carbonate, many stock cultures last years instead of months. If you don't have calcium carbonate powder try ground agriculural limestone or any limestone or blackboard chalk. To avoid settling of CaCO3, stir while filling tubes. The low level of glucose helps reduce acid production and cultures live longer.
    This is my most important medium. This is the only medium a teacher or student needs. You can do lots of interesting work using only this medium. Some bacteria do better on a more dilute medium such as PYE. Some bacteria would do better if the glucose is left out or reduced to 1 to 5 grams, I have not tried that and have no experiences to relate.

YE * Yeast Extract

Excellent growth of Streptomyces, but antibiotics are not produced on YE. Allow cultures to grow a week or two at room temperature and then store in the refrigerator. If you do not allow this time for sporulation, some cultures may be lost. Used as slants.


YIB   Yeast Infusion Broth

Yeast extract is an extremely hydroscopic powder which few schools have in stock because of the poor shelf life. Using Yeast Infusion is a suitable alternative. Yeast infusion supplies vitamins, carbohydrates, peptides, and nucleic acid fragments--everything fastidious bacteria need. Backeries often sell yeast in compressed bricks like cream cheese. Health food stores may have it.

Suspend one pound of fresh baker's yeast in two liters of water. Autoclave for one hour. You may use this crude yeast infusion water with excellent results. The cell walls will probably give stock cultures longer life. If clear broths and agars are desired, follow these additional steps:

YIB is excellent for cultivation of lactic acid bacteria, related bacteria and many other species. If you have no fresh compressed bakers yeast, grow your own in corn syrup water. Unless you have a centrifuge, you will not be able to separate the yeast cells from the medium they grew in. The main disadvantge is waste products from the yeast growth but they may not cause much problem and you can dilute the yeast infusion for use. I often add 1% powdered calcium carbonate or a few chips of limestone per tube to neutralize acidity produced by some bacteria, especially when they are groing in high sugar medium.

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 invertbrates in the water.

--- Some useful info for non-chemists -----

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. After all, 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.

You may travel the web to ATCC, NRRL, other media sites. Search for +bacteria +media. For more media formulas. However, the above are more than most students and teachers need.

Revised: 99 Jan 9


Written by Harold Eddleman, Ph. D., President, Indiana Biolab, 14045 Huff St., Palmyra IN 47164

Suggestions, corrections, and comments are appreciated: Contact Harold Eddleman (indbio@disknet.com).

Example of a diagnostic Medium

This is a diagram of a Triple Sugar Iron slant (TSI) which was inoculated with Proteus vulgaris. This medium is always used as slant. TSI medium contains cystine which is a sulfur containing amino acid andTSI slantthree sugars: glucose, lactose, and sucrose. The medium also contains 1 gram of glucose, 10 grams of lactose, and 10 grams of sucrose. This medium is used to determine whether intestinal bacteria are able to split lactose or sucrose. If the tube is red from top to bottom, it means the medium is alkaline because the bacterium forms ammonia froms ammonia from proteins. The black in the bottom indicates hydrogen sulfide was formed and reacted with the iron.