We have a new name and a new site! >>

Microbial culture media

Composition, preparation and preservation of nutritional media for culturing bacteria and fungi

(for our lab students)

The following are the microbial growth media commonly used in our lab for bacterial and fungal isolation, purification, preservation and maintenance.

General Purpose Media

Nutrient Agar (NA Medium)

Nutrient agar is a general purpose non-selective medium and supports growth of a wide variety of bacteria. NA is commonly used for bacterial isolation and maintenance.

Composition of Nutrient Agar

Peptone: 0.5 %

Beef extract or yeast extract: 0.3 %

NaCl: 0.5 %

Agar: 1.5 %

Adjust pH to 7.0

Lysogeny Broth (LB Medium)

(Also known as Luria broth, Lennox broth, or Luria–Bertani medium)

LB is also a general purpose non-selective media that supports growth of a large variety of non-fastidious bacteria. It is one of the most frequently used media for bacterial isolation and culture.

Composition of lysogeny broth

Peptone: 1.0 %

Beef extract or yeast extract: 0.5 %

NaCl: 1.0 %

Agar: 1.5 %

Adjust pH to 7.0

Trypticase Soy Agar (TSA Medium)

Trypticase Soy Agar (TSA) is another general purpose non-selective media that provides nutrients to support growth of a wide variety of bacteria.

Composition of trypticase soy agar

Tryptone: 1.5 %

Soytone: 0.5 %

NaCl: 0.5 %

Agar: 1.5 %

Reasoner's 2A (R2A) Agar Medium

R2A medium is frequently used for the isolation of slow-growing bacteria. It is also a non-selective medium but many slow-growing species that do not appear to be isolated using the above nutritionally rich media such as LB or TSA, could be isolated using R2A.

Composition of R2A agar

Peptone: 0.05 %

Casamino acids: 0.05 %

Yeast extract: 0.05 %

Dextrose: 0.05%

Soluble starch: 0.05%

Dipotassium phosphate: 0.03 %

Magnesium sulfate: 0.005 %

Sodium pyruvate: 0.03 %

Agar: 1.5 %

Media for Alkaliphilic Microbes

Alkaline Nutrient Agar (ANA Medium)

Alkaline nutrient agar media is used for the enrichment, isolation and culture of alkaliphilic and alkalitolerant bacteria.

Composition of alkaline nutrient agar

Peptone: 0.5 %

Beef extract or yeast extract: 0.3 %

Agar: 1.5 %

After autoclaving, adjust to desired alkaline pH with sterile 10% Na2CO3 solution or with the following Na-sesquicarbonate solution.

Na-sesquicarbonate solution:

NaHCO3: 0.42 %

Na2CO3 anhydrous: 0.53 %

Horikoshi-I Medium

It is another common media for the enrichment, isolation and culture of alkaliphilic bacteria.

Composition of Horikoshi-I

Glucose: 1%

Peptone: 0.5 %

Yeast extract: 0.5 %

KH2PO4: 0.1 %

MgSO4 . 7H2O: 0.02%

Agar: 2.0 %

After autoclaving, adjust to desired alkaline pH with sterile 10% Na2CO3 solution.

Horikoshi-II Medium

For the enrichment, isolation and growth of alkaliphilic bacteria.

Composition of Horikoshi-II

Replace glucose with soluble starch in Horikoshi-I media described above.

Media for Lactic Acid Bacteria

De Man, Rogosa and Sharpe (MRS) agar medium

MRS is an enriched selective medium used for the isolation, cultivation and enumeration of lactic acid bacteria.

Composition of MRS agar

Dextrose: 2 %

Peptone: 1 %

Beef extract: 1 %

Yeast extract: 0.5 %

Dipotassium hydrogen phosphate: 0.2 %

Sodium acetate 3H2O: 0.5%

Triammonium citrate: 0.2 %

Magnesium sulphate: 7H2O: 0.01 %

Manganese sulphate: 4H2O: 0.005 %

Tween-80: 0.1 %

Agar: 1-1.5 %

pH 6.25

M17 Agar Medium

M17 is also used for the cultivation and enumeration of lactic acid bacteria.

Composition of M17 agar

Tryptone: 0.5 %

Soya peptone: 0.5 %

Beef extract: 0.5 %

Yeast extract: 0.25 %

Ascorbic acid: 0.05 %

Magnesium sulphate: 0.025 %

Di-sodium-beta-glycerophosphate: 0.19 %

Agar: 1.1 %

pH 6.9

Media for Antibiotic Susceptibility Testing

Mueller Hinton Agar (MHA Medium)

Mueller Hinton Agar is most commonly used for antibiotic susceptibility testing.

Composition of Mueller Hinton Agar

Beef extract: 0.2 %

Acid hydrolysate of casein: 1.75 %

Starch: 0.15 %

Agar: 0.17 %

Fungal growth media

Sabouraud's Dextrose Agar (SDA) Medium

Sabouraud's dextrose agar (SDA) is a common fungal culture medium.

Composition of Sabouraud's Dextrose Agar

Dextrose: 4 %

Peptone: 1 %

Agar: 2 %

pH 5.6

Preparation of the Culture Media

To prepare a 1-liter media, mix and dissolve appropriate amount of all ingredients in around 800 mL of distilled water. Check and adjust pH if necessary with an appropriate acid or alkali solution. For solid or semi-solid media, add appropriate amount of agar (usually 1 to 2%). Finally add more water to make the final volume 1 liter. Sterilize by steam autoclave at 121 °C and 15 psi for 15 min.

Storage of Culture Media

Most of the prepared media can be stored for a few months at 2-8 °C, preferably away from light. Many liquid broths can also be stored at room temperature (25 °C) for weeks to months. It is, however, better to use freshly prepared media if possible. Agar plates should be stored in inverted position.

Google Sites

Report abuse