ISOLATION OF MICROORGANISMS: TECHNIQUES AND IMPORTANCE - BIOLOGY - 2024

The isolation of microorganisms involves a set of techniques used to extract and separate the species of microbes of interest from their natural habitat to an in vitro habitat. These techniques are a set of many basic and necessary tools for microbiological studies.

Most of the microorganisms that are known and that have been defined by science, are those that have been able to be isolated and kept in containers that simulate, in part, the intrinsic conditions of the places where they live.

Typical image of colonies of bacteria-like microorganisms isolated on a solid medium inside a Petri dish (Image by nadya_il at www.pixabay.com)

Perhaps one of the first men to practice the isolation of microorganisms was Anton Van Leeuwenhoek (1632-1723), who collected and isolated samples of microbes from a large number of places and ecosystems to observe them carefully under the hundreds of microscopes that he designed..

However, it was not until the times of the scientists Louis Pasteur and Robert Koch, during the course of the 19th century, that rigorous practices began to be carried out that served to isolate specific microorganisms, all in order to be able to study them in detail..

Unlike Leeuwenhoek, these researchers focused on isolating defined species from the other species of microbes in the environment. In addition, they were interested in keeping them alive as long as possible outside their natural environment.

Today, precise techniques have been developed for the isolation and growth of many different microorganisms obtained from almost any environment on the biosphere.

Microorganism isolation techniques

All microorganism isolations begin with the collection of a sample in the wild where the microorganisms of interest are found. These places can be wounds in animal or plant tissues, soils or substrates, puddles, seas, surfaces such as skin, etc.

The sample is taken by touching or supporting a container that has a medium with the appropriate requirements for the growth of microorganisms on the surface from which it is desired to isolate. In this container you will get what is known as a "culture" of microbes.

Generally, the first crop to be obtained from natural habitats is undoubtedly a "mixed crop", that is, it is composed of a large number of different species of microbes.

However, most species of microorganisms can be isolated from each other in the laboratory, seeking to obtain microorganism cultures where only the species of interest grows or, in other words, obtaining “pure cultures”.

In essence, the process that is carried out to obtain “pure cultures” is what is known as “isolation of microorganisms”.

There are a large number of techniques for the isolation of microorganisms, and there are even some specific to a certain type of microorganism in particular. In other cases, it is possible to obtain a pure culture only by collecting the sample from the natural environment.

Among the most used isolation techniques to separate a species of interest found in mixed culture media are:

Scratches or streaks

Perhaps this is the most widely used method for isolating microorganisms. This technique consists of preparing a sterile solid medium with all the nutritional compounds necessary for the growth of the microorganism in a glass container, such as a Petri dish.

Using a fine instrument, generally pointed, the microorganism to be isolated in the mixed culture is touched, then, in the sterile solid medium, the tip of the instrument with which the microorganism was touched is started to slide from side to side throughout the entire license plate.

This is done intensively back and forth across the surface of the solid or agarized medium, as if it were a zig-zag. It is usually done until about a third of the diameter of the agar on the plate is covered.

Fusion with the medium or coating

For this method, a dilution of the medium where the collected microbes live is carried out to the point where only a few hundred cells remain for each milliliter of the medium where they were diluted.

From this dilution a few milliliters are taken and mixed with the medium that will be added to the container before it solidifies. As a mixture is made between the agarized medium and the liquid medium where the microorganisms are, they remain immersed in the medium and are only visible until they proliferate as a colony.

As they develop as a colony, it is easier to separate them from the rest of microorganisms with other methods such as scratching, for example.

Serial dilutions

This method consists of making serial dilutions of the medium where the microorganisms are found. An example of this are the dilutions that are made to purify Lactococcus lactis or Lactobacillus acidophilus, bacteria responsible for the production of cheese and yogurt.

Approximately 1 milliliter is taken from a tube containing sour milk or previously fermented yogurt and this milliliter is inoculated into sterile milk without microorganisms. Later, approximately a milliliter of said milk is taken and the process is repeated.

This is repeated approximately three or four consecutive times, making it very likely to obtain Lactococcus lactis or Lactobacillus acidophilus in a medium isolated from contaminants that may represent other microbes.

Detailed photograph of the site and typical materials for isolating microorganisms (Image by Sintija Valucka at www.pixabay.com)

Enrichment procedure

This methodology is achieved by growing the microorganisms in culture media with conditions that stimulate or facilitate the growth of the species of interest and, in many cases, under conditions that inhibit the growth of other contaminating microorganisms.

Bacteria of the genus Salmonella grow in culture media enriched with selenite, since these microorganisms transform selenite into selenium in order to metabolize it. The selenite in the medium makes it difficult to assimilate the nutrients for microorganisms other than salmonellas.

Unique or exclusive technique

This is perhaps the most difficult and least effective technique for isolating microbes. This involves placing a drop of the medium (sample) where the microorganisms are housed on a sterile coverslip, and then placing it on the microscope stage.

Later, while observing, a single cell is removed with the help of a sterile micro-pipette. The drop is placed on another sterile coverslip that is incubated at the appropriate temperature for the microorganism. Finally, it is observed again under the microscope to show growth.

If on re-observation new cells have developed from the individual cell that was taken, these are added to sterile culture medium to obtain a fully isolated pure culture.

Custom techniques

There are countless different microbes on planet Earth that are scattered throughout almost all known ecosystems. Some microorganisms are known as Extremophiles and require unique conditions for their development and growth.

These extreme conditions are both advantageous and disadvantageous for isolation, since, although they only allow the growth of these microorganisms, they can be difficult to recreate in vitro.

Importance

The isolation of microorganisms has represented one of the most important advances for the area of ​​science and medicine. This has allowed humanity to study and develop effective treatments against different microbial pathogens.

Today it is known for sure that microorganisms form an essential part of all ecosystems, so achieving the isolation of some of them with relative importance for man allows researchers to study them intensively, in order to understand in depth its role in each ecosystem.

References

1. De Kruif, P. (1996). Microbe hunters. Houghton Mifflin Harcourt.
2. López, MJ, Nichols, NN, Dien, BS, Moreno, J., & Bothast, RJ (2004). Isolation of microorganisms for biological detoxification of lignocellulosic hydrolysates. Applied Microbiology and Biotechnology, 64 (1), 125-131.
3. Spigno, G., Tramelli, L., Galli, R., Pagella, C., & De Faveri, DM (2005). Biofiltration of dichloromethane vapours: isolation of microorganisms.
4. Tresner, HD, & Hayes, JA (1970). Improved methodology for isolating soil microorganisms. Appl. Environ. Microbiol., 19 (1), 186-187.
5. Willey, JM, Sherwood, L., & Woolverton, CJ (2009). Prescott's principles of microbiology. Boston (MA): McGraw-Hill Higher Education.