FUNDAMENTALS OF MICROBIOLOGY

Lesson 29. ENUMERATION OF MICROORGANISMS IN AIR, CONTROL OF AIRBORNE MICROORGANISM

Module 7. Environmental microbiology

Lesson 29

ENUMERATION OF MICROORGANISMS IN AIR, CONTROL OF AIRBORNE MICROORGANISM

29.1 Enumeration of Microorganisms in Air

Various methods commonly applied for enumeration and detection of microorganisms can be subdivided into:

• Microscopic methods
• Culture methods
• Combination of both

29.2 Microscopic Methods

These consist of

• Letting air through a membrane filter or placing a glass coated with a sticky substance (e.g. vaseline), in the path of air
• Staining of the trapped microorganisms and
• Microscopic testing consisting of cell counting

Staining with acridine orange and examination under a fluorescence microscope is often applied. The final result is given as a total number of microbes in 1 m³ of air. The advantage of this method is that it allows the detection of live and dead microbes in air, as well as those, which do not abundantly flourish in culture media. Due to this, the number of microbes determined is usually higher by one order of magnitude than in culture methods. In addition, it is possible to detect and identify other biological agents e.g. plant pollen, allergenic mites, abiotic organic dust (fragments of skin, feathers, plants, etc.).

However the methods have a serious drawback: inability to determine the species of microbes (bacteria, fungi, viruses).

29.3 Culture Methods

These methods consist of transferring microbes from air onto the surface of the appropriate culture medium. After a period of incubation at optimal temperature, the formed colonies are counted and the result is given as cfu/m³ of air (colony forming units). Because a colony can form not only from a single cell, but also from a cluster of cells, the air may contain more microbes than suggested by the CFU result. Besides, the method allows the detection of only the cells that are viable and those which are able to grow upon the medium used. Microbes transferred to the culture medium require resuscitation as they were subjected to the influence of unfavourable conditions. Therefore it is recommended to supplement the culture mediums are required to be supplemented with components such as betaine and catalase. Betaine, the methylic derivative of the glycine amino acid, is utilized by bacteria to maintain osmotic balance, and as a donor of methylic groups it is essential during the processes of biosynthesis. Catalase however breaks down harmful peroxides created in air as a result of UV radiation.

However, testing of viruses differs significantly from the methods utilized for other organisms because:

• They may develop only in living cells, therefore they require tissue cultures (e.g. the epithelium of human trachea or monkey's kidney) or, in the case of bacteriophages, bacterial cultures,
• Species identification of detected viruses is meticulous and, among other things, consists of performing electrophoresis or utilizing antiserum that contains antibodies of common viruses,
• Drawing large quantities of air is essential (over 1000 dm³, at least one order of magnitude higher than in the case of bacteria), as the amount of viruses in air is rather small (this especially concerns the enteroviruses).

After transferring the viruses onto the surface of a single-layer culture, the viruses penetrate the cells, reproduce in them, and after their destruction attack the neighboring cells. Consequently, the areas around the initial places of the cell infections get cleared of cells – this clearing is called plaques. Therefore, the number of viruses detected is given as the number of units that form the plaques, in short pfu/m³ (plaque forming units). It has to be pointed out though, that such a method only allows the detection of viruses capable of infecting the utilized cells.

29.4 Sampling of Air

There are four basic ways of sampling the air for use in culture methods:

• Koch's sedimentation method
• Filtration method (also used in microscopic methods)
• Centrifugation
• Impact methods

29.5 Sedimentation Method

This ‘Settling Plate Technique’ based on this approach is the simplest and is often used by air microbiologists. The principle behind this method is that the bacteria carrying particles are allowed to settle onto the medium for a given period of time and incubated at the required temperature. A count of colonies formed shows the number of settled bacteria containing particles. In this method petridishes containing an agar medium of known surface area are selected so that the agar surface is dry without any moisture. Choice of the medium depends upon the kind of microorganisms to be enumerated. For an overall count of pathogenic, commensal and saprophytic bacteria in air blood agar can be used. For detecting a particular pathogen which may be present in only small numbers, an appropriate selective medium may be used. Malt extract agar can be used for molds. The plates are labeled appropriately about the place and time of sampling, duration of exposure etc. Then the plates are uncovered in the selected position for the required period of time. A Petri dish containing agar medium is kept covered and, at the time of sampling, the cover is removed from the Petri dish so that the agar surfaces is exposed to air for a few minutes. The Petri dish is now incubated. One can see a certain number of colonies developing on agar medium (Fig. 29.1). Each colony represents a particle carrying microorganisms which has fallen on the agar surface. The optimal duration of exposure should give a significant and readily countable number of well isolated colonies, for example about 30-100 colonies. Usually it depends on the dustiness of air being sampled. In occupied rooms and hospital wards the time would generally be between 10 to 60 m. During sampling it is better to keep the plates about I metre above the ground. Immediately after exposure for the given period of time, the plates are closed with the lids. Then the plates are incubated for 24 hrs at 37°C for aerobic bacteria and for 3 days at 22°C for saprophytic bacteria. For molds incubation temperature varies from 10-50°C for 1-2 weeks. After incubation the colonies on each plate are counted and recorded as the number of bacteria carrying particles settling on a given area in a given period of time.

The use of settle plates is not recommended when sampling air for fungal spores, because single spores can remain suspended in air indefinitely. Settle plates have been used mainly to sample for particulates and bacteria either in research studies or during epidemiologic investigations. Results of sedimentation sampling are typically expressed as numbers of viable particles or viable bacteria per unit area per the duration of sampling time (i.e. CFU/area/time); this method can not quantify the volume of air sampled. Because the survival of microorganisms during air sampling is inversely proportional to the velocity at which the air is taken into the sampler, one advantage of using a settle plate is its reliance on gravity to bring organisms and particles into contact with its surface, thus enhancing the potential for optimal survival of collected organisms. This process, however, takes several hours to complete and may be impractical for some situations.

29.5.1 Limitation

Though the method has the advantage of simplicity, it has certain limits.

• In this method only the rate of deposition of large particles from the air, not the total number of bacteria carrying particles per volume, is measured.
• Growth of bacteria in the settled particles may be affected by the medium used since not all microorganisms are growing well on all media.
• Moreover since air currents and any temporary disturbances in the sampling area can affect the count, many plates have to be used.
• Since only particles of certain dimensions tend to settle on to the agar surface and, also, the volume of air entering inside the Petri dish is not known, this technique gives only a rough estimate and can be used only to isolate air-borne microorganisms.
• However, one can gather information about the kind of air-borne microbes occurring in a particular area by repeated use of settling plate technique for a fixed period of time.

Fig. 29.1 Agar plates showing colonies of microbes present in Air

29.6 Filtration Methods

The methods consist of using an aspirator to suck in a given volume of air, passing it through a sterile absorbing substance (liquid or solid) and transferring the filtered microbes onto the appropriate culture medium. After a pre-determined time of incubation the resulting colonies are counted. Most often, a membrane filter or a physiological solution (0.85% NaCl) is utilized for the filtration of air. Filtration using liquids (sometimes classified as the impact method) is one of the most often used and highly valued techniques of sampling bioaerosol (Fig. 29.2). It results in high output of microbe isolation as well as significant survival of the filtered microbes. The method may be utilized in virus testing as long as the remaining microbes are neutralized (e.g. with chloroform) and the liquid is concentrated before its introduction into the cell culture.

The filtration process through membrane filters allows the utilization of both culture methods (filters containing microbes are placed directly upon the culture media or are rinsed and then inoculated) as well as the microscopic methods (filters are stained and observed under a microscope).

Fig. 29.2 Wash bottle for bio aerosol absorption in filtration methods

These are simple methods for collecting particles from air. The filter can be made of any fibrous or granular material like sand, glass fibre and alginate wool (in phosphate buffer). However, recovery of organisms for culture is not so easy. The membrane filter devices are adaptable to direct collection of microorganisms by filtration of air. These methods are also rather inexpensive and not complicated; they possess two significant advantages over the sedimentation methods:

• The volume of the air tested is known,
• It is possible to detect the very small aerosol that creates the respiratory fraction (nevertheless it is still impossible to determine its size

29.6.1 Tube sampler

This is one of the oldest devices for collecting and enumerating microorganisms in the air. It consists of a tube with an inlet at the top and an outlet at the bottom which is narrower than the top end. Near the bottom there is a filter of wet sand which is supported by a cotton plug below. The entire device can be sterilized. After sterilization the air to be sampled is allowed to pass through the sand and cotton. Microorganisms as well as dust particles containing microorganisms in the air are deposited in the sand filter as the air passes through it. Later the sand is washed with broth and a plate count is made from the broth by taking aliquotes of the broth.

29.6.2 Millipore filter

This type of filters is made of pure and biologically inert cellulose ethers. They are prepared as thin porous, circular membranes of about 150 µm thickness. The filters have different porosity. The assemblage contains a funnel shaped inlet and a tube like outlet. In between these two the filter is fitted. The outlet may be connected to a vacuum pump to suck known amount of air. After collecting required volume of air through the filter, it can directly be placed onto the surface of a solid medium. After incubation colonies formed can be counted.

However, the disadvantage of this method is that it has a significantly low output as the process of passing the air through pores of the filter creates resistance. That's why the method is not recommended for microbe testing, but is routinely put to use in detection of endotoxins in air.

29.7 Centrifugation Methods

29.7.1 Air centrifuge

The first primitive type of air centrifuge was developed by Wells in 1993. The principle of air centrifuge is that the particles from air are centrifuged onto the culture medium. In his air centrifuge sampled air was passed along a tube which was rotated rapidly on its long axis. The inner surface of the tube was lined with culture medium and any bacteria containing particle deposited on it grew into a colony on incubation. A modern version of this centrifuge is the Reuter centrifugal air sampler, which is portable and battery powered. It resembles a large cylindrical torch with an open ended drum at one end. The drum encloses impeller blades which can be rotated by battery power when switched on. A plastic strip coated with culture medium can be inserted along the inner side of the drum. Air is drawn into the drum and subjected to centrifugal acceleration. This causes the suspended particles to impact on the culture medium. After sampling the strip is removed from the instrument and incubated at 37°C for 48 h. Later the colonies can be counted. Advantage of this sampler is that it is very convenient for transportation and use. However, the disadvantage is that it is less efficient than the slit sampler in detecting particle below 5 mm in diameter. More over the size of the air being sampled cannot be accurately controlled.

29.7.2 Impact methods

These methods consist of using an aspirator to suck in a pre-determined amount (volume) of air, which collides with the nutrient agar at high speed. It causes the microbes in the air to stick to the surface, which after a specific time of incubation, form colonies. The impact methods are the most highly valued and most often used methods of detecting microbes in air. Their biggest advantage is the possibility of detecting and determining the respiratory fraction of the bioaerosol, in other words, determining the size distribution of its particles. The methods can be utilized to test viruses (trapped microbes are swept from the surface of the culture medium and, after the elimination of other microbes with chloroform, introduced into the cell culture).

A disadvantage for the impact method is a decline in the microbes viability caused by the shock of a sudden collision with nutrient agar and also a possibility of the nutrient culture getting overgrown in cases of high air pollution. The above stated methods are usually not cheap. The most widely known device that is based on the impact technique is the Andersen's apparatus, in which the air is drawn in passes through six vertically positioned sieves. A petri dish with nutrient agar is placed underneath each sieve. The speed of the passing air increases as it passes through the consecutive sieves, consequently causing greater impact force as it collides with the sieves. As a result, the heaviest (largest) particles settle upon the first sieve, whereas the lighter (smaller) ones are drawn in by the current of the passing air. As they pass through the consecutive sieves, the increasingly smaller and faster particles collide with the nutrient agar. Consequently the particles of the biological aerosol are sorted according to their size and the colonies are then derived from particles of particular size. This way, by counting the colonies upon the consecutive plates, it is possible to determine the ratio of particles which settle in the upper (higher positioned plates) and lower respiratory system (lower plates).

29.7.2.1 Sampling of measured volume of air

An improvised method wherein a measured volume of air is sampled has also been developed (Fig. 29.3). These are sieve and slit type devices. A sieve device has a large number of small holes in a metal cover, under which is located a petridish containing an agar medium. A measured volume of air is drawn, through these small holes. Airborne particles impinge upon the agar surface. The plates are incubated and the colonies counted. In a slit device the air is drawn through a very narrow slit onto a petridish containing agar medium. The slit is approximately the length of the petridish. The petridish is rotated at a particular speed under the slit. One complete turn is made during the sampling operation

Fig. 29.3 The sieve-impaction sampling method

29.7.2.2 Selection of air sampler

The following factors must be considered when choosing an air sampling instrument:

• Viability and type of the organism to be sampled
• Compatibility with the selected method of analysis
• Sensitivity of particles to sampling
• Assumed concentrations and particle size
• Whether airborne clumps must be broken (i.e. total viable organism count vs. particle count)
• Volume of air to be sampled and length of time sampler is to be continuously operated
• Background contamination
• Ambient conditions
• Sampler collection efficiency
• Effort and skill required to operate sampler
• Availability and cost of sampler, plus back-up samplers in case of equipment malfunction
• Availability of auxiliary equipment and utilities (e.g. vacuum pumps, electricity, and water)