Expt 3: Air/Water Microbiology

A) AIR MICROBIOLOGY
The air contains gases, dust particles, dried vapor droplets. In addition to these, air also contains
more number of microorganisms. The air has vegetative cells and spores of bacteria, fungi, algae
and protozoa cyst. The microbiology of air can be studied under two headings such as outdoor
and indoor microflora.

Outdoor microflora
The air in the microflora, which is found outside the buildings, is referred to as outside air. The
dominant microflora of outside air are fungi. The two common genera of common fungi are
clodosporium and sporoholomyces. Besides these two genera in air, other genera found are
Aspergillus, Alternaria, phytophthora and Erysipher. The outdoor air also contains
basidiospores, ascopores of yeast, fragments of mycelium and conodia of molds.
Among the bacterial genera bacillus, clostridium, sarcine, micrococcus, corynebacterium are
widely used in the outside air. The no and kind of microorganisms may vary place to place,
depending upon human population densities.

Indoor microflora
The air found in the building is referred to as indoor air. The common genera of fungi indoor are
penicillium, aspergillus. The most common genera of bacteria found in indoor air are
staphylococcus, bacillus and clostridium in case of occupants being infected.

Atmospheric air composition

The atmosphere consists of a mixture of gases and variable quantities of water and solid
particles. According to Landsberg, air has following composition.

Element Volume/ Percentage

Nitrogen 78.03
Oxygen 20.99
Argon 0.94
CO2 0.93
H2 0.01
Neon Traces
He Traces
Xe Traces
O3 Very variable
Dust Very variable
Water vapour Very variable

The compositions show slight variations with latitude and to lesser extent with altitude. The
ozone owes its existence in the atmosphere to photosynthesis from oxygen under the influence
of solar UV rays.

Air quality
The air should be free from pathogenic microorganisms. As per the definition of WHO, air
pollution is a situation which is harmful to people or environment.

Enumeration and assessment of microorganisms in air

There are several methods adopted to enumerate microbes in air. They require special devices
and design. The most important methods are solid and liquid impingement devices, filtration,
centrifugation, sedimentation. However none of these devices collect and count all the microbes
in air sample. In general, to assess the microorganisms in air, following methods are adopted.

1. Impingement in liquids
In this method, air is drawn through a very small opening or a capillary tube bubbled through
liquid. The organisms get trapped in liquid medium. Aliquote of liquid are then pated with
medium to determine its microbial content.

2. Impingement on solids
In this method, microorganisms are collected or impinged directly on solid surface of agar
medium by gravity. Colony develops on surface of medium after few days of incubation. Several
devices are used, of which settling plate technique is simplest. In this method, cover of petri
plate containing agar medium is removed and the agar surface is exposed to air sample for
several mins. A certain no of colonies develop on incubation in petri dish. This technique gives
only approximate estimate of microbes. However, it gives information about kind and no. of
microorganisms in a particular area.
Aim: To carry out quantitative analysis of air microflora

Requirements: Sterile nutrient agar plates, incubator.

Theory:
The atmosphere contains all major groups of microbes ranging from algae to viruses. The
sampling of air to determine microbiological content requires special apparatus and devices.

Liquid impingement
The air sample in the fine spray form is passed through broth medium or sterile water, where
organisms get trapped. Aliquots of liquid are then planted, cultured to find out its microbial
content. Various types of impingements are available for sampling.
A) Protein impinge
B)Pre-impinger
C)multi-stage impinge

Solid impaction method

In this method, microbes are collected or impacted directly on solid surface such as sticky
microscopic slide or agar medium in filter dish or petri plate. Various types of impactors are
available for sampling such as
A) Casella slit samplers
B) The casella impactor
C) Hirst spore trap
D) Anderson sampler

Gravity sedimentation method

In this method, petri plate containing suitable agar medium is exposed horizontally for 5-10
mins. The plates are incubated at suitable temperatures and then the number of colonies
developed are counted. Each colony represents a particle carrying microbe which has fallen on
agar surface under the influence of gravity.
Composition of nutrient 5 g/L
agar Peptone
NaCl 5 g/L
Beef extract 1.5 g/L
Yeast extract 1.5 g/l
Agar 13.5 g/L
Procedure:
The experiment is performed by gravity sedimentation method in which sterile petri plate
containing nutrient agar as medium for growth of microbe is taken. The agar plate is then
exposed to air at various locations for various time intervals.

Result:
Different number of colonies were observed using quantitative analysis of air microflora.
Sr. no. Location Time No. of colonies
1 Right side corner 1 min
2 Outside incubator 2 min
3 Inside cabin 2 min
4 In between burners 5 min
5 Inside cabin 5 min
6 Inside incubator 5 min
7 Outside incubator 5 min
8 In between burners 10 min
9 Left side corner 10 min
 B) Water Microbiology:
Various water quality tests are available to detect the number of microorganisms in water and
assist communities in keeping the microbial content of water supplies at a low level and assess
their potability. These tests vary from the most sophisticated tests to standard procedures that
have been used for decades. Various tests that are available are
Gene probe tests

Among the most sophisticated tests for water bacteriology are those that employ gene probes. Gene
probes are fragments of DNA that seek out and combine with complimentary DNA fragments. Often
the test is designed to test for presence of E. Coli in water. This gram negative rod, usually found in
human intestine, is used as an indicator organism. If it is present, then it is likely that the water has
been contaminated with human faeces. The faeces may contain microbial pathogens.

To use gene probe test for E. Coli in water, water is treated to disrupt any bacteria present and release
their nucleic acid. Then a specific E. Coli probe is added to the water like a left hand seeking a right
hand, the probe searches through all nucleic acids in water and unites with E.Coli DNA, if present. A
radioactive signal indicates the match has been made. If no radioactivity is emitted, then gene probe
has been unable to locate its matching DNA and E.Coli is probably absent from the water.

Membrane filter technique

It uses a filtration apparatus and a cellulose filter called membrane filter. A 100 ml water sample is
passed through filter and filter pad is then transferred to a bacteriological growth medium. Bacteria
trapped in filter grow on medium and form colonies. By counting the colonies, an estimate can be
made from the number of bacteria in original 100 ml sample.

Standard plate count

It is generally impractical to test for all pathogenic organisms, but the total number of bacteria can be
calculated. One test is standard plate count. In this test, samples of water are diluted in jars containing
99 ml sterile water and samples are placed in Petri dishes with nutrient agar or other nutritious
medium. After incubation, the colony count is taken and multiplied by dilution factor to obtain total
number of bacteria per ml of sample.

Indicator bacteria can be detected to give an estimate of pathogens. The most common indicator
organisms in water bacteriology are coliform bacteria. These are gram negative rods normally found
in intestine and typified by E.Coli.

Criteria for an organism to be an ideal indicator bacterium

 • The organism should be present whenever enteric pathogens are present.
• The organism should be useful for all types of water.
• The organism should have longer survival time than the hardiest enteric pathogen.
• The organism should not grow in water.
• The organism should be found in warm blooded animals intestine.

Most probable number (MPN) test

In this test, tubes of lactose broth are inoculated with water samples measuring 10 ml, 1 ml, 0.1 ml.
During incubation, coliform organisms produce gas. Depending upon which tubes from which water
sample display gas, an MPN table is consulted and statistical range of number of coliform bacteria is
determined. The MPN test is very easy to perform and interpret, but it does not determine the exact
number of bacteria as the standard plate count does.
Aim: To determine the number of colonies and study the colony characteristics in the given
water sample.

Requirements: MacConkey Agar, sterile petri plates, Nichrome loop, water samples (Tap water,
cool water, injectable water, distilled water).

Theory: MacConkeys Agar is common medium to grow microbes.

Composition of Mac Conkey Agar
Peptone 17g
Protease peptone 3g
Lactose 10g
Bile salts 1.5g
NaCl 5g
Neutral red 0.03g
Agar 13.5g

Procedure:
1. On a sterile plate, pour 20 ml of MacConkey agar and let it solidify.
2. Innoculate 4 different agar plates with the given water samples.
3. Incubate plates for 24hrs at 37°C. Observe number of colonies.

Result:

Sr. Water Sample No. of Colonies

no
1 Distilled water
2 Water for Injection
3 Drinking Water (Canteen)
4 Drinking Water (Cooler)
5 Tap Water