CULTURE MEDIA: HISTORY, FUNCTION, TYPES, PREPARATION - BIOLOGY - 2025

The culture media are special nutritional preparations for recovery, isolation and maintenance of bacterial and fungal microorganisms. These media can be solid, liquid, or semi-solid.

Louis Pasteur was the first to show that in a broth made with boiled pieces of meat, it was used for bacteria to reproduce in large numbers, to the point of clouding the broth. In this sense, Pasteur's meat broth is considered the first liquid culture medium used.

Raw and prepared culture media (solid and liquid). Source: Flickr

Then Robert Koch, with the help of his collaborators Julius Richard Petri and Walter Hesse, made great strides. The first designed the Petri dish, which is still used today; and the second occurred to him to substitute gelatin for agar-agar to prepare solid culture media, which was very relevant, since gelatin was degraded by some microorganisms.

At present there are many classes of culture media with different purposes, therefore, these are classified according to their function: among the most important we can mention the nutritive, selective, differential, transport, enrichment, and counting culture media. colonies, maintenance and for susceptibility testing.

Some culture media are special for observing chemical reactions, being very useful for the identification of the microorganism involved. Among them we can mention: Kligler medium, MIO, LIA, citrate, among others.

History

The first culture medium was prepared by Louis Pasteur when he tried to show that microbial life was not the product of spontaneous generation, but that microorganisms could multiply and also that they came from the air.

He prepared a broth with pieces of meat and observed that after a few days after being exposed to the air, it turned cloudy and there was an appreciable amount of microorganisms in the broth. At the same time, another broth with pieces of meat previously boiled and hermetically sealed remained translucent as the days passed.

This caught the attention of many researchers and they realized that these microorganisms were responsible for decomposing the meat and also causing some diseases.

For this reason, it was essential to create a way to reproduce these microorganisms in the laboratory in order to study them further.

In this sense, Robert Koch made an invaluable contribution to the improvement of certain laboratory techniques, especially those related to bacterial isolation, as he introduced the concept of a solid culture medium.

At first he used potato slices as a solid medium but later he added gelatin to the meat broths with better results. However, there were times when the jelly would melt and turn into a liquid culture. Today it is known that this happens because some bacteria are capable of hydrolyzing gelatin.

It was then that one of his collaborators came up with the idea of ​​using agar-agar, a compound his wife used to thicken her sweets.

This rudimentary culture medium has gradually become more sophisticated, until it reaches the culture media that are known today.

Composition

Each medium has a different composition, but it is essential that it contains the specific nutrients for a good development of the type of microorganism that is sought.

It may also contain specific chemical substances that help reveal the metabolic pathway that a certain strain has, or that show the presence of certain enzymes.

Another important element is the use of buffer substances. These help maintain the osmotic balance of the media, as well as the pH.

They can also contain carbohydrates and a pH indicator to show added sugar fermentation. A color change of the medium will be observed if there is acidification generated by fermentation.

Some culture media contain inhibitory substances. Depending on the substance used, the growth of some microorganisms will be restricted and that of others will be favored.

Types of culture media

Culture media are classified according to various criteria. These are: according to its consistency, its composition and its function.

- According to its consistency

Liquids

They do not contain agar-agar. Bacterial or fungal growth is evidenced by the turbidity of the broth, which is originally translucent.

Solid

They contain between 1.5 to 2% agar-agar. The solidified mixture has a surface that resists the fine movement of the platinum handle without breaking it.

Semi-solid

They contain approximately 0.5% agar-agar, therefore, it is an intermediate state between liquid and solid. Ideal in media that serve to see motility. They are also recommended for the conservation of strains, as they maintain humidity for much longer.

Biphasic

They are media that are prepared in such a way that there is a solid phase and on this a liquid medium. Widely used for blood cultures.

- According to its composition

Natural growing media

They are substances taken directly from nature to cultivate bacteria, providing them with an environment as close to how they normally develop in the ecosystem. Example, milk, juices, diluted blood, serum, etc.

Synthetic culture media

They are the most widely used nowadays, they are the dehydrated media that we acquire in commercial houses and whose entire chemical composition is known, since they have been strategically designed according to the type of microorganism to be isolated.

Semi-synthetic culture media

It is the combination of a synthetic medium to which a natural element is added to enrich the medium.

Cell culture media

They are special media for growing viruses, as these microorganisms are unable to survive outside cells, they must contain tissue or living cells from an animal or plant.

Example: monkey kidney cell cultures or embryonated eggs.

- According to its utility

Nutritive, selective, differential, transport, enrichment, identification, colony quantification, maintenance and susceptibility testing media. They will be described later.

Function

Regardless of the type of culture medium, they all have something in common and that is that they facilitate or promote the reproduction of certain microorganisms. The difference lies in the composition of each of them, which is a determining factor for the final utility they will have.

Each of the existing culture media is strategically designed for the specific function for which it was created, that is, they all have a foundation that governs the guidelines of their specific function.

It should be noted that the culture media once sown must be subjected to conditions of temperature and oxygen appropriate to the type of bacteria or fungus to be isolated.

For example, if you want to isolate mesophilic anaerobic bacteria, you could use blood agar and incubate under anaerobic conditions (without oxygen) at 37 ° C for 48 hours.

Now, if a fungus needs to be isolated, Sabouraud agar with antibiotics is used. It is incubated in aerobiosis, at room temperature for several days, since the latter are slow growing.

Nutritious Simple Culture Media

As its name indicates, these culture media contain nutritious substances, such as sources of vitamins, amino acids, nitrogen and carbon, among them we can mention: meat extract or yeast extract, corn starch, pancreatic digest, peptones, glucose, among others.

They also contain other components that provide the environment with an osmotic balance, since most crops require a pH close to 7.0. These elements can be: sodium chloride, disodium phosphate, among others.

The diluent is distilled water and the solid media have agar-agar.

The purpose of these culture media is to recover the bacterial or fungal microbiota that exists in a given sample. It does not discriminate between microorganisms, since it is capable of growing a large number of bacteria, both Gram positive and Gram negative, as well as yeast and mycelial fungi.

They are recommended for seeding samples from normally sterile sites. However, they are not suitable for fastidious microorganisms.

They are also useful for the maintenance of strains, as long as they do not contain glucose.

Enriched culture media

If blood or heated blood is added to the simple nutritive media, they become enriched media (blood agar and chocolate agar respectively).

These media are very useful for seeding normally sterile samples, for rescuing strains that are weak, and for isolating nutritionally demanding microorganisms.

Selective culture media

Selective culture media in addition to containing essential nutrients for the growth of certain microorganisms of interest, inhibitory substances are also added, such as antibiotics, antifungals, dyes, bile salts, among others.

The inhibiting substances are intended to reduce the variety of strains that can grow, favoring the growth of a particularly special group that is to be rescued.

Example: EC broth (special for total and fecal coliforms) or Sabouraud agar with antibiotics (specific for fungi).

Differential culture media

Differential media contain nutritional elements necessary for the growth of a specific group of microorganisms and also contain substances that in the presence of certain microorganisms will be metabolized or degraded.

That is, they will produce chemical reactions that in one way or another will be evidenced in the culture medium.

Some reactions alkalize or acidify the medium and, thanks to the presence of a pH indicator, these changes can be evidenced through a color shift in the medium and in the colony.

Therefore, among a large group of bacteria that will be able to grow in this medium, those that metabolize or degrade the substance and those that do not, simply by observing the color of the colony and the medium, will be distinguished.

For example, blood agar allows one to distinguish bacteria that cause beta hemolysis (clear halo) from those that cause alpha hemolysis (greenish halo) and those that do not produce hemolysis.

Selective and differential media

An example of this is what happens in MacConkey agar. It is selective since it only allows the growth of Gram negative bacilli; and it is differential, since lactose-fermenting bacteria (fuchsia colonies) can be distinguished from non-fermenting bacteria (pale pink or colorless).

Transport culture media

As their name suggests, they are means used to transport samples that have been taken in a more or less distant place to the laboratory that will process the sample. The transport medium keeps the sample in the best conditions so that reliable results are obtained.

These culture media have very special characteristics, since they cannot be exceeded in nutrients, since the bacterial population that is present is required to remain viable, but without increasing in number.

They are generally semi-solid media, allowing the sample to remain hydrated. However, no compromise should be made in getting the sample to the laboratory as soon as possible. Examples of means of transport: Stuart medium, Cary Blair and Amies.

Enrichment culture media

These culture media are liquid. They are used to rescue specific pathogens that at any given time may be present in a sample in minimal quantity.

It is also useful to rescue a pathogenic strain that may be weak from any previous treatment received. Ex: peptone water, thioglycollate broth and selenite broth.

These media have inhibitory substances that prevent the growth of the accompanying microbiota, and specific nutrients that favor the development of the microorganism of interest.

Culture media for identification purposes

These media contain substances that can be chemically metabolized by certain bacteria, producing chemical reactions that show the presence of specific enzymes or metabolic pathways.

Therefore, they are used as biochemical tests that aid in the recognition of the genus and the species of a particular group of strains. Example: The Kligler medium shows if the microorganism is capable of fermenting glucose and lactose, if it produces hydrogen sulfide and gas.

This medium contains revealing substances that allow the reaction to be observed, such as the pH indicator, and iron ions.

This simple test can differentiate two large groups of bacterial microorganisms, such as the bacteria belonging to the Enterobacteriaceae family from the so-called non-fermenting bacteria.

Media for counting colonies

These are simple, non-selective media that serve for the quantification of a microbial population, such as the standard count medium. The type of microorganism that will grow in this medium will depend on the temperature and oxygen conditions that are established.

Culture media for susceptibility testing

The standardized medium for this purpose is Müeller Hinton agar, this medium is ideal for evaluating the behavior of different antibiotics against an isolated pathogenic microorganism.

It is especially useful in undemanding bacteria, while in fastidious bacteria it can only be used if it is supplemented with blood.

Culture media for maintenance

The purpose of these means is to reproduce the microorganism and also to maintain the viability of the bacteria or fungus for as long as possible and also to preserve its physiological functions.

An important characteristic is that this type of medium should not contain glucose, because although it is an element that provides rapid growth, its fermentation also produces acids that decrease the life of the microorganism.

Some laboratories need to keep certain microorganisms viable for later use in research studies, internal controls, or for educational purposes.

Preparation

Currently there are many commercial brands that distribute the different culture media. The media comes in freeze-dried or dehydrated form, contained in airtight jars and protected from light.

Each medium comes with a label specifying the name of the medium, its components, lot number and how much to weigh to prepare a liter of culture medium.

Distilled water is used as diluent. The weighed quantity is dissolved in one liter of distilled water until the mixture is homogenized. Most media are autoclaved, at 15 pounds pressure, 121 ° C temperature, for 15 minutes.

Liquid media are autoclaved already distributed in their respective working tubes, while solid media are autoclaved in Erlenmeyer flasks.

The latter are allowed to stand until they reach a temperature of 55 ° C and are served in the Petri dishes inside a laminar flow hood or near the Bunsen burner. They are left to solidify and stored in the refrigerator upside down.

There are also solid culture media that are distributed in tubes, allowing them to solidify either in studs (straight) or in flute beak (inclined).

Before using any prepared culture medium, whether solid or liquid, it must be tempered before sowing the sample.

Importance

Culture media are undoubtedly a very valuable work tool for microbiologists, since they make it possible to recover the infectious agent that at a certain moment may be affecting an individual or contaminating a food, an environment or a surface.

In this sense, it can be said that microbiology has different fields, among them is clinical, industrial, environmental, food microbiology, among others, and culture media are used in all of them.

Of course, the type of medium used in each case may vary according to the needs and the type of sample processed. The group of microorganism sought also influences.

The isolation of the pathogenic microorganism or the cause of any contamination is essential to be able to implement an effective treatment or adopt procedures that help eliminate the contaminant in question.

In the case of clinical microbiology, it is not only important to isolate the microorganism and identify it (know the genus and species), but the antibiogram must also be performed.

This study, which also uses a culture medium, will allow us to say which antimicrobial is sensitive and which is resistant or, in short, which can be used as a treatment and which cannot.

Therefore, culture media in general cannot be lacking in a microbiology laboratory, whatever the area.

Finally, it can be said that the culture media have made it possible to investigate various aspects of both bacteria and fungi.

Quality control of culture media

The preparation and use of culture media should not be done lightly. In each laboratory there must be a department that applies quality control protocols to the media, each time new batches are prepared, and thus ensure that they are properly prepared, sterile and functional.

To assess their sterility, one or two media are taken at random from each batch and incubated at 37 ° C for several days (there should be no growth). Duly cultured and viable ATCC (American Type Culture Collection) reference strains are used to verify their function.

Disposal of culture media

After using the culture media, it must be disposed of in such a way that it does not contaminate the environment.

For this, the material is sterilized in an autoclave before it is discarded. Subsequently, the material is removed from the glassware. The latter is then washed, dried, sterilized and stored for later use. In case of using disposable plates, these are sterilized and later discarded in special bags.

References

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