Microbial Culture Media

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 %

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. 

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 and Preservation of Glycerol Stock

We prepare glycerol stocks of our cultures for long-term preservation. Here are the steps we use for preparing and preserving glycerol stocks:

1. Growing the culture:

   • Grow the microorganism of interest in an appropriate growth medium until it reaches the desired growth phase (we usually prefer late log phase cultures).

2. Preparing the glycerol stock:

   • In a sterile cryogenic vial or microfuge tube, combine an equal volume of the microbial culture and sterile 50% glycerol solution. For example, mix 500 μL of culture with 500 μL of 50% glycerol so that the final glycerol concentration reaches 25%. We usually use a final glycerol concentration of ~20%

   • Gently mix the contents by inverting the tube a few times to ensure thorough mixing.

3. Freezing the glycerol stock:

   • Immediately place the glycerol stock in a -80°C freezer, or if unavailable  in a .-40°C freezer

4. Storage and retrieval:

   • To retrieve a culture, remove the glycerol stock from the freezer, thaw it quickly at room temperature or placing in ice and then streak a small amount of the thawed culture onto an appropriate solid growth medium using a sterile tooth pick or inoculation loop. 

It is recommended to prepare multiple glycerol stocks for each culture to ensure backup samples in case of freezer failure or other issues.