CULTURE MEDIA PREPARATION: OBJECTIVES AND STEPS - BIOLOGY - 2025

The preparation of culture media is a routine methodology used in laboratories for the growth of desired microorganisms. Culture media are solid, liquid or semi-solid preparations that have all the necessary nutrients for the development of a microbial population.

In general, the means for cultivating microorganisms are rich in proteins and amino acids and usually contain some component that favors the growth of the organism to be studied, such as vitamins, blood, serum, among others.

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There is no general or universal culture medium, since its composition varies depending on the needs of the microorganism of interest. Some bacteria can grow in any culture medium, but others have special requirements.

What does it consist of?

Microorganisms, such as fungi and bacteria, cannot be studied individually because of their tiny size. For this reason, they must be cultivated in artificial means that allow a significant increase in the population.

For example, if we want to study bacteria we have to provide them with the right conditions so that they can proliferate and form a colony (which can be observed with the naked eye).

The preparation of the culture media varies widely depending on the type of microorganism to be cultivated. Before preparing it, it is necessary to know the basic nutritional needs of the work organism.

The most common components used in culture media will be described below to get a general idea of ​​their preparation:

Agar

It is used in cultures as a gelling agent and is added when looking for a solid or semi-solid medium. The first solidifying agent used in the preparation of media was gelatin, but in 1883 agar was introduced to the world of bacteriology by W. Hesse.

Bacteriological agar has as its main component a polysaccharide with complex branches extracted from algae. This compound is used as a thickener in common foods such as ice cream and jams.

It is a very valuable element in microbiology for several reasons. Mainly because microorganisms cannot degrade it, it liquefies at a temperature of 100 ° C and remains in a liquid state until it reaches 45 ° C or less.

In case you want to prepare a solid medium, the agar concentration should be around 1.5%, while the semisolids should be prepared from 0.3 to 0.5%.

Fluids

The cultivation of pathogenic organisms needs body fluids so that they can develop as they would in their natural environment. For this reason, whole or defibrillated blood is added. The fluid is taken from a healthy animal and, once sterilized, it is added to the culture medium.

Excerpts

They are obtained from different animal parts (such as meat or liver) or vegetables (seeds) and are processed to obtain a solid concentrate in the form of a paste or powder. The most common are yeast, malt, and meat.

Peptones

These organic compounds are obtained by enzymatic or chemical hydrolysis of animal or plant tissues. The purpose is to add content rich in amino acids, which are the fundamental units of proteins.

Shock absorbers

The "buffers" or buffer systems avoid sudden changes in pH and help maintain the optimal range that the body tolerates.

Most organisms can thrive well at a pH of 7, although some bacteria prefer alkaline media. However, there are bacteria that resist pH variations between the values ​​of 6 and 9.

In pH-sensitive species, the damage is not produced by the excessive amount of hydrogen or hydroxyl ions, but by the increase of weak acids or bases that can enter the cell.

Likewise, pH indicators are added to monitor it and avoid deviations caused by fermentation or other processes.

goals

The main objective when preparing a culture medium is to add all the necessary components to allow the successful development of the organism to be isolated. The most effective combination of components and nutrients to achieve the desired medium must be identified.

Both the preparation and storage of the medium are critical to ensure successful growth, as the composition of the medium and the availability of nutrients depend on these steps.

It must be taken into account that the cultivation of microorganisms is a task that is affected by several factors external to the culture medium, such as the intensity of light received, temperature and level of acidity or alkalinity of the medium. Therefore, each of these variables must be taken into account.

Media types

Based on its composition

Based on their composition, there are three main types of crops: natural or empirical, semi-synthetic, and defined synthetic or chemical media.

Natural environment

In natural environments the exact composition is unknown. These include ingredients such as milk, diluted blood, vegetable juices, extracts and infusions of meats and peptones. For economic reasons, inexpensive components such as soy extract, whey, molasses, etc. are often added.

Semi-synthetic media

It is called a semi-synthetic medium if its composition is partially known. Any medium that contains agar becomes a semi-synthetic medium.

Among them we have potato dextrose agar, czapek-dox agar, oat agar, meat peptone agar, among other examples.

Synthetic or chemical defined medium

In this case the composition of the medium - in terms of the amount of sources of carbon, nitrogen, sulfur, phosphorus and any other growth factor needed - is fully known. It is very useful if you want to obtain reproducible results for other researchers.

For the so-called "microorganisms with special growth requirements" it is necessary to add the necessary components. An example of this type is the Lactobacillus.

Based on the type of microorganism

Similarly, there is another classification for culture media based on the type of microorganism that can grow on it. Following this principle we have the following general, enrichment, selective and differential means. Each one is described below:

General media

These support the development of a wide variety of microorganisms. If any organism needs special conditions for its growth, it will not be able to develop successfully in this type of culture.

Enrichment media

The enrichment media supports the growth of a certain type of microorganism, but no substance has been added to prevent other types of microbes from growing in it.

Selective media

They look for the specific growth of a microorganism, call it fungi, bacteria, protozoa, among others. To do this, they inhibit the development of others.

To achieve this objective, chemical compounds that are deadly for a wide group of microorganisms and harmless for the organism of interest can be added or energy sources that can only be assimilated by the target microbe can be added.

Selective media are used when taking medical samples in order to cultivate a pathogenic microorganism. Here it is necessary to promote the growth of the pathogen and inhibit the development of the normal microbial flora from the patient.

Bismuth sulfite agar, for example, does not allow the growth of gram-positive bacteria and a large number of bacteria found in the gastrointestinal cavity. For this reason, it is used to cultivate the gram-negative bacteria that cause typhoid fever, Salmonella typhi in fecal samples.

Differential media

This type uses some diagnostic characteristic of the organism of interest (peculiarities in its metabolism, for example) to be able to identify them against another species that grows in the same environment.

Both differential media and selective media are very useful in the area of ​​clinical microbiology and public health, since these disciplines need to detect the presence of specific microorganisms related to pathologies or poor hygiene conditions.

Indicator substances can be added to the culture that give a distinctive characteristic to the sought colony. For example, lactose and a pH indicator are added to agar-eosin-methylene blue (abbreviated EMB) and MacConkey agar.

Thus, when a colony develops in these media with the ability to ferment lactose and produce aldehydes, they can be observed in a special color.

Steps

Currently culture media can be purchased in lyophilized form. Therefore, the preparation is easier and the only thing left to do is rehydrate the product. The content must be weighed (taking into account the final quantity to be prepared) and dissolved in distilled water following all the indications of the product.

The content of the liquid media must be divided into the desired containers (Petri dishes, tubes, etc.) for subsequent sterilization. To distribute the solid medium it is necessary to melt it using a microwave or subjecting the material to a water bath. The pH of the medium must be adjusted.

Usually the agar is used in test tubes or Petri dishes. If the agar solidifies in an inclined position, with the proper angle so that the final terminal edge is diagonal, this arrangement is known as beak or inclined tubes. When the agar solidifies in a totally vertical position, it is called "deep".

After sterilizing the media - using an autoclave - they are allowed to cool. These must be handled in an environment free of microorganisms, the most common is to work with a lit lighter that ensures an aseptic environment in their vicinity.

References

1. Celis, JE (2006). Cell biology: a laboratory handbook (Vol. 2). Elsevier.
2. Finegold, SM, Bailey, WR, Baron, EJ, Fineglod, SM, & Scott, EG (1991). Bailey Scott: microbiological diagnosis. Pan-American Medical.
3. Olivas, E. (2004). Manual of Practices of Microbiology I and II and Parasitology. Autonomous University of Ciudad Juarez.
4. Schlegel, HG, & Zaborosch, C. (1993). General microbiology. Cambridge University Press.
5. Tortora, GJ, Funke, BR, & Case, CL (2007). Introduction to microbiology. Panamerican Medical Ed.