Introduction to Microbial Media

• Introduction
• Types of Microbial Media
• FAQs
• Microbial Medial Selection Guide

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	Introduction	Application
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.
Minimal salts (M9)	M9 medium contains essential salts and nitrogen.	Minimal salts (M9) medium 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.
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.
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	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.	SOC medium enables the high efficiency transformation of competent cells.
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.	2X YT medium 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.
NZCYM Broth	NZCYM Broth is nutrient rich broth with amino acids, peptides, vitamins and low salt concentration.	NZCYM Broth 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.
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.
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.

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

Table 1 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.

Table 2 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

Table 3 Minimal Salts (M9)

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.

Table 4 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 1 L water, add 4 mL glycerol; autoclave and cool before use

Table 5 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 1 L water; autoclave and cool before use
		Y2627	EZMix™	Mix 31.6 g in 1 L water; autoclave and cool before use

Table 6 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 1 L 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.

Table 7 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.

Table 8 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 1 L water; autoclave and cool before use	Suitable for the growth of lambda bacteriophage.

References

1. Sambrook J, Fritsch E, Maniatis T. 1989. Molecular Cloning: A Laboratory Manual. 1. New York: Cold Spring Harbor Laboratory Press.

2. Parija SC. 2012. Textbook of Microbiology & Immunology. 2. Elsevier India.

3. Types of Growth Media Used to Culture Bacteria. [Internet].[updated 07 Aug 2016; cited 17 Jul 2020]. Available from: https://www.scienceprofonline.org/microbiology/types-culture-media-for-growing-bacteria