Comparative studies on the effect of locally made black soap and conventional medicated soaps on isolated human skin microflora

Abstract

The comparative study on the effect of locally made black soap and conventional medicated soaps Dettol, Dudu osun, Zee and Tura were investigated on isolated human skin microflora using disk diffusion method. The organisms isolated as skin microbiota were Staphylococcus aureus (30%), Staphylococcus epidermidis (26%), Corynebacterium spp (22%), Bacillus spp (16%) and Echerichia coli (6%). The results obtained revealed that the traditional black soap exhibited a high level of antimicrobial activities resulting in greater zones of inhibition (10.65) against other medicated soaps such as Dettol, Dudu Osun, Zee, and Tura that show the following zones of inhibition 7.58, 9.78, 10.57 and 9.29 respectively. Significant differences (p< 0.05) were also observed on the concentrations used. African black soap through this study can thus, be recommended for use since it has the potentials of treating skin diseases.

Full text

Journal of Microbiology and Biotechnology Research
Scholars Research Library
J. Microbiol. Biotech. Res., 2012, 2 (4):533-537
(http://scholarsresearchlibrary.com/archive.html)
ISSN : 2231 –3168
CODEN (USA) : JMBRB4
533
Available online at www.scholarsresearchlibrary.com
Comparative studies on the effect of locally made black soap and conventional
medicated soaps on isolated human skin microflora
1
Ikpoh, I.S.,
1
Lennox, J.A.,
1
Agbo, B. E.,
3
Udoekong, N. S.,
2
Ekpo I.A and
1
Iyam, S.O.
1
Department of Microbiology, University of Calabar, Calabar, Cross River State, Nigeria
2
Department of Genetics and Biotechnology, University of Calabar, Calabar, Cross River State, Nigeria
3
Department of Science and Technology, Akwa Ibom State Polytechnic, Ikot Osurua, Ikot Ekpene, Akwa
Ibom State, Nigeria
_____________________________________________________________________________________________
ABSTRACT
The comparative study on the effect of locally made black soap and conventional medicated soaps Dettol, Dudu
osun, Zee and Tura were investigated on isolated human skin microflora using disk diffusion method. The organisms
isolated as skin microbiota were Staphylococcus aureus (30%), Staphylococcus epidermidis (26%),
Corynebacterium spp (22%), Bacillus spp (16%) and Echerichia coli (6%). The results obtained revealed that the
traditional black soap exhibited a high level of antimicrobial activities resulting in greater zones of inhibition
(10.65) against other medicated soaps such as Dettol, Dudu Osun, Zee, and Tura that show the following zones of
inhibition 7.58, 9.78, 10.57 and 9.29 respectively. Significant differences (p< 0.05) were also observed on the
concentrations used. African black soap through this study can thus, be recommended for use since it has the
potentials of treating skin diseases.
Keywords: Black soap, Microflora, Human skin, medicated soap
_____________________________________________________________________________________________
INTRODUCTION
The term normal microbial flora or microbiota denotes the population of microorganism that inhibits the skin and
mucous membranes of healthy normal persons. Research has shown that this normal flora now referred to as normal
microbiota provide a first line of defense against microbial pathogens, assist in digestion, play a role in toxin-
degradation, and contribute to maturation of the immune system. Shifts in the normal microbiota or stimulations of
inflammation by these commensals may cause disease as inflammatory bowel disease [1]. The density and
composition of the normal flora of skin vary with anatomical location. The makeup of the normal flora depends,
upon various factors including genetic, sex, age, stress, nutrition and diet of the individuals. Human skin is subject to
degenerative changes due to daily exposure to environment and the impact of microorganisms. Several studies have
been carried out to determine the normal flora of the skin. [2] presented a comprehensive review of the normal flora
of the foot. He stated that the flora of the foot is similar to that found in the other skin site. [3] believe that some
differences exist between foot flora and other skin site. Developmental changes in humans such as wearing, the
eruption of the teeth and the inset and cessation of ovarian function invariably affect the normal flora in the
intestinal tract, the oral cavity, and the vagina respectively. However, within the limits to these fluctuations, the
bacteria flora of human is sufficiently constant to a given general descriptions of the situation. The efficacy of
antimicrobial soaps in reducing the number of organisms can be tested in the axilla. If different soaps are used on
each axilla in the same volunteer, the transfer of antimicrobial agent from one site to another may cause difficulties

Ikpoh, I.S et al J. Microbiol. Biotech. Res., 2012, 2 (4):533-537
______________________________________________________________________________
534
Available online at www.scholarsresearchlibrary.com
in interpretation [4]. Black soap can be used on the hair, face and body. Black soap has anti-aging properties and can
reduce fine lines and wrinkles for youthful, smooth skin. Dark spots and blemishes are evened out and the natural
ingredients effectively cleanse and deodorize. Black soap does not contain specific antimicrobial ingredient, many
people prefer this soap because it does not cause resistant bacteria growth. Black soap is also a natural source of
vitamins A, E and irons which helps to strengthen the skin and hair. Medicated soaps to a large extend remove dirts
and disrupt cytoplasmic membrane to kill microorganisms [1]. It also works against enveloped virus like human
immunodeficiency virus (HIV). Several antimicrobial substances are found in medicated soaps and they have
various mode of action on various skin microflora. This study is aimed at identifying microorganisms commonly
present on the human skin and also to compare the effectiveness of some medicated soaps and locally prepared
black soaps on the isolates obtained from the skin.
MATERIALS AND METHODS
Collection of swabs and Isolation of microorganisms
Sterile swab stick were purchase from pharmaceutical stores in Calabar. Samples were taken from biological science
students. One swab stick was used for each student to collect samples. The sterile swab sticks were damped with
sterile peptone water before the samples were collected from the hand, armpit, face and legs. The area of skin to be
swab was first swabbed with methylated spirit so as to remove some of the transcient microflora. The samples
collected with swab sticks were then used to inoculate already poured and set petri-dishes. The culture plates were
then incubated in an inverted position to prevent condensed moisture from dripping into the media or bacteria
colony. The plates were incubated at a 37
0
C for 24 hours. Subculturing of isolates was done on nutrient agar and
blood agar media. Bacteria isolates were maintained on agar slant at 37
0
C for 48 hours. Biochemical
characterization and identification of the organisms was also carried out using the Bergey’s Manuel of
Determinative Bacteriology, 9
th
edition (1994).
Collection of medicated soap and treatment
Medicated soaps were obtained from Watt market Calabar. The soaps were treated in the laboratory in the following
ways. A sterile blade was used to scrap on gram (1g) of each soap. Each of the scraped soaps dilution was then
dissolved in 10mls of sterile distilled water to give a stock solution of 10
-1
. These stock solutions were then stored in
a refrigerator in well-sealed containers to avoid further dilution.
Preparation and impregnation with soap
Disks of diameter 6.5mm were bored in the laboratory. The disks were then wrapped in foil paper and sterilized in a
hot air oven at 100
0
C for 1hour. The disks were then soaked in the different soap solutions for a period of 1 hour to
ensure that the disks were fully saturated. The disks were aseptically transferred directly into the sensitivity plates. If
not to be used immediately, the disks were aseptically removed from soap solution and allowed to dry in a drying
oven at a 25
0
C. They were then packed into sterile bottles, corked and stored in the refrigerator for use from time to
time for susceptibility test. The impregnated disks were applied immediately to the surface using sterile fill contact
with the inoculated medium and to moisten the disk. The plates were then incubated at 37
0
C for 18-24 hours
inverted position.
Minimal inhibitory concentration and minimal bactericidal concentration test
The dilution susceptibility test was used to determine the MIC and MBC values
Procedures:
A series of Mueller-Hinton broth tubes containing varying concentrations of the various soap samples in the range of
500mg/ml to 62.5mg/ml was prepared and incubated with a standared density of the test organisms. The lowest
concentration of the soap sample resulting in no growth after 18-24 hours of incubation is the MIC as illustrated in
figure 4.5-4.9. The MBC can be ascertained if the tubes showing no growth are subcultureed into a fresh medium
lacking the varying concentrations of the soap sample. The lowest soap sample concentration from which the
microorganisms do not grow when transferred to a fresh medium is the MBC.
Statistcal Analysis
Data collected were subjected to a 2 factor factorial CRD experiment and means separated using LSD test.

Ikpoh, I.S et al J. Microbiol. Biotech. Res., 2012, 2 (4):533-537
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RESULTS AND DISCUSSION
The result on the zones of inhibition using the organisms shows that there were no significant differences (p> 0.05)
on the various microorganisms used for the study. Staphylococcus epidermidis have more zones of inhibition (10.2)
than Staphylococcus aureus (9.9), followed by Bacillus spp (9.83), Corynebacterium spp (9.05) and Escherichia coli
(8.87) (Fig.1). Significant differences (p< 0.05) in the zones of inhibition were observed in Dudu (9.78), Tura (9.27),
and Dettol (7.58) but no significant difference (p>0.05) exist between African black soap (10.65) and Zee (10.57)
(Fig.2). It was also observed that significant differences (p<0.05) exist among the different concentrations used for
the study with 500mg/ml (10.73) having more zones of inhibitions than other concentrations 250mg/ml (9.81),
125mg/ml (9.04) and 62.5mg/ml (8.69) (Fig.3). The result of susceptibility test was carried out on the following
organisms Corynebacterium spp, E.coli, Bacillus spp, Staphylococcus aureus, and Staphylococcus epidermidis. The
organisms showed different levels of sensitivity to the different soap samples with traditional African soap being
the most effective, with zones of inhibition (10.65) followed by Zee (10.57), Dudu osun (9.78), Tura (9.27) and
Dettol (7.58) being the least (Table 1).
RESULTS AND DISCUSSION
Table 1. MIC and MBC of some selected medicated soaps in Calabar on human skin microflora
Microorganisms
BLACKSOAP
DETTOL
DUDU OSUN
ZEE
TURA
MIC
(mg/ml)
MBC
(mg/ml)
MIC
(mg/ml)
MBC
(mg/mg)
MIC
(mg/mg)
MBC
(mg/ml)
MIC
(mg/ml)
MBC
(mg/ml)
MIC
(mg/ml)
MBC
(mg/ml)
Corynebacterium spp
NA
NA
≤125
≤500
≤62.5
≤125
NA
≤125
≤125
≤500
Escherichia coli ≤62.5 ≤125 ≤500 ≤500 ≤62.5 ≤125 ≤62.5 ≤125 ≤125 ≤500
Bacillus spp NA ≤500 ≤125 ≤500 NA ≤500 NA ≤500 NA ≤500
Staphylococcus aureus NA ≤500 ≤250 ≤500 ≤62.5 ≤250 ≤62.5 ≤125 ≤125 ≤500
Staphylococcus epidermidis ≤62.5 ≤125 ≤125 ≤500 NA ≤250 ≤62.5 ≤125 ≤125 ≤500
MIC Minimal inhibitory concentration
MBC Minimal bactericidal concentration
NA Not active
The normal human skin habours microorganisms that can be grouped into transient and resident flora [1].
Microorganisms differ in their nutritional requirements and level of susceptibility to antimicrobial agents. The effect
of soaps on the skin microflora has not been widely studied. There is no soap that contains the required ingredient
that suits all individual skin. Here skin is the main site of exposure to soap come into focus. The human skin is the
main site of exposure to soaps, therefore reactions exhibited by individual skin differs from soap to soap. The
different concentration tested on the different organisms isolated from human skin shows that the soaps contained
antimicrobial activities which inhibited the growth of the organisms, Corynebacterium spp, E.coli, Bacillus spp,
Staphylococcus aureus and Staphylococcus epidermidis to different degrees 9.05, 8.87, 9.83, 9.9 and 10.2
respectively.
The inhibition of the growth pattern of the isolates indicates the varying abilities of the organism to resist the
antimicrobial effect of the soaps. However these variations could be due to the differences in the nature and
structures of the bacteria cell wall since it is the ultimate target of any antimicrobial agent or disinfectant. The result
shows that the black soaps exhibited high levels of antimicrobial activity which is the ability of the soaps to inhibit
the growth or destroy the normal microbiota. The active ingredient in the soap is what distinguishes one type of the
soap from another. The medicated soaps in this study were found to contain trichlocarban and triclosan as the active
antimicrobial agents. These chemical compounds function by denaturing all disrupting cell activity and interfering
with microbial metabolism. These depend on a number of factors such as the inherent properties of the organisms,
contact time, the compostion of the soaps (e.g triclosan), concentration of individual formulation and skin
sensitivity. Traditional black soap lacks a key ingredient used in killing microorganisms such as triclosan, instead
when the soap is scrubbed into the skin; it helps release oils on the surface of the skin that can kill bacteria and
rinsing microorganisms away on the skin and preventing the emergence of mutating bacteria. It was also observed
that black soap has the largest antimicrobial action against the isolates with zones of inhibition (10.65). However,
the other soap samples possess very efficient antimicrobial agent but to a lesser degree. For the other medicated
soaps used in the study, effectiveness in inhibition of growth of the different organisms decreased in the order Zee,
Dudu osun, Tura and Dettol with zones of inhibition 10.57, 9.78, 9.27 and 7.58 respectively.

Ikpoh, I.S et al J. Microbiol. Biotech. Res., 2012, 2 (4):533-537
______________________________________________________________________________
536
Available online at www.scholarsresearchlibrary.com
Fig. 1 Interaction of microorganisms and the soap samples on the zone of inhibitions
Fig. 2 interactions of concentrations of the extract and microorganisms on the zone of inhibition
0
2
4
6
8
10
12
14
Black soap Dettol Dudu Zee Tura
Zone of inhibition
Samples of soap
Corynebacterium spp E.coli
Bacillus spp Staphylococcus aureus
Staphylococcus epidermidis
0
2
4
6
8
10
12
14
500ml 250ml 125ml 62.5ml
Zones of inhibitions
Concentrations of the extract
Corynebacterium spp E.coli
Bacillus spp Staphylococcus aureus
Staphylococcus epidermidis

Ikpoh, I.S et al J. Microbiol. Biotech. Res., 2012, 2 (4):533-537
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Available online at www.scholarsresearchlibrary.com
Fig. 3 Interactions of soap sample and concentrations on the zone of inhibition
REFERENCES
[1] DK Tachibana, 1976, Journal annual review of microbiology. 30: 350-375.
[2] J Marhall, JP Leeming, and KT Holland, 1987, The Cutaneous microbiology of normal human skin. In:
Antibacterial cosmetics and microflora of human skin. New internation Ed. Longman publishers, New York pp 50-
52.
[3] RR Marples, 1992, British medical journal 88-2611
[4] AM Jawetz, KA Melnick’s, and RJ Adelberg, 2010, Normal human microbiota in: medical microbiology. (25
th
ed.) McGraw- Hill companies inc. USA pp. 159-164.
0
2
4
6
8
10
12
14
Black soap Dettol Dudu Zee Tura
Zones of inhibitions
Samples
500ml 250ml 125ml 62.5ml