STRAIN (MICROBIAL): CHARACTERISTICS, IDENTIFICATION, ISOLATION - BIOLOGY - 2025

A microbial strain is the set of descendants from a single microbial isolate, which is grown in a pure medium and is usually composed of a succession of organisms that derive from the same initial colony.

A strain also represents a set of individuals of a population of a microbial species that share certain phenotypic and / or genotypic characteristics that slightly differentiate it from others of the same species, but whose differences are not enough to categorize them as different species.

Photograph of a Petri dish with solid culture medium supplemented with antibiotics where resistant microbes grow (Source: Microrao / CC BY-SA (https://creativecommons.org/licenses/by-sa/4.0) via Wikimedia Commons)

The strain is the "basis" for any microbiological study, as it guarantees scientists that the parameters and characteristics that are investigated about a species of microbe are specific only to that species. In addition, it allows them to ensure, in a certain way, the reproducibility of the investigations.

For example, for taxonomic studies in microbiology the first objective is to obtain "the strain" of the organism to be classified, since in this way it is possible to precisely define which are each of the taxonomic characteristics that differentiate this subset within of a population of one species of any other species of microbe.

The strain allows a species of microbe to be kept alive and isolated in vitro for long periods of time, that is, far from its natural environment. Strains of many microorganisms of different types can be obtained, such as bacteria, fungi, viruses, protozoa, algae, among others.

For the maintenance of the strains, they must be kept in strict isolation, which avoids the strain having contact with any contaminating agent such as fungal spores or any external microorganism agent.

Strain characteristics

All strains, regardless of the type of microorganism (the species) they represent, must meet some basic parameters, among which are:

- They must be stable genetic lines or have high genetic fidelity

It is important that all the individuals that are kept within the culture medium are as close as possible to each other, genetically speaking. That is, they all derive from the same individual or, at least, from the same population.

- They must be easy to maintain or grow

Individuals belonging to a strain must be easy to maintain in an in vitro environment. In other words, not all microbes are capable of isolating themselves from their natural environment. If these are difficult to grow in external media, their biology can easily be altered with minimal changes to the environment in which they are kept isolated in the laboratory.

- They need to have rapid growth and development under optimal conditions

If isolated microbes do not develop rapidly within the culture medium used for this purpose, they can be difficult to preserve for study, as they can deplete the nutrients in their environment, change phase, or compromise their survival under these conditions..

- They must present defined characteristics and parameters

A strain of isolated microorganisms must have common characteristics that relate it identically and specifically to individuals who are identical to it. These characteristics must be constant over time.

- Easy to handle

In general, the strains used in routine investigations do not require overly rigorous or complicated tools or protocols. This ensures that both students and new researchers can maintain the continuity of studies over time.

ID

Molecular identification

There are different methods to identify a newly isolated strain. However, at present the most accurate, fast and simple technique to determine the identity of almost any species is the analysis of a few regions of the genetic sequences that make up the genome of the individual.

Usually these analyzes are carried out by amplifying specific regions of DNA with the PCR technique (Polymerase Chain Reaction). These techniques vary according to the edge, the family and the type of microorganism whose identity is desired. These regions are generally:

- The regions that code for ribosomal RNAs

- The genes that code for the protein subunits that participate in respiration (especially if the organism is aerobic)

- The genetic region that codes for actin microfilaments (part of the cytoskeleton)

- Some genetic regions of the chloroplast or protein subunits that participate in photosynthesis (for some algae and cyanobacteria and for all plants)

Once these genome fragments have been successfully amplified, they are sequenced to determine the order of the nucleotides that make up these regions of the genome. This is done through NGS (Next Generation Sequencing) techniques with specialized equipment known as sequencers.

The sequenced regions are compared with the sequences of microorganisms of this type already reported previously, which is possible by using, for example, the database that is deposited on the GenBank website (https: // www. ncbi.nlm.nih.gov/genbank/).

Morphological identification

In laboratories that do not have molecular biology tools to analyze genetic characteristics, other phenotypic parameters are used to identify the strains of many microorganisms. Once again, the phenotypic characteristics that are studied vary depending on the organism, the phylum, the family and the species considered. Among these parameters are studied:

- The morphological characteristics of the microbe in the culture medium. Features such as: color, shape, texture, type of growth, among other aspects are observed.

- Analysis of metabolic products using biochemical tools. The production of secondary metabolites, excreted chemical compounds, among others, is studied.

- Characterization and crystallization of proteins. The internal proteins of the microorganisms are extracted and studied independently.

The typical thing in microbiological studies is to perform the characterization of the strains with both types of identification, that is, both through morphological observations and molecular analysis.

Isolation of strains

Isolation of strains involves several techniques that are also used to separate one species of microbe from another. The ability to isolate the strain of a species of interest is essential to accurately determine its defining characteristics.

Most strain isolation techniques were created during the 19th century by the fathers of microbiology Louis Pasteur and Robert Koch. Both were obsessively striving to obtain pure cell cultures (strains) of the microorganisms they studied.

Source: Sentebrinka / CC BY (https://creativecommons.org/licenses/by/4.0) via Wikimedia Commons

To obtain these cell cultures, they explored a wide variety of techniques and tools, from the use of sterile toothpicks to variations in the composition of the culture media where the microbes they studied were prepared to grow.

Strain isolation techniques

At present, all the techniques developed and used by these researchers and some more modern ones have been gathered in 6 different types, which are:

- Scratches, scratches or scratches: using a fine and pointed instrument, the place where the microorganism is found is touched (especially for cultures grown in vitro in solid medium). A sterile nutrient-rich solid medium is scratched with the end with which the microorganism was touched.

- Immersion or fusion in the medium: a small sample of microbes is taken (it can be like the one taken in the previous technique) and it is placed inside the growth medium in liquid state, agar is added to solidify and wait for it to cool. Colonies will only be seen when the microorganism is highly developed.

- Serial dilutions: a sample from the original place where the species was collected is diluted consecutively in a sterile medium free of other microorganisms. Dilutions are "seeded" on solid media and colonies are expected to appear.

- Exclusive culture media: these are culture media that allow the growth of only the type of microbe of interest; that is, it has components or nutrients that only allow the growth of the strain to be isolated.

- Manual or mechanical separation: a small sample of the microbe to be isolated is placed and through a microscope an attempt is made to separate a single individual of the species from the rest of the individuals that surround it.

Some of these techniques are easier to use than others. However, researchers use them according to the biological characteristics of the study species.

References

1. De Kruif, P. (1996). Microbe hunters. Houghton Mifflin Harcourt.
2. Dijkshoorn, L., Ursing, BM, & Ursing, JB (2000). Strain, clone and species: comments on three basic concepts of bacteriology. Journal of medical microbiology, 49 (5), 397-401.
3. Marx, V. (2016). Microbiology: the road to strain-level identification. Nature methods, 13 (5), 401-404.
4. Willey, JM, Sherwood, L., & Woolverton, CJ (2009). Prescott's principles of microbiology. Boston (MA): McGraw-Hill Higher Education.
5. Williams, JA (Ed.). (2011). Strain engineering: methods and protocols (Vol. 765, pp. 389-407). New York: Humana Press.