Exploring the Panorama of Bacterial Tradition Media

Article reviewed by Phoebe Lostroh, PhD from Colorado School.

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What Is Tradition Media?

Tradition media are nutrient sources that allow scientists to develop microorganisms in vitro.¹ “Microbial tradition media is sort of a broth or a soup that comprises plenty of vitamins equivalent to proteins and sugars,” defined Phoebe Lostroh, a microbiologist at Colorado School. 

 

Transient historical past of tradition media in microbiology

Louis Pasteur ready the primary liquid bacterial tradition medium in 1860, containing yeast, sugar, ashes, and ammonium salt.² Whereas growing this, he noticed that particular chemical options of the tradition medium might promote or impede the expansion of sure microorganisms. The competitors between totally different microbes for vitamins in a medium led to some species outgrowing the others. 

In 1881, Robert Koch famous that micro organism grew optimally when inoculated in broth containing recent meat extract or beef serum.² He additionally encountered difficulties isolating pure cultures from liquid media, which impressed him to develop stable media that enabled the isolation of particular bacterial strains. Koch combated a number of failures when he examined starch paste, coagulated egg albumin, and potato slices as solidifying brokers, till he lastly discovered {that a} gelatinous substance referred to as agar solidified the broth media. 

Julius Richard Petri designed a round “tradition field” for stable media in 1887, which helped scientists isolate bacterial colonies with decreased contamination.³ Microbiologists nonetheless use the eponymous Petri plates immediately to develop and retailer bacterial and fungal cultures.

Necessary Tradition Media Elements for Bacterial Progress

“The principle elements are molecules that micro organism can eat as meals. So, that might be issues like amino acids and sugars equivalent to glucose. Some micro organism can even eat alcohol, like ethanol,” mentioned Lostroh. For optimum bacterial progress, a tradition medium should include appropriate vitamins and inorganic minerals (e.g., iron), have the correct pH, and be incubated at a particular temperature. For fast progress, researchers have a tendency to pick bacterial progress parameters in accordance with the microbes’ pure habitats. “As an illustration, in the event that they reside on a human physique, they’d reside at 37°C as a result of that’s our physique temperature. Micro organism that come from chickens may reside at a better temperature,” defined Lostroh. 

The important thing components of a tradition medium could embrace the next.²

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

Carbon and nitrogen

A carbon supply is important for micro organism to provide their very own carbon molecules equivalent to proteins, nucleic acids, and fat.² The important thing sources of inorganic and natural carbohydrates are carbon dioxide and sugars or alcohols, respectively. Nitrogen is one other necessary element as a result of it aids protein synthesis.⁴ Researchers generally use proteose-peptone and tryptone as nitrogen sources in tradition media.

Scientists additionally choose tradition media elements and progress situations within the laboratory to satisfy bacterial vitality. For instance, many micro organism derive vitality from natural compounds equivalent to carbohydrates, whereas sulfate-reducing micro organism require sulfate-containing chemical compounds within the media to behave as terminal electron acceptors.⁵

Progress elements

Progress elements enhance bacterial multiplication when added to the tradition medium in small portions.² There are totally different progress issue sorts equivalent to purine and pyrimidine bases, nutritional vitamins, and amino acids that affect the expansion of various bacterial strains. For instance, guanine and amino acids, particularly valine and glutamic acid, are important for Leuconostoc mesenteroides progress.⁶ L. mesenteroides can’t synthesize these elements on account of its truncated ilv and leu operons, that are important for the biosynthesis of branched-chain amino acids equivalent to valine. Due to this fact, supplementation of those components in tradition media promotes progress. 

Tradition Media Sorts

Microbiologists classify tradition media based mostly on consistency or bodily state as stable, liquid (broth), and semisolid.⁸ Cultural media consistency is necessary for various functions equivalent to pure tradition preparation and motility evaluation. 

Desk 1: Completely different media sorts based mostly on consistency ^9,10

Primarily based on the nutrient composition, scientists sub-classify tradition media as easy or advanced.¹¹ Easy media equivalent to nutrient broth and peptone water are basic goal media that help the expansion of non-fastidious micro organism that don’t require any particular nutrient complement or enrichment supply to develop. Microbiologists use these media for basic isolation and progress experiments.⁸

Complicated media, equivalent to tryptic soy broth, are wealthy in vitamins that assist within the growth of fastidious micro organism which have particular dietary wants for progress.¹¹ Enriching media with substances equivalent to blood and serum can help fastidious or tough to develop microorganisms. For instance, blood agar promotes hemolytic bacterial progress.² In addition to progress promotion, such media can also elevate attribute bioactive compound manufacturing by way of microbial fermentation. 

Artificial media are a sort of advanced media comprised of chemically-defined substances. They usually include identified portions of sugar from glycerol or glucose and nitrogen from ammonium salt or different nitrates.

Researchers use selective media to develop particular microbes whereas inhibiting the expansion of others by including components equivalent to dyes, antibiotics, and bile salts, and making pH changes. For instance, scientists developed eosin-methylene blue (EMB) agar medium, which selectively grows Gram-negative bacterial strains and inhibits Gram-positive strains.¹²

Desk 2: Selective media for particular bacterial progress ²

Tradition Media Preparation

Microbiologists put together bacterial progress media by dissolving powders comprised of yeast cells and beef extracts in distilled water at impartial pH. Subsequently, they sterilize the media in an autoclave to stop undesirable bacterial progress.¹³

The important thing steps of tradition media preparation embrace the next.¹⁴

1. Preliminary preparation: Earlier than getting ready tradition media, it is very important have sterilized containers (e.g., Petri plates and conical flasks) and the required media elements. 
2. Rehydration: Scientists dissolve the tradition media elements (powder medium) in distilled water. This rehydration ensures that necessary hydrolysis reactions happen. For instance, starch is a comparatively giant molecule to move by the plasma membrane of a cell, so some micro organism produce amylase to transform it to the smaller glucose molecule, thereby enabling it to move by. This subsequently aids bacterial progress.
3. Heating: Media containing agar should be mildly heated and stirred completely for full dissolution. If wanted, the pH is adjusted for optimum bacteria-specific progress.
4. Sterilization: Tradition media is commonly sterilized by autoclaving at 121°C for quarter-hour at 15 PSI. Filtration is one other method used to sterilize liquid tradition media, significantly if selective media components are vulnerable to warmth. For this method, scientist use membranes fabricated from 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 different aseptic situations to cut back contamination danger. If required, they additional add sterilized, warmth delicate elements, equivalent to antibiotics, into the medium earlier than pouring or plating. For stable media, gel formation happens between 32°C and 40°C. Whereas utilizing semi-automatic Petri dish fillers, the filling quantity should be periodically monitored for high quality management. 
6. Labeling: Researchers label all sterile broth bottles and stuffed Petri plates. A label typically comprises details about the media sort, expiration time, and storage situations.

Challenges and Developments in Tradition Media Improvement

Though researchers proceed to formulate new and improved tradition media, figuring out particular substances for supreme bacterial progress is just not simple. “The trickiest half is attempting to determine what kind the micro organism want the vitamins in and offering molecules which have the precise kind,” mentioned Lostroh. For instance, all micro organism require nitrogen to develop, however some might have nitrogen sources in numerous kinds than others. As an illustration, some micro organism can solely acquire nitrogen from inorganic ammonium sources whereas others accomplish that from nitrogen-containing protein molecules.

Developments in industrial biotechnology and pharmacology improve the demand for brand spanking new formulations of tradition media, based mostly on new nutrient composition wants. These formulations give attention to growing microbial yield, decreasing price, and having minimal environmental affect. A comparatively new cyanobacterial extract-based formulation has enabled the versatile progress of various micro organism originating from totally different environments.¹⁵

Scientists have categorised bacterial strains (e.g., Alphaproteobacteria members) which can be typically not simple to tradition in laboratory situations as viable however non-culturable (VBNC) microbes. To fight this limitation, they formulated a number of methods equivalent to co-culturing these species with growth-promoting microorganisms or in situ cultivation that promotes bacterial progress of their native habitat.¹⁶ Researchers developed revolutionary gadgets for in situ cultivation, equivalent to a diffusion chamber that permits vitamins from the pure surroundings emigrate to the location of bacterial inoculation.¹⁷ This technique enabled culturing of micro organism, such because the phylum Verrucomicrobiota, that are typically tough to develop in a laboratory.

“One of many new applied sciences that we try could be utilizing one thing referred to as a cell sorter,” said Lostroh. Right here, researchers combine a bacterial pattern with pure water and place it in a fluorescence-activated cell sorting (FACS) machine. The machine kinds one bacterium at a time into separate tubes, subsequently including media in every tube to advertise progress. The important thing benefit of this method is isolating and cultivating sluggish rising bacterial cells, which might have been tough to tradition conventionally.

“We’re getting higher and higher at designing media that replicate the pure surroundings, and we’re studying to tradition micro organism that we’ve not been in a position to tradition ever earlier than, partially due to this sorting method,” mentioned Lostroh. The continuous technological breakthroughs profit new tradition media growth and help bacterial progress extra effectively.