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Effects of Antiseptic on Bacteria Growth

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This project was designed to investigate the effect of different antiseptics on bacterial growth. The antiseptics are different in their content and active ingredients. Two bacterial lawns were prepared using nutrient agar, Escherichia coli and Bacillus subtilis. Paper discs dipped into the antiseptics were put on the bacterial lawns and the diameter of clear zones around the paper discs were measured after incubating the bacterial lawns for 24 hours at 30°C. The results showed that 4.8% chloroxylenol is the most effective antiseptic in inhibiting the growth of bacteria, indicated by the greatest diameter of clear zone.

The results of this experiment can be applied to the use of specific antiseptics on specific type of bacteria. Further research can be done on cetylpyridinium chloride in the usage of the compound as an external antiseptic.


A trial experiment was carried out to find out whether the antiseptics will inhibit the growth of Escherichia coli and Bacillus subtilis or not. Five fresh samples of antiseptics containing different active ingredients; which are 0.05% cetylpyridinium chloride, 4.8% chloroxylenol, 0.1% acriflavin, 70% ethanol and 0.1% chlorhexidine gluconate and sterile distilled water were put into separate sterile 50ml beakers. Distilled water was used as a control. Before conducting the experiment, the table top was wiped with alcohol and hands were washed with soap. These steps were taken to kill the bacteria present. 200µl of bacterial inoculum containing Escherichia coli was introduced into a sterile petri dish using a micropipette. The lid of the petri dish was opened as minimum as possible to avoid contamination. Molten nutrient agar was then poured into the petri dish and the petri dish was swirled gently to mix the inoculum and agar. After the mixture has solidified, a sterile filter paper disc which has been dipped into the distilled water was placed on the surface of bacterial lawn containing Escherichia coli by using a sterile forceps. This step was repeated by replacing the distilled water with the antiseptics. The paper discs were placed as further apart as possible from each other. After that, the petri dish was placed in an incubator set at 30°C. The whole procedure was repeated for bacterial inoculum containing Bacillus subtilis. After 24 hours, the petri dishes were removed from the incubator and the diameter of clear zones around each paper disc was measured using a ruler.

The whole steps in trial experiment were used in the main experiment. The experiment was repeated ten times, meaning that for each antiseptic, there will be ten readings of diameter of clear zone. Repetition is necessary to calculate the mean diameter so that the efficiency of each antiseptic is measured more accurately.

A one-way Analysis of Variance (ANOVA) test will be used to analyse the data, which will test the equality of six means at one time by using variances.[15]

[1015 words]

Risk Assessment

In this experiment, I am using chemicals and bacteria. All the chemicals used are toxic if swallowed. Therefore, it is not safe to consume them. Chloroxylenol and acriflavine can cause skin and eye irritation. Therefore, gloves and goggles were worn when conducting the experiment. There was risk of infection by the bacteria during and after conducting the experiment. Aseptic measures were taken before and during conducting the experiment, such as wash hands with soap and wipe the table top with alcohol. The petri dishes must be disposed properly (do not throw into bin) and autoclaved.





I will use a one-way Analysis of Variance (ANOVA) test to demonstrate the data statistically. All the calculations are made using Microsoft Excel 2007 software. In ANOVA test, the means, variances, sum of squares (SS), degrees of freedom (df), mean square (MS) and F-ratio are calculated in order to get the P-value.[16] P-value less than 0.05 (5% confidence level) indicates that, there is a significance difference between the groups.


Experimental Hypothesis

The diameter of clear zone around paper discs dipped into 4.8% chloroxylenol is the greatest.

Null Hypothesis

There is no significant difference in the diameter of clear zone for each of the antiseptics.

P-value for both tests is less than 0.05 (1.38-10-31 and 3.83-10-42 respectively). Therefore, null hypothesis is rejected and the experimental hypothesis is accepted. 4.8% chloroxylenol shows the greatest diameter of clear zone for both bacteria.

Data Analysis

The result of one-way ANOVA test showed that there is significant difference between the values of mean for each group. This is because the P-values are far lower (1.38-10-31 and 3.83-10-42 respectively) than confidence level of 0.05. The P-values are small because the values of mean and variance are very different with each other. From the P-values, it can be concluded that there is less than a 5% probability that the results occurred due to chance and more than a 95% chance that the results are significantly different. This does not agree with the null hypothesis which states that there is no significant difference between the groups, hence it is rejected and the experimental hypothesis is accepted.

Bar chart 1 shows the mean diameter of clear zone for each antiseptic tested on Escherichia coli. The effect of cloroxylenol on Escherichia coli is almost the same as cetylpyridinium chloride (mean diameter of clear zone: 1.28cm and 1.23cm respectively). As distilled water does not have any antibacterial properties, it does not have any effect on Escherichia coli. The least effective antiseptic when tested on Escherichia coli is acriflavin (0.75cm), followed by ethanol (0.95cm) and chlorhexidine gluconate (1.10cm).

[1916 words]The same trend is seen when the antiseptics were tested on Bacillus subtilis (bar chart 2). Chloroxylenol shows large difference in the effect on the bacteria compared to the other antiseptics (mean diameter of clear zone: 2.05cm). Acriflavin is the least effective antiseptic (0.73cm). There is small difference in the effectiveness of ethanol and chlorhexidine gluconate on Bacillus subtilis.

Bar chart 3 shows the comparison of the effectiveness of antiseptics on both bacteria. Generally, all antiseptics work better on Bacillus subtilis except for acriflavin, but the difference is small. Chloroxylenol shows clear difference in its effect on both bacteria (difference in mean diameter: 0.77cm), while the others show only small difference.

Chloroxylenol is a broad spectrum antibacterial substance which works by altering bacterial enzymes and cell walls.[17][18] Its mechanism of action have been little studied, but because of its phenolic nature, it would have effect on bacteria.[3] Phenol induces lysis of intracellular constituent, including the release of potassium ion (K+). It also lyses growing culture of Escherichia coli and Staphylococcus aureus.[3] E. coli is a Gram-negative bacterium while Staph. aureus is a Gram-positive bacterium. This proves that chloroxylenol can work on both type of bacteria.

Cetylpyridinium chloride is a quaternary ammonium compound which is highly sensitive to Gram-positive bacteria, but moderately sensitive to Gram-negative bacteria.[19] Its mode of action is based on interaction of basic cetylpyridinium ions with acid group of bacteria, which subsequently inhibits bacterial metabolism by forming weak ionic compound that interfere with bacterial respiration.[20] This proves that this compound works better on B. subtilis than on E. coli.

Chlorhexidine gluconate is a bactericidal agent.[3] It damages the outer cell layers, crosses the cell wall or membrane (probably by passive diffusion) and attacks the bacterial cytoplasmic or inner membrane.[3] It is active against wide range of Gram-positive and Gram-negative bacteria.[21] This proves that chlorhexidine gluconate can work both on E. coli and B. subtilis.

[2232 words]Not much is known for the mode of action of ethanol. It is believed that it causes membrane damage and rapid denaturation of proteins, with subsequent interference with metabolism and cell lysis.[3] Most hand cleansers contain between 65% to 70% of alcohol. Higher concentrations are less effective because proteins are not denatured easily in the absence of water.[22]

Acriflavin has the ability to intercalating with DNA and cause frame shift mutation.[23] It also affect DNA molecules of microorganisms.[24] Therefore, it inhibits bacterial reproduction. In this experiment, it is proven that acriflavin can affect the growth of E. coli and B. subtilis.


The concentration for each antiseptic is different from each other. Therefore, it is not 100% confirmed that chloroxylenol is the best antiseptic in inhibiting or killing E. coli and B. subtilis. In this experiment, I am testing which antiseptic is the best, but the concentrations of all antiseptics tested are known from the label. This is concern with our daily life chemicals which we may use frequently. It is better if the pure substances of the active ingredients are available. By having the pure substance, I can test the effectiveness of same concentration of active ingredients on bacteria.

Besides that, the clear zone area around the paper discs is not regular circle in shape. This makes the reading of diameter of clear zone is not very accurate. Therefore, a few measurements of diameter have to be taken and then, the average value is calculated.

The diameter of clear zone is measured using a ruler. The smallest scale on the ruler is 1mm. In order to get more accurate reading, a more sensitive apparatus (smaller scale) can be used, such as vernier callipers.

Although aseptic steps have been taken, the environment is not 100% free of bacteria. Some bacteria might have contaminated the apparatus and materials. This is uncontrollable. Therefore, the risk of contamination is minimise as minimum as possible by conducting the aseptic steps.

[2515 words]

Sources Evaluation

All the journals were taken from reliable sources. This is because the authors of the journals used wide range of sources when writing the journals and they cited where they extract the information. Besides that, careful methods and appropriate materials also made me feel that the journals are reliable. In this report, the main journal that I referred to was Antiseptics and Disinfectants: Activity, Action, and Resistance written by Gerald McDonnell and A. Denver Russell. The journal was taken from a government's website http://www.ncbi.nlm.nih.gov. I believe that this source is reliable as a lot of experts work for them and most of them publish their work in this website.

Source 1 was taken from a book (WHO Model Formulary) published by a well known organisation, World Health Organisation. I believe that this book provides reliable information as the publisher is a world organisation. Besides that, the writers (Mark C. Stuart, Maria Kouimtzi, Suzanne R. Hill) also wrote several other books.

Source 18 was taken from another book (Browner: Skeletal Trauma, 4th ed.) published by W. B. Saunders Company. All the authors of this book are doctors or experts with at least Medical Degree. Therefore, what they wrote in the book is not bias to any side and they also cited the sources they used when writing the book.

Source 2 was taken from a website. This website is a university's website which provides notes for their students. I believe that the writer of the article is a lecturer at the university. The university (University of South Carolina) is a well established university with a lot of achievements.

Source 10 was taken from another website administrated by a company lead by doctors (MedicineNet, Inc.). I believe that this source is reliable as the authors of the website are experts from medicine, healthcare, Internet technology and business field.

[2849 words]Source 13 was taken from a website of European Bioinformatics Institute. The organisation is part of European Molecular Biology Laboratory. This source is reliable because the organisation is a joint venture between European countries and a lot of studies have been done by this organisation.

Source 19 was taken from a corporate website. This company (Vertellus Specialties, Inc.) was established before World War 2 and mainly involve in the production of chemicals. This source seems reliable as the company involves in this field for hundred of years and has involved in many researches.

[TOTAL 2916 words]

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