Microscopic, culture, metabolic, and molecular methods are used to determine the concentration and species composition of microbiota in collected samples.

Microscopic methods

consist in direct microscopic evaluation, in appropriately prepared preparations, of the number of microbial cells and calculation of their concentration in a unit of air volume or on the tested surface.

The advantage of microscopic methods is the registration of all microorganisms, both viable (culturable and non-culturable) and non-viable, while the disadvantage is the inability to precisely determine the taxonomic affiliation of the species.

Alternaria alternata (light microscope, at 400x magnification)

Culture-based methods

allows to determine the number of viable (understood here as culturable) microorganisms; their concentration is expressed as colony forming units, CFU, in a unit of air volume or on the tested surface.

This method enables the identification of isolated microorganisms down to genus and/or species level.

Colonies of mold fungi cultivated from indoor air.

Listeria monocytogenes on ALOA medium.

Metabolic and molecular methods

in metabolic and molecular methods, the concentration of microorganisms in the air or on a contaminated surface is determined by detecting the presence of products of their metabolism, non-specific and specific DNA or a gene probe.

Metabolic methods

Metabolic methods assessing the ability of microorganisms to enzymatically decompose organic substrates (e.g. API biochemical tests) or analyzing the metabolism of microorganisms in relation to specific carbon sources (e.g. BIOLOG system) have been used in the identification of bacteria and fungi.

API (Analytical Profile Index) - biochemical tests

Chemical markers of microorganisms are also used in everyday laboratory practice, e.g. endotoxins as markers of Gram-negative bacterial contamination; muramic acid from peptidoglycans as marker of Gram-positive bacterial contamination; ergosterol, N-acetylhexosaminidase and (1-3)-β-D-glucans as markers of fungal biomass.

Depending on the nature of the tested marker, appropriate analytical techniques are used, e.g. high-performance liquid chromatography (HPLC), gas-liquid chromatography (GLC), thin-layer chromatography (TLC) or UV spectrophotometry.

Molecular techniques

These techniques are characterized by high sensitivity and specificity and are not dependent on the conditions of microbial culture on microbiological media. Molecular techniques enable the identification of microorganisms that are difficult to culture and isolate in laboratory conditions and allow for their precise and unambiguous species identification and differentiation of strains within a specific species. They consist in the analysis of the genetic material of the microorganism, which is unique and is not subject to environmental changes, similarly to phenotypic features. The most popular in this area are techniques based on the polymerase chain reaction (PCR), which allows to obtain a virtually unlimited number of copies of any fragment of the genome in a relatively short time.