EFFECT OF (NH4)2SO4 ON RESISTANCE OF E.

COLI K12 1

Effect of Ammonium Sulfate on the Resistance of Escherichia coli K12 to Amoxicillin

Maya H. Patel

Freehold High School

Medical Sciences Learning Center: Materials and Methods

EFFECT OF (NH4)2SO4 ON RESISTANCE OF E. COLI K12 2

Null hypothesis for Phase 1: Exposure to a high concentration of ammonium sulfate has no effect

on the resistance of Escherichia coli K12 (E. coli K12) to amoxicillin.

Alternate hypothesis for Phase 1: If the prevalence of ammonium sulfate is related to the

resistance of E. coli K12 to amoxicillin, E. coli K12 strains exposed to a high concentration of

ammonium sulfate will be more resistant to amoxicillin than strains not exposed to any

ammonium sulfate.

Null hypothesis for Phase 2: Exposure to a low concentration of ammonium sulfate has no effect

on the resistance of E. coli K12 to amoxicillin.

Alternate hypothesis for Phase 2: If the prevalence of ammonium sulfate is related to the

resistance of E. coli K12 to amoxicillin, E. coli K12 strains exposed to a low concentration of

ammonium sulfate will be more resistant to amoxicillin than strains not exposed to any

ammonium sulfate.

Determining Treatment Concentrations and Quantities

Before any experimentation could be done, the concentrations of ammonium sulfate (AS

were determined. This experiment contained two experimental groups: E. coli K12 exposed to a

high concentration of AS and a low concentration of AS. In a previous study, a concentration of

400mg/L was used as the high concentration exposure of NaClO2 (Dan, et, al, 2016). This was

used in this study as the high concentration. To determine the amount of AS to use per plate,

dimensional analysis was used to convert 400mg/L of AS to grams per 15 mL. (Assuming that

the ideal amount of LB agar per plate was 15 mL.) 400mg/L is equivalent to 0.006 grams per 15
EFFECT OF (NH4)2SO4 ON RESISTANCE OF E. COLI K12 3

mL. To reduce error, 0.012 grams of AS was measured and mixed with 30 mL of LB agar to

yield two plates of 400mg/L concentration.

In the same experiment, 10mg/L was used as the low concentration exposure of NaClO2

(Dan, et, al, 2016). 10mg/L is equivalent to 1.5*10-4 grams of AS per 15 mL. Since the most

advanced scale measured up to a thousandth of a gram, this quantity was too small to measure.

Therefore, it was determined that the low concentration would be 200mg/L, half of the high

concentration. 0.012 grams of AS was mixed with 60 mL of LB agar to yield four plates of

200mg/L concentration.

Control group plates contained no ammonium sulfate.

The small quantities of AS were measured with caution. A piece of sterile weigh paper

was placed onto the scale, and the scale was zeroed. A scoopula was used to remove a small

amount of AS powder from its container, and was placed slowly onto the weigh paper by gently

tapping the scoopula. After exactly 0.012 grams of AS were measured onto the weigh paper, the

powder was transferred into a 50mL Erlenmeyer flask or a 250mL Erlenmeyer flask and sealed

with Parafilm for later use.

Preparing LB Agar Plates

Every day, many agar plates were poured. Before pouring, each Petri plate was labeled

with the type of plate (Control, 400mg/L, 200mg/L, or stock), cycle letter (A or B), cycle

number, date poured, and date that bacteria would be plated on them. For the first 18 days of this

experiment, a hot plate was used to melt LB agar. A 1L beaker filled with water would be boiled

on a hot plate, and a bottle of LB agar would be placed in the hot water. After about 35 minutes,

most of the media would be liquefied and ready to pour. When using a hot plate and boiling
EFFECT OF (NH4)2SO4 ON RESISTANCE OF E. COLI K12 4

water to melt the agar, the agar would become warm. When using a microwave after Day 19 of

the experiment, the agar would become extremely hot, and therefore, the agar had to cool for at

least 5 minutes before adding any AS to it. When microwaving the agar, the bottle would be

squeezed to break up the media. The bottle would be placed in the microwave without its cap for

1 minute. The agar would then be microwaved for 45 second intervals until most of it was

liquified. The agar could not be fully melted each time, because this long exposure to heat would

cause the agar to boil. Each plate had to be poured at least a day before it would be used, since

liquid agar takes at least thirty minutes to solidify in a Petri plate.

Two control plates A and B were made per cycle, and were prepared by pouring the liquid

agar directly from the bottle and into the plate. Two 400mg/L plates A and B were made per

cycle, and were prepared by pouring 30 mL of liquid agar into one of the 50mL Erlenmeyer

flasks containing 0.012 grams of AS. The AS was dissolved into the agar by holding the neck of

the flask and swirling the contents. The agar was then poured into two plates labeled 400

mg/L. A similar method was used to prepare four 200mg/L plates. 60 mL of agar was poured

into a 250mL Erlenmeyer flask containing 0.012 grams of AS. LB agar would be poured to the

50mL mark of the 250mL Erlenmeyer flask and the 10mL mark of a 20mL graduated cylinder.

The contents of the graduated cylinder would be poured into the 250mL Erlenmeyer flask. The

AS was dissolved and poured into four plates labeled 200 mg/L. Since two plates were made

per cycle, two of the new 200mg/L plates would be used the day after pouring, and the remaining

two would be used two days after pouring. In total, six plates would be used every day. On some

days, plates were made two to four days in advance. One stock plate containing only LB agar

was poured twice a week.

EFFECT OF (NH4)2SO4 ON RESISTANCE OF E. COLI K12 5

Safety goggles were always used when handling all chemicals and hot materials. Hot

gloves were used when handling hot bottle of agar. Hot plates were kept away from any

flammable objects, and everyone working near the area was aware that there was boiling water

nearby. All glassware was kept away from the edge of the table and was rinsed after every use.

Cycles of Exposure and Maintenance of Bacteria

To start the experiment, three stock plates were made. E. coli K12 was purchased from

Carolina and arrived on Day 1 of the experiment in a tube. Three plates containing LB agar that

were prepared the day before were streaked with one loopful of E. coli each. Bacteria was

streaked by starting at the top of the plate and moving the loop in a horizontal motion while

moving the loop downward. When the entire plate was covered, the plate was rotated about

ninety degrees, and the horizontal and downward motion was repeated. This plating technique

was used throughout the experiment to encourage bacterial lawn growth. Although previous

research suggested incubating stock bacteria at 37 degrees Celsius for 16 hours, the stock

bacteria was plated on a Friday and had to incubate at 37 degrees Celsius for about 72 hours

(Dan, et, al, 2016).

After 72 hours of incubation, one loopful of stock bacteria was plated onto LB agar. This

new plate served as the isogenic seeds of the experiment. After 24 hours of incubation,

isogenic bacteria was plated onto two 400mg/L plates and two control plates. This was the first

cycle of 400mg/L and control plates. (Due to time constraints, the 200mg/L cycles could not start

until Day 7, four days after the 400mg/L and control cycles started.) Two plates of each group

were plated each cycle to serve as backup in case one of the plates got contaminated or did not

grow. This continued until each group was exposed to 25 cycles.

EFFECT OF (NH4)2SO4 ON RESISTANCE OF E. COLI K12 6

Until Day 5 of the experiment, disposable loops were used to streak bacteria. The

disposable loops would be placed in a beaker of ethanol and thrown away in the trash. For the

rest of the experiment, a metal loop was used to inoculate the bacteria. The metal loop would be

sterilized over a lit Bunsen burner until the metal turned red. To cool the metal, the loop would

be dipped into solid agar on the sides of the empty Petri plate. A loopful of bacteria from the

previous cycle would be streaked onto a new plate using the lawn method. The new plates would

be taped shut before being incubated, and the six plates from the previous cycle would be

autoclaved and thrown away. Twice a week, original stock bacteria would be plated onto LB agar

and incubated at 37 degrees Celsius. Each cycle was incubated at 37 degrees Celsius for about 24

hours, but when cycles were plated on Fridays, those plates were incubated for 72 hours and

accessed again on the following Monday.

Since an open flame was being used, this experiment was conducted at a separate lab

bench. Everyone near the flame was aware of it and was wearing safety goggles. The flame was

lit with caution by making sure that the natural gas was not on too high, and that no one burned

their hands. After the Bunsen burner was turned off, the natural gas was turned off in the

classroom. To prevent the spread of bacteria, used plates containing E. coli were destroyed in the

autoclave. All plates were taped shut to prevent contamination.

Testing for Resistance

After 25 cycles of exposure to 400 mg/L, 200 mg/L, and 0 mg/L of AS, the resistance of

the E. coli K12 to amoxicillin was measured. Resistance was defined as the radius of inhibition

around a disk. Disks that either contained 20mcg of amoxicillin and 10mcg of clavulanic acid or

no chemicals (to serve as a control) would be placed onto lawns of bacterial growth. The disks
EFFECT OF (NH4)2SO4 ON RESISTANCE OF E. COLI K12 7

containing amoxicillin would cause the surrounding bacteria to die in the shape of a circle, which

is called the zone of inhibition. The radius of this circle is called the radius of inhibition. The

radii of inhibition of each group were measured and compared using a statistical 2 sample t-test.

When the amoxcillin and sterile disks arrived, experimentation had already started. The

resistance of the stock bacteria was measured by plating stock bacteria onto 8 plates containing

LB agar. Each plate had equal quadrants drawn onto them using a ruler and a permanent marker.

After lawns had grown onto each plate, four antibiotic disks were placed onto each plate. They

were each placed into the center of a quadrant of the petri dish. All plates were incubated

overnight and the radii of inhibition were measured In total, 32 antibiotic disks were used and 32

radii were collected. This process was repeated for the blank disks that served as a control.

After 25 cycles of each treatment group and the control group, 24 plates of regular LB

agar were poured and split into 3 groups of 8. These plates were called the test plates and labeled

as such. All of these plates had equal quadrants drawn onto them using a ruler and a permanent

marker. The 25th cycle of each group was plated onto 8 of these plates. A coin was flipped to

determine which cycle letter to plate from: for the control cycle, if the coin landed on heads, a

loopful of control cycle 25A was plated onto one of the eight LB test plates. If the coin landed on

tails, a loopful of control cycle 25B was plated onto one of the eight LB test plates. This was

repeated until 8 of the 24 LB test plates contained control group bacteria on its 25th cycle, 8 of

the 24 plates contained 400mg/L bacteria on its 25th cycle, and the remaining 8 contained

200mg/L bacteria on its 25th cycle. One disk containing antibiotics was placed into one

quadrant, which meant there were 32 disks being used per group. These plates were incubated

overnight and the radii of inhibition were collected. This process was repeated for the blank disks
EFFECT OF (NH4)2SO4 ON RESISTANCE OF E. COLI K12 8

that served as a control. A two-sample T test was used to determine the statistical significance of

the radii of the bacteria before and after the exposure cycles.