Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jun 20;9(12):e3273.
doi: 10.21769/BioProtoc.3273.

Detachment Procedure of Bacteria from Atmospheric Particles for Flow-cytometry Counting

Carolina M Araya  1 , Alberto Cazorla  2   3 , Isabel Reche  1   4
Affiliations

Detachment Procedure of Bacteria from Atmospheric Particles for Flow-cytometry Counting

Carolina M Araya et al. Bio Protoc. .

Abstract

The protocol separates bacteria from atmospheric particles, obtaining with greater precision their abundance in the atmospheric deposition. This procedure is similar to the one used to separate bacteria in streambed sediments. The detachment procedure consists of a chemical treatment with sodium pyrophosphate and Tween 20 and a physical treatment with agitation and ultrasonic bath to disperse the bacteria in the liquid sample. We recover the total (free and attached) bacteria by generating a density gradient with Nycodenz by centrifugation. The techniques prior to this procedure do not include the microorganisms that are attached to the aerosol particles and, therefore, considerably underestimate the total load and deposition of airborne microorganisms.

Keywords: Airborne bacteria; Bioaerosol; Cloud condensation nuclei; Ice condensation nuclei; Microbial dispersal.

PubMed Disclaimer

Conflict of interest statement

Competing interestsThe authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Scheme of the different steps for the detachment procedure. A. Chemical treatment using the sodium pyrophosphate as chelator and Tween 20 as detergent. B. Physical treatment shaking the samples and ultrasonic bath. C-D. Purification of the microbial layer using Nycodenz to generate a density gradient of four layers. E-F. Select the microbial layer and suspend it in sterilized Milli-Q water up to the initial sample volume in a cryovial.
Figure 2.
Figure 2.. Backlight picture of a microcentrifuge tube after microcentrifugation (after the Step 5). After the Step 5, the microcentrifuge is with four layers (top-down): supernatant, microbial layer, Nycodenz and dust pellet.
Video 1.
Video 1.. Backlight video moving the microcentrifuge tube to clearly identify the contrasted, milky structure of the microbial layer
Video 2.
Video 2.. Backlight video to show the procedure to retrieve the microbial layer
Figure 3.
Figure 3.. Comparison between cytograms of the same sample without the detachment treatment and after undergoing this treatment.
A-B. Cytograms of a wet deposition sample under a meteorological scenario of Atlantic influence without the detachment procedure (A) and after being submitted to the detachment procedure (B). C-D. Cytograms of a wet deposition sample under a meteorological scenario of Saharan dust intrusion without the detachment procedure (C) and after being submitted to the detachment procedure (D). The x-axis corresponds to side light scatter (SSC) and y-axis corresponds to green fluorescence (FL1-H). The R2 rectangle is the selected area for the bacterial counting. Events outside this area are impurities.
See this image and copyright information in PMC

References

    1. Aller J. Y., Kuznetsova M. R., Jahns C. J. and Kemp P. F.(2005). The sea surface microlayer as a source of viral and bacterial enrichment in marine aerosols. J Aerosol Sci 36(5-6): 801-812.
    1. Amalfitano S. and Fazi S.(2008). Recovery and quantification of bacterial cells associated with streambed sediments. J Microbiol Methods 75(2): 237-243. - PubMed
    1. Creamean J. M., Suski K. J., Rosenfeld D., Cazorla A., DeMott P. J., Sullivan R. C., White A. B., Ralph F. M., Minnis P., Comstock J. M., Tomlinson J. M. and Prather K. A.(2013). Dust and biological aerosols from the Sahara and Asia influence precipitation in the western U.S. Science 339(6127): 1572-1578. - PubMed
    1. Fröhlich-Nowoisky J., Kampf C. J., Weber B., Huffman J. A., Pöhlker C., Andreae M. O., Lang-Yona N., Burrows S. M., Gunthe S. S., Elbert W., Su H., Hoor P., Thines E., Hoffmann T., Després V. R. and Pöschla U.(2016). Bioaerosols in the Earth system: Climate, health, and ecosystem interactions. Atmos Res 182: 346-376.
    1. Gasol J. M., and Del Giorgio P. A.(2000). Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities. Sci Mar 64(2): 197-224.

LinkOut - more resources