Group 1 13 August 2009

Aquino, Vrenneli MCB 150 CD – 2L

Cocjin, Gideon
Repotente, April Ann
Visca, Sheila

EXERCISE III
Microorganisms in Air

I. Objectives:

At the end of the exercise, the students must be able to:

• Learn the sedimentation methods using gravity plate and impingement

technique;
• Compare the number and types of airborne microorganisms present in
different environments.

I. Results and Discussion

Table 1. Abundance and growth of air microspora on PCA, NA (incubated for 48

hours at 30°C) and PDA (incubated for five days at 25°C) collected using th
Gravity Plate Method and the Impingement technique
AREA Gravity Plate (unit particle/min) Impingement (cfu/ft3)
PCA NA PDA PCA PDA
bacteri molds
a
Laboratory 3.0 1.5 0.2 0.33 Contaminated No growth
room
Office 0.67 0.67 0.67 ----- 4.8 x 106 No growth
4
Streets 2.4 1.0 0.67 0.2 2.2 x 10 5.6 x 103
Rooftop 0.67 0.4 0.67 0.07 1.4 x 102 No growth

Legend:
PDA – Potato Dextrose Agar
NA – Nutrient Agar
PCA – Plate Count Agar

The gravity plate method and the impingement method were used to
collect samples from the air in different locations namely the rooftop, street, office
and the laboratory room.
A number of techniques have been used to understand aerobiota, each of
them has their advantages and disadvantages. Selection of the right sampler or
technique has paramount importance in assessing the contaminants at a
particular site.
Vegetative cells and spores can be collected through the use of active and
passive methods such as the gravity plate method and the impingement method.
Microorganisms can be removed from the atmosphere through many ways. They
may settle due to gravitational forces (a method used in the exercise to get
samples), or they may be removed by rain or other forms of precipitation. Despite
the factors that tend to remove microorganisms from the atmosphere and reduce
their viability during transport, some microorganisms are accomplished air
travelers.
In the gravity plate method, microorganisms were collected by
sedimentation or gravity without any form of agitation induced in order to not
disturb the system. The unit particles per minute (U. part/min) was obtained by
actual counting of the colonies that had grown into the PCA, NA, and PDA plates
divided by the length of the sampling time which is 20 minutes.
In the impingement method, the air is transmitted through a liquid medium
where the air particles become associated with the fluid and are subsequently
trapped. The usual volume of collection medium is 20 mL and the typical
sampling duration is approximately 20 minutes which prevents evaporation
during the sampling of warm climates or freezing of the liquid medium when
sampling at lower temperatures. The liquid and suspended microorganisms can
be concentrated or diluted by using this method of impingement.
Based on the computed values, results showed that the office with a
computed microbial load of 4.76x106 cfu/ft3 had the most abundant bacterial air
spora followed by the street with a computed microbial load of 2.8x104 cfu/ft3
followed by the rooftop exhibiting 1.4 x 102 cfu/ft3. The laboaratory room samples
were not quantitatively characterized due to the presence of contaminants on the
growth media. The plates were suspected to show colony growth of Serratia
wherein the pipetors were potential source of contamination. For fungal air spora,
the street registered the most abundant spore count with 5.6 x 103 cfu/ft3. (see
Table 1)
Table 1 also shows that the laboratory room exhibits the highest particle
unit of microorganism/ min with a value of 3 part/min on PCA, 1.5 part/min
bacteria in NA but with a lower computed value for mold (0.2 part/min). This is
followed by the street showing 2.4, 1.0, 0.67, 0.2 particles/min in PDA, NA
bacteria and mold and PCA respectively. The rooftop has a computed 0.67, 0.4,
0.67, 0.07 particle/minute in PDA, NA bacteria and mold and PCA respectively.
While the office
The gravity plate method is valued for its precision because the collection
of airborne microorganisms is affected by size and shape of the particles and
surrounding air motion. Large particles are more likely to be deposited. The force
of gravity acts upon all particles heavier than air, pulling them down and
essentially providing spatial limitations on the spread of airborne particles.
Downward molecular diffusion is a randomly occurring process caused by natural
air currents that promote the downward movement of airborne particulates (in still
as well as turbulent air). Molecular diffusion is also influenced by the force of
wind. Although molecular diffusion can occur in any direction, due to the effects
of gravity, the overall trend of the process results in net downward movement
and deposition.
Many factors can influence which spores will be collected, including air
currents, temperature, humidity, size and weight of the airborne spores. Larger
spores may be routinely overestimated using this collection method while the
smaller and lighter spores may not even be collected. The microbes are
sometimes so battered from their long journeys that they can't reproduce. Those
that do manage to grow may take days or weeks to turn up in detectable
numbers. And many microorganisms simply refuse to grow unless they are
feeding on their particular living target—an apple leaf, say, or a human lung.

A major drawback when using an impinger is that there is no particle size

discrimination which prevents accurate characterization of the sizes of the
airborne particles that are collected.

Airborne bacteria could be rooted from soil and bodies of water and for
fungal air spora originated from molds, pathogens of plans or any pre-existing
fungi that has the capacity to liberate their spores into the air layer. As expected,
the street would have the most number of bacterial spora since it has a moving
air plus it is exposed to different human activities that are potential carriers of the
microorganisms thus spore concentration is very high compared to that of the
remaining sites in which access of outdoor air and human intervention are
somehow limited.

More bacterial air spora grew on PCA compared to that of the

NA mainly because PCA contain glucose making it richer in terms of nutrient
content and carbon source thus, enabling it to support the growth of greater
number of organisms.

For the predominant fungus observed in the sedimentation of PDA plates,

presence of Penicillium and Cladosporium would be generally expected since
they are the most common fungi present in the air microspora.

With this, cells thus cause some problems. In order to correct it,
dispersants such as marbles and glass beads are used to prevent bubble
formation which could have affect the microbial cell diffusion, it breaks
aggregates of microorganisms thus uniformly releasing them into the solution
and further trap the microorganisms in the solution because of its pores. Another
problem with this method is that the adverse effect of aerodynamincs which
affects spore deposition. This could be corrected by putting the Petri dish in a
hole which has similar of that of the dish.

To compute for the air microspora load, the following formula were used:

a. No. of liter per sample = sampling time (min.) X capacity of the limiting
orifice (L/min.)

b. Cubic feet of sample = L of sample/28

c. CFU/ ft3 of air = N x V in flask + V of diluents

ft3 of sample
Where:
N= number of colonies per mL of the impingement fluid

Based on the obtained values, bacterial and fungal spora was most
abundant in the street.
For the impingement technique, liquid medium was used to collect
particles and required an input of power.
The chemical and physical parameters of the atmosphere do not favor
microbial growth and survival which is why the microorganisms present in the air
are relatively low in number. Different factors cause the microorganisms’ lethal
mutations and death (Atlas and Bartha, 1993). The atmosphere, accompanied
with different factors, serve as a barrier for the survival of microorganisms

Reference:

Microsoft ® Encarta ® 2007. © 1993-2006 Microsoft Corporation. All rights

reserved.
©2000 Environmental Microbiology Laboratory, Inc.