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AEROMICROBIOLOGY/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
vapour 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 characterised 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 number 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 source 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 ploughing the soil may also release soilborne 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

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, 1-4m, 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.

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.

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.
nvironmenEtal atmospheric temperature, factors that affect air microflora
include ch the microorganisms arhumidity, air current, the height at whie
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 -18C to 49C. Viruses in aerosols show a similar behaviour. Particles of
influenza, poliomyelitis and vaccinia viruses survive better at low temperature
from 7 to 24C.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.

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.
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.
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.

ALLERGIC DISORDERS BY AIR MICROFLORA


Microflora of air is responsible for several allergic disorders. A range of airborne
particles, such as pollen, fungal spores, insect debris, animal danders, mites etc.
are recognised allergens.
They bring about such allergic disorders as bronchial asthma, allergic rhinitis and
atopic dermatitis. In most countries pollen has been implicated and studied in
relation to these allergic disorders. However, it has now been established that
fungal spores are also equally important allergic agents. Fungal aerosols are
present in much higher densities than pollen in some environments under some
climatic conditions.
A thorough screening of diurnal, seasonal and annual variations in both indoor as
well as outdoor environments is necessary for diagnosis and therapeutic
management of these disorders. In indoor environment, such as home and
occupational surroundings, persons are exposed to fungal allergens. Fungal
aeroallergens in the outdoor environments originate from such sources as cereal
crops, decaying vegetable matter and organic debris.
In indoor environment damp walls, dustbins, mattresses, window frames;
humidifiers etc. are chief source of fungal allergens. Since first report of a fungus
implicated in asthma in 1726 by J. Floyer, spores of a range of fungal taxa have
been shown of clinical interest and being studied world over for their implications
in respiratory allergic reactions. Aeromycology has now become an established

field of research, involving scientists of basic sciences as well as professional


areas.
Several molds (species of Aspergillus, Penicillium, Mucor, Rhizopus),yeasts and
rust and smut spores are shown to be implicated in occupational allergy. On the
basis of prik and intradermal skin tests, several fungi have been identified as
agents of Type I hypersensitivity disorders.
These include species of Alternaria, Aspergillus, Candida, Cladosporium,
Curvularia, Drechslera, Epicoccum, Fusarium, Mucor, Nigrospora, Penicillium,
Rhizopus and Trichoderma.
Besides skin test, fungal allergy is diagnosed by the use of inhalation challenge,
RAST, ELISA, immunodor and immunoblot techniques. These immunological
techniques have proved to be useful in the diagnosis and treatment of pollen and
fungal allergic disorders.
India is not only a sub continent of rich biodiversity but also has various geo
climatic zones. In a given zone there are experienced marked seasonal
fluctuations in temperature and relative humidity. Consequently the sources and
nature of aeroallergens differ in different areas and in different seasons of a given
area.
For instance, pollen becomes important inhalant allergens chiefly in two seasons
1.months of September- November, dominated by pollens of weeds and grasses,
and
2.months of March-May, dominated by pollens of trees

COLLECTION AND ENUMERATION OF AEROALLERGENS

Various methods have been developed to collect and quantify the pollen and
fungal aeroallergens. Durham's gravitational sampler, developed in 1940s is the
simplest and most commonly used sampler even today in some parts of the
world including India. It is a simple device consisting of two metallic discs
separated by three struts.
The microscope slide coated with adhesive is mounted an inch above the centre
of the lower disc, upper disc acting as rain shield. The slides are exposed for 24
hr. The sampler can be used for qualitative analysis of various kinds of spores,
particularly larger ones, giving a rough idea of seasonal trend.
However, some sophisticated samplers based on impaction and suction devices
(battery - as well as power-operated) have now been developed. These provide
more accurate data on species-composition as well as density of aeroallergens.
Based on suction device with definite amount of air sucked in and impacting the
bio particles on the adhesive material on the sampler is the most acceptable
device world over.
The Burkard volumetric traps and Rotorod aeroallergen models are such
samplers. Burkard personal slide sampler (suction), battery operated, suitable for
indoor environment is 10 cm high having a rectangular orifice at the top. The
micro slide is coated with glycerine jelly. The sampler sucks air at flow rate of
10/minute through the orifice.
The particles get impacted on the slide forming a band. Slide is mounted and
scanned. Burkard personal Petri dish sampler is similar to the slide sampler
except that it has a stage to hold a Petri dish and a sieve to cover it. The orifice is
circular. Petri dish containing suitable growth medium become exposed to a
definite volume of air through the sieve on to the Petri dishes. These two
samplers arc suitable for indoor environment. Burkard continuous 7 day trap
(continuous impaction) uses a glycerine jelly coated tape mounted on a rotating
drum.

The drum is connected to a timer and rotates at a constant speed. Tape exposed
for a week is cut into seven strips, one for each day, mounted on a slide in
glycerine jelly and scanned. Each strip can also be divided into 24 vertical bands,
each band representing the particular hour of the day.
Rotorod Aeroallergen Intermitted Sampler - This sampler has an intermittent
rotating impactor, is power operated and ideal for both indoor and outdoor
studies. It is light weight, portable sampler. It has an arm that holds two cubical
rods coated with silicon grease. Unexposed rods remain folded. Arm has a shield
at top for protection from rains. Exposure time can be regulated. The rods are
mounted on a grooved slide in a basic Fuschin stain and scanned.
Anderson Six Stage Volumetric Sampler - This sampler based on suction is
power operated and ideal for indoor environment, though can also be used for
outdoor studies. There are six Petri dishes containing suitable growth medium,
kept under sieves of different pore size. Each size has 400 pores.
The circular orifice at the top sucks air at flow rate of 28.3 l/min. passing it
through the sieve arranged in gradually decreasing order of pore size. Air
impacting on last Petri dish goes out. Particles of similar size impact on a given
Petri dish only. In this way spores/pollens of six dimension orders are impacted
on agar surface of individual Petri dish.
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.
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 m 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 m 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.
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 m 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.
MAJOR DISEASES TRANSMITTED BY AIR
Lists the major diseases transmitted via air. Since air enters the body through the
respiratory tract and since such diseases frequently localize in the nose and
throat, they are called respiratory diseases as a group.
1.Bacterialo
2.Viral
3. Fungal

Diphtheria, Septic sore tnroat, Scarlet fever, Rheumatic fever.


Tuberculosis, pneumonia, Meningitis, Whooping cough.
Small pox, Chicken pox, Measles, German Measles, Mumps,
influenzea, Common cold, psittacosis.
Systemic mycoses.

Source Isolation
Where patients, who may act as source of infection are separated from others.
Protective Isolation
Where susceptible or immunocompromised patients are separated from the
sources.
Cubicle Isolation
Where patients are nursed alone in a room separated by a door and corridor
from others. In cubicle isolation clean, filtered air is supplied to the room and
usually the room is at negative pressure to the corridor for source isolation or at
positive pressure to the corridor for protective isolation.
In the former case it is said to be exhaust ventilated and in the latter case

pressure ventilated. In critical situation, for example in bone marrow transplant


units, the efficiency of air filtration may be increased. Laminar air flow can also be
maintained as a barrier around the patient.
Stringent Isolation
Where patients with highly contagious infections, such as those due to Ebola
virus, are protected by plastic tents. Other control measures include proper
ventilation, use of chemical agents and other sanitation methods.

Plenum Ventilation
This is the most frequently used system in general purpose operating rooms.
Here atmospheric air is filtered in two stages. First stage uses a coarse filter to
remove dust and debris. Secondly a bacterial filter of about 2m pore size with
95 percent efficiency is used inside the inlet grill. Some air may be recirculated
within the suite. An exhaust system removes the air to the outside.
Laminar Flow Ventilation
This is an ultra clean ventilation system but is very expensive. Air is drawn in
from the atmosphere and passes through a 5m filter with 95 percent efficiency.
About 80% of the air in the room is recycled through this filter. Before the air is
delivered to the operating site it is passed through high efficiency particulate air
(HEPA) filters with 0.3m pore size and 99.97 percent efficiency.
Thus, bacterial contamination is prevented. Coarse filters should be changed
every 3-4 months depending upon the climatic conditions while HEPA filters
should be changed every six months.

CONTROLLING IN INDUSTRY

The significance of air flora is well understood in industries in which sterile


products are prepared, processed, packaged or stored aseptically. These include
food industries, pharmaceutical industries and other fermentation industries.
In food industries, contamination of the food products by microorganisms may
lead to spoilage of the products and may cause food borne illness in consumers
who take the spoiled foods. This can be prevented by providing aseptic
atmosphere for the processing and packaging of food products.
In pharmaceutical and allied industries where aseptic filling is done or aseptic
assembly of sterile products is done, sterile atmosphere is required which can be
met by providing filtered sterile air. In fermentation industries, where
microorganisms are used for the production of antibiotics, enzymes or organic
acids, the significance of air microflora has been well recognized.
These industries employ pure cultures of microorganisms and they have to
prevent any further contamination during the fermentation. In aerobic
fermentations, where additional aeration is not required, air in the head space is
enough to carry out the process. Sterilization of the air in the head space is done
along with the sterilization of fermentor.
In aerobic fermentations, where additional supply of air is required, air is
presterilized and introduced into the sterile fermentor. Thus, even with the
addition of large volume of air contamination is prevented by air sterilization. In
certain cases, where the pH of the fermentation medium is very low, sterilization
of air is not required as the contaminants can not grow at this pH. In industrial
fermentation, aerobic processes require the continuous addition of considerable
quantities of sterile air.
Although sterilization by heating is technically possible, it has been regarded as a
costly process for full-scale operation. The most common method of air
sterilization is filtration. Filters for the removal of microorganisms from an

environment can be divided into two large groups, absolute filters and fibrous
filters.
Absolute filters are those in which the pores are smaller than the microorganisms
to be removed. Thus, absolute filters are 100% efficient in removing
microorganisms.
Fibrous filters are made up of fibrous materials such as cotton, glass, slag or
steel wool and the pores are larger than the particles which are to be removed.
For example, the gaps between the fibers are in the range of 0.5 to l5m.
Hence there is always a possibility for the microorganisms to pass through the
filter irrespective of the filter depth. Thus the removal of microorganisms by
fibrous filter can not be absolute.
Despite the efficiency of absolute filters over that of fibrous filters, most
fermentation industries are using fibrous filters because they are cheaper and
have a lower pressure drop. The simplest air filters consist of a steel casing with
an air inlet at the bottom and an outlet at the top. The fibrous material is held in
position by grids or perforated plates on either sides.
HESS'S TUBE METHOD
This sampler is made of a horizontal glass tube which contains a layer of solid
medium at the bottom. As air is drawn in to the tube through the inlet end, the
particles settle onto the medium. Upon incubation colonies develop on the
medium. If the tube is long enough or the flow is sufficiently slow, almost all
particles will settle out before reaching the outlet end.
Settle Plate Method - 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 labelled 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.
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 'minutes.
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 37C for aerobic bacteria
and for 3 days at 22C for saprophytic bacteria.
For molds incubation temperature varies from 1O-50C 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.
Air Centrifuge

The first primitive type of air centrifuge was developed by Wells (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.
Filtration
This is a simple method 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.
Tube Sampler - This is one of the oldest device 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.
Millipore Filter

This type of filters are made of pure and biologically inert cellulose ethers. They
are prepared as thin porous, circular membranes of about 150 m thickness. The
filters have different porosity. Grades from 10nm to 811m. 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.
Impingement into Liquid
Impingement on to Liquid is divided into three types, they are following:
(i) Raised Impinger
(ii) Bead- Buffer Device
(iii) Lemon Sampler
Raised Impinger
In this type of sampler impingement is made within bulk fluid by a jet of air.
Bead Bubbler Device
It is also an oldest device for sampling air. It consists of a 250ml suction flask
which has an outlet on the side connected to suction pump. A glass bubbler,
which is nothing but a glass tube with minute openings at the bottom, is kept in
place inside the flask by a rubber stopper. Glass beads of size about 5mm in
diameter are kept around the glass bubbler.
In addition the flask contains known volume of broth. Air is drawn into the flask
through the glass bubbler when the flask is continuously shaken. The incoming
air escapes into the broth in the form of bubbles through the holes at the bottom
of glass bubbler.

The shaking action of the flask and hence the glass beads facilitate the formation
of bubbles. Thus, microorganisms in the air are dispersed in the broth and after
sampling, an aliquot from the broth is plated for count.
Lemon Sampler
It consists of a glass Folin aeration tube with a perforated bulb with six holes at
one end. The bulb end of aeration tube is contained within a test tube by a two
hole rubber stopper. The bulb is actually centered near the bottom of the tube
and is immersed in 20ml of broth. Two or three drops of olive oil is added to the
broth to prevent foaming.
A kjeldahl trap with square glass baffle is shortened at both ends for
convenience. The intake end is slightly bent and inserted into the other hole of
the stopper. A flow meter measures the rate of airflow entering the upper open
end of the Folin tube. An air pump is attached to the exhaust end of the kjeldahl
trap.
The entire bubbler should be sterilized by autoclaving. Alternatively it can be
sterilized by rinsing with 70% alcohol and dried afterwards. Air is drawn at the
rate of 25-30 liters per minute and dispersed through the broth.
IMPINGEMENT ONTO SOLIDS
They are divided into two types
(i)Hollaender and Dalla Valle Sampler
(ii)Slit Sampler
Hollaender and Dalla Valle Sampler
This sampler consists of a brass container with removable bottom. The container
is fitted with an inverted glass funnel. In the lower portion of container a petridish
base with medium is placed which can be screwed tightly against the gasket

during sampling.
The funnel is kept just above the petridish without touching it. The inside of the
funnel and rim are swabbed with alcohol before use. The air sample passes
through the funnel stem and the airborne microorganisms are impinged upon the
agar medium.
The air is drawn by means of a pump in series with a flow meter. Effective
sampling rate is found to be 28 liters per minute. The method is simple, portable
and efficient.
Slit Sampler
Slit sampler is an efficient and convenient device for the enumeration of bacteria
carrying particles in a unit volume of air. It was introduced by Bourdillon et al. in
1941. It works on the principle that when air is drawn from the environment at a
fixed rate and the suspended particles are allowed to impinge on the surface of
an agar plate, on incubation each particle will form a colony.
The equipment consists of a box closed by an air tight door. There is a slit of
0.33 mm width and 27.5 mm length with vertical parallel sides of about 3mm
deep at the top through which the sampled air enters. The box is connected to a
suction pump which maintains it at a negative pressure of 22.6 mm mercury. At
the correct negative pressure air will enter through a slit of above dimension at
the rate of one cubic foot (28.3 lit.) per minute.
Inside the box at the bottom there is a rotating circular platform for keeping the
agar plate. The platform is usually covered with adhesive or gripping material to
ensure the agar plate being rotated with the platform and is not slipping out of
position while rotating. When the agar plate is correctly positioned on the
platform the slit will be exactly 2mm above and along the radius of the plate.
Thus when the plate is rotated along with platform.

Slit Sampling Procedure


Agar plates with dry, even surface are selected and marked to record the
sampling area, time of sampling, duration of exposure, volume of sampled air
etc. The slit should be unblocked and free from dust and if necessary may be
cleaned with alcohol and by inserting the edge of a stiff paper. The door of the
box is opened and the plate is placed at the centre on the platform.
The distance between the agar surface and the slit is adjusted to be 2mm. At the
correct time, the motor that rotates the plate and the suction pump that
evacuates the sampler are switched on and the negative pressure is maintained
at -22.6 mm mercury. After sampling for the time necessary to collect the
required volume of air the suction pump and the rotor are switched off.
The door is opened and the plate is taken out. The plate is covered with the lid
immediately and incubated at 37C for 24 - 48 hours. After incubation the
colonies are counted and the result is expressed as the number of bacteria
carrying particles per given volume of air.