Table of Contents

Bacterial Culture Media

Petri dishes. Image courtesy of Norm Baker, MS, MA, RBP, Johns Hopkins Univ. School of Medicine

Depending on their composition or use, culture media can be categorized into several groups; these include defined, complex, selective, and enrichment medium. In a defined medium, the exact chemical composition is known. These types of media are usually composed of pure biochemicals, and are often used to study the minimal nutrient requirement of a microorganism. In contrast, the exact chemical composition of a complex medium is not known. This latter medium type often contains reagents of a biological origin, such as yeast extract and peptone, where the exact chemical composition is unknown. Complex media usually provide a large range of growth factors that assist in the cultivation of unknown and fastidious bacterial species.3

Media may also be formulated as selective or enriched. A selective medium is formulated to inhibit the growth of certain bacterial species and/or promote the growth of a specific species. These media can consist of additional selective reagents, such as high salt concentration to select for halophiles, or can be used under selective growth conditions. An enrichment medium also allows for the growth of specific bacterial species; however, enrichment media are supplemented with a reagent that permits, rather than inhibits, the growth of a particular species.3

Generally, bacterial culture media are mixtures of proteins, salts, trace elements, amino acids, and carbohydrates. The presence and volume of these components can vary significantly among bacterial species depending on the macro- and micro-nutrient requirements of each strain. The manner in which bacterial strains are cultured also varies widely. Liquid media are often used for the growth and propagation of pure batch cultures, while solid agar-based media are used for the isolation of pure cultures.

In addition to supplying nutrients, liquid medium can assist in the maintenance of pH. The pH can be sustained through one or more buffering systems such as 3-(N-morpholino)propanesulfonic acid (MOPS) or potassium phosphate. The osmotic environment can be maintained through the addition of salts, such as sodium chloride. ATCC uses numerous types of media in order to provide the optimal growth conditions for each bacterial species. The formulations for these media can be found on the ATCC website (See: NOTE 5). Several bacterial media commonly used by ATCC include the following:

Brain Heart Infusion Agarose (BHI) (ATCC medium formulation 44) is a complex, nutrient-rich, general-purpose growth medium used for culturing fastidious and nonfastidious microorganisms, including streptococci and pneumococci. It is generated from the dehydrated infusions of bovine or porcine brain and heart tissue. This medium can be supplemented with sodium chloride and disodium phosphate for osmotic and pH maintenance, respectively. ATCC currently uses both BHI agar and broth medium supplied from Becton, Dickinson and Company (BD).

Gonococcal (GC) Medium (ATCC medium formulation 814) is a growth medium used for the cultivation of Neisseria gonorrhoeae and other fastidious organisms. It can be employed as a basal medium in the preparation of Chocolate Agar, Thayer-Martin Medium, Martin-Lewis Agar, and Transgrow Agar. GC medium is prepared from a mixture of GC agar base, Dried Bovine Hemoglobin, and IsoVitaleX™ available from BD. For the selective growth of specific organisms, this medium can be supplemented with antibiotics including chloramphenicol, streptomycin, tetracycline, and ampicillin.

Haemophilus Test Medium (HTM) (ATCC medium formulation 5129) consists of a complex mixture of yeast extract, Mueller Hinton Broth, Porcine Hematin, and nicotinamide adenine dinucleotide (NAD). The main component, Mueller Hinton Agar, was originally prepared as a solid media used to test the antimicrobial susceptibility testing of common, non-fastidious, rapidly growing organisms. This initial media, however, was not suitable for fastidious organisms such as streptococci, gonococci, and Haemophilus species.5 HTM was subsequently developed for the testing of fastidious organisms.

Luria-Bertani Broth Medium (LB) (ATCC medium formulation 1065) is an all-purpose medium used by ATCC for the propagation and maintenance of Escherichia coli for molecular biology applications. It is prepared in the Miller Composition using an LB broth mixture supplied by BD, which consists of tryptone, yeast extract, and sodium chloride. LB can also be prepared as a solid medium through the addition of agar. This medium is often supplemented with various antibiotics for the maintenance of plasmid DNA.

Marine Broth (ATCC medium formulation 2) is a selective, complex growth medium used for the cultivation of heterotrophic marine bacteria. The marine environment offers a unique set of growth conditions consisting of high salinity and low temperatures. To simulate this environment, marine broth is formulated with peptone, yeast extract, and a high salt content. ATCC generates this medium using the Marine Broth 2216 preparation from BD. Marine broth may also be prepared as a solid medium through the addition of agar.

Middlebrook 7H9 Broth (ATCC medium formulation 2714) is formulated to support the growth and propagation of Mycobacterium species. This medium is prepared as a mixture of Middlebrook 7H9 broth, glycerol, sodium pyruvate, and albumin-dextrose-catalase (ADC) enrichment.

Middlebrook 7H10 Agar (ATCC medium formulation 173) is formulated for the isolation and cultivation of mycobacteria from clinical and non-clinical specimens. This medium is prepared as a mixture of Middlebrook 7H10 broth, glycerol, and oleic acid-albumin-dextrose-catalase (OADC) enrichment. It differs from Middlebrook 7H9 broth in concentration of several salts as well as the addition of OADC enrichment rather than ADC enrichment. This medium is based on an improved formulation that was developed to promote the early growth of mycobacteria in vitro.6

Modified Chopped Meat Medium (ATCC medium formulation 1490) is a non-selective, complex medium that supports the growth of most spore-forming and non-spore-forming obligate anaerobes. This medium consists of a complex mixture of ground beef (fat-free), sodium hydroxide, trypticase peptone, yeast extract, Hemin, dipotassium phosphate, and vitamin K1 solution. Depending on the required growth conditions of each anaerobic strain, ATCC supplements this medium with various reagents such as glucose, arginine, or a combination of formate and fumarate.

Nutrient Agar/Broth (ATCC medium formulation 3) is a general-purpose medium for the cultivation of non-fastidious bacterial strains. ATCC prepares this media from a dehydrated stock provided by BD, which consists of beef extract and peptone. This medium can be further enriched by the addition of heart infusion broth. Alternatively, this medium can also be used for the selective growth of specific strains through the addition of varying concentrations of sodium chloride.

Reinforced Clostridial Medium (ATCC media formulations 1053, 2107) is used by ATCC for the cultivation and recovery of anaerobes, particularly Clostridium species, from a variety of sources. ATCC prepares this medium using dehydrated culture media from either Oxoid Limited or BD. This media consists of a complex mixture of salts, tryptose, beef extract, yeast extract, dextrose, starch, and L-Cysteine HCl.

Tryptic Soy Agar (ATCC medium formulation 18) is a medium used for the isolation and propagation of a variety of bacterial strains. ATCC often prepares this medium using a tryptic soy base, provided by BD, supplemented with 5% defibrinated sheep’s blood (ATCC medium formulation 260). The tryptic soy base mixture consists of a combination of tryptone, soytone, and sodium chloride. The addition of sheep’s blood helps to facilitate the growth of more fastidious bacteria.

Microbial Media Formulations

The formulations for media used by ATCC can be found on the ATCC website. Please note that most microbial media are not currently sold at ATCC.

Media Ingredients

IsoVitaleX™

IsoVitaleX™ (BD) is a chemically defined enrichment additive used for the cultivation of fastidious bacteria. This reagent is used in lieu of yeast concentrate to function as a nutrient supplement for the growth of bacteria including gonococci and Haemophilus. IsoVitaleX™ consists of a mixture of Vitamin B12, L-Glutamine, adenine, guanine hydrochloride p-Aminobenzoic acid, nicotinamide adenine dinucleotide, thiamine pyrophosphate, ferric nitrate, thiamine hydrochloride, L-cysteine hydrochloride, L-cysteine, and dextrose.

Nicotinamide adenine dinucleotide (NAD)

NAD is a dinucleotide compound found in all living cells. It functions as a coenzyme involved in oxidation/reduction reactions, transporting electrons from one reaction to another. NAD can also be used as a substrate in several biochemical reactions such as mono- and poly-ADP-ribosylation.7 The addition of NAD to bacterial culturing medium provides an essential role in metabolism as a coenzyme in redox reactions and can additionally function as a substrate for bacterial DNA ligases.8

Peptone

Peptone is a water-soluble protein derivative used in bacteriology culture media. This reagent is prepared via the partial hydrolysis of an animal protein by an enzyme or acid. Generally, not all bacterial species can use free atmospheric nitrogen. Many species require either organic or inorganic fixed nitrogen.9 Peptone is used in bacterial media as an organic source of nitrogen and is often used in serum-free medium. The nutritional value of peptone is dependent on the amino acid content that supplies the essential nitrogen. The starting material for peptone can range from animal to plant; these can include meat, soybean, casein, and whey.10

Yeast Extract

Yeast extract is prepared as a water-soluble extract of autolyzed Saccharomyces cerevisiae yeast cells. During autolysis, endogenous yeast digestive enzymes break down protein content into peptides and amino acids which can be used by bacteria as a source of nitrogen. Additionally, yeast extract provides an essential source of water-soluble B-complex vitamins, carbohydrates, and free glutamic acid.11, 12

ADC

ADC enrichment is used in Middlebrook 7H9 media for the selective growth of mycobacteria. It is formulated with sodium chloride, bovine albumin (fraction V), dextrose, and catalase. The addition of dextrose provides an extra source of energy. Both the albumin and catalase have protective roles via binding free fatty acids which may be toxic to mycobacteria and ridding the environment of toxic peroxides, respectively.12

OADC

OADC is an enrichment supplement added to Middlebrook 7H10 agar for the cultivation of mycobacteria. This additive provides the same reagents as ADC in addition to oleic acid. Addition of OADC provides faster and more robust growth of Mycobacterium species.6, 12

Sodium Pyruvate

Pyruvate is an intermediary organic acid metabolite produced as an end product of glycolysis and the Entner-Doudoroff Pathway.13 The addition of sodium pyruvate in bacterial culture media provides both an energy source and a carbon skeleton for anabolic processes. This reagent has also been shown to provide protection against hydrogen peroxide.14

Dextrose

Dextrose is a highly nutritious supplement used for the cultivation of fastidious organisms, older cultures, and cultures with small inoculums. This reagent can be used as a dextrose broth/agar medium or as a supplement within a complex or defined medium. Generally, dextrose provides bacteria with a carbon source, and can be supplemented with 5% blood to provide additional growth factors for fastidious microorganisms.15

Media Supplements

The growth media recommended for some bacterial species may require the addition of components not already available in the base medium. These components may include antibiotics, serum, blood, or chemical supplements.

After supplements have been added to the base medium, the shelf life of the medium should be determined on a case-by-case basis. Media containing antibiotics tend to degrade faster than base media alone. Media containing supplements should not be frozen as this may cause certain compounds to precipitate out of solution; media should be stored at 2°C to 8°C. For additional information regarding the preparation, storage, or usage of specific additives, contact your local supplier or consult with the manufacturer’s product information sheet.

Chemical Supplements

  1. Trace Elements

    Trace elements are micronutrients required by microorganisms in small amounts. These nutrients are metal ions required by most microorganisms for survival as they usually function as cofactors for essential enzymatic reactions. It is often not necessary to provide trace elements in media as they can be present in minute amounts within the water or other media reagents. Trace elements are, however, added to minimal media as it contains only the minimum addition of nutrients required for microbial growth. Examples of trace elements required for bacterial nutrition include zinc, copper, manganese, molybdenum, and cobalt.3 ATCC offers a Trace Mineral Supplement (ATCC® MD-TMS™) that is based on Wolfe’s mineral solution.

  2. Salts Pile of salt

    Various salts, such as magnesium, calcium, iron, and potassium salts, are required for bacterial growth as they provide major elements that can function as cofactors for certain enzymatic reactions. In addition to the aforementioned function, calcium and iron form major components of endospores and cytochromes, respectively.3 Furthermore, the presence of salts, particularly sodium chloride, within a medium assists in the maintenance of osmolality. Most bacteria require either an isotonic or hypotonic environment for optimum growth. In these environments, water either flows in and out of the cell at equal rates or water only flows into the cell, respectively.3

  3. Glucose

    Microbial media are often supplemented with glucose as a source of carbohydrates. In addition to this nutritional role, glucose may also be added as a means to repress an array of inducible enzymes in many different bacterial species. This operon repression is accomplished via the inhibition of cyclic AMP (cAMP) synthesis. The activity of adenylate cyclase, the enzyme required for cAMP synthesis, is blocked in the presence of glucose. In bacterial species such as Escherichia coli, this inhibition requires the cells to use glucose as the primary energy source rather than metabolizing poorer sources of energy.3

  4. Blood

    Blood is often added to growth medium to enhance the cultivation of highly fastidious microbial species. Examples of such media include Tryptic Soy Agar and Dextrose Agar, both of which are often supplemented with 5% defibrinated sheep blood. The enrichment of a medium with blood supplies nutrients, such as growth factors, as well as assists in the determination of hemolytic reactions and pigmentation12

Antibiotics

Antibiotics can be added to bacterial culture media to select for the growth of a specific variant. This type of medium is often used for the selection of strains that have inherent antibiotic resistance, harbor plasmids, or are genetically engineered.3 The addition of antibiotics often decreases the shelf life of a medium as compared to the base medium. To extend the shelf life, media containing antibiotics should be stored at 2°C to 8°C in the dark.

ATCC serum in bottle

Serum

Sera can serve as a source of growth factors, proteins, vitamins, hormones, carbohydrates, lipids, amino acids, minerals, and trace elements. Additionally, serum can function as a pH buffer and can inactivate proteolytic enzymes. The exact composition of sera is unknown and varies from lot to lot, although lot-to-lot consistency has improved in recent years.

Sera from rabbit, horse, fetal bovine, and calf bovine sources are used to support the growth of some bacterial species in culture. Many pathogens, such as members of the genera Corynebacterium, Borrelia, Leptospira, and Mycoplasma, require the addition of serum to support growth. Media formulated to support these aforementioned strains are commonly supplemented with heat-inactivated rabbit or horse serum.

Unfortunately, naturally derived products from animals, such as sera, may contain adventitious microorganisms. All reputable suppliers routinely test their products for infectious virus by several methods including fluorescent antibody, cytopathic effect, and hemadsorption. These products are also screened for the standard microbial contaminants such as bacteria, fungi, and mycoplasma. To reduce the risk of any possible contamination, ATCC recommends that all serum should be triple filtered through 0.1 µm filters before use.

ATCC offers the following four types of animal sera:

  • Fetal Bovine Serum (also known as fetal calf) -- ATCC® 30-2020
  • Fetal Bovine Serum qualified for embryonic stem cells -- ATCC® SCRR-30-2020™ 
  • Iron-supplemented Calf Bovine Serum -- ATCC® 30-2030™ 
  • Horse Serum -- ATCC® 30-2040

These products are rigorously tested for adventitious infective agents and sourced only from U.S. herds. Furthermore, each lot is tested for its ability to support cell growth and is the same sera used in ATCC labs.

  1. Storage

    Store sera at -20°C or colder for storage over 30 days. ATCC sera are routinely stored at -70°C. Do not store sera at temperatures above -20°C for any length of time. Avoid repeated freeze-thaws by dispensing and storing sera in aliquots.

  2. Thawing

    The following procedure is used to thaw serum:

    1. Place the frozen serum in a refrigerator at 2°C to 8°C overnight.
    2. Put the bottles in a 37°C water bath and gently agitate occasionally to mix the solutes that tend to concentrate at the bottom of the bottle.

    Do not keep the serum at 37°C any longer than necessary to thaw it, and do not thaw the serum at higher temperatures. Thawing serum in a bath above 40°C without mixing may lead to the formation of a precipitate inside the bottle.

  1. Turbidity and precipitates

    All sera may retain some fibrinogen. Because external factors may initiate the conversion of fibrinogen to fibrin, flocculent material or turbidity may be observed after the serum is thawed. The presence of this material does not alter the serum’s performance. If the presence of flocculent material or turbidity is a concern, it can be removed by filtration through a 0.45 µm filter.

    A precipitate can form in serum when incubated at 37°C or higher for prolonged periods of time. This is often mistaken for microbial contamination. This precipitate may include crystals of calcium phosphate, but this does not alter the performance of the serum as a supplement. Heat inactivation of sera can also cause the formation of precipitates.

  2. Heat Inactivation
  3. Because serum is a blood component, it contains complements, which are a group of proteins that are part of the immune response. These proteins can lead to complement-mediated cell lysis of bacterial cells. To reduce the risk of bacterial lysis, serum used in microbial media can be heat inactivated. Heat inactivation, however, will reduce or destroy growth factors present in the serum; this may be detrimental to the growth of some microbial cells.

    The following procedure can be used to heat-inactivate serum:

    1. Thaw the serum.
    2. Preheat a water bath to 56°C. Use sufficient water to immerse the bottle above the level of serum.
    3. Mix the thawed serum by gentle inversion and place it in the 56°C water bath. The temperature of the water bath will drop.
    4. When the temperature of the water bath reaches 56°C again, continue to heat for an additional 30 minutes. Mix the serum gently every 5 minutes to ensure uniform heating.
    5. Remove the serum from the water bath, and cool quickly. Store the serum at -20°C or colder.