Direct quantification of Aspergillus niger spore adhesion to mica in air using an atomic force microscope

Abstract

An atomic force microscope (AFM) has been used to quantify directly the adhesion between single Aspergillus niger spores and freshly cleaved mica surfaces in air, a system that is relevant to the removal of spores by filtration. The measurements used ‘spore probes’ constructed by immobilising a single spore at the end of a tipless AFM cantilever. It was found that adhesion was reproducible at a relative humidity of 64%, but that it showed substantial variability at a relative humidity of 33%, findings that are consistent with capillary forces playing a dominant role in the adhesion process. The technique should prove useful both in the evaluation of different types of filter materials and in assessing the effect of environmental conditions on spore removal efficiency.

Introduction

Aerial transmission of bacterial and fungal spores has serious implications for the food industry, agriculture, biotechnology and medicine [1], [2], [3], [4]. Spores are reproductive propagules that are able to survive extreme conditions, resisting chemical and physical attack until the environment is favourable for growth. Success of aerial transmission relies on the large numbers of spores that are produced. Typical concentrations of airborne biological particles such as bacterial and fungal spores are within the range 10–1000 m⁻³, but may reach as high as 10⁶ m⁻³ [5]. However, only small numbers are needed to survive cleaning and filtering regimes to compromise production processes and human health. For example, in the food industry, only a small microbiological innoculum is required to contaminate a sterilised environment and thus ruin the product [6]. Serious hospital-acquired infections can result from the inhalation of bacterial spores [6], [7], [8]. Airborne fungal spores can cause allergies and have been blamed for building-related illness [5]. For these reasons, it is important to use effective means for the removal of spores from such environments.

Fibrous filters are used extensively to remove spores and other biological particles from air. Such filters are most commonly fabricated from inorganic fibres. The efficiency of such filters in the removal of spores has been the subject of substantial research [5], [9], [10]. Effective operation requires a high collision efficiency of spores with fibres and also a high adhesion efficiency once collision has taken place [9]. Collision efficiency has been the subject of extensive mathematical modelling. The adhesion of spores at filter materials has been less studied and then inferred indirectly from counting measurements. The availability of a direct method for measuring spore adhesion could be of significant benefit in the selection of the most efficient filter media or the development of improved media. Such a method would also allow direct quantification of the effect of environmental conditions, such as humidity, on filter effectiveness.

Recent advances in the use of atomic force microscopes [11] have permitted the direct measurement of the force of adhesion of a single particle in a direction normal to the interacting surfaces. The technique involves the immobilisation of a single particle at the end of a cantilever, creating a colloid probe [12]. This technique has been used to study double-layer and London–van der Waals interactions in solution [12], [13] and also adhesion of inorganic particles [14], [15]. We have developed this technique to study the adhesion of particles to polymer membranes used in liquid filtration processes [16], [17]. The adhesion of latex spheres, Saccharomyces cerevisiae cells and proteins to membranes that exhibit different fouling properties was investigated in solution. Quantification of adhesion allowed identification of the membrane showing lowest fouling properties, i.e. minimum adhesion was a requirement in these cases.

In the present paper, we have applied the colloid probe technique to study spore adhesion to mica in air. Aspergillus niger spores have been used for initial studies for a number of reasons. They are unicellular, without any differentiation and with little variation. The genus Aspergillus is one of the most common groups of fungi, and causes problems in the food industry and medicine [18]. A. niger spores have previously been used for fundamental studies of air filtration. Mica has been chosen as being representative of inorganic materials that may be used in air filters. For this first study, it has the advantage of providing a very flat surface, the properties of which have been extensively documented. The adhesion has been studied at two different values of relative humidity to show the effect of environmental conditions.