How will the pit of the date fruit, when extracted with acetone, affect the growth of lactobacillus

bacteria?
Name: Suzanna Shafi Candidate Number: 002391031 Supervisor: Dr. Amit Khanna Subject: Biology

Word Count: 3,971 1/29/2012

1 |Page

ABSTRACT The pit of the date fruit is a waste product which has proven to maintain antibacterial effects and thus provides for a cheap method of patenting of potential antibiotics. On a global scale, this can help improve the health of under developed countries in need of cheap antibiotics. As research began in the United Arab Emirates regarding the potential antibacterial effects of the pit of the date fruit, I was lead to my research question: How will the pit of the date fruit, when extracted with acetone, affect the growth of lactobacillus bacteria? I predict that the pit of the date fruit will have antibacterial effects on the lactobacillus bacteria because it has proven to do so against other bacteria during the research conducted by UAE University. The experimental procedure will consist of allowing the bacteria grow in its optimal environment in five different test tubes in order to observe where the bacteria growth is least apparent. A spectrophotometer will be used to measure the light transparency through the solution to test where the bacteria has been inhibited or grown (if the bacteria has been inhibited or killed, the percent transparency will increase). I have found that the bacteria was inhibited and killed to the greatest extent when in presence of the date pit and acetone. From this, I can conclude that the date pit can be used as an antibiotic as it has proven to maintain antibacterial properties and perhaps antioxidant properties. Thus, I can accept my hypothesis. Future studies of the date pit may include testing different concentrations of acetone and the date pit in order to produce the most optimal effects.

2 |Page

TABLE OF CONTENTS Abstract..........................................................................................1 Introduction....................................................................................3 Background Information...................................................................4 Method Development (early experiments & failures)..............................5 Data Collection & Processing..............................................................9 Discussion of Results....................................................................15 Conclusion..................................................................................17 Evaluation..................................................................................18 Works Cited................................................................................21

Works Cited

3 |Page

INTRODUCTION One day while reading The National, Abu Dhabis local newspaper, a friend of mine came across an intriguing article which investigated the idea that the pit of the date fruit, when extracted with acetone, could be used as an antibiotic for chickens by placing this formula into their daily feed. In the subsequent discussion with my Higher Level Biology class, I knew it was my potential extended essay topic. Not only does it provide for a cultural glimpse into my home of Abu Dhabi, but also shows how a waste product can be used to improve the lives of animal stock and perhaps with further research into the topic; mankind. After more research, I found that there is global concern regarding the use of antibiotics in animal feed to not only cure illnesses, but to promote the growth of animals. The remnants of antibiotics in poultry even after slaughter have caused the antibiotics to enter the human food chain and hence increase the proportion of resistant bacteria which have undergone a genetic mutation (Brown, 617-67). This increase in resistant bacteria then causes the development of superbugs which carry several resistant genes and remain a very pertinent problem in the world today (Brown, 617-67). Therefore, the reason why the date pit as an antibiotic is such an important discovery is because it provides for a natural, non-pharmaceutical alternative to antibiotics. Researchers at United Arab Emirates (UAE) University also say that since the pit of the date is a waste product, the patenting of this discovery comes at almost no cost and therefore, can be beneficial towards under-developed countries in need of cheap antibiotics (Detrie). The discovery of antibiotics provided for a major decrease in mortality rates and soon after also showed an increase in life expectancy in the world population (Kumar). Most antibiotics specifically inhibit bacterial growth, thus causing the bacteria to die during reproductive stages. Therefore, both humans and animals need antibiotics in order to fight off pertinent bacterial infections. Doctor Ahmed Soliman Hussein, the head of research and a professor at the UAE University says that early date pit feed tests are very promising as they have shown to protect the poultry from common ailments such as E. Coli, Campylobacter, Shigella and Salmonella (Detrie).

4 |Page

BACKGROUND INFORMATION For many centuries, the pit of several different types of fruits has been known to have powerful effects in curing, preventing and treating different diseases. More specifically, the date pit has been found to contain edible oils and pharmaceuticals (Al-Shahib). Doctor Hussein says that the date pit contains a broken down dietary fiber which may be the reason for a decrease of infectivity within the chickens (Detrie, The National). However, the extent to which the broken down dietary fibers help prevent such diseases is not fully confirmed with evidence and is therefore simply an unknown. A medical journal written by A.A. Al-Qawari researched the ameliorative effects of dates on ethanol-induced gastric ulcers in rats (Al-Qawari). Since gastric ulcers are commonly caused by different species of bacteria which create infections in the lining of the stomach, this study is specifically relevant to my research at hand. The results of the experiment showed that when the date fruit and pit were orally given to the gastric ulcerated rats, the severity of the ulceration was mitigated. His conclusion was that the date extracts may include an anti-oxidant action which caused the gastro-protective action (Al-Qawari). In another medical journal written by Sabah A.A. Jassim includes a procedure to an experiment testing the antiviral activity of the date pit is discussed and has shown to be successful (S.A.A. Jassim),. This procedure includes extracting blended date pits with acetone and thereafter, exposing it to different viruses and bacteria. However, as viruses are potentially dangerous to test and bacteria are highly prevalent in world poultry farms today, I have chosen to specifically concentrate on the date pit and its effect on lactobacillus bacteria; a generally harmless, anaerobic bacterium found in different types of fermented foods. I will specifically plan a procedure which will test these effects in order to conclude whether or not the date pit does have antibacterial properties. Thus, I have chosen to focus this paper on the research question: How will the pit of the date fruit, when extracted with acetone, affect the growth of lactobacillus bacteria? And I will also discuss the method development, early experiments, and failures within the procedure. Based on the results I gather from the experiment, I will then evaluate the validity of 5 |Page

the date pit as a natural, non-therapeutic antibiotic and conclude the advantages and disadvantages for future usage. METHOD DEVELOPMENT (EARLY EXPERIMENTS & FAILURES) Variables Chosen The method development of this experiment required a process that would allow me to out rule each variable that could be affecting the growth or decay of bacteria and to ensure that the date powder was the sole contributor to bacterial inhibition. Included within these variables were the acetone and date powder separately and together, the environment in which the bacteria was being tested which ended up being the beef stew, and to ensure that the bacteria would grow without the date powder, the substance amoxicillin needed to be tested, too. Thus, a series of five variables in total needed to be tested; with and without the presence of bacteria. First Method: Bacteria Plates (Plain Agar & Beef Agar) Seeing as how bacterial growth is generally tested using bacteria plates containing agar, this was my first approach. I obtained five bacteria plates, for each of the five variables mentioned, and split each plate into two sections; with and without bacteria. After leaving the plates in the desiccator for two days, I found that no bacteria grew in any plates. After more research, I found that the lactobacillus bacteria would be best grown in an anaerobic environment of pH 6.0 (Hardy Diagnostics) This information provided us with a series of ingredients in order to create agar containing beef extract in order to provide this environment. The ingredients are as follows: Fructose Beef Extract Yeast Extract Sodium Acetate Dipotassium Phosphate Ammonium Citrate Magnesium Sulfate Manganese Sulfate Agar 10.0gm 5.0gm 2.5gm 2.5gm 1.0gm 1.0gm 0.05gm 0.025gm

6 |Page

Thereafter, I went through the same procedure of using the five bacteria plates, each split into two sections and found that although the bacteria did grow, it could not be accurately measured with a standard ruler due to miniscule visible growth and decay of bacteria when placed with the date-feed. Second Method: Light Intensity (Beef Stew) Finally, it was decided to use a spectrophotometer an apparatus that measures light absorbance such that if the bacteria were to grow, the percentage of light emitted through the solution would decrease and if inhibited, the percentage emitted would increase. After a few test trials, it could be seen that this method was most suitable because even the most miniscule changes in bacteria could be measured. Preparing the Feed The basis of preparing the date feed was provided by a medical journal (S.A.A. Jassim) researching alternative medicines. Within this journal, the preparation of the plant extract was as follows: 1. Date fruits needed to be obtained from a local market in Abu Dhabi and manually stripped of their flesh and rinsed thoroughly with water. The pits were then left to dry for 18 hours. 2. 100g of dried date pits were then blended and crushed and thereafter, extracted with acetone using a 1:1 ratio (weight to volume) between the crushed date pit and the acetone 3. After 48 hours, the extract should then be filtered through Whatman No. 2 filter paper and thus, the leftover filtrate was then evaporated into dryness at room temperature. My first step was then to make sure that this process was plausible and that the lab at my institution maintained the materials needed. The first issue I ran into was the ratio suggested. I found that a 1:1 ratio did not suffice (not enough acetone was present to fully extract the powder) so instead, I found that a 1:2 ratio was sufficient. The second issue presented itself why trying to filter the extract through the filter paper. When blending the date pits, it was difficult to come by more than 3.0 grams of powder due to the loud nature of the blending process as opposed to the suggested 100.0 grams. Therefore, instead of filtering the extract, I placed the 3.0 grams of powder and 6.0 mL of acetone into a 15 mL graduated cylinder and covered the cylinder with an evaporating dish (due to the volatility of acetone, the cylinder needed to be

7 |Page

covered). After only three hours, I found myself with a dark brown liquid and thus, my date-feed was successfully created.

Final Procedure in Detail Day 1 1. Peel the date fruit of its fruity overcoat, thoroughly wash the pit and leave it to dry for 24 hours Day 2 2. Place the dried pits in a blender and crush the pits for approximately one minute. Separate the solid pit pieces from the powder and weigh the mass of the powder using a balance such that 6.0g of date powder remains. 3. Separate the 6.0g of date powder into two 50 mL beakers such that 3.0g remains in each 4. In a 1:2 ratio (weight:volume), add 6mL of acetone based on the mass of the date to only one of the two 50 mL beakers and cover the top with an evaporating dish (due to the volatility of acetone, the feed will otherwise evaporate) 5. Wait 3-4 hours for the date to soak in the acetone Day 2 (after 3-4 hours) 1. To begin, measure 500 mL of distilled water into a 1500 mL flask and add the ingredients stated above according to Hardy Diagnostics. Next, boil the mixture for about 20 minutes in order to kill any stray bacteria. 2. During these 20 minutes, obtain 30 test tubes and separate them such that there are 10 test tubes in each test tube rack in 2 rows of 5. 3. In each test tube, measure out 9mL of distilled water in each in order to dilute the beef solution such that light can be emitted through when placed in the spectrophotometer. 4. Next, label each rack Trial One, Trial Two, and Trial Three. Then, on the other side of the rack, label one row of five Without Bacteria and the other subsequent row With Bacteria. Place the racks so that they are in three rows, front to back. The set up should look similar to this: 6 7 10 1 4 5 8 |Page 8 2 3 9

6 7 10 1 4 5

8 2 3

6 7 10 1 4 5

8 2 3

Row 2: With Bacteria Row 1: Without Bacteria Trial One

Row 2: With Bacteria Row 1: Without Bacteria Trial Two

Row 2: With Bacteria Row 1: Without Bacteria Trial Three

Day 2 (after 20 minutes) 1. Once the 20 minutes is up, place the following solutions into the designated test tubes: a. Test tube 1 + 6: 1 mL Plain Beef Mixture b. Test tube 2 + 7: 1 mL Beef Mixture + 3 mL Acetone i. Since we used 6 mL of acetone with the date feed and split into two test tubes, we need to keep the variables constant and use 3 mL in each, too c. Test tube 3 + 8: 1 mL Beef Mixture + 1.5 g Plain Date Powder i. Use the same amount of date powder that was added to the date feed on Day 1 and that was not mixed with the acetone ii.Since we blended 3.0g of date powder that was NOT mixed with acetone, we can use 1.5g in each test tube d. Test tube 4 + 9: 1 mL Beef Mixture + 3 mL Date Feed (created a day earlier) i. Since we made a total of 6mL the previous day we can use 3mL in each test tube e. Test tube 5 + 10: 1mL Beef Mixture + 1mL Amoxicillin i. Amoxicillin should kill the bacteria 2. Once the set-up is done correctly, add 1 mL of lactobacilli bacteria into test tubes in Row 2 3. Using the photometer, place a clear test tube into the slot and calibrate the machine to 100%. After doing so, take the reading of the percentage of light emitted through each solution in each of the thirty test tubes and record the data. 4. Next, store the test tubes overnight such that bacteria can grow within the test tubes Day 3

9 |Page

After 24 hours, repeat the process in steps 3 and 4 of day two and record the difference of light emitted. Especially take note the difference between the test tube with just date feed and mixture and the test tube with date feed, mixture, and bacteria.

10 | P a g e

DATA COLLECTION & PROCESSING In order to show how the data was processed, a sample calculation originating from the raw data of the plain beef solution will be used. The series of steps that were taken are as follows: Processing of Raw Values Trial One Day 1 Day 2 Day 3 Without Bacteria 94 79 80 With Bacteria Trial Two Without Bacteria 94 97 95 With Bacteria Trial Three Without Bacteria 94 90 92 With Bacteria 92 76 78

93 Day 1 88 Day 2 86 Day 3

94 Day 1 77 Day 2 73 Day 3

Trial One Day 1 to Day 2 Day 2 to Day 3 Trial One 24 Hours 48 Hours

Without With Trial Two Without With Trial Three Without Step One: The raw percentages of Day 1 were subtracted from Bacteria Bacteria Bacteria Bacteria Bacteria -15 Day 2 and those of Day to were3subtracted from Day 3 to produce -5 Day 1 2 -17 Day 1 to -4 the difference* Day 2 Day 2 1 -2 Day 2 to -2 -4 Day 2 to 2 Day 3 Day 3 Without Bacteria -15 -14 With Bacteria Trial Two Without Bacteria 3 1 With Bacteria Trial Three Without Bacteria -4 -2

With Bacteria -16 2

With Bacteria -16 -14

-5 24 Hours -7 48 Hours

-17 24 Hours -21 48 Hours

11 | P a g e

Trial One 24 Hours 48 Hours

Step Two: The differences of Day 1 to Day 2 were added to Day 2 to Day 3 in order to create a representation of growth Without 24With to 48 hours Without Trial With Trial Without from hours Bacteria Bacteria Two Bacteria Bacteria Three Bacteria -15 -5 24 Hours 3 -17 24 Hours -21 48 Hours -4 -2

With Bacteria -16 -14

-14 -7 48 1 Step Three: the Hours 48 hour 24 and growth with and without bacteria for each trial was averaged

Step Four: the Standard Deviation was calculated via Microsoft Excel using the formula:

Without Bacteria 24 Hours 48 Hours 9.073772 7.937254

With Bacteria 6.658328 7.000000

Without Bacteria 24 Hours 48 Hours -5.3333 -5.0833

With Bacteria -12.6667 -14.0000

*Originally, it seemed optimal to calculate the percent change between each day instead of simply subtracting each value from the other. However, I found that when calculating the percent change, the way the data would be viewed was altered. For example, when inserting acetone into the beef solution (trial two: day one was 92% and decreased to 88% on day two), the percentage of light emitted was already higher due to acetones transparency as opposed to when inserting the date powder (trial two: day one was 66% and grew to 70% on day two), the percentage of light emitted was lower due to its opaqueness If percent change is calculated between day 1 and day 2 of each solution, the acetone percent change would be -4.5% whereas the percent change of the date powder would be 5.7%. Although both of the solutions have a raw absolute difference of 4.0%, their percent changes are different

12 | P a g e

because they do not maintain the same initial value this value does not affect the bacterial inhibition Therefore, in order to place each solution on the same spectrum, it is optimal to subtract the percentages such that the initial value is zero and the reader can interpret the bacterial inhibition over the course of 48 hours

13 | P a g e

14 | P a g e

15 | P a g e

Aa

The label Aa indicates that the beef extract solution with date powder is not quite statistically different from the plain beef extract solution. However, it must again be observed that based on processed data, the date powder and amoxicillin maintain the only increases in light emission

The label A indicates that those three A solutions are not statistically different from the plain beef extract solution. However, it must be taken into consideration the obvious increase in Indications of Graph emission of light 1: 1. There is a possible presence of stray to amoxicillin as opposed bacteria causing the decrease in light emission 2. The date powder and beef solution act similarly by inhibiting the possible stray

16 | P a g e

Over 48 hours, the total inhibition in bacteria can be indicated through the percentage of light emitted; if the percentage emitted decreased, this indicates that the bacteria reproduced whereas if the percentage emitted increased, it can be inferred that the bacteria

Aa

Bbb

Bb Indications of Graph 2: 1. The date powder & acetone have the strongest inhibition of bacteria together, not separately 2. Together, they are stronger than the antibiotic, amoxicillin 3. The lactobacillus bacteria does grow in the optimal

Label A signifies that by conventional criteria, the difference between the labeled value and the control, the plain beef solution, is considered to be not statistically different (A) or not quite statistically different (Aa)

Label B signifies that by conventional criteria, the difference between the labeled value and the control, the plain beef solution, is considered to be statistically different (B), very statistically different (Bb), or extremely statistically different (Bbb)

17 | P a g e

Label A indicates that the decrease in percentage of light emitted in the plain beef extract solution is the statistically the same with and without the addition of bacteria.

18 | P a g e

Indications of Graph 3 1. It can be further confirmed that stray bacteria does exist as amoxicillin shows similar results with and without addition of lactobacillus 2. Even further confirmed as the beef extract solution shows decreased light emission without the addition of bacteria

Label A indicates that the increase in percentage of light emitted in the beef extract and amoxicillin solution is statistically the same with and without the

Label Bb indicates that the beef extract solution containing acetone with bacteria is very statistically different than without bacteria. Label Bbb indicates that the beef extract solution containing acetone and date powder with bacteria is extremely statistically different

Bb

Bbb

Label A indicates that the beef extract solution containing date powder with bacteria is not statistically different than without bacteria. 19 | P a g e

Indications of Graph 4: 1. The date powder may be inhibiting other substances in the beef extract solution 2. Acetone does inhibit lactobacillus, but perhaps not the stray bacteria 3. Together, acetone and date powder exhibit the greatest inhibiting effect

20 | P a g e

DISCUSSION OF RESULTS *Each result discussed pertains to the indications stated below each graph

When comparing the five different solutions without the addition bacteria in Graph 1, it can be seen that both the amoxicillin and date powder solutions exhibit an increase in light emission and that the other three exhibit a decrease in light emission. Both the increase and decrease in light emission indicate two plausible conclusions; that perhaps there is another source of bacteria growing within the test tubes causing a decrease in light emission and that this bacteria is being inhibited by the date powder in the same way that amoxicillin would inhibit bacteria and thus, causing an increase in light emission. As can be seen in Graph 2, the greatest increase in light emission, 27.67 percent, over the course of 48 hours was with the addition of date powder and acetone to the beef solution. The next greatest increases were upon the addition of date powder and acetone separately to the beef solution where there were increases of 5.33 and 9.33 percent. It can be duly noted that the difference between these increases is extremely significant. This suggests that the date powder and acetone separately do have inhibiting effects upon the bacteria, but that when in addition together, inhibition of the bacteria occurs to a much greater extent. It can also be seen that the percentage of light emitted decreases by 14 percent when neither is added to the plain beef solution, indicating that the bacteria does grow in the environment provided. In Graph 3, the amoxicillin solution increases in percentage of light emitted, with and without bacteria and the plain beef solution decreases in percentage of light emitted with and without bacteria. The percent differences over 48 hours in both solutions without bacteria are not statistically different than the percent difference with bacteria. This presents more evidence that there is another source of bacteria being killed by the amoxicillin when lactobacillus is not added as the T-Test indicates that the data with bacteria is the same as the data without bacteria.

In Graph 4, it can be seen that upon the addition of date powder into the beef solution, the percentage of light emitted increases by 9.33 percent without bacteria; a greater increase than with bacteria; 6.33 percent. In contrast, the other two solutions with acetone and with date powder and acetone have a percentage decrease of 3.33 and 0.33 percent without bacteria. By conventional criteria using the T-Test, it can also be noted that upon the addition of solely date powder with and without bacteria, the data is not statistically different indicating that the date powder may be inhibiting other substances within the solution, therefore increasing the percentage of light emitted to a greater extent.

22 | P a g e

CONCLUSION The results indicate that the date powder affects the growth of lactobacillus bacteria by acting as an inhibitor. The data represented supports the hypothesis, in varying degrees, that with the addition of date powder, the percentage of light emitted through the beef solution with bacteria increases; and with the addition of date powder and acetone, the percentage of light emitted increases at greater lengths; and without the addition of date powder nor acetone, the percentage of light emitted will decrease as the bacteria grows. A further conclusion can be made between the actions of amoxicillin and the date powder. Amoxicillin works as an antibiotic by inhibiting the synthesis of the bacterial cell walls. It inhibits the cross-linkage between the polymer chains (main component of cell walls). The data suggests that perhaps the date powder maintains the same inhibiting properties during the synthesis of cell walls of both the bacteria and perhaps the cell walls of the other ingredients within the solution such as yeast and beef cells. However, since neither solution containing amoxicillin nor date powder is statistically different from the plain beef solution, which endured a decrease in percentage, more evidence is needed to support these conclusions. Furthermore, acetone may not maintain the same inhibiting properties as amoxicillin or the date powder, but is commonly defined as toxic in high doses (NLM) as it irritates, damages, and kills certain cells. This provides an explanation as to why acetone and the date powder exhibit the greatest effects together.

23 | P a g e

EVALUATION ASSUMPTIONS During the length of this experiment, there are several assumptions that were accepted. The first assumption that must be addressed is that no stray bacteria grew in test tubes where no lactobacillus bacteria were added. The beef extract inserted into each of the test tubes was boiled to 100*C and each test tube, beaker, and flask was sterilized in order to eliminate the variable of the presence of stray bacteria. However, Graph 1 shows that even without the addition of the lactobacillus bacteria, the possibility of bacterial growth is probable in the test tube containing solely beef extract as it showed a decreased light emission. This bacterial growth could have originated from the distilled water used to dilute the beef extract solution which was not boiled. In addition, we must assume that the date pit does maintain the antioxidant and inhibiting effects. The variables that were tested; the beef extract solution, date powder, and acetone, were inserted into each test tube in order to eliminate them as possibilities of the cause of the bacterial growth. However, while trying to eliminate these variables, the data has shown that acetone is also a fair contributor to the inhibition of bacteria. Otherwise, other variables such as the ingredients used in the preparing of the beef extract could have been tested with the lactobacillus bacteria to ensure this. LIMITATIONS Limitations to this experiment were the apparatus available. When converting the solid date pit into its powder, a grinding machine would have been optimal in order to assure an appropriate mass and usage of the pit to its entirety. However, a blender was used instead which was only able to shave off the outer layers of the pit and produced very little mass. Although the 24 | P a g e

powder made up of the outer layers proved to inhibit the bacteria successfully, it would have been optimal to observe the effect of the core. ERROR BARS & STATISTICAL SIGNIFICANCE The standard deviation bars and their extent must also be addressed. Since the experiment consisted of only three trials per given test tube, the calculation of standard deviation and the T-Test is borderline appropriate. As only three values are considered for each calculation, any mild discrepancy in raw data indicates a weak correlation. On top of that, the method used to process the raw data included subtracting the values instead of calculating percent change. This method, although optimal when presenting the data via bar graphs (explained in Processing of Raw Values), did not prove optimal in regards to standard deviation as the values were not converted and averaged as percentages of change. However, the T-Test was carried out namely for the purpose of showing how three values can be extremely statistically different to another three values. In regards to the control compared to the addition of both the date pit and the acetone, the T-Test showed extreme statistical significance indicating that the latter test tube exhibited a truly opposite effect. IMPROVEMENTS & FUTURE STUDIES Improvement that could be made would be to leave the solutions, with and without bacteria, for a longer period of time. By allowing the solutions without bacteria to sit for longer periods of time, more evidence could arise regarding which volume to mass ratio of acetone to date powder inhibits any certain amount of bacteria and also, if the solution will leave any stray bacteria behind. Another improvement includes more repeated trials in regards to the experiment that was conducted here or in any future experiment. More trials provide for a better statistical analysis and solidified validity of the results.

25 | P a g e

In the future, it would be interesting to experiment with different volume to mass ratios of acetone and the date powder. After confirming the hypothesis that the date pit does inhibit the lactobacillus bacteria, more conclusions could be made regarding which ratio works best against other pertinent contemporary super bugs. Depending on the results, industries could use the pit for further patenting and thereafter, selling to less economically developed countries. With these improvements and changes to the experiment, it seems plausible to conduct future studies on the antibacterial effects of the date pit.

26 | P a g e

WORKS CITED "Acetone." Tox Town. National Library of Medicine, 27 Oct. 2011. Web. 22 Jan. 2012. <http://toxtown.nlm.nih.gov/text_version/chemicals.php?id=1> Al-Shahib W, Marshall RJ. Fatty acid content of the seeds from 14 varieties of date palm Phoenix dactylifera L. Int J Food Sci Tech. 2003;38:70912. Brown, Catrin. "Medicines & Drugs." Higher Level Chemistry: Developed Specifically for the IB Diploma. Pearson, 2009. 617-67. Print. Detrie, Megan. "Researchers Test Date Pits as Replacement for Antibiotics." The National. Abu Dhabi Media Company, 7 Apr. 2011. Web. 17 Sept. 2011. <http://www.thenational.ae/news/uaenews/science/researchers-test-date-pits-as-replacement-for-antibiotics>. "Lactobacilli MRS Agar." Hardy Diagnostics. Web. 17 Sept. 2011. <https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/LactobacilliMRSAgar.html>. A.A. Al-Qarawi, H. Abdel-Rahman, B.H. Ali, H.M. Mousa and S.A. El-Mougy. Journal of Ethnopharmacology, Volume 98, Issue 3, 26 April 2005, Pages 313-317 Kumar, V. "When Do We Need Antibiotics?" Wikinut Limited. 22 June 2010. Web. 17 Sept. 2011. <http://health.wikinut.com/When-do-we-need-antibiotics/1dg96v94/>. Sabah A. A. Jassim and Mazen A. Naji, In Vitro Evaluation of the Antiviral Activity of an Extract of Date Palm (Phoenix dactylifera L.) Pits on a Pseudomonas Phage, Evidence-Based Complementary and Alternative Medicine, vol. 7, no. 1, pp. 57-62, 2010. doi:10.1093/ecam/nem160

27 | P a g e