Comparison between effectiveness of antibacterial and normal soap against

Bacillus megaterium

Introduction

The purpose of this investigation is to compare the effectiveness of antibacterial and

normal soap against Bacillus megaterium. Antibacterial soaps contain anti-microbial agents
that are not found in normal hand-washing soap. Today, more than 75% of liquid soaps in
the market contain some kind of anti-microbial agent. However, the FDA has said that there
is “no evidence” that antibacterial soaps can keep people in better health than regular soap.
Instead, the agency now even requires manufacturers to prove the chemicals are safe for
long-term daily use.1 Based on this, I hereby questioned the effectiveness of antibacterial
soap. This became my motivation to investigate with the question: “How does antibacterial
soap affect the growth of Bacillus megaterium colonies in comparison to normal soap?” B.
megaterium is a Gram-positive, spore producing bacteria. It is found in the soil. It has a rod
shape and is one of the largest eubacteria which exists.2 The antibacterial soap which will be
used is Safeguard Antibacterial White with Aloe, which contains triclocarban,3 while normal
soap is Coconut soap: savon noix de coco from Body Shop.4

Background

Such agents found in antibacterial soap are as triclosan and/or triclocarban. Both
triclocarban and triclosan are chemically similar compounds. Triclocarban (which Safeguard
contains) is not active against Gram-negative bacteria (Heinze and Yackovish, 1988), but is
predominantly active against Gram-positive bacteria (such as B. megaterium) (Schebb,
Inceoglu, Chang Ahn, Morisseau, Gee & Hammock, 2012.). The Coconut soap does not
contain any triclocarban and triclosan. Another study indicates that Gram-positive bacteria
were killed at lower concentration of soaps than Gram negative bacteria. It also proves that
antibacterial soaps kill bacteria at a specific concentration; they also have bacteristatic
activity and can inhibit the growth of bacteria.5

There has been problems with antibacterial soap in the past. A study states that lack
of an additional health benefit associated with the use of triclosan-containing consumer

1 Hospital, Blank Children's. "Antibacterial Soap vs. Regular Soap: Which One Is Better?"
Www.unitypoint.org/blankchildrens/. N.p., 08 July 2014. Web. 7 Oct. 2016.
2 "Bacillus megaterium." Bacillus megaterium. N.p., n.d. Web. 8 Oct. 2016.
3"Safeguard Antibacterial White with Aloe (Procter & Gamble Manufacturing Company)

TRICLOCARBAN 1.356g in 113g soap." Drugs.com. Drugs.com, n.d. Web. 10 Oct. 2016.
4 "Coconut Soap." The Body Shop. N.p., n.d. Web. 10 Oct. 2016.

5 Riaz, Sabia, Adeel Ahmad, and Shahida Hasnain. "Antibacterial activity of soaps against daily

encountered bacteria." African Journal of Biotechnology 8 (2009): n. pag. Web. 10 Oct. 2016.

1
soaps over regular soap, coupled with laboratory data demonstrating a potential risk of
selecting for drug resistance, warrants further evaluation by governmental regulators
regarding antibacterial product claims and advertising, while encouraging further studies.6

Variables

i. Independent variable : type of soap

The purpose of the investigation is to compare the effects of both types of soap
(antibacterial and normal) on B. megaterium growth.
ii. Dependent variable : size of zone of inhibition
The size of zone of inhibition, created by soap-soaked paper discs, shows the
tolerance of B. megaterium towards the type of soap, hence indicating the size of the
effect on B. megaterium growth.
iii. Control variables :

Variable Method of Control Impact on Experiment

Concentration of diluted soap 10 grams of soap and 150 Different concentrations of

millilitres of water were used diluted soap would have
for both trials. different levels of bacterial
inhibition.

Time period of B. B. megaterium was allowed If B. megaterium is not

megaterium initial growth
to initially grow for 4 days
before exposure to soap-
soaked discs.
allowed to initially grow prior
to exposure to soap-soaked
discs, there is a chance that
any effect recorded will be
the result of growth in time
instead of the soap.

Contamination of agar The surface of the Any microorganism which

solution workstation was cleaned enters the agar solution
using paper towel and along with B. megaterium
denatured alcohol. The wire- may change the resulting

6
Aiello, Allison E., Elaine L. Larson, and Stuart B. Levy. "Consumer Antibacterial Soaps: Effective or
Just Risky?" Clinical Infectious Diseases 45.2 (2007): n. pag. Web. 10 Oct. 2016.
<https://academic.oup.com/cid/article/45/Supplement_2/S137/285530/Consumer-Antibacterial-Soaps-
Effective-or-Just>

2
 inoculating loop was passed zone of inhibition, by either
through the flame of a competing, supporting,
Bunsen burner to sterilze it reacting, etc. with the B.
before usage. The Petri dish megaterium itself.
was closed with a lid. Oxygen
was restricted by taping the
dish with the lid (with oxygen,
microorganisms are able to
undergo aerobic respiration).

External conditions Temperature and humidity According to research, the

were kept constant by air-
conditioning.
temperature might affect the
resistance of the bacteria.
Plates stored in different
environments with different
temperatures would show
different zones of inhibition
unrelated to type of soap.

Materials

 Agar solution  Normal soap (Coconut soap:

 B. megaterium cultured source savon noix de coco)
 Antibacterial soap (Safeguard  Paper sheets and paper towels
Antibacterial White with Aloe)  Tape
 Water
Instruments

 Petri dishes with  Inoculation loop  Paper puncher

lids  Microscope  Denatured alcohol

Method

This investigation revolves around the idea of the Kirby-Bauer antibiotic testing,
where antibiotic-containing disks are used to test whether particular bacteria are susceptible
to specific antibiotics. In this test, a pure culture of bacteria is isolated. Then, a known
quantity of bacteria are grown overnight on agar plates in the presence of a thin wafer that
contains a known amount of a relevant antibiotic. If the bacteria are susceptible to the

3
particular antibiotic from a wafer, an area of clear media where bacteria are not able to grow
surrounds the wafer, which is known as the zone of inhibition. A larger zone of inhibition
around an antibiotic-containing disk indicates that the bacteria are more sensitive to the
antibiotic in the disk.7

1. The agar solutions were prepared. Then, each type of agar was poured into 8 petri
dishes (85 ± 1 mm in diameter), providing 8 dishes for each agar composition.
2. 4 dishes were labelled “Antibacterial” and the other 4 were labelled “Normal”. The
dishes had their lids on until the B. megaterium was placed.
3. An inoculation loop was used to transfer bacteria samples from the cultured source to
the agar. The inoculation loop was then dipped into B. megaterium in order for the
bacteria to attach to the loop. The loop was then dragged across the agar plate.
4. The plates were then sealed shut using tape.
5. Steps 3 and 4 were repeated for every plate of agar.
6. The plates were all stored in a tray.
7. After four days, the growth of the bacteria colony was viewed using a microscope.
8. A paper puncher was used to make paper discs out of paper sheets. Each of the
paper discs had a diameter of 6 ± 0.5 mm.
9. 50 grams of the antibacterial bar soap and the normal bar soap were placed into two
separate beakers with 150mL of water and was diluted until completely liquid.
10. 4 discs were dipped into the diluted antibacterial soap solution and 4 discs were
dipped into the diluted normal soap solution.
11. The discs were then placed into the respected agar plates using a pincer.
12. After 4 days, the response of B. megaterium towards the two different types of soap
was recorded by measuring the width of the zone of inhibition (images were taken
using microscope).

The initial amount of water supposed to be used was 50 millilitres but the antibacterial
soap did not dilute properly so more water was added in order secure a complete dilution
– 150 millilitres

7
Boundless. "Kirby-Bauer Disk Susceptibility Test - Boundless Open Textbook." Boundless.
Boundless, 13 Aug. 2016. Web. 22 Nov. 2016.

4
Safety and Ethical Concern

The Bacillus genus includes both non-parasitic and parasitic pathogenic species,8 and B.
megaterium is considered to be non-pathogenic9. Despite this consideration, the experiment
was still conducted using safety equipment such as gloves and lab coats to avoid direct skin
contact with the bacteria. Sterile masks were used for the most part to ensure that the
bacteria will not enter the oral cavity. The surface of the workstation was cleaned using
denatured alcohol and paper towel.The agar solution was disposed as chemical waste, so
not to disrupt the environment in regards to ecosystems of microorganic level.

Data Processing and Analysis

1) Antibacterial Soap - Safeguard

Below are the photos which have been taken of the bacterial colony before and after
exposure to soap-solution. Photo 1 and Photo 2 display the bacterial colony before and after
the exposure to soap solution. Both are collages of 4 photos taken of different sides of the
gram-positive bacteria colony of B. megaterium. It can be seen as the lighter part of each
photo. The dark circular part is shows the soap-soaked discs, where the colony is inhibited
from growing.

Photo 1: Rims of discs before exposure

8 "Bacillus megaterium QM B1551 - eol.org." N.p., n.d. Web. 24 Nov. 2017.

9"Bacillus megaterium." Bacillus megaterium - encyclopedia article - Citizendium. N.p., n.d. Web. 26
Nov. 2016.

5
Photo 2: Rims of discs after exposure

Photo 2 clearly shows the increasing dark area and therefore a zone of inhibition in which
the bacteria colony can no longer grow.

The zone of inhibition was then measured by aligning the zone with the measurement Fig 1.

Fig. 1:

Measuring tool for zone of

inhibition

Table 1 shows the size of these zones of inhibitions in each of the trials. While the size of
paper discs is constant at 6 mm, the size of these zones differ.

Table 1: Size of zones of inhibition of B. megaterium after exposure to antibacterial soap

Plate 1 2 3 4

Zone of Inhibition (μm) 5.5 5.5 7.5 6.0

Size of paper discs (mm) 6 6 6 6

6
Mean of zone of inhibition

A mean of the zone of inhibition is performed using Equation 1.

𝑠𝑖𝑧𝑒 𝑜𝑓 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 𝑇𝑟𝑖𝑎𝑙 1 + 𝑠𝑖𝑧𝑒 𝑜𝑓 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 𝑇𝑟𝑖𝑎𝑙 2 …

𝑀𝑒𝑎𝑛 =
𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑇𝑟𝑖𝑎𝑙𝑠

Equation 1: finding the mean of zone of inhibition

Using equation 1, the mean of zone of inhibition with antibacterial soap Safeguard is:

5.5μm + 5.5μm + 7.5μm + 6μm

𝑚𝑒𝑎𝑛 𝑜𝑓 𝑧𝑜𝑛𝑒 𝑜𝑓 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = = 6.125μm
4

The mean is 6.125 μm.

However, I recognize that a better display of such value, for comparison with the effects of
other soap is by using percentages. Hence, I calculated the percentage of zone of inhibition
of each trial over the size of the paper discs used, using Equation 2. I performed the
calculations on all trials. Calculations performed on Trial 1 are used as sample. Results are
kept to 3 significant figures.

Percentage of Zone of Inhibition over Paper Disc

𝑠𝑖𝑧𝑒 𝑜𝑓 𝑧𝑜𝑛𝑒 𝑜𝑓 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛

% 𝑧𝑜𝑛𝑒 𝑜𝑓 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = × 100
𝑠𝑖𝑧𝑒 𝑜𝑓 𝑝𝑎𝑝𝑒𝑟 𝑑𝑖𝑠𝑐

Equation 2: finding the percentage of zone of inhibition in relation to size of paper disc

0.0055 𝑚𝑚 (= 5.5 μm)

% 𝑧𝑜𝑛𝑒 𝑜𝑓 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 𝑜𝑣𝑒𝑟 𝑝𝑎𝑝𝑒𝑟 𝑑𝑖𝑠𝑐 = × 100
6 𝑚𝑚

% 𝑧𝑜𝑛𝑒 𝑜𝑓 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 𝑜𝑣𝑒𝑟 𝑝𝑎𝑝𝑒𝑟 𝑑𝑖𝑠𝑐 = 0.0917%

Mean Percentage of Zone of Inhibition over Paper Disc

I then find the mean percentage of zone of inhibition over the size of paper discs used, for all
trials of antibacterial soap Safeguard (equation 3).

%𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 𝑇𝑟𝑖𝑎𝑙 1 + % 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 𝑇𝑟𝑖𝑎𝑙 2 …

𝑀𝑒𝑎𝑛% =
𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑇𝑟𝑖𝑎𝑙𝑠

Equation 3: determining the mean percentage of zone of inhibition for antibacterial soap

0.0917% + 0.0917% + 0.125% + 0.100%

𝑀𝑒𝑎𝑛% = = 0.102%
4

7
The uncertainty of this percenage is then calculated using equation 4.

Percentage Covered Uncertainty

𝑢𝑛𝑐𝑒𝑟𝑡𝑎𝑖𝑛𝑡𝑦 𝑜𝑓 𝑝𝑎𝑝𝑒𝑟 𝑑𝑖𝑠𝑐 𝑠𝑖𝑧𝑒

𝑈𝑛𝑐𝑒𝑟𝑡𝑎𝑖𝑛𝑡𝑦 𝑜𝑓 %𝑧𝑜𝑛𝑒 𝑜𝑓 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 𝑡𝑜 𝑝𝑎𝑝𝑒𝑟 𝑑𝑖𝑠𝑐 = × 100
𝑠𝑖𝑧𝑒 𝑜𝑓 𝑝𝑎𝑝𝑒𝑟 𝑑𝑖𝑠𝑐

Equation 4: determining uncertainty of the percentage of zone of inhibition over disc size

0.5 μm
𝑈𝑛𝑐𝑒𝑟𝑡𝑎𝑖𝑛𝑡𝑦 𝑜𝑓 %𝑧𝑜𝑛𝑒 𝑜𝑓 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 𝑡𝑜 𝑝𝑎𝑝𝑒𝑟 𝑑𝑖𝑠𝑐 = × 100 = ±0,003%
6 𝑚𝑚

Standard Deviation

The data was input into Microsoft Excel (Fig. 2).

Fig.2 :

Data input into Microsoft Excel

Using Microsoft Excel, the standard deviation was calculated from all the percentages
calculated (equation 5; specific formula for Microsoft Excel based on data visible in Fig. 2)

𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑑𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛: STDEV(A2:A5)

Based on this, the standard deviation ]narrowed down to 3 significant figures: 0.0158%.

Table 2: Results regarding zone of inhibition induced by antibacterial soap on B. megaterium

Trial 1 2 3 4

Mean size of zone of inhibition 6.125 μm

Percentage of zone of inhibition over 0,0917 0,0817 0,125 0,1

disc size(±0,003%)

Mean percentage (±0,003%) 0,102%

Standard Deviation 0,0158%

2) Normal Soap – Coconut Soap: Savon noix de coco – Body Shop

8
The same routine is repeated with the results from normal soap Coconut soap... –Body
Shop. The photos from this one are displayed in Photo 3 and Photo 4.

Photo 3: Rims of discs before cross- Photo 4: Rims of discs after cross-
resistance (Normal Soap) resistance (Normal Soap)

The size of zones of inhibition are placed into Table 3.

Table 3: Size of zones of inhibition of B. megaterium after exposure to normal soap

Plate 1 2 3 4

Zone of Inhibition (μm) 0.5 1 0.5 1

Size of paper discs (mm) 6 6 6 6

These measurements then undergo the same process as with the antibacterial trials, using
the same equations and Excel calculations. Hence, after undergoing this process, the final
results are shown in Table 4.

Table 4: Results regarding zone of inhibition induced by normal soap on B. megaterium

Trial 1 2 3 4

Mean size of zone of inhibition 0.75 μm

Percentage of zone of inhibition over 0,0083% 0,0167% 0,0083% 0,0167%

9
disc size (±0.003%)

Mean percentage (±0.003%) 0,0125%

Standard Deviation 0,00485%

The results from table 2 and 4 regarding percentages are represented as a graph (Fig. 2) for
better representation of data. The standard deviations are shown as error bars.

%zone of inhibition/paper disc size in

antibacterial and normal soap
0.16
% zone of inhibition/disc size (%)

0.14
0.12
0.1
0.08
Antibacterial soap
0.06
Normal soap
0.04
0.02
0
1 2 3 4
Trial

Fig. 2: %zone of inhibition/paper disc size in antibacterial and normal soap

In all trials, antibacterial soap shows a significantly higher percentage in zone of inhibition
over paper disc size than normal soap. The overall uncertainty of the data for both
antibacterial and normal soap was ±0.003%. The standard deviations for both antibacterial
(0.0158%) and normal soap (0.00485%) differ significantly.

Statistical Tests

In order to make sure that the results were accurate, GraphPad was used to calculate the P-
value. An unpaired t-test was undertaken with resulting P-value beingless than 0.0001 (p <
0.0001). By conventional criteria, the results state that this difference between antibacterial

10
soap and normal soap is considered to be extremely statistically significant. Hence, the
result shows that the results do show a relationship that is extremely not up to chance.

Conclusion

Based on the results, which are shown to be extremely statistically significant, there
is a relationship between the type of soap that B. megaterium colony is exposed to, and its
zone of inhibition. Based on the results, B. megaterium colony exposed antibacterial soap
shows a much larger zone of inhibition than that which is exposed to normal soap. This then
asks the question of why so. The main difference between both soaps is that Safeguard, the
antibacterial soap, contains triclocarban, a antimicrobial agent that antibacterial soaps
commonly have. It is then concluded, in theoretical manner, that the antimicrobial agent
inside Safeguard induces a higher inhibition of bacterial life, and hence a wider zone of
inhibition in B. megaterium colony. My investigation is inspired through questioning the
effectiveness of antibacterial soap. Based on my results, it is concluded that antibacterial
soap is effective in inhibiting bacterial growth.

Evaluation

i. Comparison of Conclusion to Related Literature

Studies mentioned in the background have suggested several things: triclocarban is

predominantly active against Gram-positive bacteria and that there is lack of additional
health benefit in using antimicrobial-containing soaps over regular soap. Based on my
conclusion, it is suggested that triclocarban is indeed predominantly active against Gram-
positive bacteria, as when triclocarban-containing Safeguard is added rather than Coconut
soap, a higher zone of inhibition in the bacterial colony can be seen. However, my
investigation does suggest that there is an additional health benefit in using such soaps over
regular soap, as it inhibits the growth of bacteria. However, it is worth noting that the latter-
mentioned study does specify on triclosan-containing soaps, of which Safeguard does not
fall into. In addition, in the former-mentioned study focuses in comparing the activity of
triclocarban against Gram-negative and Gram-positive bacteria. My investigation only
explores its effects on Gram-positive bacteria.

ii. Validity of Conclusion

The conclusion is valid as the investigation is conducted in a precise and accurate

manner. As mentioned in the control variables, external environmental conditions and
potential contamination are each regulated and restricted respectively. However, there can
be errors arising from this investigation. The external environmental conditions are only

11
regulated in qualitative manner; hence, it is possible for these conditions to fluctuate without
any quantitative notice. These are temperature, pH and humidity. The contents of Coconut
soap are not specified in this investigation. It is possible that this soap contains components
which promote bacterial growth or compete with those which inhibit bacterial growth.

iv. Suggestions for Improvement and Further Studies

It will be interesting to see further studies regarding the effect of antibacterial and
normal soap on bacteria. This investigation did not investigate another component of
antibacterial soaps, which is triclosan, which is more the source of the debate. Hence, an
idea for a further study is to explore the effect of triclosan-containing anti-bacterial soap on
the growth of bacteria. Another thing this investigation did not investigate is the effect of such
antibacterial soaps on Gram-negative bacteria. Using Gram-negative bacteria on a further
study would provide interesting insight. Another thing this investigation did not investigate is
the effect of such soaps on pathogenic bacteria, which is more of the bacteria one would use
soap against. However, such an experiment would be risky in a school environment and
should be conducted by professionals.

Bibliography

Drugeon, H. B., B. Rouveix, and A. Michaud Merard. "Triclocarban antibacterial activity on

resistant staphylococci, streptococci, and enterococci." Medecine et Maladies Infectieuses
(n.d.): n. pag. Web. 5 Feb. 2017.

Aiello, Allison E., Elaine L. Larson, and Stuart B. Levy. "Consumer Antibacterial Soaps:
Effective or Just Risky?" Clinical Infectious Diseases 45.2 (2007): n. pag. Web. 10 Oct.
2016. <https://academic.oup.com/cid/article/45/Supplement_2/S137/285530/Consumer-
Antibacterial-Soaps-Effective-or-Just>.

"Bacillus megaterium." Bacillus megaterium. N.p., n.d. Web. 8 Oct. 2016.

"Bacillus megaterium." Bacillus megaterium - encyclopedia article - Citizendium. N.p., n.d.
Web. 26 Nov. 2016.

"Bacillus megaterium QM B1551 - eol.org." N.p., n.d. Web. 24 Nov. 2017.

"Bacillus species." Bacillus species - Infectious Disease and Antimicrobial Agents. N.p., n.d.
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"Coconut Soap." The Body Shop. N.p., n.d. Web. 10 Oct. 2016.

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Use Plain Soap and Water." U S Food and Drug Administration Home Page. Office of the
Commissioner, n.d. Web. 15 Jan. 2017.

12
Hospital, Blank Children's. "Antibacterial Soap vs. Regular Soap: Which One Is Better?"
Www.unitypoint.org/blankchildrens/. N.p., 08 July 2014. Web. 7 Oct. 2016.

Boundless. "Kirby-Bauer Disk Susceptibility Test - Boundless Open Textbook." Boundless.

Boundless, 13 Aug. 2016. Web. 22 Nov. 2016.

March 15, 2016 Nicole Greenfield. "The Dirt on Antibacterial Soaps." NRDC. N.p., 15 Dec.
2016. Web. 2 Feb. 2017.

Riaz, Sabia, Adeel Ahmad, and Shahida Hasnain. "Antibacterial activity of soaps against
daily encountered bacteria." African Journal of Biotechnology 8 (2009): n. pag. Web. 10 Oct.
2016.

Rose, Joan B., and Charles N. Haas. "A risk assessment framework for the evaluation of
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Infection Control (1999): n. pag. Web. 2 Feb. 2017.

"Safeguard Antibacterial White with Aloe (Procter & Gamble Manufacturing Company)
TRICLOCARBAN 1.356g in 113g soap." Drugs.com. Drugs.com, n.d. Web. 10 Oct. 2016.

13