Exploring the Landscape of Bacterial Culture Media

Article reviewed by Phoebe Lostroh, PhD from Colorado College.

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What Is Culture Media?

Culture media are nutrient sources that enable scientists to grow microorganisms in vitro.¹ “Microbial culture media is like a broth or a soup that contains lots of nutrients such as proteins and sugars,” explained Phoebe Lostroh, a microbiologist at Colorado College. 

 

Brief history of culture media in microbiology

Louis Pasteur prepared the first liquid bacterial culture medium in 1860, containing yeast, sugar, ashes, and ammonium salt.² While developing this, he observed that specific chemical features of the culture medium could promote or impede the growth of certain microorganisms. The competition between different microbes for nutrients in a medium led to some species outgrowing the others. 

In 1881, Robert Koch noted that bacteria grew optimally when inoculated in broth containing fresh meat extract or beef serum.² He also encountered difficulties isolating pure cultures from liquid media, which inspired him to develop solid media that enabled the isolation of specific bacterial strains. Koch combated multiple failures when he tested starch paste, coagulated egg albumin, and potato slices as solidifying agents, until he finally found that a gelatinous substance called agar solidified the broth media. 

Julius Richard Petri designed a circular “culture box” for solid media in 1887, which helped scientists isolate bacterial colonies with reduced contamination.³ Microbiologists still use the eponymous Petri plates today to grow and store bacterial and fungal cultures.

Important Culture Media Components for Bacterial Growth

“The main components are molecules that bacteria can eat as food. So, that will be things like amino acids and sugars such as glucose. Some bacteria can also eat alcohol, like ethanol,” said Lostroh. For optimal bacterial growth, a culture medium must contain suitable nutrients and inorganic minerals (e.g., iron), have the proper pH, and be incubated at a specific temperature. For rapid growth, researchers tend to select bacterial growth parameters in accordance with the microbes’ natural habitats. “For instance, if they live on a human body, they would live at 37°C because that’s our body temperature. Bacteria that come from chickens might live at a higher temperature,” explained Lostroh. 

The key ingredients of a culture medium may include the following.²

• Nutrients: proteins and amino acids (e.g., yeast extract)
• Energy: carbohydrates (e.g., glucose)
• Minerals: calcium, magnesium, iron, potassium, chlorine, phosphate, and sulfate
• Buffering agents (e.g. phosphates and acetates)
• Selective agents: antimicrobial compounds (e.g., ampicillin)
• Indicator for pH change (e.g., bromocresol purple and phenol red)
• Gelling agent: agar
• Distilled water

Carbon and nitrogen

A carbon source is essential for bacteria to produce their own carbon molecules such as proteins, nucleic acids, and fats.² The key sources of inorganic and organic carbohydrates are carbon dioxide and sugars or alcohols, respectively. Nitrogen is another important component because it aids protein synthesis.⁴ Researchers commonly use proteose-peptone and tryptone as nitrogen sources in culture media.

Scientists also select culture media components and growth conditions in the laboratory to meet bacterial energy. For example, many bacteria derive energy from organic compounds such as carbohydrates, while sulfate-reducing bacteria require sulfate-containing chemicals in the media to act as terminal electron acceptors.⁵

Growth factors

Growth factors boost bacterial multiplication when added to the culture medium in small quantities.² There are different growth factor types such as purine and pyrimidine bases, vitamins, and amino acids that influence the growth of different bacterial strains. For example, guanine and amino acids, namely valine and glutamic acid, are essential for Leuconostoc mesenteroides growth.⁶ L. mesenteroides cannot synthesize these components due to its truncated ilv and leu operons, which are essential for the biosynthesis of branched-chain amino acids such as valine. Therefore, supplementation of these ingredients in culture media promotes growth. 

Culture Media Types

Microbiologists classify culture media based on consistency or physical state as solid, liquid (broth), and semisolid.⁸ Cultural media consistency is important for different purposes such as pure culture preparation and motility assessment. 

Table 1: Different media types based on consistency ^9,10

Based on the nutrient composition, scientists sub-classify culture media as simple or complex.¹¹ Simple media such as nutrient broth and peptone water are general purpose media that support the growth of non-fastidious bacteria that do not require any special nutrient supplement or enrichment source to grow. Microbiologists use these media for general isolation and growth experiments.⁸

Complex media, such as tryptic soy broth, are rich in nutrients that aid in the development of fastidious bacteria that have special nutritional needs for growth.¹¹ Enriching media with substances such as blood and serum can support fastidious or difficult to grow microorganisms. For example, blood agar promotes hemolytic bacterial growth.² Besides growth promotion, such media may also elevate characteristic bioactive compound production via microbial fermentation. 

Synthetic media are a type of complex media made from chemically-defined substances. They typically contain known quantities of sugar from glycerol or glucose and nitrogen from ammonium salt or other nitrates.

Researchers use selective media to grow specific microbes while inhibiting the growth of others by adding ingredients such as dyes, antibiotics, and bile salts, and making pH adjustments. For example, scientists developed eosin-methylene blue (EMB) agar medium, which selectively grows Gram-negative bacterial strains and inhibits Gram-positive strains.¹²

Table 2: Selective media for specific bacterial growth ²

Culture Media Preparation

Microbiologists prepare bacterial growth media by dissolving powders made from yeast cells and beef extracts in distilled water at neutral pH. Subsequently, they sterilize the media in an autoclave to prevent unwanted bacterial growth.¹³

The key steps of culture media preparation include the following.¹⁴

1. Initial preparation: Before preparing culture media, it is important to have sterilized containers (e.g., Petri plates and conical flasks) and the required media components. 
2. Rehydration: Scientists dissolve the culture media components (powder medium) in distilled water. This rehydration ensures that important hydrolysis reactions occur. For example, starch is a relatively large molecule to pass through the plasma membrane of a cell, so some bacteria produce amylase to convert it to the smaller glucose molecule, thereby enabling it to pass through. This subsequently aids bacterial growth.
3. Heating: Media containing agar must be mildly heated and stirred thoroughly for complete dissolution. If needed, the pH is adjusted for optimal bacteria-specific growth.
4. Sterilization: Culture media is often sterilized by autoclaving at 121°C for 15 minutes at 15 PSI. Filtration is another technique used to sterilize liquid culture media, particularly if selective media ingredients are susceptible to heat. For this technique, scientist use membranes made of cellulose or polymers with pore diameter ranging between 0.2 and 0.45µm. 
5. Filling: Researchers pour sterilized media into bottles or Petri plates in laminar airflow or other aseptic conditions to reduce contamination risk. If required, they further add sterilized, heat sensitive components, such as antibiotics, into the medium before pouring or plating. For solid media, gel formation occurs between 32°C and 40°C. While using semi-automatic Petri dish fillers, the filling volume must be periodically monitored for quality control. 
6. Labeling: Researchers label all sterile broth bottles and filled Petri plates. A label generally contains information about the media type, expiration time, and storage conditions.

Challenges and Advancements in Culture Media Development

Although researchers continue to formulate new and improved culture media, identifying specific substances for ideal bacterial growth is not easy. “The trickiest part is trying to figure out what form the bacteria need the nutrients in and providing molecules that have the exact form,” said Lostroh. For example, all bacteria require nitrogen to grow, but some may need nitrogen sources in different forms than others. For instance, some bacteria can only obtain nitrogen from inorganic ammonium sources while others do so from nitrogen-containing protein molecules.

Advancements in industrial biotechnology and pharmacology increase the demand for new formulations of culture media, based on new nutrient composition needs. These formulations focus on increasing microbial yield, reducing cost, and having minimal environmental impact. A relatively new cyanobacterial extract-based formulation has enabled the versatile growth of diverse bacteria originating from different environments.¹⁵

Scientists have classified bacterial strains (e.g., Alphaproteobacteria members) that are generally not easy to culture in laboratory conditions as viable but non-culturable (VBNC) microbes. To combat this limitation, they formulated several strategies such as co-culturing these species with growth-promoting microorganisms or in situ cultivation that promotes bacterial growth in their native habitat.¹⁶ Researchers developed innovative devices for in situ cultivation, such as a diffusion chamber that allows nutrients from the natural environment to migrate to the site of bacterial inoculation.¹⁷ This strategy enabled culturing of bacteria, such as the phylum Verrucomicrobiota, which are generally difficult to grow in a laboratory.

“One of the new technologies that we are trying would be using something called a cell sorter,” stated Lostroh. Here, researchers mix a bacterial sample with pure water and place it in a fluorescence-activated cell sorting (FACS) machine. The machine sorts one bacterium at a time into separate tubes, subsequently adding media in each tube to promote growth. The key advantage of this technique is isolating and cultivating slow growing bacterial cells, which would have been difficult to culture conventionally.

“We are getting better and better at designing media that reflect the natural environment, and we are learning to culture bacteria that we’ve not been able to culture ever before, in part because of this sorting technique,” said Lostroh. The continual technological breakthroughs benefit new culture media development and assist bacterial growth more efficiently.