Sample Preparation Protocols for Air Microbiome Sequencing

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Air, one of the disproportionally understudied ecosystems, is composed of ubiquitous microorganisms which were considered as passive dwellers moving with the wind. Although, in some research airborne microorganisms were strongly suggested to be metabolically active. This is in line with some findings that air is also suitable as the habitat for such airborne microorganisms.

In terms of abundance, the air has an extremely low density of biological material and only a small of portion these microbes are said to be cultivable, mostly bacteria and fungi. In fact, the concentration of atmospheric bacteria over land is around 104 cells/m3 while 109 cells/m3 over the marine surface, 1010–1011 per gram of soil (in between soil particles), and 1013–1014 per human (in the human distal gut).

The knowledge of air microbiome research has increased significantly for the past years through molecular analysis of the genetic material of the airborne microbes. There are two known common approaches in investigating microbial diversity, as well as their metabolic potential in the environment. The first approach is known as the polymerase chain reaction (PCR)-based rRNA, both 16S and 18S, gene sequencing approach which uses rRNA gene as the phylogenetic marker in comparing the relationship between the microorganisms. The second approach is called the whole-genome shotgun metagenomics approach where the entire genome of the microorganisms are broken down into smaller fragments, sequenced, and reconstructed in assessing their diversity, as well as their metabolic potential.

Air Microbial Samplers

The procedures and protocols, as well as the air samplers used in extracting the genetic material of the microorganism in air are all important factors on the outcome of metagenomic studies. Since metagenomic studies are conducted through direct extraction of nucleic acids from the environment, the quantity and quality of the extracted nucleic acid would determine the outcome of these studies. On the other hand, air samplers affect the outcome as they are significantly different from one another in terms of collection efficiency. Some of the common air samplers are impactor sampler, liquid impinger sampler, and filtration sampler.

Impactor Sampler

Impactor samplers use a solid or adhesive medium for particle collection. They are much more commonly used in commercial applications for their convenience. In a typical impactor sampler, air is drawn into a sampling head by a pump or fan and accelerated, producing laminar air flow onto the collection surface. When the correct volume of air has been passed through the sampling head, the agar plate can be removed and incubated directly without further treatment before incubation.

Liquid Impinger Sampler

Liquid impinger sampler, also known as bubblers, are tiny bottles which use an air sample pump in collecting airborne microorganisms into specific chemical liquids used for analysis. An example of this sampler is the all-glass impinger or the Greenburg-Smith impinger which has a sampling capacity of 11.5 liters/min. Its collecting fluid is phosphate-buffered distilled water with a pH of 7.2. This sampling fluid is to be stored at +4 degrees Celsius and to be quantitated in less than 4 hours. The sampling fluid will be later filter through a 0.45 micrometer cellulose membrane and will be placed directly onto an agar plate.

Filtration Sampler

Filtration Sampler is one of the most used air samplers. There are different types of filtration sampler and two of these are the gelatin filter which has a pore size of three picometers and a flow rate of 15 liters/minute, and the cellulose membrane filter which has a pore size of 0.45 picometer and a flow rate of 15-17 liters/minute. Usually, after sampling this type of filter is placed on agar plates where it dissolves.

Air Sampling Using Wet Cyclone Portable Air Sampler

During sampling, a wet cyclone air sampler is operated at a flow rate of 450 liters/minute. Its sampling buffer is comprised of 1X PBS supplemented with 0.25 mg/ml each of tetracycline and gentamycin, and 0.125 mg/mL chloramphenicol in one liter of sterile water for injection. Throughout the sampling cycle, about 10 milliliters of sample buffer must be maintained and the sampling time is around two hours. This sampler automatically turned off after each 2-hour collection cycle and drains the collected sample into a 250 mL corning bottle that contains sodium azide. After that, the solution must be placed inside a modified 4°C mini-refrigerator. After the draining process, the sampler will also automatically refill its chamber with the sample buffer and will restart the collection cycle again. The sample must be removed from the refrigerator and must be added with biology grade glycerol every 12 hours. Finally, the sample must be placed back in the refrigerator and transport back to the laboratory, and store at −80°C.


  1. Bharti R, Grimm DG. Current challenges and best-practice protocols for microbiome analysis. Briefings in Bioinformatics. 2019.
  2. Yooseph S, Andrews-Pfannkoch C, Tenney A, et al. A metagenomic framework for the study of airborne microbial communities. PloS one. 2013, 8(12):e81862.
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