Title

Effect of Different Dettol Concentrations on the Inhibition Zone of Escherichia Coli

K-12’s
Literature Review

Bacteria are microscopic single-celled organisms. Countless bacteria are on Earth, some of
which play a significant role in every environment (Lamorte, 2016). Bacteria have always been
known to cause numerous human diseases.

Escherichia coli (E. coli) is a bacterium commonly found in the gastrointestinal system of
humans and other warm-blooded animals (World Health Organisation 2018). E. coli is part of
the taxonomic family known as Enterobacteriaceae. E. coli has several strains, many of which
are harmless and can keep the digestive tract healthy. However, some strains can cause
serious illness, such as Shiga- toxin-producing E.coli (Felson, 2017). This experiment focuses
on E. coli K-12, a prokaryotic, rod-shaped bacterium approximately 0.5 µm wide. E. coli K-12 is
one of the safe strains of E. coli, and it does not colonise the human intestine (Lim et al., 2010).
E. coli K-12 performs inadequately in the environment and does not survive. Many researchers
have conducted experiments on E. coli K-12, determining that it is safe for commercial use and
does not have disadvantageous effects on microorganisms or plants. For this reason, E. coli
K-12 is widely used as a model organism for research in microbial genetics and physiology. E.
coli K-12 is used in a significant number of medical applications. These productions are
specialty chemicals (e.g., L- L-aspartic, inosinic and adenylic acids) and human medications
such as insulin and somatostatin. E. coli K-12 was first isolated from a convalescent diphtheria
patient in 1922. It was extensively researched by Charles Clitfton and Edward Tantum as a
model organism (Browning et al., 2023). Thus, E. coli K-12 has become the standard
bacteriological strain used in microbiological research and learning (Kuhnert et al., 1995). The
optimal conditions for E.coli growth are approximately 37 °C with a pH between 7.4 and 7.9 in a
protein-rich environment (Tuttle et al., 2021). Using E. coli K-12 for this study ensures a
controlled and standardised experimental system, which allows researchers to draw meaningful
conclusions about the effects of different Dettol concentrations on bacterial growth.

Dettol is an antiseptic and disinfectant that kills bacteria. The main active ingredient in Dettol is
a modified form of phenol named Chloroxylenol. It is an antimicrobial agent used to treat cuts,
bites, stings, abrasions and sterilise surgical instruments. It is a small molecule commonly used
in antibacterial soaps. Chloroxylenol is a substitute for phenol, which is historically known to be
used as an antiseptic (Hugo, 1978). A study shows that the mechanism of Chloroxylenol is that
the hydroxyl part of Chloroxylenol connects to specific proteins in the cell membrane of bacteria
(Damiano et al., 2023). This mechanism disrupts the membrane of bacteria, causing the
contents in the cell to come out through abrupt leakage. The Chloroxylenol can then enter the
bacterial cell and bind to more proteins and enzymes to control the cell's functioning. High
concentrations of Chloroxylenol can coagulate and stop the function of the protein and nucleic
acid content, leading to rapid apoptosis (PubChem 2019).

E.coli can enter bodies through the mouth, nose, and eyes and break into the skin without one
knowing that they have been infected. Promoting antibacterial products, such as Dettol, is
necessary to keep the environment healthy. Cleaning and disinfecting products with an active
antibacterial ingredient can ensure extra protection against bacteria. It is essential to clearly
understand the effects of antibacterial products like Dettol. Antibacterial resistance occurs when
conducted to aim for bacteria to develop the ability to defeat the antibacterial products designed
to kill them. The consequences of antibacterial resistance are that the antibacterial product will
become ineffective and increase the risks of spreading diseases and severe illnesses. This
understanding becomes even more crucial in managing and preventing the potential
development of antibacterial resistance. A study claims that Dettol is most effective at 100%
concentration (Prasad et al., 2020). The key finding of this study proved that Dettol works
effectively on the growth of bacteria. However, the Dettol company recommends diluting Dettol
for disinfection. The overuse or misuse of antibacterial products, such as Dettol, can contribute
to the potential development of resistant bacterial strains. This experiment aims to reconcile
these contradictory claims by investigating the effect of different Dettol concentrations on
Escherichia coli K-12's Inhibition Zone. By understanding the optimal concentration for Dettol's
effectiveness, researchers can understand the most efficient way of using Dettol as a
disinfectant.

This research question is placed into action through an experiment. Firstly, 70% and 25% Dettol
solutions were made by direct dilution. Beakers were labelled Control, 70%, 25%, and 100%
pure Dettol solution. For the 70% solution, 7mL Dettol and 3mL distilled water were mixed. The
25% solution was made with 2.5mL Dettol and 7.5mL distilled water. Agar plates were divided
and labelled. Secondly, using the Lawn plating technique, E. coli K-12 broth was evenly spread
on agar plates. Lastly, the disc diffusion method was used. Paper discs were dipped into
respective Dettol solutions and placed on labelled quadrants of agar plates. Plates were
incubated for 24 hours, and steps were repeated twice for new trials.

Aim- To investigate the effect of different Dettol contractions 100%, 70% and 25%) on
the inhibition zone of E.coli K-12.

Hypothesis-
It is hypothesised that if Dettol concentration increases, the inhibition zone of E. coli K-12 will
increase. This is because higher concentrations of Dettol contain a greater concentration of
Chloroxylenol, which can better denature proteins and cells coagulate and lead to rapid
apoptosis. 100% will be the most effective on E.coli K-12, followed by 70%, and 25% will be
least effective against E.coli K-12 compared to the other concentrations. In the 100%
concentration, the large amount of Chloroxylenol content is hypothesised to target a more
significant number of cells of E.coli K-12. Thus, the most effective concentration is 100% to
increase the inhibition growth of E. coli K-12.

Variables-

Independent - Concentrations Dettol ( 100%, 70% and 25% concentrations)

Dependent- Diameter of E.coli K-12’s inhibition zone (mm)

Controlled - Incubation time duration of E.coli K-12 was kept constant for all samples (24
hours), temperature (37°C) of incubation was consistent for all E.coli K-12 samples and
type of medium to culture the bacteria (Agar plates were used for all samples)

Risk Assessment:

Identify Asses Control

Risk 1 Poisoning due to High Display

consumption of appropriate
Dettol. behaviour and
follow the rules and
regulations of the
laboratory, by doing
so conductors of
the experiment will
take precautions
towards the
consumption of
Dettol.

Risk 2 Breakage of High Place all glassware

equipment leading to away from the edge
severe wounds like and do not touch
deep cuts. the broken glass.
Call for the teacher
to manage the
situation.

Risk 3 Indigestion due to Medium Wear gloves while

E.coli K-12 contact handling E.coli
with the mouth. K-12, and wash
hands thoroughly
after handling E.coli
K-12.
Methodology:

The 70% Dettol concentration was made using 7mL of Dettol mixed with 3mL of distilled water
in a 10mL beaker solution. Then, 2.5mL of Dettol and 7.5mL of water were combined to make
the 25% Dettol concentration. Three agar plates were divided into quadrants and labelled
Control, 70%,25% and 100%.

Sterile cotton was dipped into the E. coli K-12 broth, and the swab was streaked onto the entire
surface using the lawn plating technique.

The disk diffusion technique is used in this experiment to determine the resistance of bacteria to
various antimicrobial compounds. Small paper discs were placed into Dettol concentrations and
distilled water. The discs were applied on the agar plates and incubated for 24 hours after the
ceiling of all the plates. Inhibition zone diameters were measured, and the procedure was
repeated for two more trials.

Results-
Table- .
Table heading:
Inhibition Zone Diameters of E.coli K-12 Trials at Different Dettol Concentrations

Inhibition Zones (mm)

Dettol Trial 1 Trial 2 Trial 3 Average

concentrations
(%)

100 32 32 30.7 31.57

70 30.7 23.3 25.3 26.43

25 19.3 21 20.3 20.2

0 (experimental 0 0 0 0
control)

Figure 1: The data shows the mean diameter of the inhibition zones of E.coli K-12 observed in
each trial across different Dettol concentrations.
Graph-

Legend-
X-axis- Represents Dettol concentrations (%)
Y-axis- Represents zone of inhibition (mm)
Line- Represents the average inhibition zone diameter of the E. coli K-12 Trials.
Blue Dot- Average Inhibition Zone for each Dettol Concentration.

Figure 2: A graph showing the average diameter of inhibition zones of E.coli K-12

Summary of Results-
In trials 1, 2 and 3, 100% Dettol concentration had 32 mm, 32 mm and 30.7 results. This results
in an average of 31.57 mm inhibition zone size. For 70% Dettol concentration, the inhibition
zone for trial 1 was 30.7mm, trial 2 23.4mm and trial 3 25.3 mm, which resulted in an average
size of inhibition zone being 26.43 mm. 25% Dettol concentration diameters in trials 1,2 and 3
were 19.3 mm, 21 mm, and 20.3 mm. This results in the average inhibition zone of 25% Dettol
concentration being 20.2 mm. The Experimental Control, which was 0% Dettol, showed no
results. The graph shows that as Dettol concentrations increase, the zones of inhibition
increase.

Discussion-
Interpretation of Results -
The hypothesis stated that higher concentrations of Dettol would increase the zone of inhibition
of E. coli K-12, which was proven correct. The results present that the average inhibition zone
demonstrates a clear trend: 100% Dettol concentrations have shown the most significant
inhibition zones (31.57mm on average), followed by 70% (26.43mm) and 25% (20.2mm). The
experimental Control (0% Dettol) exhibited no inhibition zones, reinstating the specificity of the
observed effects of the Dettol concentration on E. coli K-12. This solidifies the idea through the
results that the anticipated mechanism that helped with this trend was the greater concentration
of Chloroxylenol. It has been proven that it has accelerated Dettol's antibacterial properties. This
higher concentration was assumed to denature proteins more effectively, resulting in faster cell
deaths of E. coli K-12, which was also proven by the results of the average 100% (Dettol
concentration) being at 31.57. The 100% Dettol concentration, with the most Chloroxylenol
content, was hypothesised to target a more significant number of cells, making it the most
effective in decreasing the growth of E. coli K-12, as supported by the larger inhibition zones
observed in the results. Thus accurately proving the hypothesis.

Reliability-
The experiment is reliable but to an extent. This is because the experiment yields similar results
but differs every time. There are no significant outliers in the data. The results are consistent in
each trial. For 100% Dettol concentration, trials 1 and 2 are the same size of inhibition zone
(32mm). In trial three, the size of the inhibition zone deviates by being 1.3 mm smaller. For 70%
Dettol concentration, each trial gave different results; trial 1 gave 30.7mm, then 23.3 mm in trial
2, a significant gap from trial 1, and trial 3 showed 25.3 mm, similar to trial 2. This inconsistency
in the 70% Dettol concentration results is likely due to human error. 5 to 10 more repeats of this
experiment will improve the discrepancies. For 25% Dettol concentration, 19.3 mm was in trial 1,
then 21mm for trial 2 and 20.3mm for trial 3. This concentration has no significant gaps; all the
results are close. However, they are not the same. To improve this experiment's reliability, more
repetition of the experiment must be repeated. Furthermore, three agar plates were used per
trial, the outliers were dismissed, and averages were calculated. This increases the reliability of
the experiment.

Validity-
Validity is the extent to which the experiment addresses the aim. The validity of this experiment
is high. This experiment included a Control (0% Dettol Concentration). This Control helped
provide a basis for comparison by isolating and characterising any observed effects on bacterial
growth related explicitly to the different Dettol concentrations. Thus, catering is a reference point
to assess the baseline growth of E. coli K-12 without the effect of Dettol. Using the Lawn Plating
technique addresses the aim of investigating the growth of bacteria by the effect of different
Dettol concentrations. This technique minimises bias in bacterial distribution and ensures that
the growth is assessed uniformly.

Issues and Solutions-

The first issue encountered was prolonged exposure to the external environment while
performing the Lawn plating technique. This causes a potential for contamination. This
contamination could introduce variability in bacterial growth and inhibition. A solution is to open
the lid slightly. Even Though the standard practice is 45 degrees in angle, it is too inconvenient
to spread the E.coli K-12 broth with precision. Thus, experimenting in a non-ventilated and
closed space will improve the accuracy of the experiment significantly. Another issue
encountered was the paper discs sliding off to the edge of the agar plate during the incubation
process, which comprises the inhibition zone's accurate size and contaminates the other
quadrats. A solution for this would be repeating the experiment 10 more times because, ideally,
the results should avoid ambiguity.

Future Directions-
One future direction will be the range of various concentrations being assessed. For example, in
this experiment, only three concentrations were experimented on; therefore, future research
should have a minor difference between the ranges of the Dettol concentrations. Another future
direction is testing each Dettol concentration in different agar plates. This will allow more
accuracy in results. Another direction will be comparing the results with similar studies and
refining the Lawn plating technique's application not to comprise potential exposure to air for
prolonged periods.

Conclusion :
In conclusion, this experiment has successfully investigated the effect of Dettol concentrations
(100%,70% and 25%) on the growth of E.coli K-12 with insightful results. The main point that
was hypothesised was that higher concentrations of Dettol would decrease E. coli K-12 growth,
with 100% Dettol concentration being the most effective, followed by 70% Dettol concentration
and 25% being the least effective. The results reflected this claim by presenting a clear trend in
the average inhibition zones, confirming the hypothesis. Clearly stated, 100% Dettol
concentrations showed the most significant inhibition zones ( average 31.37mm), then 70%
(average 26.43 mm), and lastly, 25% (20.2mm). The Control's results did not have a zone of
inhibition, supporting the hypothesis by reinforcing the specificity of the observed effects
attributed to the Dettol concentrations. This trend from results supports the hypothesis by
aligning with the claim that because of the presence of more significant concentrations of
Chloroxylenol in higher concentrations of Dettol, it has enhanced its antibacterial properties and
denaturation of proteins, which leads to cell deaths of E.coli K-12. The experiment has efficiently
displayed the correlation between Dettol Concentrations and the inhibition of E. coli growth.
References:

Balloux, F & van Dorp, L 2017, ‘Q&A: What are pathogens, and what have they done to and for
us?’, BMC Biology, vol. 15, no. 1.

Browning, DF, Hobman, JL & Busby, SJW 2023, ‘Laboratory Strains of Escherichia Coli K-12:
Things Are Seldom What They Seem’, MICROBIOLOGY SOCIETY , vol. 9, no. 2.

Damiano, P, Silago, V, Nyawale, HA, Mushi, MF, Mirambo, MM, Kimaro, EE & Mshana, SE
2023, ‘Efficacy of disinfectants on control and clinical bacteria strains at a zonal referral hospital
in Mwanza, Tanzania: a cross sectional hospital-based study’, Scientific Reports, vol. 13, no. 1,
p. 17998, viewed 30 November 2023, <https://www.nature.com/articles/s41598-023-45228-7>.

Felson, S 2017, What is E. Coli?, WebMD, WebMD.

Hamdallah, I, Torok, N, Bischof, KM, Majdalani, N, Chadalavada, S, Mdluli, N, Creamer, KE,

Clark, M, Holdener, C, Basting, PJ, Gottesman, S & Slonczewski, JL 2018, ‘Experimental
Evolution of Escherichia coli K-12 at High pH and with RpoS Induction’, in RE Parales (ed.),
Applied and Environmental Microbiology, vol. 84, no. 15.

Hugo, WB 1978, ‘Phenols: a review of their history and development as antimicrobial agents’,
Microbios, vol. 23, no. 92, pp. 83–85.

Kuhnert, P, Nicolet, J & Frey, J 1995, ‘Rapid and accurate identification of Escherichia coli K-12
strains.’, Applied and environmental microbiology, vol. 61, no. 11, pp. 4135–4139.

Lamorte, WW 2016, Bacteria as Pathogens, sphweb.bumc.bu.edu.

Lim, JY, Yoon, J & Hovde, CJ 2010, ‘A Brief Overview of Escherichia coli O157:H7 and its
Plasmid O157’, Journal of microbiology and biotechnology, vol. 20, no. 1, pp. 5–14.

Prasad, SMV, Venkatachalam, N, Ramesh, N, Turuvekere, P, Ramees, M & Kumar, C 2020,

‘Evaluation of efficacy of four disinfectants on striated and non-striated orthodontic instruments:
An in vitro study’, Journal of Pharmacy And Bioallied Sciences, vol. 12, no. 5, p. 254.

PubChem 2019, Chloroxylenol, Nih.gov, PubChem.

Santos-Longhurst, A 2019, What is a Pathogen? 4 Types and How They Spread Disease,
Healthline, Healthline.

Sharma, AK, Dhasmana, N, Dubey, N, Kumar, N, Gangwal, A, Gupta, M & Singh, Y 2016,
‘Bacterial Virulence Factors: Secreted for Survival’, Indian Journal of Microbiology, vol. 57, no.
1, pp. 1–10.

Tuttle, AR, Trahan, ND & Son, MS 2021, ‘Growth and Maintenance of Escherichia coli
Laboratory Strains’, Current Protocols, vol. 1, no. 1.