Alcohol Disinfectant Efficacy on Escherichia coli.

Introduction

Alcohol’s effect in terms of disinfectant use is something to consider given the various types of bacteria and their overwhelming abundance. Of these various types of microbes that happen to exist, Escherichia coli (E. coli), a gram-negative, bacilli shaped bacteria specifically, is known for its common infestation and high degree of diversity across a wide range of environmental and ecological conditions (Blount, 2015). E. coli, therefore, becomes a microbe in which careful attention must be placed simply due to the various and potentially harmful strains of E. coli present. Not to mention, the pathogenic nature of this microbe alone is also a cause for concern as well (Blount, 2015). With this in mind, one might ask the question as to why E. coli and gram- negative bacteria in general are more concerning than perhaps some other bacterial groups presently out there. For this reason, observing its cell wall structure more in depth becomes quite important for understanding its significance.

When referring to the cell wall of this microbe alone, E. coli (and gram-negative bacteria specifically) is very unique in comparison to the other non gram-negative microbes in proximity. Containing a thin cell wall known as peptidoglycan, as well as a unique, toxic like outer membrane in conjunction, E. coli and most gram-negative bacteria in general tend to make it hard for specific antibiotics to do their job (Parker, 2016). When looking at what is known as the“outer leaflet” of the gram-negative outer member more specifically, unique structures known as Lipopolysaccharides (LPS) can be found on this surface which swiftly act as an endotoxin causing harm to its inhabited host (Parker, 2016). These LPS are unique to the gram-negative bacterial structure. Aside from this, certain strains or “types” of E. coli can be deemed more aggressive than others which makes their prevention a very important tactic (“Questions andAnswers”, 2014). Escherichia coli O157:H7, an E. coli strain commonly found in undercooked meat, is a good example of this commonly seen (E. coli (Escherichia coli), 2014) (Escherichia coli (E. coli), 2019).

Therefore, when it comes to microbe disinfection and infestation prevention, alcohol, among many other disinfectants, has been shown to be a promising tool in reducing the presence of E. coli on surfaces (Infection Control, 2016). When referring to how this disinfecting agent inhibits E. coli’s growth, alcohol specifically has a mode of action that works in the process of denaturing/inhibiting protein synthesis important in E. coli’s physiology (Infection Control, 2016) (Parker, 2016). Alcohol has also been shown to be effective in killing mycobacterium (cause of tuberculosis), fungi, and certain viruses alike (Infection Control, 2016).

Therefore, when it comes to sanitation, sickness prevention, and avoiding E. coli infection in total, the use of various cleaners, such as alcohol alone, are shown to be effective and important in preventing disease. Thus, when thinking of a way to avoid E. coli infection initially, the use of alcohol might just be valid tool for the general public and community to consider. In the experiment that follows, the microbe E. coli was put to the test against isopropyl rubbing alcohol (70%). First being suspended in 9mL of isopropyl alcohol for 30 seconds, and then being serial diluted into succeeding test tubes of water, samples from the respective serial diluted test tubes were then plated on trypticase soy yeast agar (TSY) and incubated for 24 hours at 37 °C. The end product was to observe if bacteria would grow. This experiment was also compared to two control agents which comprised of the following: a serial dilution of E. coli in sterilized water which was then plated on TSY and incubated for colony formation in the same fashion as isopropyl alcohol. Bacterial growth on TSY from the serial dilution of water was used as a positive control, while an empty, non-sampled TSY plate indicative of no growth, was representative of a negative control as well. The goal of this experiment was to show the disinfectant efficacy of isopropyl rubbing alcohol against E. coli in order to present more sanitary options.

Hypothesis

The exposure of E. coli in isopropyl rubbing alcohol (70%) for 30 seconds will not produce bacterial growth on TSY due to the mode of action of alcohol alone.

Methods & Materials

Materials

● 9 TSY plates.

● E. coli turbid suspension.

● Micropipettes.

● Pipette tips.

● 16 test tubes of 9mL saline.

● 2 sterile test tubes.

● Isopropyl Rubbing Alcohol, 70%.

● Bunsen burner.

● Beads.

● 10 mL pipette.

● Sterile cotton swab.

● Pipette pump.

● Timer.

● Sterilized water.

Methods

Put on a lab coat, tie hair back if needed, wash hands, and disinfect lab area.
Acquire 9 TSY plates.
Light Bunsen burner.
Make a turbid suspension of E. coli in a test tube of sterile water. Do this by using a sterile cotton
swab to obtain the bacteria from the labeled test tube. Then place the cotton swab in the sterile water and swirl around the sides to make the suspension. Be sure to flame the top of the tube when removing the lid and replacing the lid.
Acquire 6 test tubes each of 9mL sterile water and label 10−1through 10−6.
Using a 10mL pipette transfer 1mL of the turbid suspension to a test tube labeled 10−1, flaming
the top of the tube when removing and replacing the lid.
Tickle test tube to make sure it is mixed thoroughly.
Discard the pipette tip in the biohazard and replace with a sterile one to transfer 1mL from the
10−1to the test tube labeled 10−2.
Repeat this process for test tubes 10−3through 10−6, tickling each dilution and flaming the tops of
the tubes.
Label 3 of the TSY plates 10−7, 10−6, and 10−5.
Using the micropipette transfer 0.1mL from the dilution labeled 10−6to the TSY plate labeled
10−7, 0.1mL from the dilution of 10−5to the TSY plate labeled 10−6, and 0.1mL from the dilution
of 10−4to the TSY plate labeled 10−5.
Place beads in each of the three plates and use the copacabana method to spread the liquid across
the entire media.
Label 5 test tubes of sterile water 10−2through 10−6.
Make a suspension in a test tube of 9mL of alcohol and 1mL of the turbid suspension of E. coli.
Tickle test tube then let sit for 30 seconds.
Use same steps as 8-12 to complete this dilution and transferring to the TSY plates.
Repeat the process of steps 13, 14, and 15 a second time to have more media to back up the
results.
Place all TSY plates in the “save” bin to be looked at after a week of incubation when the results will be interpreted.

Results

Data Analysis

In the serial dilution of the turbid suspension of E. coli in water (Table 1, Figure 1), the data showed uncountable bacterial colonies on the following TSY dilution plates:10−4 and10−5respectively. The cells/mL were therefore uncountable. However, on the 10−6 TSY dilution plate in regards to this data, 182 colonies were counted with a total cells/mL of 18,200,000 (182 multiplied by 100,000). This data explains that water alone is not effective at inhibiting E. coli and its growth as well.

In Trial 1 of the serial dilution of E. coli suspended in 9mL of isopropyl rubbing alcohol (70%) (Table 2, Figure 2), no bacterial growth was seen on any given TSY plate. Thus, after a careful serial dilution and after plating each respective liquid dilution sample onto TSY, no bacterial colonies and no total cell counts (cell/mL) were therefore recorded. This data explains that isopropyl rubbing alcohol (70%) in Trial 1 is extremely effective at preventing the colonization of the microbe E. coli.

Trial 2 of the serial dilution of E. coli suspended in 9mL of isopropyl rubbing alcohol (70%) (Table 3, Figure 3) produced identical results as seen in Trial 1 of the serial dilution of E. coli suspended in 9mL of isopropyl rubbing alcohol (70%). Therefore, the data in Trial 2 of the serial dilution of E. coli suspended in 9mL of isopropyl rubbing alcohol (70%) is also supportive in alcohol’s effect at inhibiting E. coli and preventing its growth.

This data is therefore supportive of the hypothesis that the exposure of E. coli in isopropyl rubbing alcohol (70%) for 30 seconds will not produce bacterial growth on TSY due to the mode of action of alcohol alone. Implications from this data analysis bring about the effect of alcohol’s mode of action. It’s very apparent that alcohol has an underlying mechanism that is particularly effective at inhibiting E. coli’s function. As compared to water which was sterile in this experiment, E. coli clearly had no resistance in growing when exposed to this neutral environment. This may also imply that E. coli’s structure plays a role in protecting it from osmotic pressures.

Conclusion

The disinfectant efficacy of isopropyl rubbing alcohol against E. coli was shown to be present based upon this study. To reiterate once more, the goal of this study was in fact to show alcohol’s effectiveness at inhibiting E. coli’s growth given alcohol’s mode of action and the concept behind its significance (inhibiting protein synthesis in E. coli specifically). Regarding the question that often follows in regards to this experiment and whether or not alcohol, or isopropyl rubbing alcohol in this case, is in fact effective at disinfecting E. coli, this study does show conclusive evidence in support of alcohol’s disinfecting power. The main findings of this study showed very alarming evidence that alcohol’s mode of action on E. coli specifically, easily inhibited its growth upon submersion. No growth was seen on TSY after a mere 30 seconds of exposure of E. coli in 9mL of isopropyl rubbing alcohol, concluding that this disinfectant is in fact effective. The positive control of E. coli suspended in water, on the contrary, showed the opposite results with abundant E. coli growth on TSY. Therefore, when considering the potential and more aggressive strains of E. coli in existence today, optimally using a disinfecting agent like alcohol becomes a viable and very important tool as a means of preventing disease. Water alone as a disinfecting tool is clearly not a good option as this experiment here shows unfavorable outcomes. Although errors in this experiment were kept to a minimum, misuse of aseptic techniques can easily take place if focus is lost or carelessness sets in throughout a laboratory experiment. As a result, surrounding microbes can easily contaminate a TSY plate which, in the end, may result in skewed data. In regards to future studies that may impact various community and clinical settings especially, research continuation should very well be initiated using this experiment as a catalyst for discovering new and specific disinfecting agents. Aside from E. coli, and the many other harmful gram-negative bacteria in existence, many other pathogenic bacteria outside of this gram-negative classification also exist and are capable of causing harm. Alcohol therefore may or may not be effective at stopping their existence which should be looked into in order to know for certain. Unseen microbes tend to put human health at constant risk. Therefore, disinfecting agents should be continually researched in order to keep up with the constant bacterial evolution.

Works Cited Blount, Z. (2015). The unexhausted potential of E. coli. eLIFE, 1-12.

Carey, E., Huerta, E., & Hull, Kalamazoo.

Carey, E., Huerta, E., & Hull, College, Kalamazoo.

Carey, E., Huerta, E., & Hull, Kalamazoo.

Carey, E., Huerta, E., & Hull, College, Kalamazoo.

G. (n.d.). E. coli in alcohol. Kalamazoo Valley Community College, G. (n.d.). E. coli in alcohol, trial 2. Kalamazoo Valley Community G. (n.d.). E. coli in water. Kalamazoo Valley Community College, G. (n.d.). E. coli in water graph. Kalamazoo Valley Community

G. (n.d.). Graph of E. coli in alcohol. Kalamazoo Valley Community

Carey, E., Huerta, E., & Hull, Community College, Kalamazoo.

G. (n.d.). Results of trial 2 of E. coli in alcohol. Kalamazoo Valley

E. coli (Escherichia coli). (2014, December 1). From Center for Disease Control and Prevention: https://www.cdc.gov/ecoli/general/index.html

Escherichia coli (E. coli). (2019, January 29). From Minnesota Department of Health: https://www.health.state.mn.us/diseases/ecoli/index.html

Infection Control. (2016, September 18). From Centers for Disease Control and Prevention: https://www.cdc.gov/infectioncontrol/guidelines/disinfection/disinfection- methods/chemical.html

Parker, N. (2016). Microbiology. Houston: OpenStax.