Title: Plan and Design

Aim:

To investigate whether continuous exposure to common indirect radiation will significantly

affect the phenotype of fungal cells compared to bacterial cells by altering their growth and
development which can be used to deduced its effect on human and at large avoid the use of lab
animals for these testing.

Background:

Studies have shown that microorganisms that are exposed to low frequency radiation are likely to

be enhanced in growth (non-thermal effect). However high frequency radiation tends to have

thermal consequences where it completely destroys the microorganisms. In a study in which

Escherichia coli cultures were exposed to microwaves at temperatures below 40°C, transient

morphological changes (dehydrated appearance) and openings of pores in the cell membrane

were observed. It seems that this effect is electro kinetic in nature (caused by increased

movements of anions and cations), causing localized structural disarrangements of the cell

membrane, which results in the emergence of pores. The pore increase in size due to the cell

molecules absorption of the microwave or cell phone energy. Large pores in the membrane cause

leakage of vital intracellular molecules; which led to their death.

Another study determines that radiation can affect the rate of growth of S. Cerevisiae cells

cultures. The experimental yeast solutions were exposed to the applied exposures under the

controlled conditions with the changes in yeast cell growth pattern and temperature being

continuously monitored. The results obtained show that the microwave radiation at the selected

frequencies and powers induces modulating effects in yeast cells. The findings clearly reveal that
microwaves at a particular frequency and power can increase or inhibit the proliferation of S.

Cerevisiae cells and these effects are not caused by any elevation in the temperature. Ultraviolet

radiation does not kill yeast cells outright. Instead, it damages their DNA. In fact, at lower

exposure times, most yeast cells might not die at all, but many will become mutated. Therefore,

it is expected that the cultures exposed to radiation will exhibit several change. These changes

includes, but are not limited to: colour change and decrease in culture size and height of the

mold; irregularly shaped microbe organisms, damaged DNA resulting in organism not

performing biological functions; crystallization or loss of water in cells resulting lysis or

damaged cell wall; destruction of cytoplasmic structures; changes of intracellular ion

homeostasis and slowed or accelerated growth.

Material/Apparatus:

Nutrient Gelatin:

 4 petri dishes
 7 g unflavored gelatin
 1000 ml distilled water
 1 bouillon cube
 3.5 g sugar
1. Direct swab
 Fungus: Black bread mold (Rhizopus stolonifer)
 Bacteria: mouth swab (Epidermidis, S. aureus)
2. Paper towels
3. Magnifying glass
4. Actively used Microwave oven
5. Pipette
6. Sterilized swabs
 7. Gloves
8. Face mask
9. Notepad and pen
10. Camera

Methodology:

Procedure 1: making nutrient gelatin growth media

1. Safety precaution was taken from the onset of this experiment, therefore gloves and face
mask were used when handling the petri dishes and other materials to keep it as sterile as
possible.
2. Ideally nutrient agar is the best growth medium but it was not available so in its place a
growth medium was made by adding 7 grams unflavored gelatin to 1000 ml distilled
water.
3. After the gelatin was completely dissolved in the water the solution was place over low
heat, after it came to a boil a bouillon cube and 3.5 g sugar was added.
4. After all the ingredients dissolved completely, the solute was taken off the heat and set
aside.
5. Four petri dishes with lids were sterilized in hot water for a few minutes after which the
gelatin was poured into the dish to half-fill it.
6. Each petri dish was set aside to cool and solidify.

Procedure 2:

1. The 4 prepare media were labelled 1 – 4.

2. A pipette was used to suction the water off the nutrient gelatin media followed by
blotting the petri dish lid to with a paper towel to remove any condensation formed.
3. With the use of a sterilized swab, the growth medium was streaked and labelled. Plates 1
and 2 were swabbed with bacterial swat with a swab from the mouth and plates 3 – 4
 were directly swatted from a free formed black bread mold. The petri dishes were closed
immediately to after to avoid contamination.
4. Plates 1 and 3 were designated as the control samples and were placed in an environment
that was conducive to their growth; away from any form of direct radiation.
5. Plates 2 and 4 were set near an actively used microwave oven over a period of 7 days.
6. After the week ended using gloves, the covers were removed the covers from the dishes
to be examined and documented.
7. The bacterial and fungal plates were examined to observe and identify any changes using
a magnifying glass.
8. Using a good camera pictures were taken of the two cultures, the control samples and
radiation affected samples, of bacteria and fungi with care not to mislabel the pictures.
9. Finally observations of characteristics of each individual samples were recorded in a
notepad using a pen.
 Results:

Organism Colour of Size and Growth pattern:

fauna distribution of form, elevation and
colony margin

Bacteria #1 – Controlled sample Opaque yellow Evenly scattered, Circular, raised

Epidermidis, S. aureus small colonies colonies with entire
margin
#2 – Radiation affected Could not Randomly scattered Colonies sank into
sample observed colonies of various the gelatin.
Epidermidis, S. aureus without the use sizes
of a
microscope
Fungi #3 – Controlled sample Black tread- 3 Large colonies filamentous,
(yeast) Rhizopus stolonifer like spores umbonate colonies
with filiform
margins
#4 – Radiation affected Pale, Randomly disperse Barely decipherable
sample dehydrated needle point-size rhizoid, flat colonies
Rhizopus stolonifer yellow colonies with labale margin

Analysis/Discussion:
The aim of this lab was to investigate whether continuous exposure to common indirect radiation

will significantly affect the phenotype of fungal cells compared to bacterial cells by altering their

growth and development which can be used to deduced its effect on human and at large avoid the

use of lab animals for these testing. Presently the results of this lab is inconclusive since the

cultures need to be examine under a microscope, however a few assumptions can be made based

on what is known about the data.

First off, the effect of radiation was more pronounce in the fungal sample than it was in the

bacterial plate. The controlled sample with the mold had 3 large colonies of black bread mold

had healthy tread-like spores; and the morphology was filamentous, umbonate with filiform

margin. The radiation–affected plate was comparatively different: the colonies were very small

and barely decipherable.

Conclusion

If the fungal cultures (being eukaryotic like humans) that are exposed to radiation is found to

exhibit signs of mutation, we can deduce humans can develop mutations from exposure too.

Furthermore, since the parameter of this study is limited in terms of available equipment for

testing genetic variables this experiment is confined to creating a framework for future research

by providing a methodological outline and recommendations for further explorations.

How microbes are affected by low and high frequency radiation

Studies have shown that Microorganisms that are exposed to low frequency radiation are likely
to be enhanced in growth (non-thermal effect). However high frequency radiation tends to have
thermal consequences where it completely destroys the microorganisms. In a study in which
Escherichia coli cultures were exposed to microwaves (18 GHz, absorbed power 1500 kW/m2,
electric field 300 V/m) at temperatures below 40°C (to avoid the thermal effects of microwaves),
transient morphological changes (dehydrated appearance) and openings of pores in the cell
membrane (approximately 20 nm in diameter, enabling passage of dextran molecules) were
observed. It seems that this effect is electro kinetic in nature (caused by increased movements of
anions and cations), causing localized structural disarrangements of the cell membrane, which
results in the emergence of pores. The pore increase in size due to the cell molecules absorption
of the microwave or cell phone energy. Large pores in the membrane cause leakage of vital
intracellular molecules; which may lead to their death.
Expected Signs of phenotypic changes in microbes which indicted that mutation has
occurred
• Change in colour and cell number
• Irregular shape
• Damaged DNA
• Crystallization or loss of water in cells
• destruction of cytoplasmic structures
• changes of intracellular ion homeostasis
• damaged cell wall
• slow growth
• alter protein conformation resulting in organism not performing biological functions.
Effects will vary among different microbes and How radiation inhibits microbial growth
fungi- yeast
A study conducted by XXX from the University of YYY in ZZZ which aimed to test the
hypothesis that low power MW radiation at the selected frequencies and powers (1800 MHz and
2100 MHz; and -10, 0, 17 dBm) can affect the rate of growth of S. Cerevisiae cells cultures. The
experimental yeast solutions were exposed to the applied exposures under the controlled
conditions with the changes in yeast cell growth pattern and temperature being continuously
monitored. The results obtained show that the MW radiation at the selected frequencies and
powers induces modulating effects in yeast cells. The findings clearly reveal that MW at the
particular frequency and power can increase or inhibit the proliferation of S. Cerevisiae cells and
these effects are no caused by any elevation in the temperature. Ultraviolet radiation does not kill
yeast cells outright. Instead, it damages their DNA. In fact, at lower exposure times, most yeast
cells might not die at all, but many will become mutated.
Bacteria
When microorganisms are subjected to UV light, cellular DNA absorbs the energy by purines
and pyrimidine bases, and adjacent thymine molecules link together, as figure illustrates. Linked
thymine molecules are unable to encode adenine on messenger RNA molecules during the
process of protein synthesis. Moreover, replication of the chromosome in binary fission is
impaired. The damaged organism can no longer produce critical proteins or reproduce, and it
quickly dies. Ultraviolet light is especially effective in inactivating viruses. However, it kills far
fewer bacteria because of DNA repair mechanisms. Once DNA is repaired, new molecules of
RNA and protein can be synthesized to replace the damaged molecules.
Are both microbes equally sensitive to non-ionizing radiation?
The fungi tends to be more resistant to non-ionizing radiation than bacteria
The observations on the effect of temperature upon the sensitivity of bacteria to ultraviolet light
lead to the conclusion that the growth of the cultures, which depends decidedly upon incubation
time and temperature, may be one of the prime factors that determine the bacterial sensitivity to
ultraviolet light.
What same effect does non ionizing radiation on the microbes?
It penetrates poorly however in damages Dna
Causes bonds to form between adjacent pyrimidine bases, usually thymines, in DNA
chains.These thymine dimmers inhibit correct replication of DNA during cellular reproduction.
What structures may be able to protect microbes from radiation
There are several microorganism with extremophilic features which allows them to resistant to
extreme conditions (extremophile). A structure found in bacteria that provides the ability for it to
thrive in physical extreme conditions is the Bacterial endospore. An endospore is a bacterial
survival strategy in response to an unfavorable change in the environment. Endospores are
produced by bacteria from the phylum Firmicute and are generally gram-positive. These spore
forming bacteria include Bacillus and Clostridium species which are known for their
pathogenicity. The endospore is a tough protective structure encasing the chromosomal DNA of
the bacteria. Comprised of multiple layers including an enzyme resistant spore coat and protein
enriched core wall they are highly resistant to UV, heat and chemical disinfectants, making it one
of the most challenging organisms to eradicate in healthcare environments. A structure existing
in fungi that support them to be resistant to extreme conditions is melanin. Melanins are
enigmatic pigments that are produced by a wide variety of microorganisms including several
species of pathogenic bacteria, fungi and helminthes. Melanins form a diverse group of pigments
synthesized in living organisms in the course of hydroxylation and polymerization of organic
compounds. Melanogenesis has probably evolved parallelly in various groups of free living
organisms to provide protection from environmental stress conditions such as ultraviolet (UV)
light, oxidizing agents and ionizing radiation .Most fungi in soil are melanized and the
production of this pigment may protect or contribute to the ability of fungi to survive from
diverse environmental insults. The importance of fungal melanization as a metabolic product is
suggested by the fact that melanotic fungi often produce large quantities of melanin. For
example, melanin accounts for 30% of the dry weight of Agaricus bisporus. In fungi, melanin
granules are localized to the cell wall where they are likely cross-linked to polysaccharides.
Recent studies suggest the fungal melanin may be synthesized in internal vesicles akin to
mammalian melanosomes and transported to the cell wall.
Which microbes should be able to survive the longest
Micro‐organisms surviving under radiation are commonly referred to as radioresistants.
Extraordinarily, some of these organisms are able to survive in both ionizing and nonionizing
radiation environments, which could otherwise be lethal to others
How microwave radiation (UV) reacts with cells
Ultraviolet light is the light between X-Rays and Visible Infrared on the light spectrum, with
wavelengths between 100 nanometers and 400 nanometers. Ultraviolet is additionally divided
into three distinct bands: UV-C, which ranges from 100 to 280 nanometers; UV-B, running from
280 nm to 315 nm; and UV-A, which finishes off that segment of the spectrum from 315nm to
400 nm. The UV-C wavelength range is the germicidal part of the ultraviolet light area of the
light spectrum which can deactivate the DNA in viruses and bacteria, successfully taking out the
chances of reproduction. All the more granularly, the nucleic acid in the cells of an infection
ends up noticeably harmed by the ultraviolet light because of the formation of covalent bonds.
When this happens, the bacteria’s DNA can’t duplicate itself; subsequently, it can’t reproduce
and in this way dies. Mutation is a genetic variation in the base grouping of DNA. Ultraviolet
(UV) light kills cells by harming their DNA. The light initiates the reaction between two
particles of thymine, one of the bases that make up DNA. The subsequent thymine dimer is
extremely stable. However repair of this type of DNA damage- more often than not by extracting
or removing the two bases and filling in the gaps with new nucleotides – is somewhat possible
and effective. All things considered, it breaks down when the harm is extensive. Ultraviolet (UV)
light, applies its mutagenic impact by energizing electrons in particles. The excitation of
electrons in DNA particles frequently brings about the formation of additional bonds between
adjacent pyrimidine (particularly thymine) in DNA. Whenever two pyrimidines are bound
together along these lines, it is known as a pyrimidine dimer. These dimers frequently change the
state of the DNA in the cell and can lead to problems during replication. The cell regularly tries
to repair pyrimidine dimers before replication. However, the repair mechanism can prompt
mutations too. The more the exposure to UV light, the more thymine dimers are formed in the
DNA and the more prominent the danger of an off base repair or a “missed” dimer. On the off
chance that cellular procedures are disrupted on account of an erroneous repair or remaining
damage, the cell can’t complete its normal functions. Now, there is two likely outcomes,
contingent upon the degree and area of the harm. If the harm is not very extensive, dangerous
cells are created from healthy cells. If it is not, the cell will die.

Conclusion

If the fungal cultures (being eukaryotic like humans) that are exposed to radiation is found to
exhibit signs of mutation, we can deduce humans can develop mutations from exposure too.
Furthermore, since the parameter of this study is limited in terms of available equipment for
testing genetic variables this experiment is confined to creating a framework for future research
by providing a methodological outline and recommendations for further explorations.