# Case Study Testing the Antibacterial Efficacy Between Alcohol-Based and Alcohol-Free Sanitiser on the Human Hand

#### Arjun Subramanian

Reviewed by: Dr. Chris Baysdorfer, Cal State East Bay

## ABSTRACT

The novel Severe Acute Respiratory Syndrome Coronavirus 2 has augmented the regular use of hand sanitiser, redefining its role in preventing viral transmissions and killing bacteria which can promote viral replication. As a result, the two major types of sanitiser, alcohol-based, made with Ethyl Alcohol, and alcohol-free/natural, made with tea tree oil and aloe vera, have experienced significant price surges. This case study was aimed to determine the antibacterial efficacy between alcohol-based Germ-X Original Hand Sanitizer and alcohol-free M.D. Science Lab Premium Hand Sanitizer. A single individual’s hands without sanitiser were sampled for controls; afterward, both sanitisers were applied on separate hands, producing two experimental sets. The cotton swabs used to sample were swiped on Petri dishes in 15-second increments to compare both products’ effects on bacterial growth. After viewing the bacterial trends on the Petri dishes, Germ-X Original Hand Sanitizer was found to be most effective at killing hand bacteria, yet dried faster than M.D. Science Lab Premium Hand Sanitizer. It may be concluded that Germ-X Original Hand Sanitizer has a greater efficacy overall, therefore agreeing with the CDC’s recommendation for a hand sanitiser with at least 60% alcohol concentration1.

## INTRODUCTION

Throughout the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic, the regular use of hand sanitiser has significantly surged. Both the World Health Organization (WHO) and the Center for Disease Control (CDC) have recommended hand sanitisers with alcohol concentrations over 75-80% and 60%, respectively1. Given that no antiviral drugs or vaccines are available for the novel virus, prevention of infection through containment is the highest priority as of now.

In February of 2020, increasing demand for hand hygiene to reduce the SARS-CoV-2 spread led to year-on-year 255% growth rates in hand sanitiser, 10% increased spending on household cleaners, and a 7% increase in liquid hand soap purchasing in the United Kingdom2. Many stores and restaurants have also been enforcing mandatory hand sanitizing before and after entering, demonstrating the newfound importance SARS-CoV-2 has placed upon these sanitisers. As a result of these combining factors, the global market for hand sanitiser is expected to exponentially grow from $1.08 billion in 2019 to$1.51 billion by 2024, a far quicker increase than years prior3. Evidently, hand sanitiser’s role as an efficient mode by which to limit SARS-CoV-2 transmissions is gaining traction. Since many transmissions between humans spread via contaminated hands, it is essential to know the more effective type of sanitiser to protect against the virus: alcohol-based, made with Ethyl Alcohol (EtOH) or alcohol-free, made with aloe vera (AV) and tea tree oil (TTO).

Germ-X Original Hand Sanitizer, the alcohol-based product used in this test, relies on its 63% EtOH concentration to kill pathogens present on the hand. EtOH can kill bacteria living on the hand (e.g. Staphylococcus aureus, Pseudomonas fluorescens/putida, and Staphylococcus warneri) due to its amphiphile chemical compounds, allowing it to bond with and break down the bacteria’s cellular membranes and promote protein denaturation4. By increasing the lag phase of bacterial growth, EtOH can also inhibit rapid bacterial cell division. By contrast, M.D. Science Lab Premium Hand Sanitizer, the alcohol-free product, utilises Melaleuca alternifolia (TTO) and Aloe Barbadensis (AV) as its antimicrobial agents. TTO has a hydrocarbon structure which allows it to partition into bacterial membranes and disrupt their vital functions5. Data showing that TTO permeabilizes model liposomal systems further supported these results6. AV, the sanitiser’s other key ingredient, contains natural anthraquinones which serve as its main antibacterial property. Consequently, the bacteriostatic, or bacteria inhibiting, plant can suppress bacteria such as Staphylococcus aureus, Streptococcus pyogenes, and Salmonella paratyphi7. The common, yet infectious, bacteria located on the hand often make humans ill, weakening the immune system and making it increasingly likely to contract the novel coronavirus.

In addition to preventing the weakening of immune systems, hand sanitisers have been proven to inactivate viruses. A recent study found that all sanitiser formulations, including ethanol, the active ingredient in the sanitiser used in this experiment, with dilutions of 40% or more killed the coronavirus and reduced the virus to background levels within 30 seconds. The authors noted that while 30 seconds is the recommended time to rub hand sanitisers into the skin and was the time used in this study, most people do not spend that much time using it8. Moreover, tea tree oil, one of the main ingredients in M.D. Science Lab Premium Hand Sanitizer, is capable of strong antiviral action and inactivated model viruses with an efficiency of 95% or more within an exposure time of 5–15 min9.

The antibacterial and antiviral properties of ethanol, tea tree oil, and aloe vera are evident, however, testing the differences in bacterial colony numbers and sizes between the two hand sanitisers will provide a clearer understanding of which is more effective. The above-mentioned studies focused on an understanding of the antibacterial and antiviral properties of an isolated ingredient, such as ethanol, TTO, or AV. Similarly, earlier studies have not compared effects on bacterial colony growth from Germ-X to M.D Science Lab Premium, the specific brands of sanitiser used in this experiment. The current paper is aimed at determining the antibacterial efficacy between alcohol-free and alcohol-based sanitiser on the human hand through a comparative case study.

## METHODS

This study was a case study involving a single participant from an American home. The individual was without any clinical signs of infection nor irregular hand hygiene patterns to produce typical dominant species of hand bacteria. Both the participant’s left and right hand’s ventral side was sampled, the former corresponding to Germ-X Original Hand Sanitizer and the latter to M.D. Science Lab Premium Hand Sanitizer. Ten sterile Petri dishes were labeled in four categories: 2 left-hand alcohol control samples (L.C1,2,), 3 left-hand alcohol experimental samples (L.A1,2,3), 2 right-hand natural control samples (R.C1,2), and 3 right-hand natural experimental samples (R.N1,2,3).

After marking each plate, a nutrient agar mix was prepared and poured into the ten Petri dishes to augment bacterial growth. One ½ inch radius circle was marked on each of the participant’s hands; the area in each circle was swabbed with sterile cotton swabs lightly dampened with distilled water and swiped onto the control plates in the same manner (L.C and R.C). In each marked circle, the participant had approximately 2ml of alcohol sanitiser rubbed on the left-hand by another person using sterilized gloves, and the same amount of natural sanitiser applied on the right-hand, both for 30 seconds at the same time. Three swabs from both hands were taken in 15-second increments after one another and swiped on the individual Petri dishes (L.A1,2,3 and R.N1,2,3) marked with subscripts indicating the order in which the dish was swiped.

The dishes were placed in sterile plastic bags and stored in a dark, warm environment for 48 hours each, after which they were examined for bacterial presence. After removing the Petri dishes from the plastic bags, the bacterial colonies were approximated using visible visual differences based on the type and pattern of the colonies. The areas of the alcohol-based sanitiser solution were compared with those of the natural sanitiser to find varying trends in bacterial deficiency and growth patterns.

Considering that the participant’s hands were sterilized and the person sampling wore sterilized gloves, there is not much room for error. However, the bottles in which the hand sanitisers were stored could have been a source of contamination, although there is no visible contamination present in the figures.

## RESULTS

Figure 1: Left-Hand Controlled Bacterial Growth (No Sanitiser). L.C1 and L.C2 both demonstrate abundant bacterial presence but the latter has a denser presence.

Figure 2: Bacterial Growth After Alcohol Sanitiser Application

Figure 3: Right-Hand Controlled Bacterial Growth (No Sanitiser). R.C1 and R.C2 both show ample bacterial growth.

Figure 4: Bacterial Growth After Natural Sanitiser Application

In the left-hand control Petri dishes (L.C1, L.C2), the bacteria visibly fully filled the shape created by the swiping motion (Figure 1). The colonies ranged in size in plate L.C1, however, plate L.C2 not only demonstrated the swiping shape but also contained a significantly larger colony on the side of the plate. Additionally, the second plate displayed a much more uniform pattern of larger colonies as opposed to the first plate’s many small spaces between the colonies. After the alcohol-based sanitiser was applied on the hand, the swabbed Petri dishes exhibited a notable decline in bacteria growth (Figure 2). The swiping outline of bacteria is completely absent in plate L.A1, with only a few colonies present. As the time after the initial application of sanitiser passed, plates L.A2 and L.A3 revealed an increasing amount of bacteria, yet none replicated the defined swipe of the control.

The right-hand control Petri dishes (R.C1, R.C2) presented similar results to the control dishes from the left (Figure 3); both had bacteria filling the lines where the dish was swabbed. The bacteria on R.C1 grew outside of the pattern and less uniformly while R.C2 contained bacteria in a far more precise pattern. Following the natural sanitiser’s application to the hand, the observed bacteria growth also declined in their respective Petri dishes (Figure 4). Plate R.N1 showed few colonies, yet a faint outline of the original swab pattern. By contrast, plate R.N2 and R.N3 began to more accurately reflect the control plates, the former having more spaces between colonies and the latter with almost negligible bacterial decline.

## DISCUSSION:

The goal of hand sanitisers, concerning the current circumstances surrounding the SARS-CoV-2 pandemic, is to provide an accessible avenue through which to destroy the novel coronavirus. Since Staphylococcus aureus, a common hand species of bacteria likely present in this study, can promote respiratory virus replication and pathogenicity through co-infection, the bacterial decline from each sanitiser could also be a potential indicator of viral killing efficacy10. Either sequencing or enzymatic tests could confirm the specific bacteria present on the hand.

Current hand hygiene recommendations advise hand washing as the most effective option to prevent SARS-CoV-2 through its mechanical (rubbing hands) and chemical processes (hand soap). However, even this presents risks. Dirty faucets and water from the sink can contaminate hands to additional microorganisms; these surfaces harbor many bacteria from communal use and tap water often contains pathogens such as Pseudomonas aeruginosa11. By contrast, both types of hand sanitisers work to kill these microorganisms instead of simply removing them, lowering the risk of contamination.

Both Germ-X and M.D. Science Lab sanitisers possess risks as well as these benefits, largely due to the ingredients that make up each respective formula. A CDC report analyzed data from the National Poison Data System (NPDS) regarding hand sanitiser exposures to children aged 12 years or younger from 2011–1412. From the total 70,669 age-group exposures reported to the NPDS, 92% were alcohol-based exposures and 8% were non-alcohol-based exposures12. Younger children, often attracted to the scent of the sanitiser, tend to be increasingly susceptible to adverse effects from ingestion as their livers do not contain ample glycogen to break down carbohydrates (such as EtOH found in alcohol sanitisers). Additionally, TTO from natural sanitisers when swallowed is toxic and potentially fatal. Adverse health effects commonly found for both alcohol- and non-alcohol-based hand sanitisers are ocular irritation, vomiting, conjunctivitis, oral irritation, cough, and abdominal pain, while rare effects included coma, seizure, hypoglycemia, metabolic acidosis, and respiratory depression12. In sum, these harmful effects are more likely to occur after ingesting alcohol-based sanitiser, but are worse with natural sanitisers.

The results of this study are mainly evident in the first Petri dish of each experimental collection (L.A1 and R.N1) and more loosely suggested throughout the rest of the Petri dishes (L.A2, L.A3 and R.N2, R.N3). On L.N1, Germ-X proved to kill the most bacteria from the enclosed circle on the participant’s hand, leaving only two isolated colonies and few other smaller colonies, whereas the immediate application of M.D. Science Lab Premium on the right-hand yielded considerably more colonies of about the same size, approximately thirty-two, as well as a faint streak of developing bacteria. As the time after the original application increased (in increments of 15 seconds as previously stated), both sanitisers’ effectiveness deteriorated as bacteria in the latter two Petri dishes in each group developed an increasing trend of bacteria. This upward trend is likely due to the sanitiser drying quickly on the hand and losing its ability to kill as much bacteria, demonstrating that neither sanitiser maintains its active ingredients’ abilities for long periods. When comparing the two growth rates in the experimental groups, the figures suggest that bacteria treated with natural sanitiser increase at a slower rate than those treated with the alcohol-based sanitiser. This observation is consistent with previous studies as AV and TTO, the main ingredients in M.D. Science Lab, have proven to maintain moisture for longer than EtOH as the former two have moisturizing properties while the latter has rapid drying time on skin 5,13-14.

Multiple studies have also proven the efficacy of rubs and sanitisers made with alcohol, tea tree oil, and aloe vera 11,15. A study testing an alcohol-based (EtOH) sanitiser from the PureHands brand demonstrated its reliability in killing all common bacteria (Escherichia coli, Proteus mirabilis, Shigella sonnei, and Staphylococcus aureus) over a 7-day period11. Another paper evaluated the role of herbal agents (TTO and AV) as cavity disinfectants, revealing that they could be effective in minimising the risk of secondary caries15. These both proved the positive effects of each products’ ingredients, however, neglected to carry out a direct comparison of both under similar conditions as present in this study.

It should be noted that the results of this study are limited to a single individual, and additional experiments should be conducted with a greater number of participants in order to more accurately determine the best sanitiser solution to kill hand bacteria. Additionally, the participant in this study is right-hand dominant, so additional participants with left-hand dominance should be sampled to determine if the dominant hand plays a role in the amount of bacteria present on the hand.

## CONCLUSION

In this case study, while M.D. Science Premium Lab Hand Sanitizer’s ingredients dried slower on the hand, Germ-X Original Sanitizer killed a greater amount of bacteria, implying the latter is more effective. These findings support the CDC’s recommendation for a hand sanitiser with at least 60% alcohol concentration. The TTO and AV evidently did not kill bacteria as efficiently as the EtOH in this participant. Considering this study simply served observational purposes in identifying visual differences between the sanitisers’ effects, further experiments are needed to determine bacteria type and if any other factors contribute to bacterial presence.

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