Airborne microorganisms are ubiquitously present in various indoor and outdoor environments. The potential implication of fungal contaminants in bio-aerosols on occupational health is recognized as a problem in several working environments. There is a concern on the exposure of workers to bio-aerosols especially in composting facilities, in agriculture, and in municipal waste treatment. The European Commission has therefore guiding rules protecting employees in the workplace from airborne biological hazards. In fact, there are an increasing number of incidents of building-related sickness, especially in offices and residential buildings. Some of these problems are attributed to biological agents, especially in relation to airborne fungal spores. However, the knowledge of health effects of indoor fungal contaminants is still limited. One of the reasons for this limitation is that appropriate methods for rapid and long-time monitoring of airborne microorganisms are not available.

Airborne Microorganisms

Besides the detection of parameters relevant to occupational and public health, in many controlled environments the number of airborne microorganisms has to be kept below the permissible or recommended values, e.g. in clean rooms, in operating theatres, and in domains of the food and pharmaceutical industry. Consequently, the continuous monitoring of airborne biological agents is a necessity for the detection of risks of human health as well as for the flawless operation of technological processes.
At present a variety of methods are used for the detection of fungal spores. The culture-based methods depend on the growth of spores on an agar plate and on the counting of colony-forming units. Culture-independent methods are based on the enumeration of spores ­under a microscope, the use of a polymerase chain reaction or on DNA hybridization for the detection of fungi. However, all these methods are limited by time-consuming procedures of sample preparation in the laboratory.

Automated Detection of Dangerous Bio-Substances

We have developed an automated image-acquisition and probe handling unit of biologically dangerous substances and the automated analysis and interpretation of microscope images of these substances.

In the system contaminated air containing bio-aerosols is collected in a defined volume via a carrier agent. They are recorded by an image-acquisition unit, counted, and classified. Their nature is determined by means of an automated image-analysis and interpretation system. Air samples are automatically acquired, prepared and transferred by a multi-axis servo-system to an image-­acquisition unit based on a standard ­optical microscope with a digital color camera. By a novel image analysis methods fungi spores are recognized in the image, described by features, and classified in one of the different fungi spore classes. The following parameters are calculated by the image analysis:

• Total number of airborne particles
• Classification of all particles according to the calculated image features
• Classification of biological particles, e.g. spores, fragments of fungal my­celia, and fragments of insects
• Number of respirable particles
• Total number of airborne particles of biological origin
• Number of dead particles of biological origin
• Number of viable and augmentable particles of biological origin
• Identification of species or geni
• Proportion of airborne abiotic and ­biotic particles
• Proportion of dead and viable airborne microorganisms.

The probe handling and image acquisition unit works as follows: In the slit ­impactor the air (fig. 3), potentially containing airborne germs, is guided on the sticky area of the object slide by the air stream generated by an air pump. After a few tens of seconds which can be ­adjusted accordingly, the pump is switched off and the object slide is transported to the pipetting unit driven by the dosing pump. To this aim it has to change its transporting axis and thus its direction of movement. From a thin nozzle one drop of lactophenol is deposited on the sticky area of the object slide which is afterwards transported via the axis crossing to the cover-slip gripper unit. This gripper acts as a low-pressure sucker and takes one cover glass from the deposit and puts it with one edge first on the object slide. Then the cover glass falls down on the object slide and flattens the drop so that it will be distributed all over the sticky area forming a thin layer. In this way the airborne germs collected in the sticky layer are immersed in the lactophenol. In lactophenol living germs get a blue color. The object slide is then transported back to an axis crossing-point where it again changes its direction of movement by 90° and is transported to the xy-table of the microscope which takes over the slide and transports it ­directly under the lens. After the object slide has reached the image acquisition position, the microscope camera then grabs the images at the programmed slide positions after auto-focusing of the microscope lens at each position. After having finished the imaging sequence, the slide is transported away from the xy-table with a special arm and falls into a box. When the image grabbing procedure by the microscope unit is still under way, the object-slide preparation unit ­already starts with the preparation of a new object slide. Once an image has been taken it is given to the image-analysis unit for further processing.