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 world’s 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 Mozart’s 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 μm in width by 2 μm 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 one’s 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 α-helix and a decrease in β-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 ½ 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
0
6
59
0
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
Bibliography
1.bp.blogspot.com. (2016). Growth Phase. [online] Available at: http://1.bp.blogspot.com/-LwBs2GFE8OQ/TkQ39zNfmUI/AAAAAAAAARg/rIuqvrx4dQ/s1600/Bacterial+Growth+phases.jpg [Accessed 11 May 2016]. Academia.edu. (2016). Mozart Effect: A Class Study on the Effects of Music on Memory. [online] Available at: http://www.academia.edu/2044356/Mozart_Effect_A_Class_Study_on_the_Effects _of_Music_on_Memory [Accessed 12 May 2016]. Alfred Tomatis. (2016). The mozart effect. [online] Available at: https://www.marshallstyler.com/alfred-tomatis.html [Accessed 22 Dec. 2015]. Bats.ch. (2016). [online] Available at: http://www.bats.ch/bats/pics/pathogenic_escherichia_coli.gif [Accessed 13 May 2016]. Chow, D. (2014). Music and bacterial growth. [online] p.3. Available at: https://prezi.com/a9rjqo4ldvp9/music-bacterial-growth/ [Accessed 13 May 2016]. Ecoliwiki.net. (2016). Escherichia coli - EcoliWiki. [online] Available at: http://www.ecoliwiki.net/colipedia/index.php/Escherichia_coli [Accessed 13 May 2016]. Foodsafetysite.com. (2016). Food Safety Education | For Educators | Competencies | General | Bacteria | Describe the conditions favorable to the growth of bacteria in food.. [online] Available at: http://www.foodsafetysite.com/educators/competencies/general/bacteria/bac2. html [Accessed 13 May 2016]. Goldstein, R. and Soyer, O. (2008). Evolution of Taxis Responses in Virtual Bacteria: Non-Adaptive Dynamics. PLoS Computational Biology, 4(5), p.e1000084. Hayashi, K., Morooka, N., Yamamoto, Y., Fujita, K., Isono, K., Choi, S., Ohtsubo, E., Baba, T., Wanner, B., Mori, H. and Horiuchi, T. (2006). Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110. Mol Syst Biol, 2. Jang, K. (2013). Effect of music on bacteria. [online] p.1. Available at: https://prezi.com/2euxfiu65ggz/effect-of-music-on-bacteria/ [Accessed 12 May 2016]. Jenkins, J. (2001). The Mozart effect. Journal of the Royal Society of Medicine, [online] 94(4), p.170. Available at:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1281386/ [Accessed 13 May 2016]. Jones, H., Feth, L., Rumpf, D., Hefti, A. and Mariotti, A. (2000). Acoustic energy affects human gingival fibroblast proliferation but leaves protein production unchanged. Journal of Clinical Periodontology, 27(11), pp.832-838. Lee Ying, J., Dayou, J. and Phin, C. (2009). Experimental Investigation on the Effects of Audible Sound to the Growth of Escherichia coli. Modern Applied Science, 3(3). Lrs.ed.uiuc.edu. (2016). The Mozart Effect: A Closer Look. [online] Available at: http://lrs.ed.uiuc.edu/students/lerch1/edpsy/mozart_effect.html#Scientific Explanations for the [Accessed 12 May 2016]. Mackert, T. (2016). KIT - Departments - Interface microbiology - AG Overhage: Bacterial Stress Response. [online] Ifg.kit.edu. Available at: https://www.ifg.kit.edu/english/911.php [Accessed 10 May 2016]. Microbes.ucsc.edu. (2016). Escherichia coli K12 (Escherichia coli K12 substr. MG1655) Genome Browser Gateway. [online] Available at: http://microbes.ucsc.edu/cgi-bin/hgGateway?db=eschColi_K12 [Accessed 1 Mar. 2016]. Mortazavian, Ph.D, A. (2016). Music affects survival and activity of microorganisms. Journal of Paramedical Sciences (JPS), [online] vol 3(2008-4978), p.1. Available at: http://journals.sbmu.ac.ir/jps/article/viewFile/3501/3141 [Accessed 11 May 2016]. Pavlov, M. and Ehrenberg, M. (2013). Optimal control of gene expression for fast proteome adaptation to environmental change. Proceedings of the National Academy of Sciences, 110(51), pp.20527-20532.
PBS LearningMedia. (2016). Sound Vibrations. [online] Available at: http://www.pbslearningmedia.org/resource/phy03.sci.phys.howmove.lp_sound/ sound-vibrations/ [Accessed 13 May 2016]. Scienceblogs.com. (2016). [online] Available at: http://scienceblogs.com/cognitivedaily/wp-content/blogs.dir/262/files/2012/04/i5750d333e73e2a00dc7e7636a96bdd6b-mozart.gif [Accessed 13 May 2016]. ScienceDaily. (2016). How bacteria respond so quickly to external changes. [online] Available at: https://www.sciencedaily.com/releases/2013/12/131202161952.htm [Accessed 10 May 2016]. Shaobin G, e. (2016). A pilot study of the effect of audible sound on the growth of Escherichia coli. - PubMed - NCBI. [online] Ncbi.nlm.nih.gov. Available at: http://www.ncbi.nlm.nih.gov/pubmed/20338730 [Accessed 13 May 2016].
Sigma-Aldrich. (2016). Bacterial Cell Lysis. [online] Available at: http://www.sigmaaldrich.com/life-science/proteomics/recombinant-proteinexpression/cell-lysis/bacterial-cell-lysis.html [Accessed 12 May 2016]. Speigel Online, (2016). Symphonic Sewage. [online] Available at: http://www.spiegel.de/international/zeitgeist/symphonic-sewage-wastetreatment-plant-plays-mozart-to-microbes-a-698040.html [Accessed 11 May 2016]. Vlab.amrita.edu. (2016). Bacterial Growth Curve (Theory) : Microbiology Virtual Lab I : Biotechnology and Biomedical Engineering : Amrita Vishwa Vidyapeetham Virtual Lab. [online] Available at: http://vlab.amrita.edu/? sub=3&brch=73&sim=1105&cnt=1 [Accessed 13 May 2016]. Xiujuan, W., Bochu, W. and Yi, J. (2002). Effect of sound stimulation on cell cycle of chrysanthemum. Colloids and Surfaces B: Biointerfaces, 29, pp.103-108. Ying, J., Dayou, J. and Khim Phin, C. (2010). Experimental Investigation on the Effects of Audible Soundto the Growth of Escherichia coli. Mod applied Science, 3(124), p.7. Zhao, H., J, W. and Wang, B. (2003). Effect of sound stimulation on Dendranthema morifolium callus growth. Effect of sound stimulation on Dendranthema morifolium callus growth., 29(143), p.7. Zhao, H., Wu, J. and Wang, B. (2001). Colloids and Surfaces B: Biointerfaces. Science Direct, 25(1), pp.29-32.