Microbiology

NAME: SUBHRAJYOTI BANERJEE

COURSE: MSc. BIOTECHNOLOGY
SEMISTER: 2ND
SUBJECT: MICROBIOLOGY ASSIGNMENT
YEAR: 2018-20
 1

ABSCTRACT:
The most commonly utilized
procedure for sterilization in
laboratories are either autoclaving or
hot-air oven. But what is not
considered is the fact that another
device can also be used for this pure
pose of sterilization, disinfection and
many more and it is microwave
devices. Microwaves (MW) are that part of
the electromagnetic spectrum which
occupy a frequency of 0.3 to 30 GHz,
corresponding wavelength range is 0.01 m
to 1 m. For commercial use, a frequency of
.45 GHz has been allotted by International
Commission for Domestic and Industrial
Ovens.
Thus in the following experiments it has
been tried to utilize the microwave ovens to
work in par with the current standing
technology for sterilization. And how or at
which points is this microwave technology
can hold their ground to the current
standing sterilization technology.
 2

Working principle of microwave Molecules of all food are consist of a dipole

ovens: and have positive charge in one side and
Microwave Oven is an electronic device. It have negative charge in another side. If we
uses microwave radiation to heat and cook put electromagnetic fields in this, all
food. The microwave has a wavelength molecules are rearranged: “+” charge is to

Microwave oven
12cm compartment

range of 0.1 cm to 100 cm. A microwave negative pole and “–“ charge is to positive
oven uses a microwave with a wavelength pole. In this process molecules heat is
of around 12 cm. produced by friction. The frequency of
microwave oven is 2,500 megahertz as we
saw before. Then microwave of this
A microwave oven uses microwaves to heat
frequency change the direction of
substance. Microwaves are radio waves. In
electromagnetic fields 2,500,000,000 times
the
in 1 second. Consequently the heat
case of microwave ovens, the commonly
efficiency of a microwave oven
used radio wave frequency is roughly 2,500
megahertz (2.5 gigahertz). Radio waves in is greatly high.
this frequency range have an interesting The microwave ovens have a key
property : they are absorbed by water or component responsible for the production
any polar molecules. When they are of microwaves and its magnetron.
absorbed they are converted directly into
atomic motion and motion is converted into
heat.
Microwaves in this frequency range have
another interesting property: they are not
absorbed by most plastics, glass or
ceramics. And metal reflects microwaves,
which is
why metals cause spark in a microwave
oven. The reason that metal reflects
microwaves is that no electronic waves
resident in inside of conductor because
conductor’s conductivity is very high. The
standard working frequency of a
microwave oven is 2,500 megahertz as it is
the resonance frequency of water.
 3

Magnetron works by creating an Every electromagnetic radiation has an

electric field and a magnetic field. electric and magnetic field
A cathode and an anode produces the perpendicularly. As both fields present
electric field. It helps to throw out the there, there will be microwave radiation.
electrons. There are a copper rod and a
copper ring in the magnetron.
A thorium-tungsten filament coils around An antenna is connected to the copper ring
the rod. Thorium is a good source of cavity. It delivers the microwave to a kind of
electrons. Tungsten has a very high boiling funnel called waveguide. The waveguide
temperature. So, this filament is used as guides the wave to the target compartment.
cathode– negatively charged electrode.
The copper ring is the anode– positively
charged electrode. When the magnetron
gets the voltage, cathode becomes very hot
and releases electrons.
The name of this type of emission is
thermionic emission. So, the electrons move
towards the anode.
There are two magnets in a joint with the
copper ring. Because of the magnetic field
force, the electrons turn their direction.
As a result, electrons start rolling around
the filament in the vacuum of the ring. This
continuous movement of electrons makes a
resonance in a cavity. All the electrons
move at a specific frequency.
 4
Working principle of autoclaves:
Most autoclaves contain a sterilizing
chamber into which articles are place and a
steam jacket where steam is maintained. As
steam flows from the steam jacket into the
sterilizing chamber, cool air is forced out
and a special valve increases the pressure
to 15 pounds/square inch above normal
atmospheric pressure. The temperature
rises to 121.5oC, and the superheated water
molecules rapidly conduct heat into
microorganisms. The time for destruction of
the most resistant bacterial spore is now
reduced to about 15 minutes. For denser
objects, up to 30 minutes of exposure may
be required. The conditions must be
carefully controlled or serious problems
may occur.
Uses of Autoclave: Autoclaving is
used to sterilize culture media, instruments,
dressings, intravenous equipment,
applicators, solutions, syringes, transfusion
equipment, and numerous other items that
can withstand high temperatures and
pressures. The laboratory technician uses it
to sterilize bacteriological media and
destroy pathogenic cultures. The autoclave
is equally valuable for glassware and
metalware, and is among the first
instruments ordered when a microbiology
laboratory is established. Autoclaves are
also used on large industrial scale. Large
industrial autoclaves are called retorts, but
the same principle applies for common
household pressure cooker used in the
home canning of foods
Limitations and Disadvantages of
Autoclave: The autoclave also has
certain limitations. For example, some
plastic ware melts in the high heat, and
sharp instruments often become dull.
Moreover, many chemicals breakdown
during the sterilization process and oily
substances cannot be treated because they
do not mix with water.
Heat requires extra time to reach the center
of solid materials, such as canned meats,
because such materials do not develop the
efficient heat-distributing convection
currents that occur in liquids. Heating large
containers also requires extra time.
Why Steam? In order to kill a cell
through heat, its temperature must be
raised to the point where the proteins in the
cell wall break down and coagulate. Steam
is a very efficient medium for transferring
heat, therefore it is an excellent way to
destroy microbes. Air, on the other hand, is
a very inefficient way to transfer heat when
compared to steam because of a concept
called the Heat of Evaporation.
To bring one liter of water to the
boiling point (100˚C) requires 80kcal of
heat energy. Converting that liter of water
to steam requires 540kcal — this means that
steam at 100˚C contains seven times as
much energy as water at 100˚C.
It’s that energy that makes steam so much
more efficient at destroying microbes.
When steam encounters a cooler object, it
condenses into water and transfers all the
energy that was required to boil it directly
 5
into it, heating it up far more efficiently than
air at similar temperatures.
Microwave sterilization in moisture
presence
The efficiency of microwave sterilization is
supposed to perform its prime under the
presence of moisture as most of the
microwave ovens and devices use 2.5GHz
as their base frequency and this very
frequency is the resonance frequency for
the (polar molecules) water. As we have
already discussed about the working
principle of the microwave devices so we
can say that the microwave ovens come
with some nodes and anti-nodes where the
anti-nodes are the locations highest
efficiency of heating.
Materials and method :-
Test organism : The following microbial
strains were procured from the Lactobacil
symbiotic sachet – Lactobacillus
acidophilus , Lactobacillus rhamnosus ,
Bifidobacterium longum , Bifidobacterium
bifidum , Saccharomyces bouladil.
Media : For the growth media we have used
the local cow milk.
Procedure : The experiment was performed
by the following process –
1. The milk was taken and
autoclaved for 15-20 min at
around 121o C which is supposed
to make approximately 15psi
pressure.
2. Then this batch was divided into
groups as per requirement like
six for lactobacil spore for
different intervals of microwave
exposure, one for blank which
will not be treated with
microwave but will be inoculated
with the spores. Each of 50ml in
quantity.
3. Now, the spores from the sachet is
inoculated to a separate batch for
the purpose of their
intermediation for the
experiment. The sufficiently
required time was provided to the
intermediation culture for it to be
ready for the experiment.
4. Once the intermediation culture
was ready for the experiment it
was inoculated to their respective
batches as described in the 2nd
point.
5. Upon inoculation these were kept
for 10 minutes and then they were
subjected to microwave as the
quantity was less thus the time
frame was also reduced as we
know that lesser the quantity
lesser the time required for it to
heat up.
6. As for that it took no less than
1minute for it to come to boil.
7. Thus the six time frames were
each of 1 minute which came to
be as 1,2,3,4,5,6 minutes
respectively
8. Each of these were then
incubated for a range of 9hours to
see whether they are able to grow
into curd or not.
9. The data for each of these
scenarios were recorded and
microscopic pictures were taken.
10. These data were then compared
with the blank and conclusions
were drawn.
Observation :
It was observed that the sample that was
microwaved for more than 5 to 6 min
showed no curdling or the curding was so
late that it was longer than the experimental
observation i.e. 9 to 10 hrs.
 6
Microwave sterilization moisture
absence:
The efficiency of microwave sterilization is
supposed to perform its prime under the
presence of moisture as most of the
microwave ovens and devices use 2.5GHz
as their base frequency and this very
frequency is the resonance frequency for
the (polar molecules) water. As we know
the efficiency of microwave sterilization is
essentially a function of both the
electromagnetic field strength and the
exposure time. The electromagnetic energy
is expressed largely in two forms:
(i) the factors that depend on the
dielectric properties of the dipole
molecules of the irradiated materials
in the form of heat (thermal effect)
and
(ii) the factors that do not depend on
the dipole molecules in the form of a
direct effect of the radiofrequency
(non-thermal effect).
Thus to ensure this the experimentation was
performed with dry spores and air dried
curd.
Test organism : The following microbial
strains were procured from the Lactobacil
symbiotic sachet – Lactobacillus
acidophilus , Lactobacillus rhamnosus ,
Bifidobacterium longum , Bifidobacterium
bifidum , Saccharomyces bouladil.
Media : For the growth media we have used
the local cow milk.
Procedure : The experiment was performed
by the following process –
1. The milk was taken and autoclaved
for 15-20 min at around 121o C which
is supposed to make approximately
15psi pressure.
2. Then this batch was divided into
groups as per requirement like six
for lactobacil spore for different
intervals of microwave exposure, one
for blank which will not be treated
with microwave but will be
inoculated with the spores. Each of
50ml in quantity.
3. Now the spores are placed on a glass
slide and were subjected to heat for
different intervals of time and as it’s a
dry heat procedure so it is expected
to require a longer span of time in
comparison to the moisture presence
batch. For this it required about
20mins for the sample to come up
around 130oC
4. Thus for this they are subjected to
microwave in 6 different sets which
was below 130oC,above 130o C and at
130o C.
5. Each of these sets were 30, 40 , 50,
60, 70,80 minutes.
6. After the above step the was over the
spores were inoculated to the
sterilized (via. autoclaving) milk and
incubated for the desired time of
about 8hrs.
7. Later the batch was observed for the
curdling effects. Comparing against
the blank which was not exposed to
microwave and was only inoculated
to the sterilized milk.
Observation :
It was observed that the sample that was
microwaved for more than 50to 80 min
showed no curdling or the curding was so
late that it was longer than the experimental
observation i.e. 9 to 10 hrs. And this is
mostly because of the thermal effect.
 7

Superior microbial growth support of good amount. Thus for this the
against autoclaved media: incubation time which is
supposed to be minimum of 4 to 7
Microwaves (MW) are that part of the hrs depending on the inoculation
electromagnetic spectrum which occupy a amount and atmospheric
frequency of 0.3 to 30 GHz, corresponding temperature, we took the
wavelength range is 0.01 m to 1 m. For temperature for 3 hrs to test the
commercial use, a frequency of 2.45 GHz proficiency.
has been allotted by International 5. After this the results were
Commission for Domestic and Industrial observed and the data was
Ovens. collected for conclusions
Microwaves have already been Observation:
applied for sterilization of injection It was observed that the microwaved media
ampoules(Sasaki et al. 1995; Sasaki et al. showed more growth than the autoclaved
1998). Through present study we show that one.
the MW method of sterilization is not just an
alternative method of sterilization but also From this it can be said that as the
promotes faster microbial growth and microwave only works on the polar
higher cell density. molecule and mostly the water components
Thus for that we have compared the growth as for its resonance frequency, so , only its
of microbes in autoclaved sterilized media thermal factor involves with the substance
against microwaved media. microwaved and this might be the cause for
Test organism : The following microbial the superior microbial growth support.
strains were procured from the Lactobacil
symbiotic sachet – Lactobacillus
acidophilus , Lactobacillus rhamnosus ,
Bifidobacterium longum , Bifidobacterium
bifidum , Saccharomyces bouladil.
Media : For the growth media we have used
the local cow milk.
Procedure : The experiment was performed
by the following process –
1. To perform this we are to take
media into three containers of
same quantity.
2. The first container was taken for
autoclaving , the second container
was taken for microwaving, and
the last container was just boiled.
3. To all the containers we add the
spores and provided them
suitable time for incubation.
4. The aim of this experiment was to
observe the curding time which is
expected to be lesser and the
formation of the curd is supposed
to be thick if the culture count is
 8

Discussion :
It was found that the sterility factor of the
microwave system was for their thermal
component and very less dependent for
their non thermal component as the
temperature was required to be the the
similar one which is required in the hot air
oven.
But it can surely be agreed from the given
experiment that the microwave technology
can surely be used for the sterilization
purpose and already beig used in aur
modern days in the sterilization of injection
ampules 1998 as metioned by Sasaki et al.
1995; Sasaki et al.
Another point is that the normal household
microwave ovens are set to a temperature
of 2.5GHz which he resonance temperature
of the water and that is the main reason that
these are taking so much time for objects of
non resonance to heat up which can easily
be fixed if the microwaves were adjusted
for the biological sterility purposes.

But as microwaves release their

residues in the atmosphere so this
wuld have been a disadvantage for
some biological sterility scenarios as
often these kind of action are not
desirable.
But the main boon of this technology I
the shorter time period for the
sterility if the components are in the
presence of components with
resonance ot its working frequency.
This could have been great in
working if the working frequency was
changed to the resonenc frequency
of the cell membranethenthis could
be used as a fast and non thermally
effective to sterilization which it is
now only working for its thermal
factor.
 9

Reference :
1. Microwave sterilized media
supports better microbial
growth than autoclaved media
Vijay Kothari*, Mohini Patadia
and Neha Trivedi
2. Mechanism of Microwave
Sterilization in the Dry State
DAVID K. H. JENG,* KURT A.
KACZMAREK,t ARCHIE G.
WOODWORTH, AND GLENN
BALASKY
3. Biological Applications of
Microwaves Neha Trivedi,
Mohini Patadia, Vijay Kothari