Air Microbiology/Aeromicobiology

Air Microbiology
Of all environments, air is the simplest one and it occurs in a single phase gas. The relative quantities of various gases in air, by volume percentage are nitrogen 78%, oxygen 21 %, argon 0.9%, carbon dioxide 0.03%, hydrogen 0.01 % and other gases in trace amounts. In addition to various gases, dust and condensed vapor may also be found in air
Various layers can be recognized in the atmosphere upto a height of about 1000km. The layer nearest to the earth is called as troposphere. In temperate regions, troposphere extends upto about 11 km whereas in tropics up to about 16km. This troposphere is characterized by a heavy load of microorganisms. The temperature of the atmosphere varies near the earth's surface. However, there is a steady decrease of about 1 DC per 150m until the top of the troposphere. Above the troposphere, the temperature starts to increase. The atmosphere as a habitat is characterized by high light intensities, extreme temperature variations, low amount of organic matter and a scarcity of available water making it a non hospitable environment for microorganisms and generally unsuitable habitat for their growth. Nevertheless, substantial numbers of microbes are found in the lower regions of the atmosphere.

Microbes Found in Air- In addition to gases, dust particles and water vapour, air also contains microorganisms. There are vegetative cells and spores of bacteria, fungi and algae, viruses and protozoan cysts. Since air is often exposed to sunlight, it has a higher temperature and less moisture. So, if not protected from desiccation, most of these microbial forms will die.Air is mainly it transport or dispersal medium for microorganisms. They occur in relatively small numbers in air when compared with soil or water. The microflora of air can be studied under two headings outdoor and indoor microflora.

Sources of Microorganisms in Air - Although a number of microorganisms are present in air, it doesn't have an indigenous flora. Air is not a natural environment for microorganisms as it doesn't contain enough moisture and nutrients to support their growth and reproduction.
Quite a number of sources have been studied in this connection and almost all of them have been found to be responsible for the air microflora. One of the most common sources of air microflora is the soil.
Soil microorganisms when disturbed by the wind blow, liberated into the air and remain suspended there for a long period of time. Man made actions like digging or plaguing the soil may also release soil borne microbes into the air. Similarly microorganisms found in water may also be released into the air in the form of water droplets or aerosols. Splashing of water by wind action or tidal action may also produce droplets or aerosols. Air currents may bring the microorganisms from plant or animal surfaces into air. These organisms may be either commensals or plant or animal pathogens. Studies show that plant pathogenic microorganisms are spread over very long distances through air. For example, spores of Puccinia graminis travel over a thousand kilometers. However, the transmission of animal diseases is not usually important in outside air.
The main source of airborne microorganisms is human beings. Their surface flora may be shed at times and may be disseminated into the air. Similarly, the commensal as well as pathogenic flora of the upper respiratory tract and the mouth are constantly discharged into the air by activities like coughing, sneezing, talking and laughing.
The microorganisms are discharged out in three different forms which are grouped on the basis of their relative size and moisture content. They are droplets, droplet nuclei and infectious dust. It was Wells, who described the formation of droplet nuclei. This initiated the studies on the significance of airborne transmission. A brief description of these agents is given below
Droplets

Droplets are usually formed by sneezing, coughing or talking. Each consists of saliva and mucus. Droplets may also contain hundreds of microorganisms which may be pathogenic if discharged from diseased persons. Pathogens will be mostly of respiratory tract origin. The size of the droplet determines the time period during which they can remain suspended.
Most droplets are relatively large, and they tend to settle rapidly in still air. When inhaled these droplets are trapped on the moist surfaces of the respiratory tract. Thus, the droplets containing pathogenic microorganisms may be a source of infectious disease.
Droplet Nuclei
Small droplets in a warm, dry atmosphere tend to evaporate rapidly and become droplet nuclei. Thus, the residue of solid material left after drying up of a droplet is known as droplet nuclei. These are small, 1-4µm, and light. They can remain suspended in air for hours or days, traveling long distances. They may serve as a continuing source of infection if the bacteria remain viable when dry. Viability is determined by a set of complex factors including, the atmospheric conditions like humidity, sunlight and temperature, the size of the particles bearing the organisms, and the degree of susceptibility or resistance of the particular microbial species to the new physical environment. If inhaled droplet nuclei tend to escape the mechanical traps of the upper respiratory tract and enter the lungs. Thus, droplet nuclei may act as more potential agents of infectious diseases than droplets.
Infectious Dust
Large aerosol droplets settle out rapidly from air on to various surfaces and get dried. Nasal and throat discharges from a patient can also contaminate surfaces and become dry. Disturbance of this dried material by bed making, handling a handkerchief having dried secretions or sweeping floors in the patient's room can generate dust particles which add microorganisms to the circulating air. Microorganisms can survive for relatively longer periods in dust. This creates a significant hazard, especially in hospital areas. Infective dust can also be produced during laboratory practices like opening the containers of freeze dried cultures or withdrawal of cotton plugs that have dried after being wetted by culture fluids. These pose a threat to the people working in laboratories

Significance of Air Microflora - Although, when compared with the microorganisms of other environments, air microflora are very low in number, they playa very significant role. This is due to the fact that the air is in contact with almost all animate and inanimate objects.
The significance of air flora has been studied since 1799, in which year Lazaro Spallanzani attempted to disprove spontaneous generation. In t 837, Theodore Schwann, in his experiment to support the view of Spallanzani, introduced fresh heated air into a sterilized meat broth and demonstrated that microbial growth couldn't occur. This formed the basis of modern day forced aeration fermentations. It was Pasteur in 1861, which first showed that microorganisms could occur as airborne contaminants. He used special cotton in his air sampler onto which the microorganisms were deposited.
He microscopically demonstrated the presence of microorganisms in the cotton. In his famous swan necked flask experiment, he showed that growth could not occur in sterile media unless airborne contamination had occurred.

Factors Affecting Air Microflora - A number of intrinsic and environmental factors influences the kinds and distribution of the microflora in air. Intrinsic factors include the nature and physiological state of microorganisms and also the state of suspension. Spores are relatively more abundant than the vegetative bacterial cells.
This is mainly due to the dormant nature of spores which enables them to tolerate unfavourable conditions like desiccation, lack of enough nutrients and ultraviolet radiation. Similarly fungal spores are abundant in the air since they are meant for the dispersal of fungi.
The size of the microorganisms is another factor that determines the period of time for which they remain suspended in air. Generally smaller microorganisms are easily liberated into the air and remain there for longer period. Fungal mycelia have a larger size and hence mainly fragments of mycelia will be present in air. The state of suspension plays an important role in the settling of microorganisms in air. Organisms in the free state are slightly heavier than air and settle out slowly in a quiet atmosphere. However, microorganisms suspended in air are only rarely found in the free state.
Usually they are attached to dust particles and saliva. Microorganisms embedded in dust particle settle out rapidly and in a quiet atmosphere they remain airborne only for a short period of time. Droplets which are discharged into the air by coughing or sneezing are also remain suspended in air for a short period of time. When their size decreases by evaporation they remain for a longer period in air.
Environmental factors that affect air microflora include atmospheric temperature, humidity, air current, the height at which the microorganisms are found etc. Temperature and relative humidity are the two important factors that determine the viability of microorganisms in aerosol. Studies with Serratia marcesens and E. coli show that the airborne survival is closely related to the temperature.
There is a progressive increase in the death rate with an increase in temperature from -18°C to 49°C. Viruses in aerosols show a similar behaviour. Particles of influenza, poliomyelitis and vaccinia viruses survive better at low temperature from 7 to 24°C.The optimum rate of relative humidity (RH) for the survival of most microorganisms is between 40 and 80 percent. Low and high relative humidity cause the death of most microorganisms. Almost all viruses survive better at a RH of 17 to 25 percent.
A notable exception is that of poliomyelitis which survives better at 80 to 81 percent. survival has been found to be a function of both RH and temperature. At all temperatures, survival is best at the extremes of RH. Irrespective of RH, an increase in temperature leads to decrease in survival time.Air current influences the time for which either the microorganisms or the particles laden with microorganisms remain suspended in air. In still air the particles tend to settle down. But a gentle air current can keep them in suspension for relatively long periods. Air current is also important in the dispersal of microorganisms as it carries them over a long distance.
Air currents also produce turbulence which causes a vertical distribution of air flora. Global weather patterns also influence the vertical distribution. High altitudes have a limiting effect on the air microflora. High altitudes are characterized by severe conditions like desiccation, ultraviolet radiation and low temperature. Only resistant forms like spores can survive these conditions. Thus high attitudes are characterized by the presence of spores and other resistant forms.

Distribution of Microbes in Air - No microbes are indigenous to the atmosphere rather they represent allochthonous populations transported from aquatic and terrestrial habitats into the atmosphere. Microbes of air within 300-1,000 or more feet of the earth's surface are the organisms of soil that have become attached to fragments of dried leaves, straw or dust particles, being blown away by the wind. Species vary greatly in their sensitivity to a given value of relative humidity, temperature and radiation exposures.
More microbes are found in air over land masses than far at sea. Spores of fungi, especially Alternaria, Cladosporium, Penicillium and Aspergillus are more numerous than other forms over sea within about 400 miles of land in both polar and tropical air masses at all altitudes up to about 10,000 feet.
Microbes found in air over populated land areas below altitude of 500 feet in clear weather include spores of Bacillus and Clostridium, ascos­pores of yeasts, fragments of myceilium and spores of molds and strepto­mycetaceae, pollen, protozoan cysts, algae, Micrococcus, Corynebacterium etc.
In the dust and air of schools and hospital wards or the rooms of persons suffering from infectious diseases, microbes such as tubercle bacilli, streptococci, pneumococci and staphylococci have been demonstrated.
These respiratory bacteria are dispersed in air in the droplets of saliva and mucus produced by coughing, sneezing, talking and laughing. Viruses of respiratory tract and some enteric tract are also transmitted by dust and air. Pathogens in dust are primarily derived from the objects contaminated with infectious secretions that after drying become infectious dust.
Droplets are usually formed by sneezing, coughing and talking. Each droplet consists of saliva and mucus and each may contain thousands of microbes. It has been estimated that the number of bacteria in a single sneeze may be between 10,000 and 100,000. Small droplets in a warm, dry atmosphere are dry before they reach the floor and thus quickly become droplet nuclei.
Many plant pathogens are also transported from one field to another through air and the spread of many fungal diseases of plants can be predicted by measuring the concentration of airborne fungal spores. Human bacterial pathogens which cause important airborne diseases such as diphtheria, meningitis, pneumonia, tuberculosis and whooping cough are described in the chapter "Bacterial Diseases of Man".

Air Microflora Significance in Hospitals - Although hospitals are the war fields for combating against diseases, there are certain occasions in which additional new infectious diseases can be acquired during hospitalization. Air within the hospital may act as a reservoir of pathogenic microorganisms which are transmitted by the patients.Infection acquired during the hospitalization are called nosocomial infections and the pathogens involved are called as nosocomial pathogens. Infections, manifested by the corresponding symptoms, after three days of hospitalization can be regarded as nosocomial infection (Gleckman & Hibert, 1982 and Bonten& Stobberingh, 1995). Nosocomial infection may arise in a hospital unit or may be brought in by the staff or patients admitted to the hospital.The common microorganisms associated with hospital infection are Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, members of Enterobacteriaceae and respiratory viruses. Development of high antibiotic resistance is a potential problem among nosocomial pathogens. For example, Methicillin Resistant Staphylococcus aureus (MRSA) and gentamicin resistant Gram-negative bacilli are of common occurrence. Even antiseptic liquids used would contain bacteria, for example Pseudomonas, due to their natural resistance to certain disinfectants and antiseptics and to many antibiotics.
Nosocomial pathogens may cause or spread hospital outbreaks. Nosocomial pneumonia is becoming a serious problem nowadays and a number of pathogens have been associated with it. (Bonten & Stobberingh, 1995). Frequent agents are Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Enterobacter, Klebsiella, Escherichia coli and Haemophilus influenzae. Other less frequent agents are enterococci, streptococci other than S. pneumoniae, Serratia marcescens, Citrobacter freundii, Acinetobacter sp. and Xanthomonas sp.
In addition Legionella, Chlamydia pneumoniae and Mycobacterium tuberculosis have also been reported. Nosocomial transmissions of tuberculosis from patients to patients and from patients to health care workers have also been well documented (Wenger et a/., 1995). There are two main routes of transmission for nosocomial pathogens, contact (either direct or indirect) and airborne spread. Airborne spread is less common than the spread by direct or indirect contact. It occurs by the following mechanisms. The source may be either from persons or from inanimate objects.
In case of spread from persons the droplets from mouth, skin scales from nose, skin exudates and infected lesion transmit diseases such as measles, tuberculosis, pneumonia, staphylococcal sepsis and streptococcal sepsis. Talking, coughing and sneezing produce droplets. Skin scales are shed during wound dressing or bed making.
In case of inanimate sources particles from respiratory equipment and air-conditioning plant may transmit diseases. These include Gram-negative respiratory infection, Legionnaire's disease and fungal infections.

Air Microflora Significance in Human Health - The significance of air microflora in human health relies on the fact that air acts as a medium for the transmission of infectious agents. An adult man inhales about '5m3 of air per day. Although most of the microorganisms present in air are harmless saprophytes and commensals, less than I % of the airborne bacteria are pathogens.
Eventhough the contamination level is very low, the probability of a person becoming infected will be greatest if he is exposed to a high concentration of airborne pathogens. Carriers, either with the manifestation of corresponding symptoms or without any apparent symptoms, may continuously release respiratory pathogens in the exhaled air.
Staphylococcus aureus is the most commonly found pathogen in air since the carriers are commonly present. The number of S. aureus in air may vary between 0-l/m3 and 50/m3.
Practically speaking, outdoor air doesn't contain disease causing pathogen in a significant number to cause any infection. The purity of outdoor air, however, is an essential part of man's environment. Dispersion and dilution by large volume of air is an inherent mechanism of air sanitation in outside air.
In the case of indoor air chance for the spread of infectious disease is more, especially in areas where people gather in large numbers. For example, in theatres, schools etc.

Air-Borne Microorganisms and Human Diseases
Air-borne microorganisms cause dangerous diseases in human beings. A detailed study of these diseases falls under the preview of a text book of medical microbiology. A chart representing air-borne diseases is given below for ready reference :

Pathogen Diseases
Viral diseases/Causative agents
Mumps/Mumps virus
Influenza/Myxovirus influenzae (A, B, C)
Poliomyelitis/Poliovirus
Common cold/Rhinovirus
German measles/Rubella virus
Measles/Rubeola virus
Chickenpox/Varicella virus
Small pox/Variola poxvirus

Bacterial diseases/Causative agents
Whooping cough/Bordetella pertussis
Psittacosis/Chlamydia psittaci
Diphtheria/Corynebacterium diphtheriae
Q. fever/Coxiella burnettii
Sinusitis, Bronchitis/Haemophilus influenzae
Tuberculosis/Mycobacterium tuberculosis
Primary atypical pneumonia/Mycoplasma pneumoniae
Pneumococcal pneumoni/Neisseria meningitidis (=Pnemococcus pneumoniae)
Scarlet fever and others/Streptococcus pyrogenes
Pneumonic plague/Yersinia pestis

Fungal diseases (Systemic mycoses)/Causative agents
Aspergillosis/Aspergillus funigatus
Blastomycosis/Blastomyces dermitidis
Gilchrist’s disease/B. braziliensis
Candidiasis/Candida albicans
Coccidiomycosis/Coccidioides immitis
Cryptococcosis/Cryptococcus neoformans
Histoplasmosis/Histoplasma capsulatlum

Air-borne microorganisms cause two types of hypersensitivity : immediate allergic reactions and delayed allergic reactions. The immediate allergy causing microorganisms include large fungal spores such as those of Puccinia and Alternaria spp. which would get deposited in the nose, and Cladosporium sp. which can reach the larger bronchi. Contrary to it, the microorganisms causing delayed allergic reactions are generally smaller than 5 mm and consist of actionomycetes, Aspergillus and Penicillium sp. The air-borne fungal spores get dehydrated thus reducing their size and density while they are in air. On inhalation, these spores quickly absorb moisture from the saturated air of the nose, increase in their size and modify the site of their disposition inside the human body.

Enumeration of Microorganisms in Air - There are several methods, which require special devices, designed for the enumeration of microorganisms in air. The most important ones are solid and liquid impingement devices, filtration, sedimentation, centrifugation, electrostatic precipitation, etc.
However, none of these devices collects and counts all the microorganisms in the air sample tested. Some microbial cells are destroyed and some entirely pass through in all the processes.Some of the methods are described below.

Impingement in liquids: In this method, the air drawn is through a very small opening or a capillary tube and bubbled through the liquid. The organisms get trapped in the liquid medium. Aliquots of the liquid are then plated to determine its microbial content. Aliquots of the broth are then plated to determine microbial content.

Impingement on solids: In this method, the microorganisms are collected, or impinged directly on the solid surface of agar medium. Colonies develop on the medium where the organism impinges.
Several devices are used, of which the settling-plate technique is the simplest, In this method the cover of the pertridish containing an. agar medium is removed, and the agar surface is exposed to the air for several minutes. A certain number of colonies develop on incubation of the petridish.Each colony represents particle carrying microorganisms. Since the technique does not record the volume of air actually sampled, it gives only a rough estimate. However, it does give information about the kind of microorganisms in a particular area. Techniques wherein a measured. Volume of air is sampled have also been developed. 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.
Filtration: The membrane filter devices are adaptable to direct collection of microorganisms by filtration of air. The method is similar in principle to that described for water sampling.

Sieve Sampler - This is a mechanically simpler form of impinger. The instrument is more or less similar to that of slit sampler with an enclosed chamber. The particles containing microorganisms are distributed over the plate as separate air jets through several holes. Upon incubation these particles form colonies which can be counted.
For more efficient sampling and size grading of particles Anderson developed a multistage sieve device in which several impingers with holes of different sizes are arranged in series.

Electrostatic Precipitation - Electrostatic precipitation is an efficient method of removing particles from air. In Litton large volume air sampler the air is allowed to pass through the electrodes.The charged particles fall on a rotating disc which is fed with collecting fluid at a rate of 10ml per, minute. Air is sucked into the chamber by a rotating fan at the bottom. The low resistance of the system enables high rates of air flow. They are suitable for large volumes of air. Luckiesh et al. devised a sampler which contains two removable covers. Each unit has one upper electrode and one lower electrode. In one unit the upper electrode is negative and the lower electrode is positive and in the other unit the electrical condition is reversed. Air is drawn at equal rates in both the units. Charged microorganisms are collected in the petridishes placed on opposite electrodes.

Significance of Microorganisms in Air - As long as microorganisms remain in the air they are of little importance. When they come to rest they may develop and become beneficial or harmful. Knowledge of the microorganisms in air is of importance in several aspects.
Food manufacture:Microorganisms that have been transporated through the air and have settled on, or in, the material are involved in various fermentation products. Production of alcoholic beverages, vinegar, sauerkraut, ensilage, dairy products, etc., are often due to microbial activity.

Spoilage of foods and fermentation products:Microorganisms are often troublesome in the home and in industry where foods and other fermentation products are prepared. In industrial processes, where particular organisms are to be grown, to supply sterile air free from contaminating organisms is a considerable problem.
Airborne diseases:There are two main sources of microorganisms in air. These are saprophytic soil organisms raised as dust, and organisms from body tissues introduced into the air during coughing, sneezing talking, and singing.Most dust particles laden with microorganisms are relatively large and tend to settle rapidly. Droplets expelled during coughing, sneezing, etc consist of sativa and mucus, and each of them may contain thousands of microorganisms.Most droplets are large, and, like dust, tend to settle rapidly. Some droplets are of such size that complete evaporation occurs in a warm, dry climate, and before they reach the floor quickly become droplet nuclei. These are small and light, and may float about for a relatively long period.
Airborne diseases are transmitted by two types of droplets, depending upon their size.(1) Droplet infection proper applies to, droplets larger than 100 µm in diameter.(2) The other type may be called airborne infection, and applies to dried residues of droplets. Droplet infection remains localized and concentrated, whereas airborne infection may be carried long distances arid is dilute.

Control of Air borne Microorganisms - Various methods for the removal or destruction of microorganisms have been employed and found to be practicable. Airborne microorganisms are controlled through the application of physical techniques or chemical agents.
Air merely represents a special environment for their application. Under certain conditions disinfection or sterilization of air is desirable. Several general methods are available for the control of microorganisms in the air of rooms and buildings, and are described in the following paragraph.

Dust control: Dust found in homes, offices, schools, factories and hospitals arises from airborne sand, ash, and soot, soil and lint from bedding, clothing and carpets. Most dust particles are laden with a variety of microorganisms, and have been studied particularly in relation to infections of respiratory tract and skin, and secondary infections of burns and wound. Suppression of dust in room cleaning operations is therefore, extremely important. Oiling floors, bedclothes, and other textiles is a highly effective method for the control of dust. Use of dry vacuum pick up, followed by the application of an appropriate disinfectant-detergent solution has been recommended for dust removal. Where vacuum cleaning facilities are not available, some material such as oiled saw dust should be applied before sweeping. This prevents the scattering of dust.

Ultraviolet radiation: The lethal effect of ultraviolet radiation on microorganisms is discussed in the chapter 'Microbial control'. Application of this killing effect has been made in the irradiation of air with ultraviolet light using a wavelength of 254 nm, which is microbicidal but not too irritating. These radiations are effective only when they make direct contact with the particles carrying the organisms, as they have little peneterating power. Secondly ultraviolet 91Ys are irritating to human eyes arid skin. Practical application, therefore, requires skillful installation of the lamps. Rooms which are either unoccupied, or occupied for short periods of time, are exposed to direct irradiations. When the rooms are not occupied, ultraviolet lights are left on. In occupied rooms indirect irradiations ate used, and the occupants are shielded from direct exposure to the rays. In some situations air can be treated apart from the room or space. In air -circulating systems, air is first filtered and then passed through a tube, where it is irradiated by powerful ultraviolet sources.

Bactericidal vapours: Many airborne microorganisms are killed when certain chemical substances are vaporised or sprayed into the air of a room. Germicidal substances are dispersed as aerosols. Vapours of propylene glycol and triethylene glycol are strongly germicidal. These are colourless, tasteless, non-irritating, nontoxic, and not explosive or corrosive. The vapour from as little as 0.5 mg of propylene glycol can kill nearly all the microorganisms in a liter of heavily contaminated air within 15 seconds. Triethylene glycol is nearly 10 times as germicidal.

Laminar air flow system: In this system air passes through high efficiency particulate air, (HEPA) filters. These consist of cellulose acetate (filter medium) pleated around aluminium foil. Particles as small as 0.3 µm are removed by this filter system. Air is passed through a bank of these filters and into the enclosure, so that the entire body of air moves with uniform velocity along parallel flow lines. Many other methods and practices are useful in controlling microorganisms in air.Ventilation is one such method which is very effective in controlling airborne diseases indoors. With extensive development in space technology, electronics, and the aerospace industry an extremely high degree of cleanliness is required. In recent years much attention is DOW paid to aerobiology, particularly air hygiene.