c c Hoang 1

Bryan Hoang, Brian Ponce, Wesley Aladume

Mrs. Adams

Chemistry Pre-AP A3

12/02/10

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Bacteria are microorganisms that linger everywhere on earth. There are some bacteria beneficial

to our health, yet dreadful ones that influence one¶s internal equilibrium systemic functions

negatively. From this fact, my group began investigating analytically into the sciences of

Bacteria itself and its reactions to certain subjections. We hence came across an article called

³Why Is It So Difficult to Eradicate Salmonella?´ (ScienceDaily) Within this article, which our

project is based primarily on, elucidates how salmonella bacterium is derived from the

contamination of food products/liquids. It shows how researchers tested potential growth of

salmonella on agar plates, concluding in its formation of biofilm. From our readings, biofilm are

usually composed of synthetic materials in forms of slime. This biofilm induces a resistance

coating, making it harder for chemicals to eradicate Salmonella. We all were baffled by the

sophisticated structures of the bacterium¶s creation of a resistant against growth inhibition. We

all started conceiving the fact whether applications are practical in destroying the biofilm. We

came to a result having the notion of subjecting the bacteria to certain conditions. And since

Salmonella was already hard to eradicate, we pondered whether our hypothesis was valid upon

various bacteria including E. coli. We summarized potential occurrences to that the bacteria may

develop an adaption to certain allocations of pH and temperature and become resistant (similar to
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antibiotic resistant bacteria). And at low ph (acidic) and elevated temperatures, E. coli, at first,

may have a potential of growth rate inhibition, but can mutate to their contiguous conditions for

survival and multiply. Another idea we proposed was how the utilization of acidic conditions

within the nutrients may induce deficiencies for the bacteria, such as a deoxydation or

dehydration mechanism. We came to a conclusion that these bacteria would then be void of

oxidation and water, ceasing reproduction at a quicker rate. This general science article was very

essential in commencing our notions of eliminating bacteria.


 
 

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Upon the conclusion of our science fair project to universally be in regard to microbiology

(bacteria specifically), we have gathered four professionally research articles in relevance to our

general topic. These articles include mainly the bacterial internal functions subjected to

artificially generated environments through uses of chemical manipulation, temperature

fluctuations, and growth models for bacterial cells. In one article, ³The Combination of Zinc

Compounds and Acidic pH Limits Aerobic Growth of a Salmonella Typhimurium Poultry

Marker Strain in Rich and Minimal Media´ (Park, S.Y., et al. 199-207), researchers¶ utilized

Amalgamated compounds of zinc and acidic pH levels which influenced aerobic growth on a

certain bacteria known as Salmonella Typhimurium. With Ph levels below .05 significantly

inhibited or ceased growth of this bacterium. The growth rates of S. Typhimurium significantly

decreased as a combination of poultry isolate was present in zinc acetate than zinc sulfate.

Growth rates of the bacteria itself were also decreased in minimal Medias than in comparisons to

rich Medias. In pertinence to this article mentioned, our group figured to see how an alternate
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bacterium situated within intestines of organism, Escherichia coli, may be influenced chemically

when pressured at certain pH values between .01 and 1 with strong corrosive acids. Another

article we took into consideration was "Growth and genetic responses of Salmonella

Typhimurium to pH-shifts in an anaerobic continuous culture." (Dunkley, K.D., et al. 35-42)

Within this article, we saw researchers perform an experimentation of pH variability in the

gastrointestinal tracts of poultry. This fact and event is hence analyzed to see any potential of

Salmonella influence. The researchers evaluated growth responses of Salmonella affected by pH

shifts of 6.17 to 7.35 which was critical in recording the outcome at that variable. By doing so,

they observed how increases in pH values of chemicals/ substances, resulted in higher cell

protein concentrations, glucose disappearance, and glucose and ATP yields. At high pH

influences, Salmonella Typhimurium survives in the tract compared to lower pH, which wasn¶t

able to withstand the environmental condition. Overall in conclusion, changes in pH do affect

Salmonella Typhimurium, where the higher and augmented the acid is, and the more influence it

can induce, while lower standards of pH values (at high pH value) somehow inhibit growth of

the bacterium. From reading this article, we concluded how strong, corrosive substances can

greatly affect a bacterium¶s growth rate and how the surrounding temperatures are also a key

factor for variables to be taken into consideration. It also occurred that we had questions whether

our target of E. coli can also be affected similarly in terms of the Salmonella Typhimurium. We

came to a thought of subjecting E. coli to a strong acid such as sulfuric acid as well as other acids

less in strength to compare the growth rates and potential alternations or dissimilarities. Another

article that we used was called "Comparative acid stress response of Listeria monocytogenes,

Escherichia coli O157:H7 and Salmonella Typhimurium after habituation at different pH

conditions." (Koutsoumanis, K. P., and J. N. Sofos. 321-326). This article greatly relates to our
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project because it includes reactions of various bacteria like Listeria monocytogenes, Escherichia

coli O157:H7 and Salmonella enterica serotype Typhimurium. All these bacteria are shown to be

subjected to various pH conditions to be compared in regard to their adaptive responses to acidic

conditions. This article elucidated how acidic environments provided a resistance against lethal

acidic conditions. Low pH habituation showed a different case where each response was

different of each pathogenic bacteria. Within the pH ranges of 5·0±6·0, 4·0±5·5 and 4·0±5·0,

Salmonella showed the most resistance among the rest. This article was ideal in giving a ³bigger

picture´ to our project. By showing the different kinds of working and failed bacterial

resistances, helped brought a slight conclusion to whether low pH values of chemical substances

can affect bacteria greatly. The pH variability shown in the article was also beneficial to our

project; for we are trying to find if low pH valued substances can influence Escherichia coli upon

growth response, rate or reactions, including values where the substances can inhibit growth if

possible or applicable. Our last article we took into consideration was "Adaptive responses of

Salmonella enterica serovar Typhimurium DT104 and other S. Typhimurium strains and

Escherichia coli O157 to low pH environments."(Jonge, R., W.S. Ritmeester, and F.M. Leusden.

625) So far we have discussed how the last three articles mentioned and explained the

comparative acidic conditions of several bacteria, the growth rates and response of Salmonella

Typhimurium, and three, how Acidic substances affect growth of anaerobic Salmonella. This

final article completes our variables we are searching for and is explain the adaptive responses of

the Salmonella bacterium. The article shows how Salmonella Typhimurium was subjected to

extreme acidic conditions. Using its current condition, it was compared to its growth and/or

adaptive response to that of acid-resistant E. coli O157 and other S. Typhimurium phage types.

At a certain phase of S. Typhimurium on the culture dish, the bacterium showed resistance at the
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pH of 2.5 hence concluding that the ability of S. Typhimurium to survive at pH value of 2.5 was

shown to be dependent upon other variables, considerably amino acids. This article was crucial

to our project because it provided the last of the three variables needed to be taken into

consideration; Salmonella Typhimurium adaptive response to acidic conditions itself. All four of

the professionally reviewed journal articles provided an essential outline for our project. In

provided the many variables for our project to be taken into consideration. As a result, our group

concurred to base our project upon Escherichia coli and its effects when subjected to low pH

values using acids and varying pH chemicals.

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The focal purpose of this experiment, primarily, is to observe growth responses of Escherichia

coli affected by ph variability (mainly upon low pH valued substances such as sulfuric acid).

Upon this, we are to see the effects of the bacterium itself and address the problem statement or

question as: If low pH, caused by the addition of sulfuric acid with amalgamation of Tryptic Soy

Agar, cause changes in the growth rates of E. coli or if possible induce growth inhibition?

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If Escherichia coli are subjected within sulfuric conditions at 37 degrees Celsius, then the

bacteria will most likely have its cell membrane ruptured in a way that it will cease growth or die

off progressively due to the cell¶s high intake of low pH acid.


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  :

mc Sulfuric acid ± website (The Science Company)

mc E. coli ± website (Science Stuff)

mc  Petri dishes (or more if needed for more test variables) ± provided by laboratory.

mc Latex gloves ± provided by laboratory.

mc Sterile swabs (in packets) ± provided by laboratory.

mc Tryptic soy agar dishes ± website (WARD'S Natural Science)

mc Bacterial incubator (Universal temperature of 37°C) - provided by supervising

scientist/laboratory

mc Autoclave ± provided by supervising scientist/laboratory

mc Lab coats ± website (Science Company)/laboratory and supervising scientist.

mc Alternate nutrient Agar/Tryptic dishes (10 Petri dishes with agar) ± website (Science

Stuff)

mc Distilled water ± provided at home

mc Safety goggles ± website (Science Company)

mc Lab aprons ± provided by supervisor/laboratory

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Control group:

- E. coli being subjected to nutrients itself (No ph mixture).

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Test group:

- E. coli cultured within sulfuric acid with nutrients.

Measurements:

- Bacterial growth will be algebraically analyzed and concluded through approximate formulas

(Exponential/logarithmic growth rate). Each of the formulas/diameters/area obtained from one -

observation will be compared to other cultures.

Frequency:

- Once a week for a month.

Independent Variable:

- pH conditions applied.

Dependent Variable:

- Growth response or rate of bacteria.

1.c Put on lab coat, latex gloves, and take out  cotton swabs and  Petri dishes.

2.c Fill up Petri dishes with necessary nutrients or use one that is already present.

3.c Using a sterilized cotton swap, place a small sample of E. coli (2 grams for adaption to

dish) on Agar dish.

4.c Repeat this step for replications of eight more dishes.

5.c Label four of the dishes as ³C´ for control.

6.c Set ³C´ cultures with pH value of 7 using distilled water mixed with the nutrients.

7.c Label the other four Petri dishes as ³pH ± 0.5´

.c Determine amount of sulfuric acid to be added to make the pH level of 0.5 using the

formula pH = -log10[H3O+]. (10ml for approximate quantity)

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9.c Add the determined amount of sulfuric acid (10ml) to Petri dishes labeled ³pH ± 0.5´ and

put them inside incubator at 37 degrees Celsius.

10.cAt the same time, add the ³C´ control group Petri dishes in incubator at 37 degrees

Celsius.

11.cRecord the point of time where cultures are initially incubated.

12.cLeave the incubator for the time being and record any observations of initial growth rates.

13.cMonitor the growth from all Petri dishes for the duration of one month. Make sure to take

pictures of the cultures and to note any changes on the area on the Petri dish that the

culture has covered. In order to prevent any corrupted data, the cultures will be monitored

weekly.

14.cWhen one month has passed, carefully take out dishes and dispose upon supervision.

15.cAt the end of the experiment, use disinfectants, such as 70% ethanol, a 10% bleach

solution, or a commercial antibacterial kitchen/bath cleaning solution, to thoroughly clean

any surfaces used.

16.cRecord data and observations into a table.

17.cCreate logarithmic graph or exponential delineating the average growth rate of each

bacterial culture.

1.cOnce calculated for each culture, plot the functions on a y ± axis versus x ± axis graph.

19.cCompare average graphs of ³C´ cultures to ³pH ± 0.5´ cultures and record observations.

20.cFinally, gather a final conclusion about E. coli growth rates and record in a research

report or table.


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Norwegian School of Veterinary Science. "Why is it so difficult to eradicate salmonella?."

ScienceDaily 3 February 2010. 2 December 2010 <http://www.sciencedaily.com

/releases/2010/02/10020210392.htm>.

Park, S.Y., et al. "The Combination of Zinc Compounds and Acidic pH Limits Aerobic Growth

of a Salmonella Typhimurium Poultry Marker Strain in Rich and Minimal Media." X
 


  
  

   
 




  39.1 (2004): 199-207. 

  . EBSCO. Web. 1 Aug. 2010.

Dunkley, K.D., et al. "Growth and genetic responses of Salmonella Typhimurium to pH-shifts in

an anaerobic continuous culture."   14.1 (200): 35-42. 

  .

EBSCO. Web. 1 Aug. 2010

Koutsoumanis, K. P., and J. N. Sofos. "Comparative acid stress response of Listeria

monocytogenes, Escherichia coli O157:H7 and Salmonella Typhimurium after habituation at

different pH conditions."  


!
 
 " 3.4 (2004): 321-326. 


  . EBSCO. Web. 1 Aug. 2010

Jonge, R., W.S. Ritmeester, and F.M. Leusden. "Adaptive responses of Salmonella enterica

serovar Typhimurium DT104 and otherS. Typhimurium strains andEscherichia coli O157 to low

pH environments." X
 

!
 
 " 94.4 (2003): 625. 


  . EBSCO. Web. 1 Aug. 2010.

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