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HALESWOWEN COLLEGE

DOES MUSIC ALTER


MICROBIAL GROWTH?
Jodie Parsons

Contents
Abstract................................................................................................................. 2
Introduction........................................................................................................... 2
Environmental Stimuli............................................................................................ 3
Sound.................................................................................................................... 4
E-coli K12............................................................................................................... 5
The Mozart effect................................................................................................... 6
AIM......................................................................................................................... 7
Hypothesis............................................................................................................. 7
Variables:............................................................................................................... 7
Apparatus:............................................................................................................. 7
Method................................................................................................................... 8
Aseptic technique method..................................................................................... 8
Results................................................................................................................. 10
Results................................................................................................................. 11
Conclusion........................................................................................................... 11
Other research..................................................................................................... 12
Evaluation............................................................................................................ 13
Other Applications............................................................................................... 14
Photos from the experiment................................................................................ 14
Bibliography......................................................................................................... 15

Abstract
The biological effects of electromagnetic waves have been widely studied,
mainly due to their harmful effects, such as radiation-induced cancer and to their
application in diagnosis and therapy. The biological effects of sound, which is in
the form of a longitudinal wave which we are frequently exposed to have been
considerably disregarded by the scientific community. Although a number of
studies suggest that emotions evoked by music may be useful in medical care,
alleviating stress and nociception in patients undergoing treatments in cancer
and burned patients, little is known about the mechanisms by which these
effects occur. It is generally accepted that the mechanosensory hair cells in the
ear transduce the mechanical vibrations into neural impulses, which are
interpreted by the brain and evoke the emotional effects. Recently several
studies suggest that the response to music is even more complex, and evidence
has shown that cell types other than auditory hair cells could respond to audible
sound. Hence, the aim of the present study was to evaluate the response of the
harmless bacterium e-coli k12 to music. The results obtained suggest that music
can alter the growth in cultured cells. The results suggest that audible sounds
could modulate bacterial cell growth, by causing constant stress to the cell
resulting in inhibited growth.

Introduction
Type of bacteria
Photosynthetic
Bacteria
Enterobacter
Aerogenes

Description
Photosynthetic bacteria are a unique species of
microorganisms that use the sun as a source of energy.
Enterobacter aerogenes, part of the Enterobacteriaceae
Family, is a rod-shaped bacteria that causes bacterial
infections, and is usually acquired in a hospital or hospital-type
atmospheres. It usually causes opportunistic infections,
meaning that it will usually only cause a disease in a person or
host that has a compromised immune system. Studies are now
showing it causing increased alarm in community infections. It
rarely is known to cause a disease in someone with a healthy
immune system. These types of bacteria are extremely
sensitive to antibiotics but have the ability to become resistant
through their adaptive capabilities.
Francisella Tularensis
Francisella tularensis has been known to infect small mammals
such as rabbits, muskrats, mice, and also humans.
Interestingly however, is that no case of tularemia has
appeared to be caused through human to human contact.
Infection has appeared to always be caused by contact with
infected animals or forms of transmission such as mosquitoes
or ticks that have bitten infected animals.
Helicobacter Pylori
These types of bacteria are found in the stomach. H. pylori, a
major cause of stomach and other gastro esophageal issues
such as gastric ulcers, chronic gastritis, duodenal ulcers, and
even stomach cancer flourishes in the upper gastrointestinal
tracts of at least half the worlds population.
Bacteria are microorganisms that cannot be seen by the naked eye, they exist in
all environments on Earth. The human body, hosts more bacterial cells within

than it has total cells comprising the body. There are a diverse species of
bacteria, these are summarised in the table below. (ScienceDaily, 2016)

Enterobacter bacterium respond quickly to external changes and is important in


the research of the resistance to antibiotics. These bacteria rapidly adapt to
environmental changes through smart regulation of their gene expression. A
study published in the journal PNAS (Proceedings of the National Academy of
Sciences) presented a theoretical model that can determine the ultimate limit for
how quickly bacteria can adapt their proteins to changes in their environment.
(Pavlov and Ehrenberg, 2013) The growth of bacteria is determined not only by the
composition of their surroundings but also by sudden changes in the living
environment. This has been known since the middle of the 20th century. High
levels of bacteria growth in a stable environment requires a certain kind of
physiology, but environmental changes also require rapid adjustments of the
bacteria's protein production. The newly developed model indicates the
'minimum' time such adjustments require. This is especially important in
research for antibiotics. (ScienceDaily, 2016)
The adaptation process of Enterobacter bacterium is mainly mediated by a
striking combination of transcriptional regulatory networks, which allow bacteria
to sense and convert extracellular, physical or chemical stimuli into a specific
cellular response, resulting in altered gene expression and enzyme activities
(signal transduction). Whereas some of these alterations are reversible and

disappear when the stress is over, others are maintained and can even be
passed on to surviving bacteria.
Bacterial adaption and stress response network (Mackert, 2016)

Environmental Stimuli
Research has been carried out focusing on the investigation of bacterial stress
responses and the effect of external environmental changes. (Goldstein and Soyer,
2008) A better knowledge of the bacterial stress response include the
identification of stress-inducing effectors and key molecular switches is the basis
of understanding and controlling bacterial growth in any environmental setting.
There are vasts amount of environmental stimuli that can cause a stress
response to bacteria. The bacteria are able to sense and respond to a variety of
external stimuli with responses that vary from stimuli to stimuli and from species
to species, the bacterium will move to the most favourable location where there
is plenty of food, enough light and are not stressed. (Foodsafetysite.com, 2016)The
best-understood in current research is chemotaxis in Escherichia coli, where the
dynamics and structure of the underlying pathway is well characterised. There is
increasing experimental evidence that bacteria integrate responses from
different stimuli to generate a coherent taxis response. There is currently a lack
of understanding of the different pathway structures and dynamics and how this
integration is achieved. (Goldstein and Soyer, 2008)
Examples of external stimuli include changes to temperature, light, nutrients and
sound.
Sound
Sound travels through waves which are created by the vibration of an object,
which causes the surrounding air to vibrate. The waves are unique to each sound
causing different patterns of vibration. (PBS LearningMedia, 2016) When a speaker
vibrates from emitting music, the surrounding molecules vibrate in the same
unique pattern which can affect the growth of bacteria in a specific way. Bacteria
lack the ability to hear music, but are sensitive to the environmental vibrations
that they are growing in.
There are four phases of bacterial growth.
Lag Phase: growth is very slow
Log Phase: Begins to multiply exponentially
Stationary Phase: growth stops and stabilises
Death Phase: toxic waste builds up, the cell dies. (Vlab.amrita.edu, 2016)

(1.bp.blogspot.com, 2016) Graph to show the phases through bacterial growth


Not much research has been carried out to determine whether music has a
positive or negative effect on the growth of bacteria. A study by Amir M.
Mortazavian suggests that music affects the survival and activity of
microorganisms, he concluded that sonic waves from classical music affects the
metabolism and growth of bacteria, his study suggested that classical music was
shown to render influences on acidification rate and viability of probiotics and
reduced incubation time. (Mortazavian, Ph.D, 2016) In Germany, a sewage
treatment plant has tested the idea that music could have a positive impact on
microbial growth, the plant used the ability of Mozarts music to motivate
microbes in their treatment facility hoping to drive down energy costs. They
found that sound affects cell metabolism and bought energy costs down by
1,000 euros a month. (Speigel Online, 2016)

E-coli K12

(Bats.ch, 2016)

Escherichia coli is a rod-shaped bacterium. Each bacterium measures


approximately 0.5 m in width by 2 m in length. E. coli is a Gram-negative
bacterium. (Ecoliwiki.net, 2016) E. coli cells stain Gram negative because they
have a thin cell wall with only 1 to 2 layers of peptidoglycan. E. coli is a
facultative anaerobe, which does not require oxygen, but grows better in the
presence of oxygen. (Microbes.ucsc.edu, 2016)E-coli K12 is a non-pathogenic
bacterial cell. This is because it contains no toxins. E-coli K12 is used instead of
E. coli in the lab because it is not harmful , e-coli k12 is a member of the:

Bacteria Proteobacteria gammaproteobacteria Enterobacteriaceae


Escherichia (Hayashi et al., 2006)

The Mozart effect


The term Mozart effect refers to the widely contested theory that exposure to the
music of composer Wolfgang Amadeus Mozart, particularly from an early age,
can improve ones general intelligence. This theory grew out of 1993 research
findings which showed that listening to Mozart temporarily strengthened spatial
logic among a group of college students. (Jenkins, 2001) From the time of their
publication, many members of the media and the public misinterpreted these
findings, leading to the misinformed notion that exposure to Mozart can provide
an overall boost to the intelligence. While most psychologists regard it with
scepticism, the concept of a Mozart effect persists among many members of the
public, due partly to the sale of classical audio recordings alleged to improve
intelligence.
The term Mozart Effect was first coined by Alfred A. Tomatis who used Mozart's
music as the listening stimulus in his work attempting to cure a variety of
disorders. (Alfred Tomatis, 2016) He found that mozart music not only had an effect
on humans, but on animals. Further research has shown that the Mozart effect
also has an effect on cells.
A number of studies have studied the effects of Mozart on eukaryotic cells. Jones
et al. showed that a frequency of 261 Hz altered the growth of hair cells (Jones et
al., 2000) and Zhao et al. showed that sound-wave stimulation from Mozart made
significant changes to protein structure of tobacco cells, producing an increase in
-helix and a decrease in -turn. (Zhao, Wu and Wang, 2001) Xiujuan et al. showed
a sound stimulation effect on cell cycle of chrysanthemum (Xiujuan, Bochu and Yi,
2002) an effect also observed by Zhao et al. (Zhao, J and Wang, 2003) in the callus
growth of Dendranthema morifolium. More recently, Ying et al. showed that the
tonal sound of 5 kHz gave significant increase in cell number of Escherichia coli
bacteria (Ying, Dayou and Khim Phin, 2010) and Shaobin et al. observed that a
frequency of 1 kHz also promoted the growth of E. coli. (Shaobin G, 2016)

(Scienceblogs.com, 2016) The graph shows an increase in spatial IQ test score when listening to
mozart.

Does music affect microbial growth?

AIM: To discover whether music has an effect on microbial growth in a nutrient


agar plate with colonies of e-coli K12 (Enterobacter bacterium)
E-coli K12 is not considered a human or animal pathogen, it is not toxicogenic. Ecoli-K12 is mitigated by its poor ability to colonise the colon and establish
infections.
Hypothesis: If a calm and soothing piece of music is played to colonising
bacteria, the positive rhythm will change the incubation time and growth rate of
bacteria.
Variables:

Control- The petri dish without music but contains e-coli k12
Independent- The type of music played to a petri dish containing e-coli k12
Dependant- The growth of bacteria in each sample
Constant- The temperature and location.

Apparatus:

To make
Nutrient
agar
Distilled Agar powder
water
Hotplate Magnetic
Stirrer
Flask x 1 Beaker x 1
To conductBunsen
Saucepan
theBurner
experiment
Heated Petri dishes x
container 6

To plate
streak petri
dishes

e-coli k12 2 x
inoculating
loops
6 x Sterile Verkon
petri dishes disinfectant
at 9 cm
Distilled Cloth
water
1 x Tray Marking pen
Storage at Bunsen
room burner
temperature
Paper
template

Method
Creating nutrient agar:
1.
2.
3.
4.
5.

Mix 700ml of distilled water with 17.1g of nutrient agar powder


Use a hotplate and a magnetic stirrer to mix, leave to boil for 1 minute.
Put the mix in hot water to keep at a liquid state
Autoclave at 125OC for 15 minutes.
Pour the liquid agar into a petri dish and leave to solidify.

Aseptic technique method.


The aseptic technique is applied to plate streaking bacteria to minimise the
exposure to other bacterium and dust. The goal is total asepsis.
Plate streaking method:
1. Use verkon and a cloth to sterilise the surrounding area, including the
tray. A sterilised working area was established on the tray.
2. Keeping the Bunsen burner as close as possible at all times to the
working area to prevent contamination, the inoculating loops were
flamed using the Bunsen burner to sterilise and left to cool down to
prevent killing any bacteria
3. Using one of the loops, insert into the pot of e-coli k-12 and with the
templates attached to the petri dishes, the e-coli k12 was placed onto
the agar in the petri dishes as close to the edge of the plate as
possible.
4. Flame the pot of e-coli k12 to remove any unwanted bacteria and dust
5. Flame the inoculating loop to remove any bacteria
6. With the second inoculating loop, dip in the distilled water and in the
same spot as the e-coli k12 place the distilled water onto the agar,
being careful not to make an indent in the agar.
7. Flame the pot of distilled water to remove any unwanted bacteria and
dust
8. With the same loop, follow the template underneath the petri dish to
streak the bacteria in lines, turning the petri dish 90 degrees counter
clockwise
9. Continue streaking until the middle of the plate is reached.
10.Flame the inoculating loop
11.Put the lid onto the petri dish and using the maker, label the dish.
12.Repeat the steps for all petri dishes, choose two successful plates, one
for the variable, and one for the control.
Self-drawn image to show how plate streaking was achieved

Image from the experiment


shows the layout of plate
streaking via the aseptic
technique

The experiment method:


1. Using the successfully plate-streaked
petri dishes, attach headphones with
selotape to the independent petri dish
2. Place the control petri dish in the same room, making sure the amount of
light available is the same as the independent petri dish, but also making
sure the control dish is not exposed to sound waves.
3. Attach the headphones that are attached to the independent petri dish to
a computer, and loop the Mozart track to play for 48 hours. This allows the
e-coli k12 to colonise most of the petri dish.
4. Record the results at 12, 24 and 48 hours by using a colony counter.

Image from the experiment, showing headphones attached to the independent petri
dish

Results
The bacteria were left in a silent room with plenty of light, the temperature of the
room stayed at approximately 23oC for the duration of 48 hours. The results were
collected after 12 24 and then 48 hours in order for the e-coli to establish grown
colonies. The table below shows the amount of colonies counted in the time
given.
Colonies
established
Independent
variable
Control variable

After 12 hours

After 24 Hours

After 48 Hours

59

100

283

After 12 hours of incubation time, no bacteria were found to be alive in the petri
dish, after 24 hours of incubation time very few colonies were found in the
independent petri dish, but more were found to be colonised in the control petri
dish. The results continued to show an increase in the amount of colonies in the
control petri dish and established in a 24 hour period approximately 283 live
colonies of e-coli k12, compared to approximately 58 in the petri dish that was
exposed to music.

Effect of music on microbial growth


48 Hours

24 Hours

12 Hours
0

50

100

150

Control Colonies

200

250

300

Independent Colonies

Effect of music on microbial growth


400
350
300
250
200
150
100
50
0
12 Hours

24 Hours
Control Colonies

48 Hours

Independent Colonies

The line graph


shows the number of colonial changes throughout time, the graph shows the comparison
between the two variables on the same measurement day.
The bar graph shows the data in a different way, the independent petri dish grew more
substantially than the control petri dish.

Results

Control petri dish

Independent petri dish

The inverted
image from
the
experiment
shows the
grown
cultures in
the
independent
and control
petri
dishes

Conclusion
In accordance with my hypothesis
the
results show that music does affect
microbial growth, but the data shows that it does not affect the growth in a
positive way. The control and independent petri dishes were left in exactly the
same room, at the same temperature with the same amount of light. The e-coli
k12 in the control petri dish grew more colonies than the e-coli k12 in the
independent petri dish. The incubation time stayed the same. This may be
because of overstimulation or stress , but at this time remains unknown because
of limitations to the experiment. Current research suggests that the Mozart effect
has a positive impact on the growth of bacteria (Lrs.ed.uiuc.edu, 2016), my results
do not correlate with the current research. Research also suggests that the
harmony in Mozart corresponds to the harmony that binds and breaks down the
bonds between molecules. (Academia.edu, 2016) The growth of bacteria can be
inhibited when the vibration overstimulates the cell causing cell-lysis. (SigmaAldrich, 2016) The Mozart track was played to the independent dish, for a full 48
hours with no stoppage time. This may have caused the molecules within the cell
to vibrate rapidly causing the inhibited growth. Both the control and independent
petri dish had a slow incubation time, nothing grew after 12 hours. The line
graph shows that, as the lag phase of the bacteria started to occur the difference
between the independent petri dish only established 2 colonies, whereas the
control had 50 colonies. This was after 18 hours. The log phase then occurred
after 18 hours where the cells multiplied exponentially and the growth sped up,
after 27 hours the control had established 160 colonies, and the independent
had 20 colonies. This could suggest that the vibrations in the music caused the
bacteria to stay in lag phase due to overstimulation. Overall there was a
130.99% difference in grown colonies between the control and independent. This
can be worked out by:
= ( | 59 - 283 | / ((59 + 283)/2) ) * 100
= ( | -224 | / (342/2) ) * 100

= ( 224 / 171 ) * 100


= 1.309942 * 100 = 130.99%
My results agree and disagree with other research, there are contradicting
answers as to whether audible sound affects microbial growth and more research
is needed. The experiment performed may have had a lot of inaccuracies.
Other research
An experiment into the effects of microbial growth conducted by Kye Jang, which
involved rock music and classical music also showed that bacteria do not
respond well to classical music. (Jang, 2013)

(Jang, 2013) The bar graph shows the difference in the number of bacterial colonies of
each variable.

The graph conduced in the study on the effects of microbial growth shows that
the rock music the bacteria was exposed to had a faster growth rate after the
second measurement than in the controlled petri dish. Classical music had a
negative effect on growth rate. The results show that during the lag phase (1 st
measurement) music could have been causing a prolonged period of lag phase.
Which correlates to my results, both in the classical and rock music 1 st
measurement growth was slowed down. (Jang, 2013)
A study by Shaobin G. showed that audible sound had a negative effect on e-coli
when the e-coli was exposed to a stress factor. Shaobin G. Investigated the
response of Escherichia coli cells to the stimulation by audible sound under the
normal condition and environmental stresses. The results showed that the
audible sound treatment significantly increases the colony forming of E. coli
under the normal growth condition. However, under osmotic stress induced by
the sugar, audible sound stimulation may enhance the inhibitory effect of
osmotic stress on E. coli growth. More interestingly, audible sound treatment
seems to alleviate the inhibitory effect of salt stress on E. coli growth when the
concentration of sodium chloride was increased to 30 g/l, although the action of
sound waves of audible frequency is likely to evoke an inhibition of the growth of
E. coli in the medium containing 20 g/l of sodium chloride. Some potential
mechanisms may be involved in the responses of bacterial cells to audible sound
stimulation. (Shaobin G, 2016)
An experimental investigation conducted Lee Ying on the effects of audible
sound on the growth of e-coli found that at selected frequencies of 1kHz, 5kHz,
and 15 kHz had increases the number of viable cells in a petri dish. Thus showing

that the bacteria reacted positively to the sound treatment which resulted in a
faster growth of the e-coli. (Lee Ying, Dayou and Phin, 2009)

In other research, classical music had a positive effect, in an experiment to


observe the effects of music on bacteria showed classical music had a faster and
greater growth rate than any other variable. (Chow, 2014)

The graph shows classical music grew faster than the control and heavy metal music,
suggesting the bacterial growth was not inhibited. . (Chow, 2014)

Evaluation
There were a lot of limitations with this experiment, the e-coli k12 had a very
unusual long incubation time, and was presumed dead after 12 hours, only after
18 hours did colonies become visible. The room temperature was recorded at
23OC during the daytimes after every 12 hours, but this could have changed
during the night as there was no one in the laboratory to observe the room
temperature. The change in temperature could have caused the unusually long
incubation time but could not have affected results due to the independent and
control variables staying in the same room. Light availability may have also
affected results. The control may have been left in a slightly lighter area than the
independent petri dish, causing the bacteria to multiply faster. The plate
streaking technique may have played an important role in causing error to the
experiment. This is because there could be more e-coli k12 on the inoculating
loop, meaning there was more cells on the plate than the other to start with,
making division occur more often.
The growth of bacteria was not measured accurately and the colonies counted
were an approximation, the experiment could have been made more accurate by
having several trials. The type of bacteria used in experiments may also have
different susceptibilities, pH readings were also not taken. Taking a pH reading
would have improved accuracy in the experiment because a low or high pH could
have been ruled out as an influencing factor for the inhibited microbial growth In
the independent dish.
In order to improve the experiment, there should have been many more control
and independent petri dishes, which would give better results, if music has
inhibited the growth of bacteria, all independent dishes would have much less

colonies than the control dish. Another way to improve the experiment and
prevent over stimulation is to vary the amount of music given to the independent
variable, different types of music also could have been used to determine
whether music affects microbial growth.

Other Applications
Bacteria are perceptive to changes in surrounding vibrations, which may suggest
that music has an effect on bacterial cells inside animals. A slower or faster
growth rate may affect how a cell becomes resistant to antibiotics. According to
the results of the experiment, if an animal is exposed to a lot of classical music
bacterial growth may be inhibited. Music could also speed up or slow down other
microbial assisted duties, such as those in a sewage system, decreasing energy
costs and increasing productivity. If music has a positive effect on bacteria,
insulin could be made quicker in a laboratory.

Photos from the experiment

All images were taken at Halesowen college on 20/03/16

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