Carole Gau ¨ ze `re 1 Marina Moletta-Denat 1 Faisl Bousta 2 Ste ´ phane Moularat 1 Genevie ` ve Orial 2 Se ´ bastien Ritoux 1 Jean-Jacques Godon 3 Enric Robine 1 1 Universite ´ Paris-Est, Centre Scientifique et Technique du Ba ˆ timent (CSTB), Laboratoire de Recherche et d’Innovation pour l’Hygie `ne des Ba ˆtiments, Marne-la-Valle ´e, France 2 Laboratoire de Recherche des Monuments Historiques (LRMH), Champs-sur-Marne, France 3 INRA, UR50, Laboratoire de Biotechnologie de l’Environnement (LBE), Narbonne, France Research Article Reliable Procedure for Molecular Analysis of Airborne Microflora in Three Indoor Environments: An Office and Two Different Museum Contexts Biological aerosols from air constitute a significant source of exposure to micro- organisms in public places. Airborne microorganisms are involved in the development of certain respiratory symptoms, allergies, or infections among users and occupants. Various sampling instruments have commonly been used in aerobiology to collect bacteria and fungi suspended in the air. The objective of this study was to develop a reliable procedure for sampling in indoor public environments presenting different levels of occupancy, airborne bacteria and fungi to be subjected to molecular analysis (bacteria and fungi quantitative PCR, capillary electrophoresis single strand confor- mation polymorphism fingerprinting). Four different sampling devices were tested in situ in an office building (open-plan type) and the sampling strategy chosen was tested in two museum contexts. In accordance with the drawbacks involved to our study (quantitative and qualitative aspects, cost, and overcrowding), cyclone device appeared to be most suitable. The results underline the effectiveness of this high-volume aerosol sampling device for both qualitative and quantitative molecular analysis. Four in situ sampling collections were carried out in 1 day in the Louvre Museum to study quan- titative and qualitative variations of airborne bacterial and fungal diversity. The quantitative results revealed a similar order of magnitude for the numbers of both bacteria and fungi. In the Louvre Museum, the samples yielded between 3.7  10 4 and 4.1  10 4 genome equivalent (GE) bacteria/m 3 air and between 5.0  10 4 and 5.9  10 4 GE fungi/m 3 air and in the Decorative Arts Museum between, 2.1  10 4 and 2.5  10 4 GE bacteria/m 3 air and between 1.4  10 4 and 1.7  10 4 GE fungi/m 3 air. The results also indicate that the dominant bacterial community displayed a stable structure over a short period of time whereas dominant eukaryotic airborne community appeared more variable. Keywords: Bioaerosol; CE-SSCP; Indoor air quality; Microbial aerosol; Q-PCR Received: December 12, 2011; revised: April 16, 2012; accepted: April 19, 2012 DOI: 10.1002/clen.201100699 1 Introduction In recent years, the increase of epidemic alerts has led to a reconsi- deration of the risks of microbial aerosols associated with indoor air quality and of human exposure to various pollutants, including microorganisms [1]. Sites with high occupancy rates (transport sys- tems, public buildings, etc.) appear to be particularly sensitive to these risks. Given its proven impact on both health and buildings, the microbiological quality of air in indoor environments has thus become of increasing interest. Airborne microorganisms can cause respiratory symptoms among occupants and studies have demon- strated their role in the degradation (partial or total) of the substrate they colonize (wood, textiles, paper, pigments, varnish, etc.) [2–4]. Despite the importance of this subject, the microbial content of air has been the object of much less studies than other environments such as soil or water. Different devices are used for collecting microorganisms from the air and are based on different techniques for recovering the micro- organisms (filtration, solid impaction, or impingment) [5]. The choice of a particular sampling device will be linked essentially to the type of analysis to be carried out and the environment which is involved. However, factors such as the device’s cut-off diameter (d 50 ), the non-multiplication of microorganisms in fluid collections and re- aerosolization are often considered when studying microorganisms from the air. While maintaining the viability of airborne microbes in the collection media is essential for analysis of the cultivable fraction, this is not always so when using molecular tools. Using molecular techniques, the factors which need to be taken into account are mainly the d 50 and the larger quantities of air that must be collected within a short period. A short sampling period is also required in order to take into account possible rapid variations in airborne microbial diversity [6]. The collection of micro- organisms using sedimentation methods, which is widely used in combination with culture methods, is not be used with molecular Correspondence: Dr. C. Gau ¨ze`re, Universite´ Paris-Est, Centre Scientifique et Technique du Baˆtiment (CSTB), Laboratoire de Recherche et d’Innovation pour l’Hygie`ne des Baˆtiments, 84, avenue Jean Jaure`s - Champs-sur-Marne, 77447 Marne-la-Valle´e Cedex 2, France E-mail: carole.gauzere@gmail.com 226 ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clean-journal.com Clean – Soil, Air, Water 2013, 41 (3), 226–234