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

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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

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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

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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.

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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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