June 28, 2024

Artiom Peshkur 

10th Grade

Northfield Mount Hermon

Abstract

The escalating global challenges of antibiotic resistance have prompted the search for alternative antimicrobial agents. In a previous study that explored the antimicrobial potential of apple cider vinegar (ACV) against Escherichia coli and other bacteria.1 Their investigation provided the basic framework for this experiment to compare the difference in the minimum dilution of “antibiotics” required for growth exhibition on several microbial species. The study also highlighted that microbial cultures exposed to ACV impaired cell integrity, organelles, and protein expression in the microbes. Key enzymes and proteins involved in cellular processes were found to be absent or downregulated following ACV exposure. These findings displayed the broad-spectrum antimicrobial potential of ACV and its ability to disrupt microbial physiology at the molecular level. It also underscores its potential as a therapeutic agent in the fight against microbial infection and as an alternative antimicrobial. 

Introduction

A New York Times article mentions a few additional benefits of eating apple cider vinegar, including its ability to improve gut health by killing microbes that cause bloating.2  The article mentions a paper showing that apple cider vinegar can be served as an alternative antibiotic, effectively killing bacteria such as C. albican and E. coli.3  This raises the question of whether apple cider vinegar is an effective antibiotic and whether we can make a safe antibiotic at home. 

Using the disc diffusion assay, our experiment will test the efficiency of different antibiotics against different bacteria. The main antibiotic is apple cider vinegar from a store. We will compare its efficiency with the one of homemade apple cider vinegar to answer if we can make the antibiotic by ourselves. We also compare it with the one of distilled white vinegar to test if common vinegar gives the same result or not. 

We also use various types of bacteria in this experiment. The main bacteria is E.coli representing normal bacteria in the intestine. We use E.coli which gains Ampicillin resistance to test whether apple cider vinegar works against antibiotic-resistant bacteria. Finally, we use bacteria from a doorknob, representing bacteria in general. If successfully conducted, this experiment will debunk or support the claims that we can distill a safe antibiotic at home or that we can use apple cider vinegar to alleviate bloating. 

Methodology 

I. Preparing Bacterial Sample 

a. E. coli for tube i was prepared by scooping E. coli directly from the stock using a sterile loop and quadrant streaking an LB agar plate. Afterward, the plate was incubated for a day and a single E. coli colony was scooped with a sterile loop. The sterile loop was swirled inside a 2 ml Eppendorf tube containing 500μL of LB broth and incubated at 37 degrees Celsius for two days. This tube served as gut bacteria. 

b. E. coli for tube ii was prepared by scooping an E. coli colony with a sterile loop from the same LB agar plate that was used for tube i. A 2 ml Eppendorf tube containing 250μL of transformation solution (50 mM CaCl2) was placed to rest on the ice for 10 minutes. Afterward, the E. coli colony would be swirled inside of the aforementioned tube of transformation solution that was on the ice, and 10 μL of +pGLO and Ampicillin-resistant plasmid would be added. The tube will be cooled on the ice for 10 more minutes. Then, we will heat shock the tube by exposing it to 42°C water for 50 seconds. This process of cooling and heating was repeated twice more until at last the tube was put back on the ice for two more minutes. Then 250μL of LB broth was added to the tube. It rested for 10 minutes at room temperature and incubated at 37 degrees Celsius for two days. Afterward, 200μL of the solution was pipetted and evenly streaked onto an LB agar plate with Ampicillin. After culturing this sample of bacteria, an individual colony was scooped up by a sterile loop and swirled in a 2 ml Eppendorf tube containing 500μL of LB broth and incubated at 37 degrees Celsius for two days. This tube serves as an antibiotic-resistant bacteria. 

c. E. coli for tube iii was prepared by taking a 1″ by 2″ piece of paper towel, covering it with water, and rubbing it against a commonly used door handle. The piece of paper towel was placed inside a 2 ml Eppendorf tube containing 500μL of LB broth and incubated at 37 degrees Celsius for two days. After incubation, 200μL of the solution was pipetted and evenly streaked onto an LB agar plate. After culturing this sample of bacteria, an individual colony was scooped up by a sterile loop and swirled in a 2 ml Eppendorf tube containing 500μL of LB broth and incubated at 37 degrees Celsius for two days. This tube represents general germs on a surface.

II. Preparing Prospective Antimicrobial Substances 

a. Homemade apple cider vinegar, store apple cider vinegar, white vinegar, and Ampicillin underwent serial dilution of 1X, 10X, 100X, and 1000X with deionized water. They were stored in 2 ml Eppendorf tubes containing a total of 1 ml of solution each in tubes A, B, C, and D respectively. Tube E acted as a negative control with deionized water. 

b. The preparation of the disk diffusion assay utilized 51 paper filters, seventeen for each tube of bacteria (i, ii, iii). 

I. View of Bacterial Growth 

Discussion

The first key result was the heat shock of the +pGLO plasmids with the E. Coli. If the heat shock was successful there would have been colonies, which the plate was full of. This was unexpected because usually only a few colonies get successfully transformed. However, the colonies didn’t display the fluorescent glow of GFP under UV light and we first thought that the Ampicillinin in the LB agar plate was defective. However, we decided to add Arabinose to induce the expression of GFP to make sure the heat shock wasn't a failure by pipetting 500μL of Arabinose at a concentration of 2g/L over the heat-shocked colony. Sure enough, after a couple of days, the bacteria was brightly fluorescent from the production of GFP. However, we couldn’t scoop up an individual colony because too much arabinose was added and it made the whole plate a thin evenly distributed layer of successfully heat-shocked E. coli. 

The next important result was the neat organization of the plates and the experiment as a whole, it was meticulous (Fig. 4) and tedious as we made sure that this experiment was simple, yet complicated. However since this experiment was a flop, I’ll go over possible errors that caused the null results. 

First, was the fact that we didn’t use the appropriate discs to diffuse the liquid. Since our paper filters were only capable of holding 6μL before overflowing while the recommended amount of liquid of a paper filter was supposed to be 20μL. Which could potentially explain the infectivity of the liquid on the paper filters. Along with the fact that the discs were very dry when we put them onto the LB agar plates, meaning that we should’ve used better discs for the diffusion. There was something certainly wrong with the way that the diffusion was set up because even our positive control group, Ampicillin, which was certainly supposed to display a zone of inhibition around the paper filter simply didn’t. In fact, in most of the LB agar plates, you can vividly see that the filter paper was contaminated. (Fig. 5) However, we didn’t sterilize them placing them onto the LB agar plates as we didn’t want to interfere with the prospective antimicrobial substance that would potentially disrupt its antimicrobial activity. Thus it is plausible that the contamination on the paper filters came from the antimicrobial that was meant to give inhibition against the microbes. Another possible solution was the brief exposure to air when placing the paper filters onto the plates, but the consistency of the contamination being specifically on the paper filters and none outside the area of the paper filter (Fig. 3) proves that the contamination came from the prospective antimicrobial substances. Therefore it can be concluded that the 3 vinegars tested in this experiment don’t show antimicrobial properties. 

Nonetheless, that doesn’t account for the fact that our positive control group, the Ampicillin, didn’t display any zone of inhibition when it was supposed to work. This could simply be because not enough of the liquid was held by the paper filter, making it ineffective. An essential observation to make was that the Ampicillin paper filters had no contamination on them. This means that the Ampicillin solution was able to inhibit enough antimicrobial properties to prevent contamination, unlike the other prospective antimicrobial substances used in this experiment. Further research should be done to come to accurate conclusions.