Culture Media: Classification, Types, and Relevance

What Is Culture Media?

Culture media are mediums that provide essential nutrients and minerals to support the growth of microorganisms in the laboratory.

Microorganisms have varying nature, characteristics, habitat, and even nutritional requirements, thus it is impossible to culture them with one type of culture media. However, there are also microorganisms that can’t grow on a culture media at all in any condition – these are called obligate parasites.^[1]

Culturing microorganisms is essential for diagnosing infectious diseases, obtaining antigens, developing serological assays for vaccines, genetic studies, and identification of microbial species.^[1]

Furthermore, it’s also essential for isolating pure cultures, storing culture stock, studying biochemical reactions, testing microbial contamination, checking antimicrobial agents and preservatives effect, testing viable count, and testing antibiotic sensitivity.^[2]

This article will focus on the composition, classification, and types of culture media used in microbiology labs to study a spectrum of microbial forms.

Classification and Types of Culture Media

Growing microorganisms in the lab involve mimicking the organisms’ natural habitat or environment, and this is possible in the laboratory by formulating culture media that meets their requirements. Therefore, many culture media were developed by scientists according to the microbial species to be cultured.

The basic media contains a source of carbon & energy, nitrogen source, growth factors, and some trace elements.^[1] Some commonly used media components include peptone, agar, water, casein hydrolysate, malt extract, meat extract, and yeast extract. In addition, the pH of the medium should be set accordingly.^[3]

However, some additional components or nutrients are added to the media when growing specific microorganisms.

Culture media can be classified in three ways: based on their consistency, nutritional component, and applications.^[1]

A. Classification of culture media based on consistency

Solid media: In these media, the agar which is an unbranched long chain of polysaccharides is added in the concentration of 1.5-2.0%. Most commonly, 1.3% agar is used to prepare solid media in labs. The agar-containing media solidifies at 37 ºC.^[1] Sometimes, in the place of agar, some other inert solidifying agents are used, such as gellan gum.
Solid media are used to grow microorganisms in their full physical form, prepare bacterial pure cultures, or isolate bacteria to study colony characteristics.^[1] The bacterial growth on solid media varies in appearance as mucoid, round, smooth, rough, filamentous, irregular, and punctiform. The media is not hydrolyzed by microorganisms and is free from growth-inhibiting substances.^[3] Examples of solid media are blood agar, nutrient agar, McConkey agar, and chocolate agar.
Semisolid media: This media has 0.2-0.5% agar concentration, and due to the reduced agar concentration, it appears as a soft, jelly-like substance. It’s mainly used to study the motility of microorganisms, distinguish between motile and non-motile bacterial strains (through U-tube and Cragie’s tube), and cultivate microaerophilic bacteria – bacteria on this media appear as a thick line. Examples of semi-solid media are: Hugh and Leifson’s oxidation fermentation medium, Stuart’s and Amies media, and Mannitol motility media.^[1]
Liquid media: These media do not contain any traces of solidifying agents, such as agar or gelatin, and large growth of bacterial colonies can be observed in the media. Liquid media are also called broths, they allow for uniform and turbid growth of bacterial strains when incubated at 37ºC for 24hrs. The media is used for the profuse growth of microorganisms and fermentation studies. Examples include Tryptic soy broth, phenol red carbohydrate broth, MR-VP broth, and nutrient broth.

Other than these, there are also biphasic media, which consist of both solid and liquid media. And sometimes in the place of agar, egg yolk and serum are added to the media as a solidifying agent.^[3]Learn more on how to make agar plates here.

While naturally, these substances are liquid, they are solidified by using heat, and the prepared media is sterilized using the inspissation technique. Examples are Lowenstein Jensen medium and Dorset egg medium, which contain egg yolk, and Loeffler’s serum slope, which contain serum.^[3]

B. Classification based on the nutritional component

Simple media: It’s a general-purpose media that supports the growth of non-fastidious microbes, and it is primarily used for the isolation of microorganisms. Examples are nutrient broth, peptone water, and nutrient agar.
Complex media: These are media containing nutrients in unknown quantities that are added to bring about a particular characteristic of a microbial strain. Examples are tryptic soy broth, blood agar, and nutrient broth.
Synthetic media: Synthetic media is a type of chemically defined media and is produced from pure chemical substances. A defined media refers to a medium having a known concentration of ingredients, like sugar (glucose or glycerol) and nitrogen source (such as ammonium salt or nitrate as inorganic nitrogen). It is generally used in scientific research, and an example is Czapek Dox Medium.^[1]

C. Classification of culture media based on application/chemical composition

Basal media: These are routinely used simple media having carbon and nitrogen sources that boost the growth of many microorganisms. They are also known as general-purpose media and are considered non-selective media.

The basal media do not require enrichment sources for the growth of non-fastidious bacteria and are suitable for growing Staphylococcus and Enterobacteriaceae.^[1]
They are generally used to isolate microorganisms in labs or in sub-culturing processes. Examples are nutrient broth, nutrient agar, and peptone water.
Enriched media: This media is prepared by adding additional substances like blood, serum, or egg yolk in the basal medium. It’s used to grow fastidious microorganisms as they require additional nutrients and growth-promoting substances.

Examples are chocolate agar, blood agar, and Loeffler’s serum slope. Chocolate media is used to grow N. gonorrhea while blood agar (which is prepared by adding 5-10% blood by volume to a blood agar base) is used to identify hemolytic bacteria.^[2]
Selective media: This media allows the growth of certain microbes while inhibiting the growth of others. It’s an agar-based medium that is used to isolate microorganisms in labs.

The selective growth of microbes is decided by adding substances like antibiotics, dyes, bile salts, or by pH adjustments.
Below is a list of common selective media and the bacteria they’re used to culture:^[2]

S. No	Culture media	Inhibiting substances	Bacteria
1	Thayer Martin Agar	Contains antibiotics; vancomycin, colistin, and nystatin	Used for Neisseria gonorrhoeae
2	MacConkey’s Agar	Contains bile salts	Used for Enterobacteriaceae members
3	Lowenstein Jensen Medium	Addition of malachite green	Used for M.tuberculosis
4	Mannitol Salt Agar	Contains 10% NaCl	Used to recover S.aureus
5	Crystal Violet Blood Agar	Contains 0.0002% crystal violet	Used for Streptococcus pyogenes
6	Thiosulfate citrate bile salts sucrose (TCBS) agar	Have elevated pH of about 8.5-8.6	Used for isolating Vibrio cholerae
7	Wilson and Blair’s Agar	Addition of dye brilliant green	Used for recovering S. typhi
8	Potassium tellurite medium	Contains 0.04% Potassium tellurite	Used to recover C.diphtheriae
9	Pseudosel Agar (cetrimide agar)	Contains cetrimide (antiseptic agent)	Used to recover Pseudomonas aeruginosa
10	Salmonella-Shigella Agar	Contains bile salts, brilliant green, and sodium citrate	Used for the isolation of Salmonella, which causes typhoid

Enrichment media: It’s a liquid medium, used to increase the relative concentration of certain microbes before culturing them on a solid medium plate. It’s used as a broth medium and inhibits the growth of commensal species of microorganisms (those who live in close association with each other) in the clinical specimen.
It’s also used in isolating fecal and soil microorganisms. Examples are selenite F broth which is used to isolate Salmonella typhi from a fecal sample, tetrathionate broth, and alkaline peptone water.^[1]
Differential or indicator media: It contains certain indicators like dyes or metabolic substrates in the medium composition which gives different colors to colonies of different microbial species when they utilize or react with these components.
It allows the growth of more than one microorganism, however, the bacterial colonies are differentiated based on their color when a chemical change occurs in the indicator, such as neutral red, phenol red, methylene blue. Examples are:^[1]
- Blood agar: In blood agar, three types of blood cell lysis or hemolysis are observed: alpha, beta, and gamma hemolysis.^[5] It allows the growth of many microorganisms, however, their ability to lyse blood cells differs, and this helps to distinguish the bacterial colonies.
  For example, S. pyogenes completely lyse blood cells (beta hemolysis), thus causing total clearing of the media around its colonies. S. pneumoniae partially lyse red blood cells, resulting in a greenish-colored medium, while gamma hemolytic microorganisms like Enterococcus faecalis, Staphylococcus saprophyticus, and Staphylococcus epidermidis, can’t lyse red blood cells, thus causing no color change in the medium.^[6]
- Mannitol salts agar: The fermentation of mannitol by Staphylococcus aureus causes the media to change to yellow, however, coagulase-negative staphylococci that can’t cause fermentation to appear in pink.^[7]
- MacConkey agar: It differentiates the gram-negative bacteria based on their lactose metabolism. The lactose fermenting bacteria, such as Escherichia coli, Klebsiella spp, Citrobacter, and Enterobacter forms pink-red colonies, while lactose non-fermenters, like Salmonella, Shigella, Proteus, Providencia, Pseudomonas, and Morganella form pale or colorless colonies.^[8]
- Thiosulfate citrate bile salts sucrose (TCBS) agar: The media contain sucrose, which is utilized by ferment microbes and helps to distinguish them from non-ferment microorganisms. Based on this characteristic, different colored bacterial colonies are formed on the media that help to identify and distinguish them from each other.
  For example, V. cholerae ferment the sucrose and form slightly flattened yellow colonies having opaque centers and translucent peripheries. Whereas, V. parahaemolyticus can’t ferment the sucrose and forms green to blue-green colonies.^[9]
Transport media: Transport media are useful for clinical specimens which are required to be transferred immediately to labs to maintain the viability of potential pathogens and to prevent overgrowth of commensals or contaminating microorganisms. Some of them are semi-solid in consistency, and examples include:
- Sach’s buffered glycerol saline: It’s used to transport feces from patients suspected to be suffering from bacillary dysentery.
- Cary Blair transport and Venkatraman Ramakrishnan media: Fecal samples collected from suspected cholera patients are transported using these media.
- Pike’s medium: A throat specimen containing Streptococci is transported using this medium.^[2]
Anaerobic media: This media is for anaerobic bacteria which require low oxygen levels, extra nutrients, and reduced oxidation-reduction potential. It is supplemented with hemin and vitamin K nutrients and oxygen is removed by boiling it in a water bath and sealing it with paraffin film.^[2]
Examples are: Thioglycollate broth and Robertson Cooked Meat (RCM) medium which is commonly used to grow Clostridium spp.^[2]
Assay media: It’s used for amino acids, vitamins, and antibiotics assays. For example, antibiotic assay media is used to determine the antibiotic potency of microorganisms.
Storage media: It’s used to store microorganisms for a longer period, examples are chalk cooked meat broth and egg saline medium.^[2]

Conclusion

Culture media is a source of nutrients and growth factors required for the growth of microorganisms and even plants in laboratory conditions. Every organism has different nutritional requirements based on its habitat or living conditions. Therefore, a single formulation of culture media can’t be used to grow all organisms in labs.

Many types of culture media have been developed by scientists to grow selective or desired microorganisms. These are classified based on their nutrient composition, consistency, and application or use in life science laboratories.

Culture media serve several purposes in labs like isolating specific strains of microorganisms, identifying disease-causing pathogens, preparing pure culture of a microbial species, distinguishing bacterial species, and studying their responses to certain antibiotics.

Thus, before deciding which culture media to use, it is critical to determine the purpose of your study and in some cases the type of microorganism you want to study. This narrows down your choices and helps you to choose which media is best for your experiment, without wasting your time and effort.

References:

Tankeshwar Acharya (2021). Bacterial Culture Media: Classification, Types, Uses. Retrieved from https://microbeonline.com/types-of-bacteriological-culture-medium/.
Fatima Aiman (2022). Microbial Culture Media- Definition, Types, Examples, Uses. Retrieved from https://microbenotes.com/types-of-culture-media/#application-of-culture-media.
Rao Sridhar. Bacterial Culture Media. Retrieved from https://www.microrao.com/micronotes/pg/culture_media.pdf.
Aryal Sagar (2022). Salmonella Shigella (SS) Agar- Composition, Principle, Preparation, Results, Uses. Retrieved from https://microbenotes.com/salmonella-shigella-ss-agar/.
Tankeshwar Acharya (2021). Blood Agar and Types of Hemolysis. Retrieved from http://microbeonline.com/blood-agar-composition-preparation-uses-and-types-of-hemolysis/.