Introduction to Microbial Media

Microbiological studies require the availability of different species of bacteria, yeast or viruses in the laboratory. Large scale processes such as fermentation, protein and vaccine production require large numbers of bacteria in physiologically active state. A suitable nutrient medium that provides appropriate biochemical environment and maintains all the characteristics of microbes is therefore required for various applications.

Any microbial medium should include sources of nutrition, energy, essential minerals, buffering agents and pH indicators. Sometimes they also include selective agents and solidifying agents. Obligate parasites such as bacteriophages may be cultivated in a nutrient medium that also contains host bacteria.

The following are the essential components of a typical microbial medium:

  • Nutrition sources – sources of proteins, vitamins, mineral and carbohydrates; mostly mixture of peptone and meat extract
  • Energy sources – sources of carbohydrates; mostly glucose
  • Essential minerals – sources of micro and macro minerals; mostly present in peptone and meat extract
  • Buffering agents – agents that maintain optimum pH; mostly specific amino acids, phosphates, citrates and zwitterions
  • pH indicators – agents that indicate variation in pH of the medium by change in color; mostly phenol red
  • Selective agents – agents that allow the growth of only specific bacteria; antibiotics, tellurites, azides and bile salts are examples
  • Solidifying agents – agents that maintain the medium in solid or semi-solid state, if required; widely used agent is agar though gelatin is sometimes used
  • Specific substances such as growth factors, enzymes may be incorporated into the medium for specific bacteria

Types of microbial media

  • Simple or basal media: Include nutrient broth and peptone water; used routinely to isolate and culture a variety of bacteria in a molecular biology research laboratory
  • Complex media: Contain mixture of a variety of nutrients; the exact composition of amino acid source is not defined. Examples include chocolate agar, MacConkey agar, Lowenstein-Jensen medium
  • Defined media: Contain known and defined sources of carbon and nitrogen as required by certain bacteria
  • Special media: Contain specific substances that encourage growth of fastidious bacteria, discourage the growth of unwanted bacteria or contain indicators that distinguish different types of bacteria

Table 1: Types of microbial media
 

Name Application
Popular Products
Bacterial Media
LB media Lysogeny Broth or LB is the most popular and commonly used medium to culture E. coli. It was first created by Bertani G to culture Shigella indicator strain. LB is a rich medium containing peptone, yeast extract, NaCl and agar (for solid medium). Depending on the concentration of NaCl, the LB medium may be classified as follows: LB - Miller, LB - Lennox and LB - low salt. LB media are suitable for non-selective cultivation of E. coli strains for cloning, production of DNA, plasmid DNA and recombinant proteins. It is also suitable for selective cultivation when appropriate antibiotics are added. Plates containing sterile LB agar with or without antibiotics are also available for selective culture of antibiotic-resistant strains of E. coli. Powder: L3022 (LENNOX), L3522 (MILLER), 71753-M (MILLER), L3397 (LURIA LOW SALT)
Powder with Agar: L2897 (LENNOX), L3147 (MILLER), 71752-M (MILLER)
EZMIX Powder: L7658 (LENNOX)
Liquid: L2542 (MILLER)
Tablet: L7275 (LENNOX)
Tablet with Agar: L7025 (LENNOX)
Agar plate: L5542, L5667 (Ampicillin), L0543 (Kanamycin), L0168 (Ampicillin), L0418 (Carbenicillin)
Minimal salts (M9) M9 medium contains essential salts and nitrogen. It is suitable for non-selective cultivation of E. coli strains for cloning, production of DNA, plasmid DNA and recombinant proteins. It is also suitable for selective cultivation when appropriate antibiotics are added. M9956, M6030
Terrific Broth - modified Terrific broth is a nutrient rich medium that results in higher yield of bacteria in less time.Terrific broth (modified) is suitable for non-selective cultivation of E. coli strains for cloning and high yield production of DNA, plasmid DNA and recombinant proteins. It is also suitable for selective cultivation when appropriate antibiotics are added. T5574, T9179, 71491
Hanahan’s Broth (SOB Medium) SOB medium has higher peptone, amino acids and peptides than LB medium. Hanahan’s Broth (SOB medium) enables the preparation of high efficiency competent cells.
SOC Medium SOB medium has higher peptone, amino acids and peptides than LB medium. It enables the preparation of high efficiency competent cells. SOC medium is a modified SOB medium with added glucose, a rich medium used primarily in the recovery step of E. coli competent cell transformations. H8032, S1797
2X YT medium 2X YT medium contains double the concentration of yeast extract compared to typical LB medium. It is useful for the growth and maintenance of high density of cells for a longer growth period. It is suitable for non-selective cultivation of M13 bacteriophage and E. coli strains for cloning, production of DNA, plasmid DNA and recombinant proteins. It is also suitable for selective cultivation when appropriate antibiotics are added. Y1003 (Liquid), Y2377(Powder), Y2627 (EZMIX)
NZCYM Broth NZCYM Broth is nutrient rich broth with amino acids, peptides, vitamins and low salt concentration. It  is suitable is suitable for non-selective cultivation of lambda bacteriophage and E. coli strains for cloning, production of DNA, plasmid DNA and recombinant proteins. It is also suitable for selective cultivation when appropriate antibiotics are added. N3643 (Powder), N6905 (EZMIX)
Yeast Media
Yeast Nitrogen Base with or without amino acids Yeast Nitrogen Base is nutrient-rich medium with sources of nitrogen, vitamins, trace elements and salts. It acts as the basic nutrient medium for most fermentation reactions involving yeast cultures. Yeast Nitrogen Base with and without added amino acids for the cultivation of yeast and for classification based on amino acid and carbon requirements, respectively. L0626, Y1625, Y1250, YEAST1, Y1771, Y2021
Phage Media
NZ Amine®Broth NZ Amine® Broth is nutrient rich nutrient-rich medium with amino acids, peptides, vitamins and low salt concentration.NZ Amine® Broth is suitable for the growth of lambda bacteriophage. N3518

FAQs

What are the differences among the Luria, Lennox and Miller LB formulations?

LB, (originally termed lysogeny broth) was initially composed of tryptone, yeast extract, NaCl and glucose.Soon after, the glucose was omitted (Miller's version), and later the NaCl content lowered by half (Lennox's version).For some applications, even lower salt is required (Luria's low salt version).

What is the difference among the LB - Miller products?

LB - Miller is available in many types to suit your needs.The different product formats include powder and liquid form.The powder form is also available with agar for easy LB-agar plate preparation.L2542 (LB Miller liquid)L3522 (LB Miller powder)L3147 (LB Miller powder with agar)

Which bacterial culture medium is the best choice for my application?

Each of the broths will likely grow E. coli very well, but there are still general guidelines for choosing a broth when you are working without a protocol.Generally:LB - Miller and LB - Lennox are used for E. coli growth and maintanence, DNA plasmid production and protein production.The Lennox formulation has a lower salt content required for some salt-sensitive selection antibiotics.LB - Luria low salt is used for special applications where the E. coli growth or other constraints require the lowest possible salt content.Terrific Broth is used for higher yield protein production and high yield DNA plasmid production, because of the faster growth of the E. coli in this medium.SOB is used for protein production, DNA plasmid production and the generation of high-efficiency competent cells.SOC is used for initial growth of competent cells and the transformation procedure.

Will adding magnesium to the culture medium increase cell density?

In microbial broth formulations that do not already contain magnesium, the addition of 10-20 mM MgCl2 or MgSO4 may increase cell densities. You may need to also increase the shaking speed of the incubator.

Microbial Media Selection Guide

LB media
 

Name Components Product No.     Format   Preparation Notes Application
LB – Miller • 10 g/L Tryptone
• 5 g/L yeast extract
• 10 g/L NaCl
L2542 Liquid Ready-to-use Suitable for regular E. coli culture.
•10g/L Tryptone
•10g/L NaCl
•5g/L Yeast Extract
L3522 Powder Mix 25 g in 1L water; autoclave and cool before use
•15g/L Agar
•10g/L Tryptone
•10g/L NaCl
•5g/L Yeast Extract
L3147 Powder with agar Mix 40 g in 1L water, heat to dissolve; autoclave and cool before use
•15g/L Agar
•10g/L Tryptone
•10g/L NaCl
•5g/L Yeast Extract
•100μg/ml ampicillin
L5667 Agar plates with 100 μg/ml ampicillin Ready-to-use
•15g/L Agar
•10g/L Tryptone
•10g/L NaCl
•5g/L Yeast Extract
•50μg/ml ampicillin
L0168 Agar Plate with 50 μg/ml ampicillin
•15g/L Agar
•10g/L Tryptone
•10g/L NaCl
•5g/L Yeast Extract
•50μg/ml kanamycin
L0543 Agar plates with 50 ug/ml kanamycin
•15 g/L Agar
•10 g/L Tryptone
•10 g/L NaCl
•5 g/L Yeast Extract
•100 μg/ml carbenicillin
L0418 Agar plates with 100 μg/ml carbenicillin
LB – Lennox • 10 g/L peptone
• 5 g/L yeast extract
• 10 g/L NaCl
L3022 Powder Mix 20 g in 1L water; autoclave and cool before use Suitable for culture of E. coli strains that are salt-sensitive.
•15 g/L Agar
•10 g/L Tryptone
•5 g/L Yeast Extract
•5 g/L NaCl
L2897 Powder with agar Mix 35 g in 1L water, heat to dissolve; autoclave and cool before use
•10 g/L Tryptone
•5 g/L Yeast Extract
•5 g/L NaCl
•2.2 g/L inert binding agents
L7275 Tablets Add each 1.2 g tablet to 48.3 mL water; autoclave and cool before use
•13.72g/L Agar
•9.14g/L Tryptone
•4.57 g/L Yeast Extract
•4.57 g/L NaCl
•1.6g/L inert binding agents
L7025 Tablets with agar Add each 1.68 g tablet to 48.3 mL water; autoclave and cool before use
•10g/L Tryptone
•5 g/L Yeast Extract
•5 g/L NaCl
•0.6 g/L inert binder (EZMix formulation only)
L7658 EZMix™ Mix 20.6 g in 1L water; autoclave and cool before use
•15g/L Agar
•10g/L Tryptone
•5 g/L Yeast Extract
•5 g/L NaCl
L7533 EZMix™ with agar Mix 35.6 g in 1L water, heat to dissolve; autoclave and cool before use
•15g/L Agar
•10g/L Tryptone
•5 g/L Yeast Extract
•5 g/L NaCl
L5542 Plates Ready-to-use
•10 g/L Tryptone
•5.0 g/L Yeast Extract
•5.0 g/L NaCl
L9234 Liquid Ready-to-use
LB – low salt • 10 g/L peptone
• 5 g/L yeast extract
• 0.5 g/L NaCl (and 15 g/L agar for solid medium)
L3397 Powder Mix 15.5 g in 1L water; autoclave and cool before use Suitable for culture of E. coli strains that require low salt conditions to grow.
•15g/L Agar
•10g/L Tryptone
•5g/L Yeast Extract
•0.5g/L NaCl
L3272 Powder with agar Mix 30.5 g in 1L water, heat to dissolve; autoclave and cool before use
Overnight Express Instant LB Medium • Overnight Express Autoinduction System 1 components
• Luria Bertani (LB) broth
71757-M Granulated medium Just add the EasyPak contents to 1 L sterile water, supplement with 10 ml glycerol, and microwave for 2 min to dissolve. Overnight Express Instant LB Medium is also available in 1 kg bottles. Granulated medium ensures rapid, uniform dissolution in water, prevents clumping of the medium, and avoids inhalation of airborne powder. Enables regulated protein expression in E. coli, without monitoring the culture or adding IPTG during cell growth.
Overnight Express Instant TB Medium • Overnight Express Autoinduction System 1 components
• Terrific Broth (TB) medium
71491 Granulated medium EasyPak is an aluminum foil pouch containing 60 g granulated medium, sufficient for 1 L culture. Just add the EasyPak contents to 1 L sterile water, supplement with 10 ml glycerol, and microwave for 2 min to dissolve. Overnight Express Instant TB Medium is also available in 1 kg bottles. Granulated medium ensures rapid, uniform dissolution in water, prevents clumping of the medium, and avoids inhalation of airborne powder.

 

Minimal salts (M9)
 

Name Components Product No. Format   Preparation Notes Application
Minimal salts (M9) • 33.9 g/L Na2HPO4.7H2O •15 g/L KH2PO4 • 5 g/L NH4Cl and 2.5 g/L NaCl M6030 Powder Mix 56.4 g in 1L water to obtain 5X stock solution; autoclave, cool and dilute to 1X before use Suitable for recombinant E. coli strains
•15 g/L KH2PO4
•64 g/L Na2HPO4
•2.5 g/L NaCl
•5.0 g/L NH4Cl
M9956 Liquid Ready-to-use

 

Name Components Product No. Format Preparation Notes Application
Hanahan’s Broth (SOB Medium) • 20 g/L tryptone
• 5 g/L yeast extract
• 2.4 g/L MgSO4
• 0.5 g/L NaCl
• 0.186 g/L KCl
H8032 Powder Mix 28 g in 1L water; autoclave and cool before use Enables the preparation of high efficiency competent cells.
SOC Medium • 20 g/L tryptone
• 5 g/L yeast extract
• 4.8 g/L MgSO4
• 3.603 g/L dextrose
• 0.5 g/L NaCl
• 0.186 g/L KCl
S1797 Liquid Ready-to-use SOC medium is a modified SOB medium with added glucose. In the presence of glucose the cellular processes utilize other sugars are repressed. The expression of recombinant proteins that might affect the host cell physiology is inhibited thus resulting in high efficiency transformation reactions. It enables the high efficiency transformation of competent cells.

 

Terrific Broth - modified
 

Name Components Product No. Format Preparation Notes Application
Terrific Broth - modified • 23.6 g/L yeast extract
• 11.8 g/L tryptone
• 9.4 g/L K2HPO4
• 2.2 g/L KH2PO4
T5574 Liquid Ready-to-use Suitable for non-selective cultivation of E. coli strains for cloning and high yield production of DNA, plasmid DNA and recombinant proteins. It is also suitable for selective cultivation when appropriate antibiotics are added.
T9179 EZMix™ Mix 48.2 g in 1L water, add 4 mL glycerol; autoclave and cool before use

 

2X YT medium
 

Name Components Product No. Format Preparation Notes Application
2X YT medium • 16 g/L tryptone
• 10 g/L yeast extract
• 5 g/L NaCl
Y1003 Liquid Ready-to-use Suitable for non-selective cultivation of M13 bacteriophage and E. coli strains for cloning, production of DNA, plasmid DNA and recombinant proteins. It is also suitable for selective cultivation when appropriate antibiotics are added.
Y2377 Powder Mix 31 g in 1L water; autoclave and cool before use
Y2627 EZMix™ Mix 31.6 g in 1L water; autoclave and cool before use

NZCYM Broth
 

Name Components Product No. Format Preparation Notes Application
NZCYM Broth • 10 g/L tryptone
• 5 g/L yeast extract
• 5 g/L NaCl
• 1 g/L Casamino acids
• 1 g/L MgSO4
N3643 Powder Mix 22 g in 1L water; autoclave and cool before use Suitable for non-selective cultivation of lambda bacteriophage and E. coli strains for cloning, production of DNA, plasmid DNA and recombinant proteins. It is also suitable for selective cultivation when appropriate antibiotics are added.

 

Yeast Media
 

Name Components Product No. Format Preparation Notes Application
Yeast Nitrogen Base with amino acids Nitrogen Sources:
•Ammonium sulfate, 5.0 g/L

Amino Acids:
•L-histidine, 10 mg/L
•DL-methionine, 20 mg/L
•DL-tryptophan, 20 mg/L

Vitamins:
•Biotin, 2.0
•Calcium pantothenate, 400
•Folic acid, 2.0
•Inositol, 2.0 mg/L
•Nicotinic acid, 400
•p-Aminobenzoic acid, 200
•Pyridoxine HCl, 400
•Riboflavin, 200
•Thiamine HCL, 400

Trace Elements:
•Boric acid
•Copper sulfate
•Potassium iodide
•Ferric chloride
•Magnesium sulfate
•Sodium molybdate
•Zinc sulfate

Salts:
•Potassium phosphate monobasic, 1.0 g/L
•Magnesium sulfate, 0.5 g/L
•Sodium chloride, 0.1 g/L
•Calcium chloride, 0.1 g/L
Y1250 Powder Mix 6.7 g in 100 mL water, add 5 g glucose to obtain 10X stock solution; filter sterilize and dilute to 1X with sterile water before use Yeast Nitrogen Base is a highly-referenced growth medium used for the cultivation of yeast. This nutrient-rich microbial broth contains amino acids, nitrogen, vitamins, trace elements and salts.
Yeast Nitrogen Base without amino acids Nitrogen Sources:
•Ammonium sulfate, 5.0 g/L

Vitamins:
•Biotin, 2.0
•Calcium pantothenate, 400
•Folic acid, 2.0
•Inositol, 2.0 mg/L
•Nicotinic acid, 400
•p-Aminobenzoic acid, 200
•Pyridoxine HCl, 400
•Riboflavin, 200
•Thiamine HCL, 400

Trace Elements:
•Boric acid, 500
•Copper sulfate, 40
•Potassium iodide, 100
•Ferric chloride, 200
•Manganese sulfate, 400
•Sodium molybdate, 200
•Zinc sulfate, 400

Salts:
•Potassium phosphate •monobasic, 1.0 g/L
•Magnesium sulfate, 0.5 g/L
•Sodium chloride, 0.1 g/L
•Calcium chloride, 0.1 g/L
Y0626 Powder Mix 6.8 g in 100 mL water, add 5 g glucose and 5-10 mg of appropriate amino acids to obtain 10X stock solution; filter sterilize and dilute to 1X with sterile water before use Yeast Nitrogen Base is considered ideal for culturing wild type yeast. The presence of amino acid is known to prevent selectable marker utilization. Hence, this media is formulated without amino acids and is useful for cloning and manipulation of yeast artificial chromosome.

 

Phage Media
 

Name Components Product No. Format   Preparation Notes Application
NZ Amine®Broth • 10 g/L tryptone
• 5 g/L NaCl
• 1 g/L MgSO4
N3518 Powder Mix 16 g in 1L water; autoclave and cool before use Suitable for the growth of lambda bacteriophage.

 

 References