Boback-Hassig 1 Introduction Has it ever become a thought that leaving antibiotics in the car on a road trip over the

summer could diminish the effectiveness? For most people, that is not the problem. Man people over the ears still use antibiotics after the have been e!posed to e!treme heat. "ome mothers have #uestioned if the antibiotic $ill still $ork on their child, but for others that thought does not occur %"homon&. In some severe cases, the reactions to antibiotics that have been e!posed have been fatal. In hopes of ans$ering $hether or not the antibiotic is still effective after being e!posed to e!treme heat, the researchers took on the challenge. 'epending on the storage temperature of the antibiotics, the heat ma or ma not affect it. (o test $hether or not the heat does affect the antibiotic, the researchers performed a series of trials consisting of different high temperatures and pH of the agar $here the antibiotic $as placed. )scherichia coli %). coli& is a ver common t pe of bacteria. It can be found in intestines of people and animals. ). coli reproduces b cellular division, and mutations an also occur, but since the researchers are not testing an antibiotic over a long period of time, there should not be an mutations %*$la!&. (he first variable is temperature of "treptom cin. (he researchers chose this antibiotic because it has a storage temperature of 1+-,- ./ %0Health "#uare1&. (he researchers $anted a lo$er storage temperature to see ho$ it the high temperatures affect the antibiotic. (he temperatures chosen for the e!periment ranged from ,2- ,3 ./. (hese $ere chosen because the $ould simulate a hot summer da . "ince the temperatures during the summer do not change to much da to da , the researches kept the temperatures in close range.

Boback-Hassig 4 (he second variable is the pH of the agar on $hich the ). coli is gro$n. (he normal pH of agar is 5 %Heifets&. For this reason, the researchers put the standard at 5. (he high and lo$ $ould then be at 2 and 3. (his is because the researchers $anted to sta in close range so the data is easil compared and there $ould not be a big gap. (he response variable $as ). coli. ). coli $as gro$n in a 6etri dish and the "treptom cin $as then tested on it to see ho$ the different variables affect it. (o see ho$ $ell the "treptom cin $as $orking, the researchers measured the area of non- gro$th in the 6etri dish. (he higher pH combined $ith the lo$er temperature of "treptom cin, the more effective the antibiotic should. 7fter these factors have been added to the agar, the gro$th of ). coli should decline. In preparing the agar, citric acid $as added to decrease the pH of the agar %"H8) $hile sodium bicarbonate $as added to increase the pH %"'9F ,&. 7fter, 1m: of ). coli $as added to each of the + 6etri dishes, one disc of streptom cin $as placed in the center of each disc. (hen, the dishes $ere placed in their correct incubators based on $hat temperature the had to be in. 7fter 4; hours, the + 6etri dishes $ere taken out and observed.

6roblem "tatement 6roblem "tatement< Ho$ does the temperature of "treptom cin and pH of the agar affect the gro$th of bacteria in the 6etri dish?

Boback-Hassig , H pothesis< (he higher the temperature of streptom cin and the larger amount of pH in the agar $ill decrease the amount of bacteria in the 6etri dish than lo$ering the temperature of streptom cin and decreasing the amount of pH in the agar $hich $ill increase the amount of bacteria.

'ata Measured< (he t$o independent variables are the temperature of "treptom cin %./& and pH of the agar. (he dependent variable is the =one of inhibition of bacteria in a 6etri dish. (he statistical method used to anal =e the data $as a t$o-factor 'esign of )!periment %'8)&. (he levels for temperature are< :o$-,2./ "tandard-,5./ High-,3./

(he normal temperatures on a summer da are around ,5./. "ince temperatures do not fluctuate ver much da to da , the temperatures chosen $ere ,2./ for lo$ and ,3./ for high. (he levels for pH of agar are< :o$-2 "tandard-5 High-3

(he normal pH in agar is 5. "o to sta in close range, the pHs chosen $ere 2 as our lo$ and 3 as our high.

Boback-Hassig ; )!perimental 'esign Materials< 7gar >utrient %2 g& Bacteria :oop Baking "oda %pinches& Balance 3- m: Beaker %4& BI/ :ighter Bunsen Burner /itric 7cid %pinches& Escherichia Coli Fleaker %1--- m:& Hot 6late Incubator %,2./, ,5./, ,3./& 6rocedure< 7gar 6reparation 1. 4. ,. ;. +. 2. 5. 3. Boil +--m: of $ater in a flask on the hot plate. 6lace paper to$el on balance. 7fter it $eighs the paper to$el, set the scale to =ero. 6our agar nutrients on to paper to$el to the amount of 2 g. If the amount of agar goes over 2 g, leave it? 6our agar nutrients into boiling $ater $ith magnet. 6our agar into the standard 6etri dish until bottom is full covered. 6our 2- m: of agar into t$o beakers. Magnet 6aper (o$el 6etri 'ishes %4-& )co (estr pH 4 6robe "harpie 6ermanent Marker "terile 9ater %1 m:& "treptom cin %discs& (est (ube (hermometer %./& ($ee=ers 9ater %1--- m:&

/hanging the pH of the 7gar 1. 4. 7dd pinches of baking soda to the agar in one beaker until the pH probe reads 3. 7dd pinches of citric acid to the agar in the other beaker until the pH probe reads 2.

Boback-Hassig + ,. 6our the agar $ith baking soda into the %-,@& and %@,@& 6etri dishes until bottom is full covered. ;. 6our agar $ith citric acid into the %-,-& and %@,-& 6etri dishes until bottom is full covered.

). coli "ample 6reparation 1. 4. ,. ;. +. Fill test tube $ith 1m: of $ater. 7dd ). coli to the sterile $ater in the test tube. Mi! ). coli and agar together in 6etri dish $ith loop. )mpt the e!cess out over sink. 7fter putting the ). coli in a 6etri dish sterili=e the loop.

7dding 7ntibiotic 'iscs 1. 4. ,. 7dd one of "treptom cin to each 6etri dish in the center $ith t$ee=ers. 6ress do$n on discs $ith the t$ee=ers. 6lace all 6etri dishes in correct incubator a. %@,@& goes in ,3./ b. %@,-& goes in ,3./ c. %-,-& goes in ,2./ d. %-,@& goes in ,2./ e. standard goes in ,5./ ;. +. :eave in incubator for one da . 7fter one da , take out of incubator

Boback-Hassig 2 2. Inspect each of the 6etri dishes and record data.

Finding the "urface 7rea of Ho$ the 7ntibiotics "pread 8ut %Aefer to Figure 1& 1. Find the across and height of $here the bacteria has not gro$n due to the streptom cin. 4. ,. ;. +. *sing BrC, find the area in cm. /alculate that for all five 6etri dishes. Aecord data. Aepeat steps for all four trials.

'iagram<

Figure 1. ). coli in a %@,@& environment Figure 1 sho$s the ). coli gro$th after being in the incubator for one da . (his 6etri dish had a high pH of 3 and $as placed in a high temperature of ,3C.

Boback-Hassig 5

'ata and 8bservations 'ata< (able 1 Dro$th of Bacteria '8) 1 4 , ; 7verage Dro$th of Bacteria %cm& (emperature of "treptom cin%./& and pH of 7gar %@,@& %@,-& %-,@& %-,-& "tandard ,.1; ,.5EE; ,.1; 1-.15,2 1.5224+ ;.1+42+ ,.1; 5.-2+ 1.5224+ 1.5224+ E.2124+ 1.+,32 5.-2+ 1.+,32 1.5224+ ,.1; 4.3,,3+ ,.1; 1.+,32 1.5224+ +.-1444+ 4.345E2, +.1-4+ ,.5+;42, 1.5251+

In (able 1, there $ere ; '8)Fs completed and the e!act se#uence the $ere run, standard al$a s $ent first. (hen on the 1st and ,rd trials the high pH G%@.@& and %-,@&H $as made and placed in the dishes first. 8n the 4nd and ;th trials the lo$ pH G%@,-& and %-,-&H $as made and placed in the dishes first. 7ll five dishes $ere placed in their correct incubators in random order. (able 4 'esign of )!periment Ialues (emperature of streptom cin%./& "tandard @ ,2 ,5 ,3

pH of the agar "tandard 5

@ 3

In (able 4, the pH of the agar $as chosen b finding $ith the research that the standard of agar is 5 %Heifets&. (o sta in a close range, 2 $as chosen as a lo$ and 3 $as chosen as a high. (he $ere chosen in close range so $hen the data could be more efficientl compared do to the smaller gap. (he point of the e!periment is to heat the

Boback-Hassig 3 antibiotic to see its effectiveness on a gro$th of ). coli in a 6etri dish. (he factor in this $as high temperatures. "tandard $as chosen at ,5, due to high temperatures during the summer and to sta in a close range ,2 $as chosen as a lo$ and ,3 $as chosen as a high. (he temperatures $ere chosen in close range because during the summer, da b da temperatures do not change too much. 8bservations< (able , 8bservations of (rials )!amined 'ate ,J41J4-14 ,J4,J4-14 ,J45J4-14

8bservations

(he bacteria in the %-,-& 6etri dish onl covered half of the dish in '8) 1. 7ll the other dishes had full bacteria gro$th in '8) 1. 7ll bacteria gro$th $as similar. "tandard had a denser gro$th colon . ($o trials e!amined toda . 7ll colonies $ere around the range of the same amount of thickness

In (able ,, there are observations about ho$ colonies looked after being treated $ith different pHs of the agar and different temperatures of the streptom cin. (here are gaps bet$een each trial because sometimes it took more time to prepare, and after putting the 6etri dishes into the incubator the researchers had to $ait 4; hours before e!amining.

In '8) 1, %-,-& had the largest surface area of $here ). coli did not gro$ due to the streptom cin spreading out. In '8) 4, %-,@& had the largest surface area. In '8) ,, %@,@& had the largest surface area. In the final trial, %@,-& had the largest surface area.

Boback-Hassig E

%@,-& "tandard

%@,@&

%-,@&

%-,-&

Figure 1. '8) 4 6etri 'ish "amples Figure 1 sho$s five samples from '8) 4. 7s sho$n above, the circles of $here the streptom cin spread out is in a generall close range. From the picture, the observation that can be made is that standard had a denser gro$th of ). coli than the other four 6etri dishes. (he surface area of most trials and 6etri dishes are similar. %@,@& and %-, @& have a ver similar amount of gro$th. In %@,-& and %-,-&, there are scattered areas $here the gro$th becomes more dense.

'ata 7nal sis and Interpretation

'ata 7nal sis< (able ; Main )ffect of (emperature on ). coli

Boback-Hassig 1-

In (able ;, the ). coli space $as the region measured $here ). coli did not gro$
Temperature -1 1 E. coli

;.;43,31 ,.E4--E; due to the temperatures of the "treptom cin.

Area of Non- Growth(cm)

Main Effect of Temperature on E. E. Coli coli

4.5 4.4 4.3 4.2 4.1 4 3.9 3.8 3.7 3.6 1

4.428381

3.920094

-1

Tempera ture (degrees Celsius)

Figure 4. Main )ffect of (emperature on ). coli Draph In Figure 4, the effect value of temperature on ). coli is --.+-343, so it can be concluded that $ith lo$er temperatures, the area $here no ). coli gre$, $as greater. (able + Main )ffect of pH on ). coli
pH -1 1 E. coli

3.291113 5.0573625

In (able +, it is sho$n that $ith the higher pH, the more effective the "treptom cin $as.

Boback-Hassig 11

Main Effect of pH onE. E.coli Coli

6 Area of Non- Growth(cm) 5 4 3.291113 3 2 1 0 -1 pH 1 5.0573625

Figure ,. Main )ffect of pH on ). coli Draph In Figure ,, the effect value of pH on ). coli is 1.5224;E+, so it can be concluded that the higher pH made the "treptom cin more effective.

(able 2 Interaction )ffect pH (-) Temp. "olid "egment 'otted "egment %@& %-& 4.345E2, ,.5+;42, (+) +.-1444+ +.1-4+

(able 2 sho$s the interaction effect bet$een temperature and pH.

Boback-Hassig 14 nteraction Effect

6

Area of Non- Growth (cm)

5.1025

(emp. 2.827963

%-&

5 4 3

(emp. %@&

5.012225

3.754263

2 1 0

-1 pH

Figure ;. Interaction )ffect of pH and (emperature In Figure ;, the interaction effect value of pH and temperature on ). coli is 4.;;-,5.

Figure +. "tandard 'ata "catter 6lot

Boback-Hassig 1, Interpretation< In Figure 4, "treptom cin $as more effective after being kept at a lo$ temperature. 9ith a lo$ temperature the =one of inhibition $as larger than the area of the higher temperature factor. (he reasoning for this is because "treptom cin is normall stored at a colder temperature %0Health "#uare1&. (he cause this had on the ). coli $as that it inhibited a larger portion of the bacteria than the "treptom cin that $as kept a higher temperature did. In Figure ,, "treptom cin $ith a high pH $as more effective than the "treptom cin $ith a lo$ pH. (hus concluding that the effect this contributed on the ). coli $as the higher pH killed off more of the bacteria. (he reasoning for this is because "treptom cin, $hen mi!ed $ith li#uids, is more effective $ith a higher pH. In Figure ;, the 6etri dish that had a lo$ temperature and high pH killed off the most ). coli. (his, again, is because "treptom cin is normall kept at lo$er temperatures and it is also more effective kept at a higher pH. (he 6etri dish $ith a high temperature and lo$ pH inhibited the least ). coli. (hese t$o factors combined definitel had a positive effect on the "treptom cin. In Figure +, there is no variabilit . For each trial the standards $ere al$a s the same. (his could have been because standard $as al$a s prepared first on each trial. 7ll of the values are considered to be part of the 0vital fe$1 due to the fact that our range of standards $as =ero.

Boback-Hassig 1; /onclusion (he h pothesis is the lo$er the pH of the agar and the lo$er the temperature the more effective the "treptom cin $ill be on decreasing ). coli gro$th in the 6etri dish. (he h pothesis $as reKected. (he researchers planned on testing the effect of temperature of "treptom cin and pH of agar on ). coli, in order to see ho$ the affected the effectiveness of "treptom cin. (he researchers performed this e!periment to see if heat affects the performance of antibiotics. In this e!periment, the pH of the agar and temperature of "treptom cin $as changed to create different effects on ). coli gro$th. (he researchers concluded that $ith a lo$er temperature and higher pH, the more effective "treptom cin $ould be in decreasing gro$th of ). coli in a 6etri dish. (he results $ere like this because the storage temperature for "treptom cin is about room temperature %0Health "#uare1&, so the lo$est temperature $ould be closest to the room temperature, making it the best. (he researchers did not make an design fla$s. It is proven b the standards, $hich $ere all e!actl the same. (here $ere, ho$ever, a couple errors made $hile follo$ing the procedure. (he first error happened $hile changing the pH of the agar. (he researchers took previousl made agar off of the hotplate not reali=ing that the agar $as cold. 'uring this trial, the agar solidified $hile the pH $as being adKusted. (he second error made $as $hen putting ) .coli onto the agar. 9hen the ). coli $as poured into the 6etri dish, the researchers forgot to empt the e!cess ). coli out of one dish. (he researchers could have prevented this b being more careful and observant. 7lso, the could have double check their $ork and make ne$ agar.

Boback-Hassig 1+ For future research, the researchers could test different t pes of antibiotics to see if the different ones have the same results as "treptom cin. (he research enhances the understanding of this topic because it $ould compare results and $ould more effectivel ans$er the problem statement of, 0'oes high temperatures of antibiotics and pH of agar affect the effectiveness of the antibiotic.1 (his research could benefit man people. (his is because almost ever one uses antibiotics some time in their life. (he could easil not kno$ that leaving the antibiotic in e!treme heat could dramaticall affect the antibiotic. It could also be used in the medical field, because if an antibiotic is stored in the $rong place it could be a drastic problem. 7fter reflecting on the e!periment the researchers concluded that over the course of the e!periment the should have taken better notes of certain things that the encountered throughout the e!periment. 7lso, the should have double checked all of their $ork before finishing and cleaning up that da .

7ckno$ledgement (he researchers $ould like to thank Mr. )stapa. Mr. )stapa helped the researchers develop the procedure. Mr. )stapa also helped the researchers $ith an misunderstandings or #uestions. 7nd again, thank ou Mr. )stapa.

Boback-Hassig 12 9orks /ited LBacteria Dro$ing )!periments in 6etri 6lates.L Science Company. (he "cience /ompan , n.d. 9eb. 5 Feb 4-14. B, Mord n . L7gar pH.L Newton: Ask A Scientist. *" 'epartment of )nerg , "ep 4--;. 9eb. 5 Feb 4-14. LHealth "#uare.L Health Sq are. >.p., 15 Ma 4-14. 9eb. 12 Ma 4-14. Heifets, :eonard. L>e$ 7gar Medium for (esting "usceptibilit of M cobacterium tuberculosis to 6 ra=inamide .L !o rnal o" Clinical #icro$iolo%y. >.p., n.d. 9eb. 4; Ma 4-14. Nonrad, 9alecia. LMistakes in "torage Ma 7lter Medication.L The New &ork Times. (he >e$ Oork (imes, 1+ -3 4-11. 9eb. 5 Feb 4-14. :iu, "hiKun, and :aurie *singer. L7ll 7bout 7gar.L Science ' ((ies. "cience Buddies, n.d. 9eb. 5 Feb 4-14. LpH.L Simply Hy(roponics an( )%anics. >.p., n.d. 9eb. 44 Ma 4-14. "homon, Mar . L9arning? Hot (emperatures Ma Be Ha=ardous (o Oour 'rugs.L A$o t. 7bout.com, 3 4--2. 9eb. 5 Feb 4-14. ('" and pH.L Sa"e *rinkin% +ater ,o n(ation. >.p., n.d. 9eb. 41 Ma 4-14.