What Doesn't Kill You Makes You Stronger:

An In-vitro Investigation of Facilitated Biofilm Formation in E. coli Exposed to Sublethal levels of Ampicillin

Anna Yang and Ben Kim

SCROLL TO THE END for some quality humor! But first, take a look through our website... :)

Video Abstract (enjoy!)

For more information, CLICK HERE to read our paper!

What are biofilms?

Bacteria are everywhere! They are microscopic, single-cellular organisms responsible for both the vital regulation of ecosystems and bacterial infections. Although it may seem that bacteria are anti-social creatures who float around on their own, some bacteria are actually very collaborative and form complex networks to defend against harmful, foreign substances such as antibiotics and host immune cells (2). This protective network is called a biofilm. Biofilm-associated cells display a different phenotype than planktonic, or free-floating cells, characterized mainly by the formation of an extracellular polymeric substance (EPS) matrix and the regulation of certain genes to allow for inter-cell communication known as quorum sensing (2).

What is ampicillin, and how does it work as an antibiotic?

Antibiotics are antimicrobial compounds commonly used in treating bacterial infections. They work by disrupting vital cellular processes such as the function of the cell wall and/or production of nucleic acids (DNA or RNA) and proteins, thus killing the bacteria or stopping cell division (3). Ampicillin, in particular, is in a class of antibiotics called penicillins, which itself is in a class of antibiotics called beta-lactam antibiotics (antibiotics whose core molecular structure involves beta-lactam rings). It is a broad-spectrum antibiotic, meaning that it works on multiple different types of bacteria (4). Beta-lactam rings covalently bind to PBP proteins that are responsible for catalyzing the synthesis of peptidoglycan, a fundamental structure consisting of sugar and amino acids that form bacterial cell walls (5). This covalent bonding inhibits the PBP and, consequently, cell wall (peptidoglycan) synthesis, ultimately killing the bacteria. (6)

How does biofilm formation improve antibiotic tolerance in E. coli?

An increasing concern accompanying the prevalent use of antibiotics is the acquisition of antibiotic resistance. When antibiotics fail to kill all the infectious bacteria, certain beneficial traits that arise from mutations, such as the ability to form biofilms, may be passed on. This is a crucial concern in the medical field because many medical devices such as catheters and implants provide optimal surfaces for bacteria to attach and embed in an EPS matrix, forming highly resistant biofilm communities that put patients at risk for chronic infections. Failure to target biofilm forming, pathogenic bacteria using standard antimicrobial techniques could lead to persistent infections by multi-drug or highly resistant bacteria (7).

VS.

Introduction

E. coli is a common species of potentially pathogenic bacteria responsible for intestinal infections and urinary tract infections (UTI). Previous studies have concluded that uropathogenic E. coli has become extremely resistant to multiple antibiotics. In a 2013 study by Sabir et al., 81% of uropathogenic E. coli isolates extracted from human samples were found to be multi-drug resistant (8). Additionally, E. coli isolates displayed high resistance to ampicillin, the most commonly prescribed antibiotic for UTIs (8, 9).

Despite the numerous research conducted on antibiotic resistance, these studies have not examined antibiotic resistance specifically in the context of biofilm formation. Even in the studies that involve antibiotic resistance and biofilms, the focus was mainly on the biofilm formation as an antibiotic resistance mechanism itself, instead of how certain concentrations of antibiotics may exacerbate biofilm formation -- an area of study crucial for evaluating antibiotics for therapeutic use on persistent biofilm infections. In this study, we aim to examine the effect of prolonged exposure to sub-minimum inhibitory concentrations of ampicillin (concentrations lower than the lethal threshold) on E. coli biofilm formation and antibiotic resistance.

How do antibiotics affect biofilm formation in E. coli?

Do biofilms offer bacteria increased protection from antibiotics?

We shall find out!

The ultimate goal of our study is to determine the efficacy of ampicillin in the potential treatment of E. coli biofilm infections using an in-vitro quantified approach. We aim to determine the effect of varying sub-minimum inhibitory doses of ampicillin on E. coli biofilm formation over time.

Hypothesis

We hypothesize that when grown in low concentrations of ampicillin, biofilm formation in E. coli would increase due to the selective pressure of antibiotics favoring biofilm-associated bacteria.

Higher concentrations of ampicillin, however, would decrease biofilm formation because lethal and near-lethal antibiotic concentrations may reduce cell concentrations to levels low enough to adversely affect biofilm formation.

Methods Overview

To compare growth, antibiotic resistance, and biofilm formation between bacteria selected for biofilm formation and planktonic bacteria, we ran two sets of growth experiments, one for 4 days with daily bead transfers to select for optimal biofilm-forming bacteria, and one for 1 day with no bead as a control. For each of the two growth experiments, we exposed bacteria to antibiotic concentrations of 0, 1, 2, 5, 10, 20 μg/mL (inspired by Sabir et al.) by growing single colonies of E. coli in culture media containing the 5 respective ampicillin concentrations. After finishing their journey, whether it lasts five days or one, we took their OD600 values and performed the OD595 Crystal Violet Assay to measure their final antibiotic resistance and biofilm formation. For the bacteria grown for 4 days, we transferred the Day 5 bacteria from their original concentrations to 0 or 2 μg/mL ampicillin to assay for their ability to form biofilms under controlled environments. The growth and crystal violet results collected on Days 5 and 6 were used to compare bacteria subjected to selective pressure induced by the bead with bacteria that has not.

Where We Started

E. coli K12 Starter Plate Streaked for Single Colonies

Work in Progress

Cell cultures of Various Ampicillin Concentrations After 24 hr Incubation

Final Plating

Bacteria Grown in 5 μg/mL Ampicillin Plated on LB Agar on Day 5

Keep reading for the detailed methods or CLICK HERE to see the results!

Materials and Methods

Part 1. Control and Preliminary Testing

Day 0 - Control OD600

Before beginning our 5-day sequence of biofilm selection, we measured the growth of E. coli (strain K12) in its planktonic form under varying concentrations of ampicillin to set a baseline value for antibiotic tolerance, without the selective pressure induced by the bead. E. coli K12 bacteria were streaked on an LB agar plate for single colonies and incubated at 37 degrees celsius overnight. Then, single colonies were picked from the starter plate and inoculated into culture tubes containing 5 mL of liquid LB medium with 0, 1, 2, 5, 10, and 20 µg/mL ampicillin, the same concentrations used in the experimental groups on days 1-5. After 24 hours of growth on Day 1, cell growth will be quantified for each ampicillin concentration through OD600 measurements (See Day 1 procedure for details).

Day 0 - Control Crystal Violet Assay

A crystal violet assay was performed with bacteria grown in each ampicillin concentration for 24 hours to measure the extent of biofilm formation without the selective pressure of the bead. E. coli cells were cultured in identical conditions as for the OD600 measurements. After 24 hours of growth, the liquid was pipetted out of the culture tubes, leaving only the biofilm that has formed on the walls of the tubes. 5 mL of crystal violet solution was added to each tube and incubated for 15 minutes to stain the biofilm. 10 mL of DI water was added to each tube, incubated for 1 minute, and removed to rinse away excess crystal violet solution. Then, 1 mL of 95% ethanol was added into each tube and vortexed to dissolve the stained biofilm. 200 µL was pipetted into each well of a 96 well microplate three times for each concentration (n=3). Absorbance at 590 nm was measured using a microplate reader.

Click here for a step-by-step procedure!

Part 2. Growth and Biofilm Selection

Click here for a step-by-step procedure!

Day 1 - Inoculation

OD600 of Day 1 bacteria: Before starting the 5-day sequence of growth, antibiotic tolerance of planktonic bacteria from the Day 1 control cultures (inoculated on Day 0 and grown for 24 hours) were measured by quantifying cell growth at various ampicillin concentrations. The absorbance at 600 nm (OD600) was taken using a spectrophotometer. For each concentration, 3 technical replicates were conducted by using 3 separate cuvettes (n=3).

To begin our 5-day experiment, we inoculated single colonies from the E. coli K12 starter plate into culture tubes containing 5 mL of liquid LB medium with ampicillin concentrations of 0, 1, 2, 5, 10, and 20 µg/mL, in addition to a negative control with no bacteria to ensure no contamination. A sterile black polystyrene bead was placed in the media for each of the experimental groups to provide a surface for cell attachment and biofilm formation. The culture tubes were placed in a 37 degree water bath with a shaking speed of 60 rpm overnight to facilitate growth. For each ampicillin concentration, 3 biological replicates were cultured independently (n=3). Additionally, 3 tubes of pure LB with no antibiotics were inoculated without a bead as a negative control and to prepare for an initial round of crystal violet assay on Day 2 to set a baseline biofilm formation for parental E. coli cells.

Day 2 - Bead Transfer

After 24 hours of incubation, the black beads from each culture tube were transferred to 1.5 mL microcentrifuge tubes filled with 950 µL of PBS buffer using sterile forceps. Each centrifuge tube was vortexed for 1 minute to remove all bacteria from the bead and suspend them in the surrounding PBS. 500 µL of PBS containing the cell suspension from each 1.5 mL centrifuge tube was pipetted into culture tubes containing 5 mL of LB with the corresponding ampicillin concentration. For the negative control with no bead, 50 µL was directly pipetted from the Day 1 culture tube into the Day 2 culture tube containing 5 mL of LB. A white bead was added into each of the culture tubes except the negative control. All culture tubes were incubated overnight in a 37 degree water bath with shaking at 60 rpm.

Click here for a step-by-step procedure!

Day 3 - Bead Transfer

Day 3 culture tubes with 5 mL of LB were prepared with the standard ampicillin concentrations (0-20 µg/mL) including a negative control with no bead. A sterile black bead (NOT the bead from Day 1) was added into each Day 3 culture tube excluding the negative control. Then, white beads from the Day 2 cultures, which have been incubated for 24 hours, were transferred to the Day 3 culture tubes with the existing black bead. The resulting tubes with both the black and white beads were incubated overnight in a 37 degree water bath with shaking at 60 rpm.

Day 4 - Bead Transfer

Day 4 culture tubes with 5 mL of LB were prepared with the standard ampicillin concentrations (0-20 µg/mL) including a negative control with no bead. A sterile white bead was added into each Day 4 culture tube excluding the negative control. Then, black beads from the Day 3 cultures, which have been incubated for 24 hours, were transferred to the Day 4 culture tubes with the existing white bead. The resulting tubes with both the black and white beads were incubated overnight in a 37 degree water bath at with shaking at 60 rpm.

Click here for a step-by-step procedure!

Part 3. Biofilm Quantification and Antibiotic Resistance Assays

Day 5 - Growth Preparation for Crystal Violet Assay

After 24 hours of growth, Day 4 E. coli selected for biofilm formation under antibiotic concentrations of 0, 1, 2, 5, 10, and 20 µg/mL ampicillin were transferred to new culture tubes containing no ampicillin (0 µg/mL) and low ampicillin (2 µg/mL) to assay for their respective ability to form biofilms under controlled growth conditions. High ampicillin concentrations were omitted from the final assay to ensure a sublethal dose is used. To perform the final bead transfer, the white bead from each Day 4 culture tube was transferred to a 1.5 mL centrifuge tube with 950 µL of LB and vortexed to remove all bacteria from the bead. Then, 50 µL of solution was pipetted into each corresponding Day 5 culture tube (there should be a 0 µg/mL and a 2 µg/mL tube for each sample). For the negative control with no bead, 50 µL was pipetted from the Day 4 tube directly into the Day 5 tube. The Day 5 culture tubes contain no bead to allow the bacteria to form biofilms on the inner surface of the tube. The bacteria were incubated overnight in a 37 degree water bath with shaking at 60 rpm.

Day 5 - Plating

After 24 hours of growth, white beads from the Day 4 culture tubes were transferred to 1.5 mL microcentrifuge tubes filled with 950 mL PBS buffer and vortexed until all bacteria were removed from the bead, resulting in the undiluted sample (10^0). A serial dilution was performed by pipetting 100 µL of the 10^0 cell suspension into 900 µL of PBS to make a 10^-1 suspension, and subsequently, a 10^-2 dilution by diluting 100 µL of the 10^-1 cell suspension into 900 µL of PBS. 100 µL of the 10^-2 dilution was pipetted onto a LB agar plate and spread evenly using a plate spreader. The 6 resulting plates were incubated overnight at 37 degrees.

Click here for a step-by-step procedure!

Day 6 - Final OD600

After 24 hours of growth, we determined if bacteria selected for optimal biofilm formation (Day 5) evolved antibiotic resistance compared to the control (Day 1) bacteria. Antibiotic tolerance was quantified by measuring the OD600 values of the Day 5 cultures under 0, 1, 2, 5, 10, and 20 µg/mL of ampicillin, which was compared with the control OD600 values taken on Day 0. For each ampicillin concentration, 1 mL of cell suspension was distributed into a 1.5 mL cuvette, and the optical density at 600 nm was measured using a spectrophotometer. For each concentration, 3 technical replicates were conducted by using 3 separate cuvettes and averaged (n=3).

Day 6 - Final Crystal Violet Assay

After 24 hours of growth, the biofilm forming ability of Day 5 E. coli that was transferred to 0 µg/mL and 2 µg/mL of ampicillin from antibiotic concentrations of 0, 1, 2, 5, 10, and 20 µg/mL ampicillin were quantified through a crystal violet assay. The liquid was carefully pipetted out of each culture tube. Then, 5 mL of crystal violet solution was added to each tube and incubated for 15 seconds. 10 mL of DI water was added to each tube, incubated for 1 minute, and removed to rinse away excess crystal violet solution. Then, 1 mL of 95% ethanol was added into each tube and vortexed to dissolve the stained biofilm. 200 µL was pipetted into each well of a 96 well microplate five times for each concentration (n=5). Absorbance at 595 nm was measured using a microplate reader.

Results

Figure 1. Final biofilm formation and antibiotic resistance of E. coli cells selected for optimal biofilm formation after 4 generations of bead transfer in growth media containing varying concentration of ampicillin. (A and B) Absorbance at 595 nm was taken after performing a crystal violet assay on bacterial samples grown for 4 days with daily bead transfers in LB containing 0-20 µg/mL ampicillin, in comparison to the control Day 1 bacteria (n=5). On Day 5, bacteria grown under selective pressure of each ampicillin concentration was transferred to LB with no ampicillin (A), and LB with 2 µg/mL ampicillin (B). Background absorbance not attributed to biofilms was accounted for by performing the crystal violet assay with an empty culture tube. (C) Antibiotic resistance of bacteria selected for biofilm formation on Day 5 (black) in comparison to planktonic bacteria on Day 1 (gray) was measured through OD600 assays on bacterial cultures after 24 hours of growth in media containing 0-20 µg/mL ampicillin (n=3). Unpaired, two-tailed t-tests were performed between all data points. Differences in letters indicate significance (p<0.05). Uncertainty bars represent ±SD.

Figure 2. Morphological differences arise from colonies grown in ampicillin-containing media. 100 µL from the 10^-2 dilution of Day 5 cultures were plated onto LB agar plates to observe potential changes in colony morphology. (A) Colonies of uniform size were observed in the control culture grown in 0 µg/mL ampicillin. (B) Varying colony sizes were observed in bacteria grown in 5 µg/mL ampicillin medium. Red arrows indicate two types of colonies that differ in size and morphology, suggesting potential mutations induced by exposure to ampicillin.

Key Findings

Prolonged growth in sublethal concentrations of antibiotics accelerate the evolution of biofilm forming ability in E. coli.

Higher concentrations of ampicillin have an adverse effect on biofilm formation due to its lethality.

Over time, bacteria exposed to ampicillin are selected for mutations causing observable changes in colony morphology

Biofilm-associated bacteria demonstrate higher resistance to ampicillin than planktonic bacteria.

Biofilm Formation in Standard LB Environment (0 µg/mL ampicillin)

When grown for 24 hours in LB with no ampicillin, E. coli selected for optimal biofilm formation under low concentrations of ampicillin (1, 2, 5, and 10 µg/mL) for 4 days displayed significantly higher biofilm forming abilities compared to the control bacteria that has been grown for 1 day (p<0.05) (Fig. 1A). Bacteria selected under 1 µg/mL ampicillin formed more biofilms when transferred to an environment containing no ampicillin than both bacteria selected under 0 µg/mL and higher levels of ampicillin (2, 5, 10, 20 µg/mL). When exposed to near-lethal concentrations of ampicillin, however, biofilm formation was completely eliminated, even when the bacteria were transferred to an environment containing no ampicillin. Consistent exposure to 20 µg/mL ampicillin for 4 days produced no significant increase in biofilm formation by Day 5 compared to the empty tube control (p>0.05) (Fig. 1A).

Biofilm Formation in Low Antibiotic Environment (2 µg/mL ampicillin)

When grown for 24 hours in LB containing 2 µg/mL of ampicillin, E. coli selected for optimal biofilm formation for 4 days under all ampicillin concentrations examined (1, 2, 5, 10, and 20 µg/mL) showed a significant increase in biofilm formation compared to the control bacteria that has been grown for 1 day (p<0.05) (Fig. 1B). Bacteria grown in 5 µg/mL ampicillin yielded the highest crystal violet absorbance, indicating optimal biofilm formation. Ampicillin concentrations deviating from 5 µg/mL have an adverse effect on biofilm formation, but still significantly higher compared to the Day 1 control (p<0.05). The Day 1 control culture formed no biofilms in 2 µg/mL ampicillin after 24 hours, indicated by the statistical insignificance between the Day 1 control and the empty tube blank readings (p>0.05) (Fig. 1B).

Increased Antibiotic Resistance in Biofilm-Selected E. coli

For all ampicillin concentrations examined, E. coli selected for optimal biofilm formation after 5 days of bead transfer display an increase in antibiotic resistance, reaching a significantly higher OD600 value after 24 hours of growth compared to Day 1 bacteria grown for 24 hours in their respective ampicillin concentrations (p<0.05). For antibiotic sensitive Day 1 bacteria, the OD reaches a near-zero value of 0.003 for ampicillin concentrations of 5 µg/mL and over (Fig. 1C). Bacteria selected for biofilm formation on Day 5 plateaued at an OD of around 0.487 for ampicillin concentrations of 5 µg/mL and over, a significant increase in antibiotic tolerance compared to Day 1 bacteria even for near-lethal concentrations (p<0.05) (Fig. 1C).

Morphological Differences Arise Under Selective Pressure of Ampicillin

When ampicillin is introduced to the growth medium, colonies plated on LB agar plates on Day 5 show changes in morphology compared to both colonies on the starter plate and the 0 µg/mL ampicillin Day 5 culture with bead transfers. Colonies from the 0 µg/mL culture were uniformly spaced, have generally consistent size, and are white in color (Fig. 2A). Some colonies from the 5 µg/mL culture, however, developed an increased size, an opaque yellow color, and a tendency to form clusters compared to colonies on the 0 µg/mL plate (Fig. 2B).

Discussion

EPS Matrix in Biofilms Contributes to Increased Antibiotic Resistance

The data supports our hypothesis that low concentrations of ampicillin induce additional selective pressure, causing the selection for biofilm-forming bacteria to proceed at a faster rate than bead transfers alone. Bacteria in biofilm communities obtain antibiotic resistance mechanisms distinct from planktonic cells, which rely mostly on efflux pumps, drug-degrading enzymes, and target site mutations (7). The extracellular polymeric substance (EPS) matrix acts as a physical barrier that reduces the rate at which antibiotic molecules enter the cell. Their lack of an EPS matrix as a barrier makes planktonic bacteria more susceptible to antibiotics due to a faster penetration rate of the antibiotic compounds. Thus, when low concentrations of ampicillin are present in the medium, a greater percentage of biofilm-associated bacteria survive and reproduce than planktonic bacteria. Previous studies suggest that the multicellularity of the biofilm structure allows individual bacteria within the biofilm to signal via quorum sensing, collectively up or down-regulating genes related to surface attachment and pathways differentiation (7). This multicellular communication process is responsible for the increased antibiotic resistance in biofilm bacteria since deeper layers of the biofilm are not directly exposed to antibiotic stress. This theory is consistent with our data, specifically that Day 5 bacteria produced a higher cell density in 24 hours under all ampicillin concentrations examined compared to Day 1 bacteria, suggesting that the improved biofilm forming ability observed in E. coli cultures exposed to low concentrations of ampicillin is attributable to the difference in antibiotic tolerance between biofilm and planktonic cells (Fig. 1C). At near-lethal concentrations of ampicillin, however, biofilm formation was completely eliminated to baseline value (Fig. 1A). Since EPS matrices are not completely insusceptible to penetration by antibiotics, ampicillin concentrations approaching MIC values (20 µg/mL) were able to effectively suppress biofilm formation by significantly reducing cell density.

Figure 3. Increased ampicillin resistance in biofilm-associated bacteria compared to planktonic bacteria. The EPS matrix in biofilm communities reduces antibiotic penetration rate, making biofilms less susceptible to antibiotics than planktonic bacteria, which lack protection from an EPS matrix.

Figure 4. A proposed mechanism correlating biofilm formation with growth rate in biofilm-selected E. coli. Flagella are synthesized at higher rates in biofilm communities due to a selection for motility. The up-regulation of the master regulator gene of flagella synthesis (FlhD₄C₂) in highly mobile biofilm bacteria may be responsible for observed differences in growth rate and colony size.

Upregulation of Flagella Synthesis Gene Increases Growth Rate in Biofilm-Selected Bacteria

Furthermore, biofilm-associated bacteria reached higher cell densities compared to planktonic bacteria when grown in environments without ampicillin, implying an overall increase in fitness due to selection for mutations unrelated to antibiotic resistance (Fig. 1C). A potential explanation for this phenomenon is the selection for genes involved in flagella synthesis, an organelle responsible for motility in some bacterial cells. Biofilm bacteria are selected for the ability to advance through stages of the biofilm cycle (attachment, maturation, detachment, and recolonization), requiring high motility and thus, effective synthesis of the flagella. A previous study had suggested that flagella abundance in E. coli cultures directly correlate to growth rate due to changes in regulation of the flagellar master regulator gene FlhD₄C₂, which supports our observation of increased growth rates in biofilm-selected bacteria (10). Another point that remains elusive is the cause of the different colony morphologies observed in the 5 µg/mL ampicillin plate (Fig. 2B). It is suspected that the increase in colony size corresponds with the increased OD600 values on Day 5, since both phenotypes imply an increased growth rate. However, this change is not seen in the 0 µg/mL Day 5 culture with bead transfers, demonstrating that morphological changes are induced by the presence of ampicillin, not the selection for biofilm-forming bacteria. Although our findings generally align with current theories on antibiotic resistance and biofilms, we have yet to pinpoint mutations in specific genes that allowed for these phenotypic changes. Genome sequencing of morphologically distinct colonies will provide us with a more accurate understanding of the selection for biofilm-assisting traits on a genetic basis.

Conclusion & Further Studies

Thus, we have demonstrated that instead of eliminating biofilms, antibiotics facilitate their growth! The results of our study have implications for the efficacy of ampicillin in the in-vivo treatment of E. coli biofilm infections, which can be more broadly applied to antibiotic therapy in general. The tendency for bacteria to resist antibiotics through biofilm formation remains a current medical issue with the emergence of multidrug resistant strains. Our findings show that alternative approaches would perhaps be needed to eradicate resistant biofilms. Further studies with quorum sensing inhibitors, matrix-degrading enzymes, and photodynamic therapy are needed to provide alternative solutions.

A next experimental step to determine the underlying genetic changes responsible for the observed phenotypes can be achieved by sequencing the genomes of biofilm-selected and planktonic bacteria. Since colony morphology may be indicative of biofilm forming ability and antibiotic resistance as observed on the 5 μg/mL ampicillin plate, sequencing and aligning the genomes of bacteria that formed different sized colonies can more definitively indicate the location of possible mutations induced by the selective pressure of ampicillin. Further studies include exposing bacteria selected for biofilm formation in ampicillin to media containing other types of antibiotics (e.g. streptomycin, tetracycline, gentamicin, etc.) to test whether acquired antibiotic resistance applies to a broader spectrum of antibiotic type.

If you're interested, CLICK HERE to read our paper for a more in-depth analysis!

Acknowledgements

The authors would like to thank Mr. Edgar for supporting us through every step of the scientific process. And of course, all the open lab teachers that had to deal with us at some point (which is practically every science teacher at Milton Academy) -- thank you for not kicking us out... permanently.

References

(1) Sharma, G., et al. "Escherichia Coli Biofilm: Development and Therapeutic Strategies." Society for Applied Microbiology, 26 Jan. 2016, sfamjournals.onlinelibrary.wiley.com/doi/10.1111/jam.13078. Accessed 22 Apr. 2022.

(2) Donlan, Rodney M. "Biofilms: Microbial Life on Surfaces." National Library of Medicine, Sept. 2002, www.ncbi.nlm.nih.gov/pmc/articles/PMC2732559/. Accessed 22 Apr. 2022.

(3) "How Do Antibiotics Work?" ReAct Group, www.reactgroup.org/toolbox/understand/antibiotics/how-do-antibiotics-work/. Accessed 22 Apr. 2022.

(4) "Ampicillin (Penbritin): An Antibiotic to Treat Bacterial Infections." NetDoctor, 5 July 2018, www.netdoctor.co.uk/medicines/infection/a7323/penbritin-ampicillin/. Accessed 22 Apr. 2022.

(5) Opal, Steven M., and Aurora Pop-Vicas. "Molecular Mechanisms of Antibiotic Resistance in Bacteria." Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases, 8th ed., Elsevier. Science Direct, Elsevier, www.sciencedirect.com/science/article/pii/B9781455748013000187. Accessed 22 Apr. 2022.

(6) Li, Mengchen, et al. "The Resistance Mechanism of Escherichia Coli Induced by Ampicillin in Laboratory." National Library of Medicine, www.ncbi.nlm.nih.gov/pmc/articles/PMC6750165/. Accessed 22 Apr. 2022.

(7) "Antibiotics versus Biofilm: An Emerging Battleground in Microbial Communities." Biomed Central, aricjournal.biomedcentral.com/articles/10.1186/s13756-019-0533-3. Accessed 22 Apr. 2022.

(8) Sabir, Sumera. "Isolation and Antibiotic Susceptibility of E. Coli from Urinary Tract Infections in a Tertiary Care Hospital." National Library of Medicine, 2014.

(9) Vranic, Sabina Mahmutovic. "Antimicrobial Resistance of Escherichia Coli Strains Isolated from Urine at Outpatient Population: A Single Laboratory Experience." National Library of Medicine, 25 Mar. 2016, www.ncbi.nlm.nih.gov/pmc/articles/PMC4851537/. Accessed 22 Apr. 2022.

(10) Sim, Martin, et al. "Growth Rate Control of Flagellar Assembly in Escherichia Coli Strain RP437." Nature, 24 Jan. 2017, www.nature.com/articles/srep41189. Accessed 22 Apr. 2022.

Bloopers

Warning: Memes are not scientifically accurate.

Day -3

Mr. Edgar: How did you determine your experimental antibiotic concentrations?

Ben and Anna: Totally didn't take advice from a (super reputable) NIH paper that saves time by using ug instead of µg (Sabir et al., 2013).

Day -2

We decided to discard the "data" because the negative control was the only tube that grew : /

Day -1

According to the spectrophotometer, 100 µg/mL of tetracycline yielded higher cell densities than 1 µg/mL of tetracycline... Hmm...

Day 0

Finally learned to balance the risk of "contaminación" with the risk of setting our gloves on fire with a bunsen burner. The safety of our E. coli cultures clearly takes precedence over our own.

Day 1

It's only Day 1, and our culture tube record has reached 54. Still counting...

Day 2

Anna: Don't forget to dilute the control blank in 500 µL of blank.

Ben: To make it blanker?

Day 3

Culture tube count: 118.

Data point count (excluding outliers): 6.

Today, we realized that the Law of Large Numbers does not apply to our experiment.

Day 4

Anna: It's exploding!

Ben: No, it's just boiling.

Day 5

Final culture tube count: 152.

Yes, 152.

Ben: How are you differentiating between the tubes that you poured out and tubes you didn't?

Anna: Um... there's stuff in it?!

Day 6

Anna: I'm leaving!

Mr. Edgar: That's a rare sight.

Anna: Mathematically, I've left this lab the exact number of times as I've entered it...

(1 hour later...)

Anna: Minus one.

Day 7

Mr. Edgar: Your group gets "humor points".

Ben: Visibly confused

Anna: So, what's the conversion factor from humor points to points?

Ben: Eh, probably zero.

Day 8

Anna: Serious dilemma, should I omit the 2 µg/mL to get a better regression line?

Ben: Hmm... For the love of science or for the love of grades?

Anna: (contemplating life) Probably grades.

Ben: How dare y- Yeah, probably grades. *delete*

Side note from Anna: If I genuinely cared about grades, I would be writing my (overdue) English essay right now. Don't tell Ms. Bond.

Day 9

Anna: I finished writing Day 6!

Ben: Of the procedure?

Anna: No, of the bloopers section of course!

Ben: (Working on the gif) ... Still more effort than me so far.

Ben: (An hour later, comfortably sitting on the library chair while STILL working on the gif) I think we're making great use of our time.

DYO GIF.mp4

Existential Questions:

What does it feel like to live in an orbital shaker? Is the Earth a giant orbital shaker shaking us at approximately 1.9 * 10^-6 rpm? From the perspectives of our E. coli friends, we are the ones who are orbital-shaking. Or are we? Is the E. coli really shaking???
Why does the LB smell better than Sage chicken broth?

(While ordering Starbucks on Sunday morning)

Anna: (genuinely curious) What's the difference between vanilla and french vanilla?

Ben: It's like the difference between fries and french fries...

Anna: According to merriam-webster.com, french fries are...

In all seriousness...

"Nothing is ever a waste of time." -- Anna Yang

"...Unless you think it is." -- Ben Kim

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