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1.0 CHAPTER ONE
1.1 INTRODUCTION
A cosmetic product can be defined as any substance or preparation, that can be applied onto
different parts of the human body for example, nails, face, hair and teeth. The role of the
cosmetic product is to keep the body in a good condition, change its appearance and may also
remove body odors through perfuming, cleansing or protection. (Council of the European
Communities, 1976). Depending on the application area, cosmetics may be categorized as
cosmetics for skin, hair-scalp and oral care as well as fragrances (Mitsui, 1997). Cosmetic
ingredients cover a wide range of products ranging from oily materials, surface active agents,
polymers, ultraviolet absorbents to fragrances and vitamins (Muhammed, 2011). According to
the Council Directive (76/768/EEC), established in 1976, companies producing cosmetics are
obliged to ensure safety of their products. However, cosmetics are not liable to be sterile that is,
free of pathological microorganisms.
The microbial contamination of cosmetic products may occur in the course of their production,
through raw materials, ingredients, and during handling, or through repeated use by the
consumer of the products (NakiSiviri et al., 2006). Microbial spoilage can alter physical
properties of the product such as colour, taste, odour and viscosity, and can also deactivate
crucial constituents, thus depriving the cosmetic of its features [Osungunna et al., 2011).
Microbiological contaminants may produce toxins and metabolites that can cause irritation and
allergic reaction of the skin (Yorgancioglu et al., 2013. There can be also pathogens that may
cause hazard to the human health (Lundov, 2008). Microorganisms can survive in an
environment that fulfil their physical and chemical requirements resulting in their proliferation
and further development. Most important of such physical requirements include suitable
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temperature and pH of the environment. Microorganisms also do require presence of moisture,
easily metabolized nutrients as well as oxygen to thrive (Rope, 2002). Almost all cosmetics fulfil
all of these requirements for microbial growth. Cosmetics are rich in free water, having pH close
to neutral. Consumers keep them at home, at room temperature, which is an optimum for
proliferation of some microbes. Most of the cosmetics are kept in the bathroom, where the
temperature and humidity are high (Pinon, 2007). Composition of cosmetics varies from product
to product. The specificity of cosmetic application requires that its ingredients are nourishing and
easily assimilated. Hence, such components as proteins, minerals, vitamins and glycerin are
easily metabolized sources of nitrogen, carbon, hydrogen as well as micro- and macro-elements,
necessary for microbial development (Rope, 2002).
Microorganisms such as Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans
and Aspergillus brasiliensis were listed by European Union Pharmacopoeia as the most
commonly found contaminants that cause microbial spoilage of cosmetics and constituting risk
to the consumer’s health. They may not be found in the given volume of cosmetic sample
(Siegert, 2010). In order to protect cosmetic product from microbial spoilage to which cosmetic
is subjected during use, preservatives are added. This addition of preservatives has generated the
one of the most controversial issues in the production and use of cosmetics (Draelos, 2012).
COSMETICS INVOLVED IN THIS STUDY
The three types of cosmetic products involved in this study are Powders (brown), Lotions and
Mascaras.
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POWDERS
Powder is a cosmetic product used by people to improve their looks, prevent prickly heat, which
sometimes cause body odour (Mirhosseini et al., 2011). The functions of powder include
beautification and prevention of prickly heat etc. (Tran and Hitchins, 1994). Despite these
functions, they provide a favorable condition for microbial growth. Cosmetic powders could be
contaminated either during their preparation, storage, transportation and usage (Álvarez-Lerma et
al., 2008).
LOTIONS
Lotions are applied to the external surface of the body for the purpose of smoothening,
moisturizing and softening the skin tissue. They also serve as medication delivery systems into
the body. Moisturizing creams and lotions, which contain special additives (including plant
extracts, fatty acids and vitamins) that can support bacteria growth (Okeke and Lamikanra,
2001).
MASCARA
The Collins English Dictionary defines mascara as, "a cosmetic substance for darkening,
lengthening, curling, coloring, and thickening the eyelashes, applied with a brush or rod."
Microbial organisms are normally present on human eyelashes and the application of mascara to
lashes has the potential to introduce microbes into the mascara tube which results in subsequent
users being at risk of being infected (Latricia et al., 2008).
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1.2 THE STATEMENT OF PROBLEMS
Microbial contamination of cosmetic products such as Powders, Lotions and Mascara may lead
to spoilage of the cosmetic product resulting in alteration in properties of the products such as the
spoilage which may include change in colour, odour and quality of the products and this may
pose a risk of infections and health hazards to the consumer.
1.3 GENERAL OBJECTIVE
The general objective of this study is to determine the assessment of microbial contaminants
associated with 3 types of cosmetics used by undergraduate female students of Babcock
university, Ogun State.
1.4 SPECIFIC OBJECTIVES
The specific objective of this study are:
1. To detect microbial contaminants associated with each of the three types (Powders,
Lotions and Mascaras) of cosmetics.
2. To determine the level of microbial contamination in each of the three types of cosmetics.
3. To compare the level of microbial contamination in used cosmetic products and that of
newly purchased cosmetic products.
4. To identify the specific organisms involved in the microbial contamination of each of the
three types of cosmetics.
1.5 NULL HYPOTHESIS
1. Facial powders do not possess microbial contaminants such as bacteria or fungi.
2. Lotions do not possess microbial contaminants such as bacteria or fungi.
3. Mascaras do not possess microbial contaminants such as bacteria or fungi.
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4. There is no significant difference between the level of microbial contamination
between in-use cosmetic products and newly purchased ones.
1.6 JUSTIFICATION FOR THE RESEARCH
1. Information from this study can be of benefit in assessing the quality of these cosmetics
products purchased within their expiry date and in determining their level of safety.
2. The discovery or the outcome from this study will create an awareness on the part of
consumers that their cosmetic product may be contaminated which could be responsible
for facial rashes, eczema and other dermatitis, conjunctivitis, ulceration and deep
infection of the cornea
3. This research will provide answers to the question of whether cosmetic products get more
contaminated during production or through repeated usage by consumer.
4. Information from this study will aid the proper management of infection resulting from
microbial contamination of cosmetic products.
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2.0 CHAPTER TWO
2.1 LITERATURE REVIEW
2.2 HISTORICAL BACKGROUND OF COSMETICS.
The word cosmetic was derived from the Greek word “kosm tikos” meaning having the power to
arrange, skill in decorating (Shivanand et al., 2010). The origin of cosmetics forms a continuous
narrative throughout the history of man as they developed. Man in prehistoric times 3000BC
used colours for decoration to attract the animals that he wished to hunt and also he survived
attack from the enemy by colouring his skin and adorned his body with colour for protection and
to provoke fear in an enemy (whether man or animal) (Kapoor, 2005). The origin of cosmetics
was associated with hunting, fighting, religion and superstition and was later associated with
medicine (Draelos, 2003).
In Egypt, as early as 10,000 BC, men and women used scented oils and ointments to clean and
soften their skin and eliminate body odor. Dyes and paints were used to color the skin, body and
hair. They rouged their lips and cheeks, stained their nails with henna and lined their eyes and
eyebrows heavily with kohl. Kohl is a dark-colored powder made of crushed antimony, burnt
almonds, lead, oxidized copper, ochre, ash, malachite, chrysocolla (a blue green copper ore) or
any combination thereof. It was applied with a small stick. The upper and lower eyelids were
painted in a line that extended to the sides of the face for an almond effect. In addition to
reducing sun glare, it was believed that kohl eyeliner could restore poor eyesight and reduce eye
infection. Kohl was kept in a small, flat-bottomed pot with a wide, tiny rim and a flat, disk-
shaped lid. Cosmetics were an inherent part of Egyptian hygiene and health. Oils and creams
were used for protection against the hot Egyptian sun and dry winds. Myrrh, thyme, marjoram,
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chamomile, lavender, lily, peppermint, rosemary, cedar, rose, aloe, olive oil, sesame oil and
almond oil provided the basic ingredients of most perfumes that were used in religious ritual and
embalming the dead. For the lips, cheeks and nails, a clay called red ochre was ground and
mixed with water. Henna was used to dye fingernails yellow or orange. Makeup was stored in
special jars that were kept in special makeup boxes. Women would carry their makeup boxes to
parties and keep them under their chairs. Although men also wore makeup, they did not carry
makeup kits with them (Chaudhri and Jain, 2009).
Cosmetic deodorant was invented in 1888, by an unknown inventor from Philadelphia, and was
trademarked under the name Mumm. During the early years of the 20th century, makeup became
fashionable in the United States of America and Europe owing to the influence of ballet and
theatre stars. But the most influential new development of all was that of the movie industry in
Hollywood. In1900, black entrepreneur Annie Turnbo began selling hair treatments, including
non-damaging hair straighteners, hair growers and hair conditioners door-to-door. In Los
Angeles, Max Factor started selling makeup that did not cake or crack to movie stars in 1904
(Chaudhri and Jain, 2009).
Modern synthetic hair dye was invented in 1907 by Eugene Schueller, founder of L’Oréal. He
also invented sunscreen in 1936. In 1914, T J Williams founded Maybelline, the specialized
mascara manufacturing company. After the First World War, the flapper look came into fashion
for the first time and with it came cosmetics: Dark eyes, red lipstick, red nail polish and the
suntan, invented as a fashion statement by Coco Chanel. Previously, suntans had only been
sported by agricultural workers while fashionable women kept their skins as pale as possible. In
the wake of Chanel’s adoption of the suntan, dozens of new fake tan products were produced to
help both men and women achieve the “sun-kissed” look. In Asia, skin whitening began to
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represent the ideal of beauty. Lipstick was introduced in 1915 in cylindrical metal tubes. In 1922,
the bobby pin was invented to manage short (bobbed) hair. In 1932, Charles and Joseph Revson,
nail polish distributors, and Charles Lackman, a nail polish supplier, founded Revlon. A new
method for permanent waving, using chemicals, which did not require electricity or machines,
was introduced in 1933. In 1935, pan-cake makeup, originally developed to look natural on color
film, was created by Max Factor. Aerosols were patented in 1941, paving the way for hair spray.
In 1944, a Miami Beach pharmacist, Benjamin Green, developed sunscreen to protect soldiers in
the South Pacific. Lawrence Gelb, in 1950, introduced Miss Clairol Hair Color Bath, a one-step
hair coloring product. Roll-on deodorant was launched in 1952 and mascara wands debuted in
1958, eliminating the need for applying mascara with a brush. In 1963, Revlon offered the first
powdered blush-on. Aerosol deodorant was introduced in 1965 (Chaudhri and Jain, 2009).
2.2.1 FACE POWDERS
Powder is a decorative cosmetic product used by both men and women to improve their looks. It
also inhibits the growth of bacterial pathogen which may cause unpleasant odour and sometimes
skin infections (Duke, 1978). It contains many ingredients such as zinc oxide, titanium dioxide,
essential oils which are added to provide the characteristics of a good powder, talc is also added
to help the powder to spread easily during application (Josh, 2006). Cosmetic powder comes
packaged either as a compact powder or in a loose powder container which is used for make-up.
It can also be applied throughout the day to minimize shinning of oily skin. It can be applied
with a sponge, brush or powder puff. Because of the wide variation among human skin tones,
there is a corresponding variety of colours of cosmetic powder to suit each colour of skin.
Besides toning the face, most make-up powders are available with sun protection fraction (SPF)
that helps prevent pigmentation of the skin under the sun (Ashour et al., 2008).
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Cosmetic powders have some positive effects on a person’s appearance which include; reducing
wrinkles and puffiness, it hides the blemishes and the dark circles, it also gives beautiful and
clean appearances to the skin (Stabile, 1984). However, critics have also pointed out that the
powder cannot put a stop to the ageing process as the wrinkles return after a certain period of
time, also, it was established that some cosmetic powders can be contaminated with moulds and
other microorganisms (Elane, 1989). Cosmetic powders can be contaminated with
microorganisms which include Staphylococcus aureus, Psuedomonas aeruginosa, Clostridium
tetani, and yeasts. The source of contamination may be from the raw materials or during
manufacturing, processing, or breakage/damage of the cosmetic powder container, at the retail
market. Contamination may also occur from dust entering the damaged containers (Pollack,
2000). Contamination of cosmetic powder by these microorganisms may result in severe
infection of the skin and mucous membrane which may be difficult to cure (Pollack, 2000). It has
also been reported that some of these cosmetic powders are contaminated with spores of
microorganisms which later germinate when they are poorly preserved (Duke, 1978).
Powders harbor fungi and other microbes and spread infections and a lot of women are not aware
of this, some women even share powders and applicators with others, increasing their chances of
acquiring facial infection. Others do not replace powders until it’s completely finished despite
how long they purchased and used them and this gives an advantage to microbes as they rapidly
grow and multiply the longer they stay in powder, which may lead to biodegradation of product
and hence the risk of infection to consumers of the product (Behravan et al., 2005: Anderson and
Parkin, 2007).
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Figure 1.1 : showing different types of facial powders.
Source: wikihow.com
10
Figure 1.2: showing the application of facial powders
Source: wikihow.com
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2.2.2 MASCARAS
Of the different types of make-up products, those for the eye area merit special attention because
of the proximity and contact with this region, and thus, the higher probability of causing
irritation or, if the product is contaminated, ophthalmic infections. There is a wide variety of
cosmetics for the eye area which have many different functions and contain diverse ingredients
that may cause adverse effects (Barata, 1995). The cosmetics used on the ocular region are the
main cause of eyelid dermatitis due to their pigments, resins, preservatives and vehicles for
application (Draelos, 2009). Mascara is one of the most popular cosmetic products, used to
lengthen eyelashes and make them thicker, highlighting the feminine face (Rieger, 2000).
However, this product is at a great risk of contamination, because it is an aqueous-based
formulation (Draelos, 2001). Staphylococcus epidermidis and Staphylococcus aureus proliferate
in contaminated mascaras. The most common infections caused by these microorganisms occur
especially when the surface of the eyeball is traumatized (Draelos, 2001). Pseudomonas
aeruginosa is the main causative agent of eye infections like conjunctivitis, keratitis and
ophthalmitis, and the infections may threaten the integrity of the eye by destroying tissues and
damaging visual acuity (Esteva, 2006). Infections by P. aeruginosa have been reported to occur
due to contaminated mascara, trauma to the eye or bad hygiene (O’Donoghue, 2000). Fungi can
also be found in contaminated mascaras, although less frequently than bacteria, fungal infection
is being related to immune-compromised people or those who wear contact lenses (Draelos,
2001).
The quality and performance requirements for mascara are as follows: They should
Be non-irritating as they are applied so close to the eyes
Not harden the eyelashes or form blobs
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Make the eyelashes look thick and long
Make the eyelashes curl effectively
Have an appropriate luster
Have an appropriate drying time
Not go on to the lower eyelids when dry and their appearance must not be spoiled by
sweat, tears or rain
Be easy to remove
Be easy to use throughout their period of use
Not be contaminated by microorganisms
(Mitsui, 1998).
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Figure 1.3: showing mascara with different shapes of wands for application.
Source: www.self.com
14
Figure 1.4: shows the application of a mascara.
Source: www.makeupforever.com
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2.2.3 LOTIONS
Lotions are liquid preparations applied to the skin for the purpose of smoothening the skin or to
serve as a delivery system for medication into the body. Lotions are able to increase the
hydration of the skin and to prevent the loss of hydration to restore the epidermal barrier of the
skin. Lotions contain “Moisturizing” ingredients that typically fulfill one or more of the
following three functions: humectant, occlusive, or emollient (Jeanine and Downie, 2010).
Humectants.
Water within a topical moisturizer formulation is an essential ingredient, but it typically
contributes little to the delivery of hydration to the stratum corneum (Rawlings et al., 2004). In
fact, water itself (regardless of soap or detergent use) is shown to be irritant to the skin under
occlusion (Tsai and Maibach, 1999). and can be associated with causing skin dryness. Therefore,
the primary hydrating effect of moisturizers is provided via humectant ingredients, which attract
and hold water in the skin, either by drawing it up from the dermis to the epidermis or from the
environment into the epidermis. They can cause water to be evaporated into the environment and
thus need to be used with occlusive agents to decrease or prevent more transepidermal water loss
(TEWL) (Jeanine and Downie, 2010).
Occlusives
Occlusives are ingredients that sit on the skin, creating a barrier to TEWL. Petroleum jelly is
among the best known and most widely used occlusive. It is known to reduce transepidermal
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water loss by more than 98 percent. By contrast, other oily occlusives, which include mineral oil,
silicone, and lanolin, reduce transepidermal water loss by about 20 to 30 percent (Rawlings et
al., 2004). They form a hydrophobic film between corneocytes on the skin.
Emollients.
“Emollient” refers to the “feel” of a product, as emollients are ingredients that spread easily on
the skin, helping to hold down desquamating corneocytes and fill any “gaps” between them.
Emollients are typically oils and lipids that enhance skin texture and flexibility. This results in a
smooth skin feel, or what has been termed “skin slip” in the commercial realm, and helps make
the stratum corneum soft, supple, and flexible (Rawlings et al., 2004). Emollients may be
occlusive and/or humectant or neither. As they provide the immediate feel of moisturization,
they rate high among consumers for product satisfaction.
The risk of microbial contamination occurring in present-day moisturizing creams and lotions is
increased because they contain special additives (including plant extracts, fatty acids and
vitamins) that could serve as substrates for bacteria (Okeke and Lamikanra, 2001).
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Figure 1.5: showing different brands of body lotions.
Source: BeautySouthAfrica.com
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2.3 MICROBIAL CONTAMINATION
Cosmetics are preparations normally applied externally to human body parts mainly for
beautifying, cleansing and protecting the body (Onurdag et al., 2010). These products are
formulated from an array of chemicals in the presence of plentiful amount of water and mostly
exhibit a near neutrality pH (Abu Shaqra and Al-Groom, 2012). Cosmetic products are basically
non sterile but must be completely free of high-virulence microbial pathogens. The total number
of aerobic microorganisms per gram must be at minimal stipulated standard by various
authorities in any country. For instance, International Standard Organisation (ISO), Standard
Organisation of Nigeria (SON) and National Agency for Food and Drug Administration and
Control (NAFDAC) as is the case in Nigeria. Production of stable cosmetics requires an
integrated quality management system which consists of quality raw material, proper product
formulation, hygienic design of production facilities, good production hygiene process,
packaging containers and a validated preservative system (Detmer et al., 2010 : Siergert, 2012).
The first reported contamination of cosmetics was in 1946 by several cases of neonatal death
from talcum powder containing Clostridium tetani (Baird, 2003). Since 1960s, opportunistic
organisms, such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas sp., Serratia
sp. and Enterobacter sp., have been isolated from cosmetic products (Geis, 2006).
Cosmetics may be liable to microbial contaminations either during the course of transportation,
storage of finished goods or during in use by the consumers (Gamal et al., 2015). These
contaminants could be pathogens, opportunistic pathogens or saprophytes which may in turn
result in economic loss and infection on the body (Anelich and Korsten, 1996). Since the 1960s,
opportunistic organisms, such as Klebsiella pneumoniae, Pseudomonas aeruginosa,
Pseudomonas sp., Serratia sp. and Enterobacter sp., have been isolated from cosmetic products
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to a certain level (Geis, 2006). A situation where these cosmetic products are heavily
contaminated with pathogenic organisms, could lead to biodegradation of the product and risk of
infection to consumers (Bos et al., 1989). This spoilage usually results in alteration in
organoleptic properties of cosmetic products which may bring about colour, odour changes and
biodegradation of the active component of the preparations. The products are therefore stored at
ambient temperature particularly in tropical regions. It is not acceptable that the following
potentially pathogenic microorganisms of Staphylococcus aureus, Pseudomonas aeruginosa,
Candida albicans, Escherichia coli and other members of Enterobacteriaceae are present in
cosmetics product (Detmer et al., 2010).
Microorganisms can definitely cause spoilage or chemical changes in cosmetic products that can
also result in the physical injury to the user. Unwarranted amounts of bacteria and fungus can
affect the cosmetic in several manners like, odors, destabilize the emulsion and color changes.
These microbes can affect the consumer in many unwanted ways likely from harmless itching of
the skin to the severe infections; even can lead to the permanent or temporary blindness because
of the products that include eye make-up (Haider, 2016).
2.3.1 FACTORS INFLUENCING THE GROWTH OF MICROORGANISM IN
COSMETICS
The factors that influence the growth of microorganisms in cosmetics include solutes and water
activity, hydrogen ion concentration (pH), temperature, oxygen requirements.
SOLUTES AND WATER ACTIVITY
During manufacture, the main sources of contamination are the used raw materials, including
water, and the manufacturing process itself. The microbiological quality of water depends on its
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origin. Water remains one of the most important factors in the contamination of a cosmetic
product. Species of genera such as Pseudomonas, Achromobacter, Aeromonas, Flavobacterium,
Xanthomonas, Actinobacter, and Aerobacter spp. Have been recovered from natural waters. The
presence of Escherichia coli may be a sign of recent contamination by wastewater (Neza, 2016 ;
Wallhäuser, 1985).
Usually, water is the major constituent of cosmetics, and it is an ideal growth factor for
microorganisms. Water activity is inversely related to the osmotic pressure of a solution. The
higher the water activity, the lower the osmotic pressure and the more likely organisms will
grow. Also the lower the water activity, the higher the osmotic pressure and the less likely
organisms will grow. Most organisms prefer growing at water activity rates of 0.98 to 1.0. A few
are osmotolerant and can survive water activities as low as 0.6, but these organisms are generally
nonpathogenic. The addition of a wide variety of water binding molecules in a formulation can
actually tie up the water, resulting in a low water activity rate. Thus, this method of preservation
should not be overlooked. For example, many antiperspirants and dry make-up powders have
extremely low water activity levels and so do not support microbial growth unless if water
present in skin debris is placed into the products during use (Geis, 2006)
HYDROGEN ION CONCENTRATION (pH)
The pH term refers to the measurement of hydrogen ion concentration of an environment on a
logarithmic scale from 0 to 14. A pH of 0 represents the high concentration of 1.0 M hydrogen
ions (H+) and a pH of 14 is the low concentration of 10–14 hydrogen ions. Microorganisms
grow in environments ranging from a pH of 1 to 10. The only microbes of concern for
microbiologists involved in keeping consumer products free of microorganisms are the
neutrophiles that grow at pH 5.5 to 8.0 because they are also potential pathogens. Unless
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spoilage organisms that grow below 5.5 or above 8.0 are present in a product, they
are of minimal concern to the consumer product microbiologist (Geis, 2006). Generally
speaking, microorganisms cannot proliferate or survive in a cosmetic formulation with a pH of
less than 4 or greater than 10 (Kabara, 1997).
TEMPERATURE
Temperature affects microorganisms because they have no way to control their own temperatures
and because enzymatic reactions required for growth are affected by temperature in the same
way any other chemical reaction is affected: the rate of reaction roughly doubles for every 10°C
rise in temperature. At low temperatures, the enzymes operate more slowly and thus growth is
also slower. Up to a certain temperature, growth increases until the point is reached where high
temperatures are lethal and damage cells by denaturing proteinaceous enzymes, destroying
transport proteins, and destroying lipid membranes. The key or cardinal temperatures to consider
for each species of microbe are the minimum, optimum, and maximum. Microorganisms based
on their temperature requirements can be placed into four categories;
a) Psychrophiles
They grow between 0 and 20°C and are found throughout Arctic and Antarctic regions as
well as in man-made refrigerated environments. In Alpine snowfields, the psychrophilic
alga, Chlamydomonas nivalis, produces pink snow. Psychrophilic bacteria include members
of the genera Pseudomonas, Flavobacterium, Achromobacter, and Alcaligenes. Facultative
psychrophiles grow optimally at 20 to 30°C and can spoil refrigerated foods. Few, if any, of
these microorganisms present problems for consumer products because they only act as
spoilage organisms and cosmetic products are rarely kept at such low temperatures.
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b) Mesophiles
They can grow from 20 to 45°C although their optimum temperatures are from 25 to 40°C.
Nearly all human pathogens are mesophiles because human body temperature is 37°C.
Mesophiles are the key difficult organisms for a consumer product microbiologist to control.
The key mesophile species that require control in manufacturing environments include
Pseudomonas cepacia, Pseudomonas maltophilia, Pseudomonas aeruginosa, Enterobacter
cloacae, Enterobacter agglomerans, Enterobacter gergoviae, Escherichia coli.,
Staphylococcus spp., and some yeasts and molds.
c) Thermophiles
They can grow between 45 and 110°C although the typical range is 55 to 85°C (Geis, 2006).
OXYGEN REQUIREMENTS
Many organisms encountered in a manufacturing environment are obligate aerobes. These are
organisms that grow in the presence of atmospheric oxygen (O2). Oxygen is critical for these
organisms since it serves as the terminal electron acceptor for the electron transport chain that
generates Adenosine Triphosphate (ATP) during aerobic respiration. Other organisms are
microaerophiles that require some oxygen but not at levels encountered in the atmosphere. Some
organisms are facultative anaerobes; they do not require oxygen but fare better when they have
it. Aerotolerant anaerobes grow whether or not oxygen is present because they are not affected
by it. Occasionally encountered, but relatively rarely in a manufacturing environment are
obligate anaerobes, organisms that grow in the absence of oxygen. Although strict anaerobes are
killed by any exposure to oxygen, they can be recovered from habitats that appear to be aerobic.
This is especially true for organisms that associate with facultative anaerobes in communities
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where the facultative anaerobes serve as oxygen scavenging guilds to deplete any available
oxygen (Geis, 2006).
2.3.2 MICROBIAL CONTAMINANTS USUALLY FOUND IN COSMETICS
The organisms most likely to be encountered in contaminated cosmetic products are those that
are likely to be present in an ordinary household (Geis, 2006). Microorganisms such as
Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and Aspergillus brasiliensis
were listed by European Union Pharmacopoeia as the most commonly found contaminants
posing microbial spoilage in cosmetics and risk to the consumer health. They may not be found
in the given volume of cosmetic sample (Siegert, 2010).
Pseudomonas
The genus Pseudomonas consists of strictly aerobic, non-sporing, Gram negative bacilli. The
species except Pseudomonas mallei are motile by polar flagella. The type specie is Pseudomonas
aeruginosa (formerly known as Pseudomonas pyocyanea). The other important species include
Pseudomonas pseudomallei, Pseudomonas putida, Pseudomonas fluorecens, Pseudomonas
maltophilia, Pseudomonas cepacia and Pseudomonas stutzeri. Pseudomonas aeruginosa mainly
infects wounds and burns and it also causes urinary tract opportunistic infections, usually
associated with catheterization (Ochei and Kolhatkar, 2000). In cosmetics, the organism has been
implicated in eye infections and loss of sight. When found in a cosmetic manufacturing plant,
they usually arise from failure to control and monitor water systems, formation of biofilms in the
equipment, ineffective or infrequent sanitization, and dead legs (short lengths of pipes with
closed or “dead” ends) or other sources of stagnant product (Geis, 2006).
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Staphylococci
Staphylococci are Gram-positive bacteria, with diameters of 0.5 – 1.5 µm and characterized by
individual cocci, which divide in more than one plane to form grape-like clusters. To date, there
are 32 species and eight sub-species in the genus Staphylococcus, many of which preferentially
colonise the human body (Kloos and Bannerman, 1994). However, Staphylococcus aureus and
Staphylococcus epidermidis are the two most characterized and studied strains. The
staphylococci are non-motile, non-spore forming facultative anaerobes that grow by aerobic
respiration or by fermentation. (Kloos and Schleifer, 1986; Wilkinson, 1997). Members of this
genus are catalase-positive and oxidase-negative (Wilkinson, 1997). Staphylococci are tolerant to
high concentrations of salt (Wilkinson,1997) and show resistance to heat (Kloos and Lambe,
1991). Pathogenic staphylococci are commonly identified by their ability to produce coagulase
(Kloos and Musselwhite, 1975). This distinguishes the coagulase positive strains, S. aureus (a
human pathogen), and S. intermedius and S. hyicus (two animal pathogens), from the other
staphylococcal species such as S. epidermidis, that are coagulase-negative (CoNS). Some of the
species (e.g., S. aureus) cause boils, are involved in impetigo, cause conjunctivitis, and cause
food poisoning. A common manifestation of infection is the production of pus.
Candida spp
It is the most common fungal pathogen of humans. It can grow as both yeast and filamentous
forms in the host. This is a phenomenom known as dimorphism (Romani et al., 2003). Candida
albicans colonizes the skin, genital and/or intestinal mucosae of 30-70% of healthy individuals at
any given moment, and it is therefore noteworthy that under normal circumstances the fungus
does not cause significant disease (Perlroth et al., 2007). Candida albicans is the most important
specie and it is responsible for oral thrush, vaginal candidiasis, candiduria and Candidemia
25
frequently seen in patients and it is also causes vulvovaginitis in girls at pubeteric age group
(Singh et al., 2013).
Aspergillus spp.
Aspergillus species are widespread in the environment, growing on plants, decaying organic
matter, and in soils, air/bioaerosols and in freshwater and marine habitats. Aspergilli are also
found in indoor environments (surfaces of buildings, air, household appliances, etc.) and in
drinking water and dust. The diverse species which make up the Aspergillus genus are able to
utilize a wide variety of organic substrates and adapt well to a broad range of environmental
conditions (Cray et al., 2013). Although there are several hundred species in the Aspergillus
genus, there are only a few species which have considerable impacts on human or animal health.
Infections are typically caused by Aspergillus flavus, Aspergillus fumigatus, Aspergillus
nidulans, Aspergillus niger and Aspergillus terreus, among other species (Baddley et al., 2001).
Aspergillosis includes diseases such as toxicosis (due to toxin produced by A. flavus); allergic
reactions; localized infections of the skin, external ear (otomycosis), nasal sinuses and the orbit
of the eye (keratitis); pulmonary infection; and disseminated multi-organ disease (Ochei and
Kolhatkar, 2000).
Enterobacter spp.
Enterobacter is a Gram-negative fermentative rod. Enterobacter produces butanediol, ethanol,
and carbon dioxide. It can be isolated from contaminated surfactants and particularly from
quaternary-containing conditioners. It is also a typical contaminant in households and can grow
in poorly preserved products. Some of the key species contaminating cosmetics include
Enterobacter agglomerans, Enterobacter gergoviae, and Enterobacter cloacae. These organisms
26
are found in soil and often invade plant tissues, thus causing a variety of necrosis. Enterobacter is
also found in gastrointestinal tract and it is incriminated in wound and urinary tract infection.
(Geis, 2006).
Serratia spp.
The genus Serratia, a member of the Enterobacteriaceae, is comprised of a group of bacteria that
are related both phenotypically and by DNA sequence. The type species of the genus is Serratia
marcescens. Some species and biotypes of Serratia produce a nondiffusible red pigment,
prodigiosin, or 2-methyl-3-amyl-6-methoxyprodigiosene (Williams and Qadri, 1980).
Serratia marcescens is generally an opportunistic pathogen causing infections in
immunocompromised patients. Among the possible pathogenicity factors found in Serratia
strains are the formation of fimbriae, the production of potent siderophores, the presence of cell
wall antigens, the ability to resist the bactericidal action of serum, and the production of
proteases (Grimont and Grimont, 2006).
Escherichia coli (E. coli)
E. coli, a member of the bacterial family of Enterobacteriaceae, is the most prevalent
commensal inhabitant of the gastrointestinal tracts of humans and warm-blooded animals (Kaper
et al., 2004). As a commensal it lives in a mutually beneficial association with the hosts, and
rarely causes disease. It is, however, also one of the most common human and animal pathogens
as it is responsible for a broad spectrum of diseases. The peculiar characteristics of the E. coli,
such as ease of handling, availability of the complete genome sequence, and its ability to grow
under both aerobic and anaerobic conditions, makes it an important host organism in
biotechnology. E. coli is used in a wide variety of applications both in the industrial and medical
27
fields and it is the most used microorganism in the field of recombinant DNA technology (Yoo et
al., 2009). Escherichia coli remains one of the most frequent causes of several common bacterial
infections in humans and animals. E. coli is the prominent cause of enteritis, urinary tract
infection, septicaemia and other infections, such as neonatal meningitis. E. coli is also
prominently associated with diarrhoea in pet and farm animals (Allocati et al., 2013).
Klebsiella spp.
Klebsiella is a Gram-negative rod organism that is very widespread in the environment.
Klebsiella is a human pathogen; some species are commensals. They are found in soil and water
and are plant pathogens. Klebsiella pneumoniae causes a severe fulminating pneumonia in
people who are debilitated physically or those who abuse alcohol. Unlike other members of the
Enterobacteriaceae family, Klebsiella is nonmotile. It is found routinely in households and can
contaminate cosmetics during consumer use (Geis, 2006).
Bacillus spp.
This group of organisms consists of large aerobic spore-bearing rods. They are Gram positive
bacilli which may sometimes be arranged in long chains. Most of the species are motile. Type
species is Bacillus anthracis but other species that may cause infections are Bacillus cereus and
Bacillus subtilis (Ochei and Kolhatkar, 2000). Very few Bacillus spp. are pathogenic and these
include B. anthracis and B. cereus. B. anthracis can cause anthrax, a cutaneous disease caused
by spores that enter the skin through small cuts and abrasions. The organism is invasive because
of the production of virulence factors that include polysaccharide capsules and exotoxins that
produce edema and cell death. The initial disease presents as a papule that becomes increasingly
necrotic and then ruptures to form a painless black scab called an eschar. B. anthracis can also
28
cause pulmonary anthrax from inhalation of airborne spores. In the cosmetics industry, some of
the more common raw materials that may be contaminated with Bacillus spores include Aloe
vera and a variety of thixotropic agents such as quaternized clays. Pasteurization of aloe vera gel
will not eliminate Bacillus because its spores are resistant. Instead, tyndallization (a repetitive
heating process) is required (Geis, 2006).
2.3.3 CONTROL OF MICROBIAL CONTAMINATION OF COSMETICS
Generally speaking, all products including cosmetics, containing water and organic/inorganic
compounds under appropriate physicochemical conditions, are prone to microbial contamination.
This justifies why these products require effective and adequate protection against
microorganism proliferation (Huang et al., 2003). An ideal preservation system (intrinsic or
extrinsic) should protect the product from microbial degradation, both in its original closed
packaging until use, and in an open container throughout its use (Pitt et al., 2016). In recent
years, the safety record for personal care products has been excellent, resulting in a scarce
occurrence of infections due to contaminated products (Stewart et al., 2016). Studies have shown
that the most frequent microorganisms found in cosmetics comprise Pseudomonas aeruginosa,
Klebsiella oxytoca, Burkholderia cepacia, Staphylococcus aureus, Escherichia coli, Candida
albicans, Enterobacter gergoviae, and Serratia marcescens, but also other bacteria, fungi and
yeasts. The skin and mucous membranes are protected against microorganisms; however, their
presence in these products can increase the risk of microbial infection (Scientific Committee on
Consumer Safety, 2016).
Microbial contamination may occur during manufacture (primary contamination) and/or during
consumer use (secondary contamination) (Smith and Alexander, 2005). All potential sources of
29
contamination must be identified and monitored. In order to do so, four steps must be
considered:
1. inspection and control of raw materials
2. manufacturing process
3. delivery of the final product
4. its use by the consumer.
To achieve a good protection of cosmetic products against microbial contamination, the industry
provides two stages of preservation: primary and secondary. The strategy of primary
preservation occurs during manufacturing and is based on the application of Good
Manufacturing Practices (GMP), then secondary preservation, which takes place after
manufacture, uses chemical, physical, or physicochemical means to attain an efficient protection.
Primary Preservation Strategy
Certification, ISO 22716:2007 which encompasses the Good Manufacturing Practices (GMP) for
Cosmetics, has been approved and accepted (with or without modification) by most regulatory
organizations around the world, particularly after the July 2008 meeting of the International
Cooperation on Cosmetic Regulation (ICCR) by the United States, the European Union, Japan,
and Canada) (De Boer, 2018. Good manufacturing practices must be strictly obeyed during the
production of cosmetic products. The preparation of the cosmetics under strictly aseptic
conditions must avoid their microbial contamination. Water treatment, microbial control of raw
materials, equipment disinfection, and qualification of personnel can reduce the risk of
contamination (Mitsui, 1997).
30
Physicochemical Secondary Preservation
Water Activity
Usually, water is the major constituent of cosmetics, but it is an ideal growth factor for
microorganisms. To solve this problem, certain substances such as salts, polyols, protein
hydrolysates, amino acids, and hydrocolloids (xanthan gum, guar gum, etc.), glyceryl
polyacrylate gel, sodium polyacrylate and sodium chloride can reduce the water activity (aw).
The choice of these substances depends on their toxic effect, and also the nature of the cosmetics
(Sedlewicz , 2005; Geis, 2006) Water activity can also be reduced by the use of vapour-resistant
bottles, film strip, vapour-repellent film coatings, or polyacrylamide hydrogels (Hiom. 2013).
Emulsion Form
Water-in-oil (W/O) emulsions can minimize the risk of microbial contamination more than oil-
in-water (O/W) emulsions (Varvaresou et al., 2009). The size of the emulsion droplets can
improve the cosmetics effectiveness. In many cases, the decrease in the size of the emulsion
droplets (Nano emulsion) increases the antimicrobial activity. However, the antimicrobial
activity depends also on the oil phase chemical composition, namely the type of phenolic
compounds, their concentration, and chemical structure (Char et al., 2016).
pH Control
The optimum pH for microorganism’s growth in cosmetic products is between 5 to 8, meaning
that any pH outside this range induces unfavorable conditions, thus decreasing their growth rate
(Varvaresou et al., 2009). The acidic pH of cationic hair conditioners (pH = 4, approximately)
contributes to the antimicrobial status of the products (Dias, 2015). Other formulations with
acidic pH can inhibit the growth of microorganisms, such as products containing salicylic acid
and aluminum compounds in antiperspirants (pH ranging from 3.5 to 4.5) (Lukic et al., 2016)
31
Liquid soaps having an alkaline pH (pH 9.5 to 10.5) exhibit an unfavourable environment for
microorganism to grow (e.g., destabilizing their membrane), this is due to the effects of ionized
fatty acids and free alkalinity of the existent NaOH. Generally speaking, microorganisms cannot
proliferate or survive in a cosmetic formulation with a pH of less than 4 or greater than 10
(Berthele et al., 2014).
32
3.0 CHAPTER THREE
3.1 MATERIALS AND METHOD
3.2 STUDY AREA
This study was carried out in Babcock University, Illishan Remo community, Ogun State,
Nigeria. Illishan Remo is a residential town located within the Irepodun district in Ikenne Local
Government of Ogun state, South Western Nigeria. Its geographical coordinates are
6054’00’’North and 3043’00’’ East.
3.3 STUDY DURATION
This study was carried out between the months of May and June, 2019.
3.4 STUDY POPULATION
Female undergraduate Babcock university students were the target population.
3.5 SAMPLE SIZE
For the purpose of this experiment the following brands of cosmetics were used; facial powders
– Classic, mascaras – Classic, body lotions – Nivea. Fifteen (15) samples, 5 of each of the listed
brands were used for the experiment. For study control, five (5) of each brand of cosmetic
products (5 new classic facial powder, 5 new classic mascara, 5 new nivea body lotion) was
bought from Illishan market and this was used to control the microbial load. These cosmetic
products were already in use by female students. The cosmetic products were checked to ensure
that they have not exceeded their expiry dates.
3.6 ETHICAL CONSIDERATION
Ethical clearance was obtained from the Babcock University Health Research Ethics
Committee (BUHREC).
33
3.7 CONSENT
Informed consent was obtained from each of the owners of the cosmetic products participating in
the study. The purpose of the study and the nature of the study was explained properly.
Afterwards, participants were requested to voluntarily complete the consent form in their
handwriting and this served as proof of willingness to provide the cosmetic products. They were
assured of the confidentiality associated with the study.
3.8 DATA COLLECTION
Before the sample collection, demographic and clinical information were obtained from the
participants through the administration of prepared questionnaires. Each questionnaire had a
unique participant identification number (PIDN). The first part of this questionnaire contained
the bio data of the patient e.g age, level, religion. The second part included any history of
reactions to cosmetic products such as watery eyes, facial and skin reactions. Responses to the
questionnaire were used to collect data on the possible presence of microbial contaminants in
cosmetic products. For the purpose of privacy, all information obtained from the participants
were treated confidentially.
3.9 SPECIMEN COLLECTION
A small portion of the compact facial powder was taken and put into a sterile universal bottle. A
small volume of the body lotion was poured into a sterile universal bottle. In the collection of
sample from the mascaras, the mascara applicator along with the tube was collected upon
consent been granted by participants.
3.10 SPECIMEN STORAGE
The cosmetic specimens were transported to the Laboratory unit of the Department of Medical
Laboratory Science, Babcock university.
34
3.11 LABORATORY ANALYSES
3.11.1 MEDIA TO BE USED
Nutrient agar, Macconkey agar, CLED (Cysteine Lactose Electrolyte Deficient) agar was used
for the isolation and enumeration of the bacterial load of the cosmetic samples. Sabrouad
Dextrose Agar was used for the isolation and enumeration of yeasts and molds. The media was
prepared and sterilized according to the manufacturer’s instructions.
3.11.2 BACTERIAL COUNT OF FACIAL POWDERS, BODY LOTIONS
AND MASCARAS.
1. In order to assess the degree of contamination, 1g of material was dispersed in 4 ml
sterile Ringer solution containing 0.25% tween 80.
2. Appropriate dilutions were made in the same dispersing vehicle.
3. 0.1 ml was plated out on the solid medium using the surface viable method that is 0.1ml
from the last dilution was spread across the surface of a solid dry medium using a glass
rod.
4. All the plates were incubated at 37oC for 24 hours.
5. Emergent colonies were counted after the necessary incubation.
6. Results were expressed as colony forming unit per gram (CFU/g).
7. All bacterial isolates were identified based on their Gram reaction and biochemical tests,
as described by U.S.FDA manual online (Cheesbrough, 2005).
3.11.3 FUNGAL COUNT OF THE FACIAL POWDERS, BODY LOTIONS
AND MASCARAS.
1. 1g of material was dispersed in 1ml sterile peptone water.
35
2. Appropriate dilutions were made in the peptone water, and 0.1ml of each of the dilutions
was inoculated on Sabouraud dextrose agar plates using spread plate method that is
0.1ml of each of the dilutions was spread over the surface of the dry solid medium.
3. The plates were then incubated at 25 0C for 3 (three) days. Colonies were counted after
three days.
4. Results of colony count were expressed as yeasts and molds counts per gram.
5. All fungal isolates were identified based on their macroscopic and microscopic
appearance with reference to standard manual (Larone, 1995).
3.12 SUB-CULTURING OF ISOLATES.
A sterile inoculating loop was flamed until it was red hot and it was allowed to cool after which
the loop was used to pick a distinct colony from the culture plate. The colonies were streaked out
on the surface of the sterile fresh media, flaming the loop after each series of streaks. The sub
cultured plates were incubated at 370C for 24 hours for the isolation of bacteria.
3.13 IDENTIFICATION OF MICROORGANISMS
After the incubation period, the plates were read to examine the general morphology.
3.13.1 Gram staining
Smears of the isolates from 24 hours culture were made by applying a drop of normal saline on a
clean grease free slide with a sterile inoculating loop. The smears were air dried and heat fixed
by passing the slides quickly over the flame. The fixed smears were flooded with crystal violet
(primary stain) for about a minute and them rinsed off under a running tap.
The slides were flooded with Lugol’s iodine which was also allowed to stay for 1 minute and
then rinsed off under a running tap. The slides were decolourized using acetone by adding two to
three drops to the slide. The slides were rinsed immediately under the running tap.
36
The slides were flooded with safranin and allowed to stay for a minute before rinsing off. The
slides were left to dry and then viewed under the light microscope using the oil immersion x100
objective lens.
3.13.2 Fungal staining
The fungi were microscopically and macroscopically identified. The growth pattern and
morphology of the fungi were studied in the plates. Then a drop lactophenol blue was placed at
the center of the grease free slide, the forceps was sterilized red hot with the Bunsen flame after
which it was used to pick a portion of the pure culture, it was then teased gently in the
lactophenol blue solution, after which it was covered with a coverslip. The slide was examined
under the light microscope using ×10 and ×40 objective lens.
3.14 BIOCHEMICAL TESTS
The following biochemical tests can be carried out for further identification of the bacteria
isolate from the study.
3.14.1 Catalase test
Principle of catalase test
Catalase acts as a catalyst in the breakdown of hydrogen peroxide to oxygen and water, an
organism is tested for catalase production by bringing it in contact with hydrogen peroxide.
Bubbles of oxygen are released if the organism produces the enzyme catalase.
2H2O2 → 2H2O + O2
Procedure – Slide Test Technique
Colonies of the organism was emulsified in distilled water on a clean grease free slide.
Two drops of hydrogen peroxide were added.
It was then observed immediately for effervescence.
37
Positive control
A colony of Staphylococcus aureus was emulsified in the distilled water on a clean
grease free slide.
Two drops of hydrogen peroxide were added on a glass slide
It was then observed immediately for effervescence.
3.14.2 COAGULASE TEST
Principle
The enzyme coagulase converts soluble fibrinogen to insoluble fibrin thus causing plasma to
clot.
Procedure
Slide coagulase test
Two drops of saline were placed on a grease free slide
The saline was emulsified with the selected colonies
Two drops of plasma were added and was mixed properly
It was observed for clumping within 5-10 seconds
Positive control
Two drops of saline were placed on a grease free slide
The saline was emulsified with the selected colonies of Staphylococcus aureus
Two drops of plasma were added and was mixed properly
It was observed for clumping within 5-10 seconds
3.14.3 INDOLE TEST
Principle
38
The test organism is cultured in a medium which contains tryptophan. Production of indole is
detected by Kovac’s reagent which contains 4 p-dimethyl amino –benzaldehyde. This reacts with
the indole to produce a red ring.
Procedure
The organism was grown in peptone water at 370C for 24 hours.
0.5ml of Kovac’s reagent was added to the broth.
It was then examined for a red ring which denotes a positive result.
3.14.4 OXIDASE TEST
Principle
The enzyme oxidase will oxidise a redox dye tetramethyl paraphenyldiamine dihydrochloride to
deep purple colour. This enzyme is produced by some aerobic bacteria as part of their respiratory
mechanism.
Procedure
A piece of filter paper was placed in a petri dish. 2 drops of freshly prepared oxidase
reagent were added.
A piece of stick was used to pick colonies and smeared on the filter paper.
It was observed for the development of deep purple colour.
Positive control
A piece of filter paper was placed in a petri dish. 2 drops of freshly prepared oxidase
reagent were added.
A piece of stick was used to pick colonies of Pseudomonas aeruginosa and smeared on
the filter paper.
It was observed for the development of deep purple color.
39
3.14.5 CITRATE TEST
It is based on the ability of an organism to utilize citrate as its on.ly source of carbon and
ammonium as its only source of nitrogen. The citrate is metabolized as acetoin and carbon
dioxide.
Procedure
Slopes of Simmon Citrate Agar was prepared in bijou bottles as recommended by the
manufacturer.
A sterile inoculating loop was used to first streak the slope with suspension of the test
organism after which the butt of the medium was stabbed.
It was incubated at 370C for 24 hours.
The medium was observed for colour change at the end of 24 hours incubation.
3.14.6 UREASE TEST
Principle
The test organism is cultured in a medium which contains urea and the indicator phenol red.
When the strain is urease producing the enzyme breaks down urea by hydrolysis to give
ammonia and carbon dioxide. With the release of ammonia, the medium becomes alkaline
indicated by a change in color of the indicator to pink red.
Procedure
The test organism was inoculated in a bijou bottle containing 3ml sterile Christensen
modified urea slope.
It was incubated at 350C-370C overnight in an incubator.
It was observed for a pink red colour.
40
3.14.7 TRIPLE SUGAR IRON IDENTIFICATION TEST
Principle
Triple Sugar Iron Agar is a medium used in the identification of Gram negative enteric rods. The
medium measures the ability of a bacterium to utilize three sugars glucose, sucrose and lactose.
A pH indicator included in the medium detects acid production from fermentation of the sugars
while no colour change indicates alkaline production. The tube is inoculated on the slant and
stabbed at the butt. Carbohydrate utilization can be determined through analysis of the extent of
acid production. Acid production limited to only the butt is indicative of glucose utilization.
Hydrogen sulphide production is detected by the presence of black pigment in the agar.
Production of other gases is detected by cracks in the agar as well as an air gap at the bottom of
the test tube.
Procedure
The colonies were introduced into the agar by streaking the colonies on the slant and
stabbing the agar to the butt using a sterile wire loop.
The test tubes were plugged and incubated at 370C for 24 hours.
It was observed for changes after 24 hours incubation.
TABLE 3.1 POSSIBLE FERMENTATION REACTIONS AND INTERPRETATIONS
OF RESULTS.
Slant Butt Interpretation Probable results
Red Yellow with gas
production (black
pigmentation).
Glucosee fermentation
has occurred.
Proteus species.
Yellow Yellow Lactose, sucrose and Escherichia coli,
41
glucose has fermented. Klebsiella species.
Red Red No carbohydrate
fermentation has
occurred.
Pseudomonas
aeruginosa.
3.14.8 MOTILITY TEST
This method is used to determine the motility of bacteria microscopically when it is suspended in
fluid. This is observed by the movement of bacteria from one position to another in a haphazard
manner.
Procedure
The test organism was inoculated in nutrient broth. It was incubated at 370C for 24 hours.
A ring was made using plasticine on a clean grease free slide, a loop full of culture was
placed in the center of a coverslip.
The ring of plasticine was carefully placed with the drop of culture in the center of the
ring. The slide was carefully inverted. It will be examined under the microscope.
3.15 GENERAL PRECAUTIONS
Personal protective equipment such as laboratory coat, hand gloves and face mask were worn
and all laboratory procedures were carried out aseptically to prevent any form of contamination
while working.
3.16 DISPOSAL OF ISOLATES
The plates containing the isolates and all other contaminated materials that were generated in the
course of this work were destroyed by sterilization in an autoclave at 1210C for 15 minutes at
42
15psi (pounds per square inch). After sterilizing, the plastic bag containing the used culture
plates were placed in a container set aside for laboratory waste for its disposal.
3.17 DATA ANALYSIS
Data was entered into Microsoft Excel. Statistical analysis was carried out using SPSS
(Statistical Package for Social Sciences) software package (version 21.0), Chi square and Turkey
Kramer Multiple Comparison Test. Statistical analysis outputs were presented using tables and
charts.
3.18 POST RESEARCH BENEFITS
At the end of this research work, the presence of microbial contamination of cosmetic products
of the participants will be determined and it will be passed on to the participants confidentially.
For participants with contaminated cosmetic products, they will be advised to dispose the
cosmetic products and also avoid sharing cosmetic products in future. Information gotten from
this study will be of immense help to researchers and to the community by raising awareness
regarding microbial contamination of cosmetic products.
CHAPTER FOUR
4.0 This Section Presents the Statistical Analysis and Interpretation of Frequency and
Percentage Distribution of Respondents
Table 4.1: Socio-Demographic Demographic Characteristics of the Respondents
Characteristics CategoryFrequency
(N = 5)Percentage (%)
Age range 18 Years 1 20.0
43
19 - 21 Years 4 80.0
22 – 25 Years 0 0.0
Total 5 100.0
Religion Christianity 3 60.0
Islam 2 40.0
Traditional 0 0.0
Others 0 0.0
Total 5 100.0
Tribe Yoruba 2 40.0
Igbo 3 60.0
Hausa 0 0.0
Others 0 0.0
Total 5 100.0
The result of the total viable count of bacteria isolated from cosmetic samples is shown in Table
2. The total viable counts ranged between 1.3 × 104 cfu/ml to 4.8 × 104 cfu/ml.
44
Table 4.2: Total viable counts of bacteria isolated from Cosmetic samples
SAMPLES Cfu/ml
P1 2.4 × 104
P2 2.1 × 104
P3 3.3 × 104
P4 4.8× 104
P5 1.8× 104
BL1 2.9 × 104
BL2 2.5 × 104
45
BL3 1.3 × 104
BL4 1.8 × 104
BL5 2.0 × 104
M1 2.1× 104
M2 2.3 × 104
M3 2.6 × 104
M4 2.8 × 104
M5 3.1× 104
Keys
P1, P2,P3,P4,P5 = Powder samples collected from female students; BL1,BL2,BL3,BL4,BL5 =
Body lotions collected from female students; M1,M2,M3,M4,M5 – Mascara samples collected
from female students.
Table 4.3: Comparing of the Bacterial Count between the used (old) and new cosmetics
Parameter Group N Mean Std. Deviation Std. Error Mean t-value p-value
Bacterial Count Old 15 2.520 0.827 0.214 11.796 0.000*
New 15 0.000 0.000 0.000
Key: * = statistically significant
46
Table 4.4: Distribution of Types of Cosmetics according to the Biochemical Tests Characteristics
Biochemical Test CharacteristicsPowder
N (%)
Mascaras
N (%)
Body Lotion
N (%)P-Value (X2)
Gram Reaction
Positive 0 (0) 5 (33.3) 0 (0)
.001*Negative 5 (33.3) 0 (0) 5 (33.3)
Total 5 (33.3) 5 (33.3) 5 (33.3)
Catalase Test Positive 0 (0) 5 (33.3) 0 (0) .001*
Not Applicable 5 (33.3) 0 (0) 5 (33.3)
47
Total 5 (33.3) 5 (33.3) 5 (33.3)
Coagulase Test
Positive 0 (0) 2 (13.3) 0 (0)
.099Not Applicable 5 (33.3) 3 (20.0) 5 (33.3)
Total 5 (33.3) 5 (33.3) 5 (33.3)
Morphology
Rod 5 (33.3) 3 (20.0) 5 (33.3)
.099Cocci 0 (0) 2 (13.3) 0 (0)
Total 5 (33.3) 5 (33.3) 5 (33.3)
Indole Test Positive 2 (13.3) 0 (0) 2 (13.3) .005*
Negative 3 (20.0) 0 (0) 3 (20.0)
Not Applicable 0 (0) 5 (33.3) 0 (0)
Total 5 (33.3) 5 (33.3) 5 (33.3)
Citrate Test Positive 3 (20.0) 0 (0) 3 (20.0) .082
Not Applicable 2 (13.3) 5 (33.3) 2 (13.3)
Total 5 (33.3) 5 (33.3) 5 (33.3)
Urease Test Positive 3 (20.0) 0 (0) 3 (20.0) 1.000
Not Applicable 2 (13.3) 5 (33.3) 2 (13.3)
Total 5 (33.3) 5 (33.3) 5 (33.3)
Oxidase Test Positive 3 (20.0) 3 (20.0) 3 (20.0) .099
48
Not Applicable 2 (13.3) 2 (13.3) 2 (13.3)
Total 5 (33.3) 5 (33.3) 5 (33.3)
Motility Test Motile 5 (33.3) 3 (20.0) 5 (33.3) .099
Not Applicable 0 (0) 2 (13.3) 0 (0)
Total 5 (33.3) 5 (33.3) 5 (33.3)
Table 4.5: Distribution of the Bacteria Isolated according to the Types of Cosmetics
BacteriaPowder
N (%)
Mascaras
N (%)
Body Lotion
N (%)
Total
N (%)P-Value (X2)
Escherichia coli 2 (13.3) 0 (0) 2 (13.3) 4 (26.7)
.020*
Pseudomonas spp 3 (20.0) 0 (0) 3 (20.0) 6 (40)
Staphylococcus
aureus0 (0) 2 (13.3) 0 (0) 2 (13.3)
Bacillus spp 0 (0) 3 (20.0) 0 (0) 3 (20.0)
Total 5 (33.3) 5 (33.3) 5 (33.3) 15 (100.0)
49
E.coli27%
S.aureus13%
Pseudomonas40%
Bacillus 20%
Bacterial isolates
Figure 4.1: Pie chart showing the frequency of bacterial isolation from cosmetic samples of
female babcock university students.
50
Table 4.6 shows the growth pattern of the fungi isolated from all the cosmetic samples. The
fungi isolate generally exhibited scanty, slow or profuse growth patterns. The result for the
morphological characteristics of the bacteria isolates for the cosmetic samples is shown in the
Table 3. Most of the bacteria isolates were irregular in shape, opaque and exhibited flat
elevation. Table 4 shows Gram reaction and biochemical characterization of the bacterial
isolates.
51
Table 4.6: Growth patterns of fungi on the cosmetic samples
SAMPLES GROWTH RATE
P1 PRG
P2 PRG
P3 NG
P4 PRG
P5 PRG
BL1 PRG
BL2 SCG
BL3 NG
BL4 PRG
BL5 NG
M1 SCG
M2 PRG
M3 NG
M4 NG
M5 PRG
Keys
52
P1,P2,P3,P4,P5 = Powder samples collected from female students; BL1,BL2,BL3,BL4,BL5 =
Body lotions collected from female students; M1,M2,M3,M4,M5 – Mascara samples collected
from female students.
PRG: Profuse growth
SCG: Scanty growth
SLG: Slow growth
NG: No growth
53
Table 7 shows the morphology of fungal isolates from the cosmetic samples. The fungal isolates
isolated from samples P1, P2, P4, P5, BL1, BL4, M2, M5 had colonies consisting of a compact
white basal covered by a dense layer of black conidial heads. The fungal isolates from samples
BL1 and BL4 had colonies that were fast growing, suede-like, white with greenish conidial
heads. Conidiophores were hyaline, smooth walled bearing terminal verticils of 3-5 metulae with
each bearing 3-7 phialides. Fungal isolates from M1 had colonies that were pale yellowish-
brown, the sporangiophores were brownish and are subglubose. The fungal isolates from BL2
had colonies that were slow growing, they formed a waxy, glabrous, convoluted thallus with a
cream coloured surface, they did not produce microconidia or macroconidia but there was
irregular branching hyphae with prominent cross walls and chlamydospores.
54
Table 4.7: Morphology of fungal isolates from cosmetic samples
SAMPLE Isolate Macroscopy Microscopy Presumptive
identity
Powders P1, P2, P4, P5 colonies with a
compact white
base covered by
a dense layer of
black conidial
heads
Rough walled,
Large conidial
heads, dark
brown, radiate
and biserate with
metulae twice as
long as the
philades.
Aspergillus spp
Body lotions BL1, BL4 Colonies are
rapid growing,
velvety and
cottony in
texture
Conidiophores
were hyaline,
smooth walled
bearing terminal
verticils of 3-5
metulae .
Penicillum spp
BL1, BL4 colonies with a
compact white
base covered by
a dense layer of
Aspergillus spp
55
black conidial
heads
BL2 slow growing,
waxy, glabrous,
convoluted
thallus with a
cream coloured
surface
No microconidia
or macroconidia,
irregular
branching
hyphae with
prominent cross
walls and
chlamydospores
Microsporum
spp
Mascara M1 Pale yellowish-
brown, cotton
like colonies
Hyphae with
rhizoid,
brownish
sporangiosphores
Rhizopus spp
M2, M5 colonies with a
compact white
base covered by
a dense layer of
black conidial
heads
Rough walled,
Large conidial
heads, dark
brown, radiate
and biserate with
metulae twice as
long as the
philades.
Aspergillus spp
56
Table 4.8: Distribution of the Fungi Isolated according to the Types of Cosmetics
FungiPowder
N (%)
Mascaras
N (%)
Body Lotion
N (%)
Total
N (%)P-Value (X2)
Aspergillus spp 4 (66.7) 2 (33.3) 0 (0) 6 (100.0)
.134Penicillium spp 0 (0) 0 (0) 2 (100.0) 2 (100.0)
Microsporium spp 0 (0) 0 (0) 1 (100.0) 2 (100.0)
Rhizopus spp 0 (0) 1 (100.0) 0 (0) 3 (100.0)
57
66%
16%
8%
10%
Fungal isolates
Aspergillus Penicillum Rhizopus Microsporum
Figure 4.2: Pie chart showing the frequency of fungal isolation from cosmetic samples of female
Babcock university students.
58
A B
Plate 4.1:
A- 3- day old culture of Aspergillus niger spp on Sabouraud Dextrose Agar (SDA) plate.
B- Lactophenol blue staining of Aspergillus niger spp under the microscope with ×40
objecvtive showing dark brown rough walled, large conidial heads.
59
A B
Plate 2:
A- Lactophenol blue staining of Penicillum spp under the microscope with ×40 objecvtive
showing smooth walled bearing terminal verticils of 3-5 metulae
B- 3- day old culture of Penicillum spp on Sabouraud Dextrose Agar (SDA) plate.
60
Powders Body lotions Mascaras 0
20000
40000
60000
80000
100000
120000
140000
160000
Bacterial load of the cosmetic samples
Tota
l via
ble
coun
t (cf
u/m
l)
Figure 4.3: Bacterial load of Cosmetic samples
61
CHAPTER FIVE
5.0 DISCUSSION
Cosmetics are products which people use to enhance and care for their outward appearance.
Cosmetic products are substances or preparations that are placed in contact with the various
external parts of the human body. The microbial contamination of personal care products may
already occur in the course of production, through raw materials, ingredients, and during
handling, or through repeated use by the consumer. (NakiSiviri et al., 2006)
In the cosmetics used in this study no physicochemical changes were found that could indicate
microbiological contamination, such as: a change in colour, smell, change in consistency,
appearance of sediment. Similar study results are reported by Hugbo et al., 2003.
According to European Union (EU) legislation cosmetic products must not contain more than
1,000 CFU/ml and in the present study, the bacterial count range from 1.3 × 104 to 4.8 × 104
CFU/ml.
The bacterial species recovered from the cosmetics in this study include: Pseudomonas spp
(40%), Escherichia coli (27%), Bacillus spp (20%)., Staphylococcus aureus (13%).
Pseudomonas spp and Escherichia coli were isolated from the powders, in similar study Ashour
et al. (1989) isolated Staphylococcus aureus, Escherichia coli, Enterobacter agglomerans and
Citrobacter freundii while Budecka and Kunicka-Styczynska (2014) isolated Pseudomonas
aeruginosa, Serratia liquefaciens and Candida parapsilosis in powders applied on the facial
skin.
62
Escherichia coli and Pseudomonas spp were isolated from the body lotions. This agrees with the
study of Okeke and Lamikanra (2001) who recovered Escherichia coli, Pseudomonas
aeruginosa, Klebsiella pneumoniae , Enterobacter aerogenes Staphylococcus aureus,
Streptococcus pyogenes from body creams and lotions. Bacillus spp and Staphylococcus aureus
were isolated from the mascaras used in this study and this agrees with the study of Latricia et
al., (2008) who isolated Staphylococcus spp and Bacillus spp from mascaras.
The current results agree with those obtained by many investigators (Behravan et al., 2005;
Lundov et al., 2009) who found Staphylococcus aureus, Pseudomonas aeruginosa and E. coli
the common pathogenic bacteria in cosmetic samples. Water and materials of animal, plant and
mineral origin used in production of cosmetics may cause their contamination with
microorganisms from the genus Bacillus, Clostridium, Pseudomonas, Micrococcus. (Sodjka,
2003)
In this study, Pseudomonas aeruginosa (40%) was the most prevalent microbe. There is a wide
spread exposure to potential contaminants during manufacture, particularly from raw materials
such as water. Pseudomonas spp can be found in water and soil, some strains are also isolated
from skin, animals and plants. It can also be isolated from the drainage system thus explaining
how the cosmetic samples could have been contaminated. Pseudomonas aeruginosa belongs to
one of the most commonly isolated bacterium from cosmetic products.
Staphylococcus aureus species (13%), being an element of natural human microflora, is
responsible for purulent skin infections, such as: folliculitis, sycosis, boil,
hidradenitis suppurativa and bacterial conjunctivitis. S. aureus may cause bullous
impetigo in newborn babies (SSSS – staphylococcal scalded skin syndrome) caused
by epidermolysine generated by this species (Duggal et al., 2016).
63
In the present study, Aspergillus spp. (66%), Penicillium spp. (16%), Rhizopus spp. (8%),
Microsporum spp. (10%) were the common isolated fungal types. The results in this study are
relatively similar to the results found by Hugbo et. al., 2003, Omorodion et.al., 2014, Gamal
et.al., 2015 who found Aspergillus fumigatus, Pencillium and Microsporium spp in cosmetic
samples.
The high fungal contamination of some cosmetic creams, in the present study is attributable to
the fact that products are often water in oil emulsions, with high concentrations of solutes and
lowered water activity. These conditions are favorable for fungal growth. Fungi lower the quality
of cosmetic products and they can also induce infections of the skin and mucous membranes, as
well as hair and nails.
The slow and scanty fungal growth observed in the cosmetic samples could be attributed to the
fungal spores usually at the resting stage and the subsequent profuse fungal growth of the
cosmetic samples could be attributed to the germinating period of the spores.
5.1 CONCLUSION AND RECOMMENDATION
The study confirms that microbiological contamination of cosmetic product is a
recurrent issue. The microbial contamination of personal care products may occur in
the course of production, through raw materials, ingredients and handling, or the
contamination of a final product may ensue through its repeated use by the user or
in the process of sharing cosmetics with others. Different dangerous bacterial and
fungal genera were found in cosmetic samples tested in this study. They are
pathogens which may cause skin irritation and infections, especially through
wounded epithelium. Due to application area, they can be a serious hazard and the
cause of infections of other parts of the body including eye and nails. To achieve a
64
good protection of cosmetic products against microbial contamination, the industry
must prepare cosmetics under aseptic conditions through effective water treatment,
microbial control of raw materials and equipment disinfection. This study has
identified that cosmetics in use are usually more contaminated with microbes. Users
are advised to maintain good hygiene practices and avoid sharing of cosmetic
products with others. They should also endeavor to change their cosmetics after a
period of time to prevent the harboring of microbes.
65
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