Standards Development

Scope

This part of ISO 16000 specifies the requirements of examination of total cell count in building material samples by acridine orange direct count. The acridine orange direct count enables the determination of total biomass in material samples by counting spores and bacteria as fluorescent cells (Meider, 2019). It describes the analytical procedure in the lab and the technical requirements.

Purpose

In recent years, the microscopic evaluation of samples has been established alongside CFU analysis. In air samples, adhesive microscope slides are coated via impaction and analysed microscopically (DIN ISO 16000-18, 2012). Surfaces of materials are sampled using clear strips of adhesive film and evaluated via light microscopy. This methodology has not yet been validated, but the Verein Deutscher Ingenieure, or Association of German Engineers (VDI), is currently developing a guideline on sampling and evaluation. In spite of the lack of validation, there are methods for microscopic analyses of air and of material surfaces.

CFUs from material samples are analysed by the suspension method, which takes the complete sample into account rather than just the surface. Spores, hyphae, and bacteria are transferred into a sterile suspension when preparing the sample (DIN ISO 16000-17, 2010). Adhesive film samples represent only the surface of a material. Hence, adhesive film samples do not correlate with the number of CFUs identified using the suspension method for the same material. To obtain comparable results, the same suspension must be used for the CFUs and microscopic investigation. The microscopic determination of spores, hyphae, and bacteria in the suspension is the analysis of TCC (Trautmann and Meider, 2018).

The literature contains many studies of suspensions using fluoresce molecules such as 3,6-bis[dimethylamino] acridinium chloride, or acridine orange (AO). Hobbie et al. (1977) described the staining of the suspension on Nuclepore filters to count bacteria. With AO, the RNA and DNA strands of cells contained in the suspension are stained and then counted via fluorescence microscopy. This publication was the basis for many additional investigations and the method came to be known as acridine orange direct count (AODC). This staining and counting method has been used in different areas and has quickly come to be considered the standard method for determining microorganisms in suspensions (Meider, 2019).

To determine the TCC of moulds and bacteria in air samples (filtration method), AODC was used and published as the CAMNEA method (Palmgren et al., 1986). This method was taken up and described as VDI Guideline 4253 Part 4.

The analysis of a suspension with the CFU method and the TCC method shows not only the viable microorganisms of a sample that can be cultivated, but also the moulds and bacteria that do not grow under laboratory conditions. Kepner Jr and Pratt (1994) have shown that there are also viable bacteria that did not produce as CFUs. Sherachan et al. (2018) also state that viability and the ability to be cultivated are not synonymous, and that the ability to be cultivated is reduced considerably by stress (e. g. lack of moisture). Investigations of indoor samples have revealed that the TCC can be > 100 times higher than the CFU (Trautmann and Meider, 2018).

Indoor environment samples are being investigated for microbial loading to estimate the level of health risk to inhabitants. In most stu- dies on the topic of interior space contamination, the CFU is used as the reference method. However, it is difficult to establish a link between health effects and microorganisms through qualitative detection of viable microorganisms (WHO, 2009; Jarvi et al., 2018). Newer investigations have shown that a health-endangering effect may also arise from microorganisms that cannot be cultivated (Croston et al., 2018; Korkalainen et al., 2017). It is thus becoming ever clearer that the health risk determination is independent of the viability of spores. Different cellular components and metabolic products can initiate inflammatory processes in the body (Croston et al., 2018; 

Korkalainen et al., 2017). The total amount of microorganisms is therefore more important than the composition of microflora when assessing the level of health risk.