1

CHAPTER 1

INTRODUCTION

1.1. THE MICROBIOTA OF SKIN

The normal skin of humans is subjected to colonization by various

microorganisms. These microbes are present as commensals on the

surface of the skin. The differences in the moisture content,

temperature, pH, concentration of lipids on the skin, exposure to Ultra

violet radiations and pollutants, use of skin care products are some of

the factors that contribute to the survival of the microbial flora on the

skin. The microorganisms colonize the sebaceous glands, ducts of hair

follicles and the sweat glands (Noble, 1990).

1.2. THE INDIGENOUS MICROBIOTA OF SKIN

The skin is exposed to the environment and due to this constant

contact with the environment, the skin may contain transient

microorganisms. The skin also has well- defined resident flora (Jawetz,

2007) such as Propionibacterium spp.Non- hemolytic aerobic and

anaerobic Staphylococci, Preptococcus spp, Enterococcus spp.

1.3. ACNE VULGARIS

Acne vulgaris commonly called as acne is a skin disease that is most

common during adolescence, afflicting more than 85% of teenagers and

over 40 million people in US alone (Darren et al., 2009, Fried et al., 2006,

2

Taglietti et al., 2008, Shailaja et al., 2012). Acne effects the self-esteem as

it can persist and show the detrimental effects even in adulthood. It is a

chronic inflammatory disease, which effects the pilosebaceous unit. This

condition is a result of increase in the production of sebum which is

induced by androgen, altered keratinization and colonization of hair

follicles by P.acnes. 20 % of the teenagers are affected by facial scarring

(Hywel et al., 2012).The many symptoms of acne include the formation of

pustules, nodules and cysts. Acne scarring is caused mainly due to the

inflammatory lesions and it is of great concern as it effects the patient

psychologically (Nakatsuji et al., 2009). P.acnes can also cause

endophthalmitis after an intraocular surgery. It is also known to cause the

chronic blepharitis. (Dali et al., 2001).

1.4. AETIOLOGY OF ACNE

Acne effects the sebaceous follicles in the face, chest, shoulders and back.

The aetiology is multifactorial and is caused due to the four factors which

are pathophysiological. The factors include seborrhoea, formation of

comedones, colonization of the ducts the organism, action of the host

immune system results in the inflammation.

1.4.1. SEBORRHOEA

The composition of sebum is species specific. The synthesis of sebum

lipids involves two main biosynthetic pathways which leads to the

synthesis of triglycerides, free fatty acids, esters, synthesis of squalene and

3

cholesterol (Downie et al., 2004). Sebum secretion increases the lipid

concentration and the clogged pores lower the oxygen concentration.

These conditions attract the P. acnes residing on the skin and the bacteria

multiply in the hair follicles, which results in the local inflammation of the

skin. Cysts, pustules and scars are formed due to the failure of the

immune system to kill or remove the microorganisms.

1.4.2.COMEDONES

The formation of comedones is due to the hair follicle and sebaceous gland.

The opening of the follicle is plugged due to the sebum which combines

with the excess keratin (Hywel, 2012, Downie et al., 2004).

Microcomedones are the first acne lesions which can be seen histologically

while comedones are the clinically visible lesions. The composition of the

sebaceous lipid, androgens, local cytokine production, hyper proliferation

of keratinocytes in the affected area and changes in the expression of

keratinocyte integrins of the infra infundibulum, may be some of the

factors that are involved in the induction of this process. (Oprica 2006,

Pawin et al., 2004)

1.5. ORGANISMS CAUSING ACNE

1.5.1. Propionibacterium acnes

P. acnes was first isolated from cheese (Oprica, 2006). P. acnes can also be

isolated from acne. It is aero tolerant, gram-positive and non-spore forming

4

organism present on the skin. It effects the glands of the sebum, follicles

and causes acne (Bojar et al., 2004). These rods can grow in presence of

oxygen at reduced rates since they possess oxygen de-toxifying enzymes.

The amount of Guanine and Cytosine is very high and this is a

distinguishing character of Propionibacterium spp. (Oprica, 2006; Eady et

al 1994, Johnson et al., 1972).They colonize the facial skin but are not

pathogenic and they grow in the deeper layers of skin due to the low levels

of oxygen. The peptidoglycan layer makes them more stable and protect

the organisms from shock and stress. Propionibacteria produce lipases,

that breakdown the lipids, produce proteases that liberate arginine. The

products of enzymatic action are used as sources of carbon and nitrogen

(Wilson, 2005).

1.5.2. PATHOGENESIS of Proponibacterium acnes -

P. acnes is generally considered as a harmless commensal due to its low

virulence. The organism may become pathogenic in immuno-compromised

patients. The organism can attach to oleic acid from sebum in the skin

and this fatty acid promotes the co-aggregation of the bacteria (Yu et al.,

1997).The degradation products of glucose and fructose, breakdown

products of the lipids, certain enzymatic reactions result in the

inflammation of the tissue without the involvement of the immune

response (Csukas et al.,2004). The exocellular enzymes and other bioactive

products like Lipase, Phospholipase C, Proteinase, Hyaluronidase,

5

Neuraminidase, Acid phosphatase, Bacteriocins, Histamine and

Tryptamine may act as true virulence determinants (Eady et al., 1994).

The long standing inflammation of P. acnes may be due to the resistance

of the organism to phagocytosis and its intracellular persistence in the

macrophages for a long time. This could be due to the cell wall structure

of the organism. The P. acnes persistence in tissue may explain the

longevity of inflammatory acne lesions (Webster, 1995).

The bacteria which are opportunistic, colonize the tissue, as the damage

and inflammation caused by acne causing organism makes it more

susceptible to infection by S. aureus and S. epidermidis. It is not known if

it is just a casualty, opportunistic or co-existence of the organisms

resulting in complex pathogenicity (Bek- Thomson et al., 2008).

1.5.3. Staphylococcus epidermidis

S. epidermidis which is a part of the normal flora, is gram positive and do

not produce coagulase. It effects the skin when there is a lapse in the host’s

defense mechanism or the innate immunity. It grows under aerobic and

anaerobic conditions in glucose, uracil and biotin containing complex

medium. It cannot ferment mannitol and also lacks the enzyme coagulase

(Parisi, 1985; Jones et al., 1963).

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S.epidermidis known to be associatedwith acne, causes nosocomial

infections and forms biofilm on the biomaterials due to its ability to

produce a glycocalyx. It also develops resistance to phagocytosis (Peters,

et al., 1982).

1.5.4. Staphylococcus aureus

Staphylococcus aureus is a gram-positive, facultative anaerobic cocci. It

forms a part of the normal flora on skin and is an opportunistic pathogen.

It is highly infectious and spreads by means of personal contact with

infected person or wounds with pus. It is widely seen in persons with

dermatitis.

1.6. TREATMENT OF ACNE

P.acnes is susceptible to the antibiotics like benzyl penicillin, amoxicillin,

cephalosporin, clindamycin (CL), erythromycin (EM), tetracycline (TET)

and the combination of Penicillin and β-lactamase inhibitors (Bansalet al.,

1984; Chow et al., 1978; Denys et al., 1983).

Antibacterial treatment of acne is possible by using either antibiotics,

topical application of BPO, retinoids, salicylic acid and hormonal therapy.

1.6.1. ORAL - ANTIBIOTIC TREATMENT

Systemic administered antibiotic treatment represents the most widely

used therapy. Antibiotics with therapeutic value in acne are lipid soluble

substances which may concentrate in the pilosebaceous unit. (Bojar et al.,

7

2004). The antibiotics used in the acne treatment are concentrated by the

leucocytes and are delivered at the site of inflammation where the local

concentrations can become higher than the serum concentrations

(Murdoch et al.,1991)

Cycline antibiotics such as TET hydrochloride, oxytetracycline,

doxycycline, Macrolides like erythromycin, oleandomycin, azithromycin,

clindamycin, trimethoprim, co-trimoxazole, fluoroquinolones like

Levofloxacin are used to treat acne (Oprica et al., 2006).

The antibiotics reduce the levels of P. acnes in acne by

i. Inhibiting the protein synthesis – Cyclines and macrolides

inhibit the protein synthesis. Tetracycline prevents the

association of amino acyl t- RNA with the codons. Inhibition of

enzymes in the bacterial pathway for the production of

Tetrahydrofolate (trimethoprim).

ii. Inhibition of enzyme bacterial gyrase (quinolones)

iii. Inhibition of production of pro-inflammatory mediators.

1.6.2. TOPICAL ANTIBACTERIAL TREATMENT

The topical antibiotics useful in treating mild to moderate acne. EM, CL

and TET are most widely used antibiotics for topical application. (Eady et

al., 1994). These antibiotics act as anti-inflammatory agents, reduce the

8

number of bacteria (Toyoda et al., 1998) and bring about the reduction in

lesions (Simpson et al., 2004).

1.6.3. BENZOYL PEROXIDE (BPO)

Benzoyl peroxide (BPO) brings about the reduction of acne lesions. It also

brings down the severity of the infection. It can be used alone or in

combination with antibiotics or other treatments available for treating

acne. It has a cidal effect on acne causing organisms and resistance to

BPO has not been reported (Simonartet al., 2012, Seidleret al 2010,

Tanghettiet al., 2009).

1.6.4. DRAW BACKS OF THE PRESENT TREATMENTS

1.6.4.1. Antibiotic Resistance

The management of P.acnes infections includes the combination of

intravenous, intramuscular, oral antibiotics, topical application of

antibiotics and BPO. EM or azithromycin are rarely used due to resistance

and cross resistance to CL (Dreno, 2004). Clindamycinis not so commonly

used due to a possible side effect – pseudomembranous colitis.

1.6.4.2. Factors that promote resistance in P.acnes

Low dose of antibiotic, use of antibiotics belonging to chemically distinct

groups simultaneously, poor treatment compliance are some of the factors

which cause antibiotic resistance (Oprica, 2006; Eady,2004).

9

P.acnes resistance may be due to the mutations in the peptidyl transferase

enzyme of the 23 S r RNA (Ross et al, 2003). If the organism is resistant to

a particular antibiotic, it becomes resistant to the antibiotics of that class

(Eady et al., 1998).

Antimicrobial agents (such as Benzoyl Peroxide) have been used

epicutaneously to treat acne for several decades and are still widely

prescribed for acne patients, suffering from mild to moderate acne. The

oxidizing agent BPO (Benzoyl peroxide), one of the most frequently used

epicutaneous medication has several side effects such as erythema,

scaling, burning and flare (Tanghetti et al; 2009, Castro et al., 2008).

Treatment of acne involves use of antibiotics for a long duration. The

treatment becomes difficult due to the antibiotic resistance shown by the

organism. Non – antibiotic treatment such as the use of nanoparticles

might be the best alternative to treat acne.

1.7. NANOTECHNOLOGY

It deals with the particles in the nanorange (< 10-7 m or 100nm). Unlike

bulk particles, properties of the nanomaterials vary based on the size.

(Kalaiselvam et al., 2012). Supra Para magnetism and SPR are

characteristics of the nanoparticles. The nanomaterials have vast

applications in the field of drug delivery, imaging and treatment of

infections.

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1.8. NANOPARTICLES

They can be defined as particles of nano size. A nano powder by definition

is “an agglomeration of non-crystalline nano structural subunits with at

least one dimension less than 100nm.” Nanocrystals have at least one

dimension less than 100nm and are homogenous. “Composite molecules,

including organic, polymeric inorganic materials and more advanced

nanomaterials are being developed to suit various objectives” (Ghosh et

al., 2012).

1.8.1. Applications of nanoparticles

Researchers are developing a variety of products based on nanoparticles,

some for public consumption in the market. They are presently used in

liquids used for cleaning the floor which can easily absorb grime and dust

particles (Ballauff, et al., 2007).

They also have many applications in the field of medicine due to their small

size and high toxicity towards the microorganisms. Also, nanoparticles are

known to be key players against cancer. They are useful in diagnosis,

biosensors in and in the adjustment of taste and flavor in various foods

and drinks (Sharma et al., 2009).

1.8.2. Zinc oxide nanoparticles

Nanoparticles specifically the oxides of the metal are used in the

agricultural fields, bio diagnostics and various industries (Kolodziejczak et

11

al., 2014). ZnO nanoparticles are more ionic and have more surface area

this makes it more advantageous than the other antimicrobial compounds.

It is also stable for long duration. They are also used in industries for

purification of water and in the preparation of different cosmetic products

(Ravishankar et al., 2011). The specific activity of the nanoparticles against

the microorganisms makes them good and effective antimicrobial agents

and thus have grabbed the attention of many researchers (Allahverdiyev

et al., 2011). They are excellent antibacterial agents (Huang et al., 2008)

and anti-fungal agents (He et al., 2011).

1.8.3. Antibacterial activity of the ZnO nanoparticles

Compared to the particles of bigger size, the particles in the nano range

are more effective because of the larger surface area which ensures more

interactions with the bacteria. Studies suggest that zinc oxide nano-

particles bring about bacterial cell mortality by increasing the permeability

of the bacterial cell membrane, leading to a defect in the membrane

transport systems. On entry into the bacterial cells, the zinc nano-particles

react with the proteins (enzymes, cellular proteins) and nucleic acids

(especially DNA), and denature them, thus inhibiting the replication. One

of the main advantages of employing zinc oxide nano-particles as anti-

bacterial agents is that there are very few chances of the bacteria

developing resistance, since their activity is nonspecific. However, their

inhibitory effect is based on concentration, the amount of bacterial load

12

and the type of bacteria in the sample. It has also been found that they

prevent the formation of bio-films by preventing the attachment of bacteria

onto the host cell surface. These features of zinc oxide nano- particles

suggest that they would make excellent anti- microbial agents for

treatment of various infections.

1.8.4. Selective toxicity of ZnO nanoparticles

The cancerous cells are more sensitive to the effect of the nanoparticles

than the quiescent cells which are least sensitive. Cytotoxic studies and

hemolysis carried out by Prashant et al., conclude that the ZnO

nanoparticles are biocompatible, as the interaction of ZnO nano powders

with the RBC revealed a hemolysis per cent of greater than 5 at a

concentration of 5mg ml-1 of ZnO nano powders (Prashanthet al 2015).

The research carried out aims to propose a novel way of treating acne using

creams prepared with ZnO nanoparticles as the treatments available for

acne which include application of BPO, antibiotics, retinoid etc., though

effective, either have side effects or the organism develops resistance to

these treatments. Hence it becomes very important to employ alternative

treatments like the use of nanoparticles which are very effective

antimicrobial agents. ZnO in its bulk form is an active ingredient for

dermatological application in creams, lotions (Hughes et al., 1988).

However zinc oxide nanoparticles possess excellent durability, heat

13

resistance and are more effective antibacterial agents (Sawai, 2003). “Zinc

oxide nanoparticles also have good biocompatibility to human cells” (Lili

Heet al., 2011).These nanoparticles are an important source of zinc, which

is involved in various essential biochemical reactions in the body, since

they function as enzyme co-factors (Anitha et al., 2010, Sara et al., 2014).

They also help in maintaining a healthy immune system. Zinc oxide nano-

particles are also relatively good antimicrobial agents, whose activity

increases with a reduction in the particle size (Lili Heet al., 2011, Sara et

al., 2014).

To achieve this objective the organism causing acne was isolated and

identified on the basis of cultural characters and biochemistry, molecular

characteristics. ZnO was synthesized and characterized by using different

methods and its effect against bacteria was tested. Invitro testing using

standard agar well diffusion method and in vivo testing in Mice against

P.acnes induced inflammation was done in order to prove their therapeutic

potential. Invitro cytotoxic testing was done using MTT [3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay in MCF 10

A ( human epidermal cell lines).

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GENERAL OBJECTIVE

The present research was designed to find out the possibility of treating

acne using skin products prepared with ZnO nanoparticles.

SPECIFIC OBJECTIVES

The specific aims of the studies were

• To Isolate and identifyacne causing organism

• To synthesise and Characterize Zinc oxide nanoparticles.

• To test the antibacterial activity of Zinc oxide nanoparticles against

acne causing organism.

• To test the stability of ZnO nanoparticles with time and temperature.

• To prepare cosmetic skin care products with nanoparticles to be

used on the skin.

• To study the antibacterial activity of the cosmetic skin care products

with nanoparticles invitro by standard agar well diffusion method

• To test the stability of creams and emulsions with time.

• To test the Cytotoxicity of zinc oxide nanoparticles in Mice in which

P. acnes infection is induced.

To test the invitro cytotoxicity of zinc oxide nanoparticles in human

epidermal cell lines

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CHAPTER 2.

REVIEW OF LITERATURE

2. An overview of background research- major contributions

S.No Year Authors Contribution

1. 1963 Kirschbaum et al., Proved significant role of

Propionibacterium acnes in causing acne by injecting viable organism

into sebaceous glands.

2. 1972 Martin et al., Reported the use of antibiotics to

treat acne.

3. 1977 Kligman et al., Reported that benzoyl peroxide can be used in treating acne

4. 1988 Kurokowa et al., Published the mechanism of resistance shown by

Propionibacterium acnes to antibiotics

5. 1994

Hegemann et al., Studied the mode of action of benzoyl peroxide

6. 2009 Tanglietti et al., Reported the side effects of benzoyl peroxide

7 1987 Sugimoto et al., Reported the antimicrobial activity of silver and Zinc oxide

8. 2007 Serpone et al., Reported the use of nanoparticles in dental products

9. 2012 Singh et al., Demonstrated the selective toxicity of ZnO nanoparticles to microorganisms

10. 2012

Chitra et al., Published the use of ZnO nanoparticles against food borne

pathogens

11. 2013 Raoufi et al., Reported that the ZnO nanoparticles

are cost effective

12. 2014 Sara et al., Reported that ZnO nanoparticles are good antimicrobial agents and

activity of nanoparticles increases with reduction in size

13. 2015 Prashanth et al., Carried out cytotoxic studies on ZnO nanoparticles and concluded that

they are biocompatible

16

Our analysis of the literature reveals that Propionibacterium acnes is the

main causative organism of acne and benzoyl peroxide, antibiotics,

retinoids which are available to treat this condition cause side effects.

Hence there is a need for finding an alternate treatment for acne. The

present research was designed to find out the possibility of treating acne

using nanotechnology methods. Literature also shows that ZnO

nanoparticles are effective antimicrobial agents and are biocompatible

and no studies were carried out to test the antibacterial activity of ZnO

nanoparticles against acne.

The main objective of this study is to propose a novel way of treating

acne using ZnO nanoparticles.

17

2.1.NANOPARTICLES

“Nanoscience and nanotechnology has been leading to a technological

revolution in the world, which is concerned with materials with

significantly novel and improved physical, chemical and biological

properties.” (Wani et al., 2012, Sundararajanet al.,2012).Nanosized

materials have proved to be very effective agents in treating bacterial

infections (Raghupathi et al., 2011).

Nanoparticles like Au, Ag, Fe, TiO2, ZnO etc., are inorganic in nature. Some

of them possess magnetic properties while others act as semiconductors.

Because of the excellent durability, stability, compatibility and versatility

the nanoparticles can be used in imaging, diagnosis of infections,

treatment and drug delivery. Glass was colored using the gold

nanoparticles (Xu et al., 2006, Colomban, 2009, Corti et al., 2002).

Nanometal oxides like zinc oxide, calcium oxide and magnesium oxide are

very effective against the microorganisms, do not need activation with

light, have good stability and are materials graded as GRAS(Stoimenov et

al., 2002).

The oxides of silver and zinc in the nanorange are effective against the

diseases (Sugimoto, 1987). The size depends on attachment, aggregation

and growth (Lifshitz et al., 1961). Bulk particles of TiO2 stabilize on

reduction in size. Iron, nickel, cobalt, manganese and zinc are used in

biomaterials and electrical equipments (Snelling, Eric Charles., 1989;

Willard et al., 2004).Study related to ZnO nanoparticles shows that they

18

have good antimicrobial activity against various groups of microbes (Rai et

al., 2009).

2.2. ZnO NANOPARTICLES

They have vast application in industries related to pharma and cosmetic

products (Denget al., 2008; Krishna kumar et al., 2008). It is a good metal

oxide and has wide range of uses in acoustic wave devices, can be used as

a semiconductor and is cost effective (Raoufi, 2013). It is very effective

antibacterial and antifungal agent. It also kills the spores but is not toxic

to the human cells as it is very effective against the microorganisms at a

less concentration(Karvani et al., 2011, Singh et al., 2012). The selective

toxicity of the nanoparticles is because they rupture the phospholipid

bilayer specific for the bacterial cell and this results in the release of the

cell contents (Singh et al., 2012).

2.3. SYNTHESIS OF ZINC OXIDE NANOPARTICLES-

Zinc oxide nano particles are synthesized by different methods that can be

used as anti-microbial agents against food borne particles has been

reported by Chitra and Annadurai, in their study on ‘Anti-microbial

activity of wet chemically engineered spherical shaped ZnO nano particles

on food borne pathogens’ (Chitra et al, 2012). They studied the anti-

microbial activities of ZnO nano particles against E. coli andPseudomonas

aeruginosa.

19

Sangeetha Gunalan et al., 2012 performed various microbiological tests

using different concentrations of green and chemically synthesized nano

particles and found that green synthesized nano particles showed better

biocidal activity against different pathogens and the effectiveness of nano

particles increased with increase in particle dose, duration of treatment

and synthesis method. They also reported that various factors like the size

of the particle and high surface to volume ratio influence the antimicrobial

activity of the nanoparticles (Gunalan et al., 2012).

“Various reports suggest that about 500 and 50-30,000 tons of Zinc Oxide

nanoparticles are produced annually around the world” (Piccinno et al.

2012). Zinc oxide nanoparticles are the third most produced nanoparticles

after silica and titanium dioxide nanoparticles. Zinc oxide nanoparticles,

are generally used as an ingredient in dental products (Serpone et al.

2007). They are also used in electronic appliances like Liquid crystal

display, textile industries etc., (Dastjerdi et al., 2010).

2.4. ACNE:

Stern in 1996 reported that acne also called as Acne Vulgaris is a disorder

and main reason for persons aged between 15 and 45yrs to visit a

dermatologist. The mean prevalence of the disease is between 70 to 87%.

It effects both men and women. Ladies, because they are more conscious

about their skin and beauty visit dermatologist (Stern, 1996). The

evaluation of inflammation shows that the infection is more common in

20

teenagers than in adults and it is difficult to analyze the reason for

this(Dreno et al., 2003). Same pattern of evolution regarding age of onset

was noticed which is 12 yrs in boys and 11 yrs in girls, with the

predominance of comedonal acne (Dreno et al., 2003). Significant pre-

pubertal acne is seldom found to be a sign of an endocrine abnormality

(Simpson et al., 2004).

2.5. Role of Propionibacterium acnesin causing acne:

P. acnes is the major causative organism of acne for more than 100 yrs.

The association was reinforced after the antibiotic were used in acne

treatment. However, acne is not an infection and Koch’s postulates are not

applicable to the disease. It is difficult to allocate a pathogenic role to a

bacterium when it is present on both normal skin and in a diseased

condition (Bojar et al., 2004). The bacterial population co-relates with

decrease in the content of the lipids (Mc Ginley et al., 1980), the chronic

nature of infection is not proportional to the number of bacteria (Simpson

et al., 2004).

The first evidence of P.acnes significance in acne came from an

in vivo study in which the injection of concentrated viable, but not dead P.

acnes or other bacteria into a sterile cyst of Steatocystoma multiplex ( cyst

formed in pilosebaceous glands) produced inflammation (Kirschbaun et

al., 1963). Moreover, the clinical improvement of acne with antibiotics

reinforces this hypothesis. Some authors have suggested that the

21

pilosebaceous gland itself may produce immune factors and P. acnes will

amplify this initial immune response (Zouboulis, 2001). It was

demonstrated that P. acnes stimulates the proliferation of the lymphocytes

by specific antigens and the production of non-specific mitogens (Jappe et

al., 2002). A few cases of the primary infections caused by P. acnes have

been documented. Primary purulent folliculitis dissimilar from acne

vulgaris (Maibach, 1967), acute meningitis (Schlesinger et al., 1977),

Osteomyelitis (Suter et al., 1992), Sepsis and different eye infections have

been reported (Eady et al., 1994). An outbreak of P. acnes postoperative

shoulder arthritis has been found in non-debilitated patients resulting

from a poor efficiency of the ventilation (Lutz et al., 2005).

P. acnes mainly causes acne. It is now a main concern to medical

practitioners as it is growing as a biofilm on biomedical implants (Eady &

Ingham, 1994, Brook & Frazier, 1991, Ahn et al., 1994, Delahaye et al.,

2005).

2.6. Propionibacterium acnes- resistance to antibiotics:

The long term use of antibiotics effects the microbial ecology. The normal

microflora represents a barrier against colonization by pathogenic bacteria

and colonization by already present microorganisms, i.e., colonization

resistance. Acne patients who are in general heavily treated with

antibiotics may suffer such disturbances (Sullivan et al., 2001a).

22

Antibiotics have been prescribed for more than 40 yrs for treating acne,

Erythromycin and Clindamycin are generally prescribed for acne. The

organism became resistant even before the application of these antibiotics

onto the skin. This mechanism of resistance was observed in

Propionibacteria strain collected from clinic (Martin et al.,

1972).Resistance to these antibiotics was observed in United States of

America (Leyden et al., 1983). This mechanism of resistance shown by

Propionibacterium acnes have been published across the world by various

researchers (Kurokowa et al., 1988, Ross et al., 2003).

In Europe about 50% of the people with acne were infected by

Propionibacterium strains resistant to erythromycin and clindamycin.

Twenty percent of the people were infected by Propionibacteria strains

resistant to the antibiotic tetracycline in Europe(Ross et al.,2003).

Several factors contribute to the mechanism of resistance shown by the

organism. Administration of the drug at less concentration and

development of resistance among the normal flora of the skin are some of

the reasons responsible for resistance (Dreno et al., 2004).Treatment using

chemically different antibiotics simultaneously may result in multiple

resistant P. acnes (Eady et al., 1998).

Genetic basis as one of the factors was reported by Ross et al., (Ross et al.,

1997). In a group of patients receiving oral TET( tetracycline ), pronounce

23

changes in the colon microflora and new colonization with TET resistant

strains occurred (Borglund et al.,1984).

2.7. TREATMENT OF ACNE USING BENZOYL PEROXIDE (BPO)

Benzoyl peroxide was used in treating acne as early as in 1960, when

William Pace used precipitated Sulphur cream that contained BPO. BPO

has both comedolytic and antibacterial effects. Evidence for the

comedolytic effect comes from several studies. Work with the

experimentally induced comedones by Oh and Myung (Oh and Myung,

1996) and Kaidbey and Kligman demonstrated that 5 per cent of BPO was

an effective comedolytic agent. The bactericidal properties of the drug have

been well recorded (Kaidbey et al., 1975). Bojar et al., showed that the

application of 5 per cent BPO gel removed the bacteria both from the skin

surface and follicles (Bojar et al., 1995).

Kligman has suggested that twice daily application of BPO for five days will

reduce P. acnes population more than 95 % (Kligman et al., 1977).

BPO acts against the acne causing organisms by releasing the free radicals

that act on the membrane proteins (Rod Tucker and Shenaz Walton,

2007).

In addition Fulton (Fulton et al., 1974) and Cunliffe (Cunliffe et

al.,2002,2004) reported that BPO acts on the lipids and reduces its

content significantly. It kills the polymorpho nuclear cells and removes

24

inflammation. It blocks the reactive oxygen species from being released.

(Hegemann et al., 1994).

2.7.1. Possible side effects of BPO gel

Treatment with benzoyl peroxide takes longer time. Some medicines may

interact with BPO gel. BPO use can make the skin more susceptible to sun

burn. Dryness, irritation, itching, peeling, redness, stinging, swelling of

the skin are some of the side effects. Use of BPO may also result in

excessive burning, itching, redness or tenderness (Tanghetti et al., 2009)

Due to the side effects of BPO, retinoid and development of resistance by

the organisms to antibiotics, there is a necessity to employ an alternate

treatment method for acne. The main aim of this research is to evaluate

the antibacterial activity of Zinc oxide nanoparticles against acne causing

organisms and propose the treatment of acne using cosmetic skin care

products prepared with the ZnO nanoparticles.

25

CHAPTER 3

MATERIALS AND METHODS

3.1. ISOLATION OF ACNE CAUSING ORGANISMS

BHI (Brain heart infusion )agar,tryptic soy and thioglycollate broth, ken

knight’s broth, Cassmans media, Clostridial agar were used for the

isolation and culturing of acne causing organisms. The media were

procured from Hi Media. The glassware used was of borosilicate make and

the chemicals of Analytical grade.

For the isolation of acne causing organisms, about 50 samples were

collected from students aged between 18-21 yrs. A 1 cm2 area of the facial

skin of the volunteers was cleansed and with a sterile swab the sample

was collected and then the swab was placed in a test tube containing 10ml

of thioglycollate broth and tryptic soy broth. The broth was incubated for

4days under anaerobic conditions. After incubation, the culture was then

streaked onto BHI and Clostridial agar and incubated for 7 days at 37 o C

in an anaerobic chamber with a disposable carbon dioxide generator

envelope (Gas Pak). After incubation, different colony types based on their

morphology were identified. Marples and Mc Gliney have previously shown

that Propionibacteria can be presumptively selected based on the colony

morphology upon primary sub cultivation (Marples et al., 1974).

From each isolated sample, one colony of different morphology were

cultured anaerobically into BHI broth and the pure cultures were

26

preserved and maintained on BHI agar slants and as stab cultures on BHI

agar tubes.

3.2. CHARACTERIZATION OF ACNE CAUSING MICROORGANISM

The bacteria are characterized based on gramnature, biochemical tests,

fatty acid analysis (FAME) and 16S rRNA gene sequencing.

3.2.1. Biochemical characterization (Prescott,2002)

The biochemical characteristics of the strains of anaerobes isolated from

the persons suffering with acnes were analyzed by sugar fermentation

tests (glucose, sucrose, Maltose, Esculin, Adonitole), TSI test, IMViC

reactions, nitrate reduction, urease, catalase production and Gelatin

hydrolysis

3.2.1.1. Sugar fermentation tests

Bacterial identification can be done based on their ability to ferment

different sugars. These reactions provide the required energy to the

microorganisms. Different microorganisms ferment different types of

sugars because of the presence of enzymes required to carry out that

particular reaction. The microorganisms ferment the carbohydrates and

produce various acids and alcohols like lactic acid, butyric acid, propionic

acidespecially when the culture media contains reducing sugars like

glucose. The production of these acids brings down the pH which brings

about a change in the colour of the medium when a pH indicator is

27

incorporated in to the medium.Gas released into the medium is entrapped

in the form of a bubble inside the durham tube which can be visualized.

To the peptone water, 0.5% of sugars -glucose, sucrose, maltose, esculin,

adonitole were added to each of the labelled tubes and a 0.01% of phenol

red was added as an indicator. After sterilization 0.1 ml of the culture was

added and the tubes were incubated and observed.

3.2.1.2. Esculin Hydrolysis

Esculin, a hydroxyl coumarin is hydrolysed by certain esculin positive

bacteria resulting in the production of esculetin. This compound forms a

black colored diffusiblecomplex on reaction with the ( ferric ) Fe+3 ions

present in the broth. β- D-Glucose is another product formed.This test is

performed to differentiate Propionibacterium acnes from the other species

of Propionibacterium spp. like P. avidum.

Esculin Hydrolysis test

28

3.2.1.3.Triple Sugar Iron agar (TSI Agar):

This test is performed to analyze the ability of the organism to utilize

glucose- the reducing sugar, disaccharide lactose which is made of

galactose and glucose, disaccharide sucrose – the table sugar. To perform

the test 200 ml of agar was prepared by dissolving the TSI agar – in water

(6.5%) and homogenizing it on a hot plate. It was then poured into the

tubes capped with the screw caps or plugged with cotton. The tubes were

sterilized at 121oC for 15 min. The tubes were placed in a slanting position

after autoclaving in such a way that the culture can be streaked on to the

agar slant and stabbed into the butt. It was placed for incubation at 37oC

for 3-4 days under anaerobic conditions. If the organism ferments any of

the sugars, it is indicated by a change in the color of the medium. A crack

in the butt indicates the release of gas.

3.2.1.4. Test for Indole

To 1.5 % peptone water 1 % of Tryptophan was added and the media was

mixed well and heated. 5 ml of media is added to the tubes and sterilized

for 15 min at 121 oC. A loopful of the culture was added to the tubes and

were kept at 37 oC for 4 days under anaerobic conditions. After incubation,

0.5 ml of Kovac’s reagent was added and mixed well. A pink or red ring

observed on the surface of the medium indicates that the reaction is

positive for indole.

29

3.2.1.5. Methyl red test (MR Test):

To perform the test aqueous media containing 5 % of glucose and 5 % of

peptone was prepared. Potassium biphosphate (K2HPO4) was added as a

buffering agent. The media was homogenized by heating and then

dispersed into the tubes. The tubes were autoclaved. Loopful of the culture

was added to the medium and incubated at 37 oC for 4 days. On addition

of the indicator methyl red, change in the colour of the medium to red

indicates a positive reaction.

3.2.1.6.Voges- Proskauer test (VP Test)

Medium containing 5 % of glucose and 5 % of peptone was prepared to

carry out VP test. The acne causing organisms were inoculated in to the

medium and kept at 37 oC for incubation. After 4 days of incubation 5%

of alpha naphthol and 40% KOH was added and mixed well. Change in

color to red shows that the reaction is positive.

3.2.1.7. Citrate Test

This test is performed to analyze the ability of the organisms’ ability to

utilize the citric acid as a source of carbon. The agar was prepared by

dissolving 0.025 percent of trisodium citrate (Na3C6H5O7),

Ammonium phosphate [(NH4)3PO4],Magnesium sulphate (MgSO4),0.1% of

and 0.01 % of the indicator bromothymol blue and agar. The medium was

added to the tubes, plugged and sterilized. Culture was streaked on to the

30

slants and placed in gas pak at 37 oC for 72 h. Change in the color of the

medium from green to blue indicates the utilization of citrate as a sole

source of carbon.

3.2.1.8. Urease test

Christensen’ s urea agar was used to perform the test. The media

components were weighed, dissolved in water and added to the tubes and

sterilized. Urea which was sterilized separately was added to the tubes.

The acne causing organisms were inoculated on to the agar slants and the

change in color from yellow to pink indicates a positive test.

3.2.1.9. Gelatinase test

Microorganisms can liquefy gelatin due to the secretion of

gelatinase.Gelatin hydrolysis, tests the virulence of the organism and it is

used for breaking the connecting tissue and causes infection. Gelatin agar

slants were inoculated with the culture and incubated. After incubation

the tubes are placed in the refrigerator at 4oC for 30 mins. If the test is

positive, the surface of the medium liquefies. If there is no hydrolysis then

there is no liquefaction.

3.2.1.10. Nitrate reduction

Chemoorganoheterotrophic bacteria that require organic compounds for

growth. This test is used to detect the ability of the organism to produce

enzyme, nitrate reductase that converts Nitrate (NO3-)toNitrite (NO2-)ions

and produce a red color indicating a positive reaction. To carry out this

31

test broth containing nitrate and Potassium permanganate (KMnO4)is

used.NO2- ions presence is confirmed by using 2 reagents. Reagent A and

Reagent B (details mentioned in appendix III .1). Negative test is confirmed

by the addition of Zn powder. Zinc reduces nitrates to nitrites and turns

the medium red.

The pure culture was inoculated into the nitrate broth under aseptic

conditions and incubated for 4 days at 37oC and checked for growth. 0.5ml

of the reagent A and B were added to the tubes and checked for color

change. Negative reaction is confirmed by the addition of Zn Powder.

3.2.1.11.Catalase Test

The culture was added to a drop of hydrogrn peroxide (H2O2)solution and

observed for the presence of gas bubbles.

3.2.1.12. Hemolysis:

Hemolysis is observed because of the hemolysins secreted by the bacteria.

The organisms were plated onto the Blood Agar plates containing 10%

defibrinolysed sheep blood to test for Hemolysis. The plates were incubated

anaerobically for 4 days at 37 oC.

3.2.2. TEST FOR BIOFILM FORMATION

2g of Congo red and 12.5g of Sucrose were taken and sterilized separately

in an autoclave at 10lbs for 10 mins. Sterilised BHI agar medium was

mixed with congo red and sucrose and poured into petri plates. The

organism was streaked and petri plates were incubated for 48 h.

32

Formation of the biofilm is indicated by the formation of black colored

colonies(Nabajit Deka,2014).

3.2.3. CHARACTERIZATION OF THE ORGANISM USING FAME

ANALYSIS:

Microorganisms have fatty acids in their cell membrane and are the

components of the phospholipids. These fatty acids are present either as

free fatty acids or methyl esters can be analysed by using GC. Alkylation

reagent is used for the esterification of the fatty acids. The analysis of the

fatty acids can be used as a tool to identify and classify the microorganisms

(Goodfellow et al., 1985). “The MIDI (Microbial Identification Incorporation)

Sherlock FAME analysis is a laboratory-based system that can be used on

a routine basis to identify commonly isolated bacteria from clinical and

environmental source (Leonard et al., 1995)”. FAME analysis was carried

out using Agilent 6850 Series II. Anaerobic library BHI was referred for the

analysis. FAME’s were analysed by using GC and the chromatogram was

used in the classification and identification of the pathogenic organisms.

3.2.4.MOLECULAR CHARACTERIZATION

The 16S ribosomal ribonucleic acid analysis is used to identify the

organisms especially those that cause infections and difficult to culture

due to their nutritional requirements. This is a quick method and helps in

identification of the organism upto species level. Many data bases are

33

available that are used to identify the organisms based on the 16 S rRNA

gene. In the present study, the Ez Taxon data base is used. Ez Taxon by

Chun lab is useful to identify many isolates as it has valid strains of

various species of microorganisms which are published on the web. It is

an open access database.

3.3. MTCC cultures (Microbial Type collection Centre)

Standard cultures Propionibacterium acnes MTCC 1951, MTCC 3297 were

procured from IMTECH, Chandigarh and were used as test organisms. The

cultures were grown in BHI broth under anaerobic conditions at 37oC for

4 days and were later preserved on BHI agar slants.

Staphylococcus epidermidis MTCC 435 was grown in BHI broth under

anaerobic conditions for 48h and were preserved on BHI agar slants.

S. aureus MTCC 7443 was cultured on nutrient agar plates and incubated

for 24h.

3.4. Zinc Oxide nanoparticles

The oxide of zinc in the nanorange can be synthesized by different methods

and different capping agents. The present study employed three methods

of synthesis which were feasible to synthesize in the lab.

34

3.4.1. Role of the capping agents in the synthesis of the

nanoparticles

Capping agents control properties of the particles in the nanorange. The

use of coating the nanoparticles with capping agents is to prevent the

agglomeration of the particles due to their high surface energy.

3.4.2. Method I (Shailaja et al., 2013)

The chemicals used are of Analytical grade. Zinc nitrate, sodium hydroxide

and ethanol. The chemicals have been obtained from Himedia laboratories

and these are of the highest purity available.

100ml of 1M zinc nitrate (27.948g in 100ml) was mixed with 250ml of 1M

sodium hydroxide (10g in 250ml), leading to the neutralization of the two

solutions. The pH was then set at 12 using NaOH. The precipitate is

collected after centrifugation for 3 min at 3000 rpm. It was then washed

with de-ionized water three times and twice with standard ethanol. The

cleansed pellet was heated overnight at 80oC and was weighed.

3.4.3. Method -II

The chemicals used for synthesis are Zinc nitrate (Zn(NO3)2), Sodium

hydroxide (NaOH)and starch (Yadav et al., 2006).

500ml of 0.5% starch and 14.874 g of zinc nitrate were mixed together,

and the solution mixture was neutralized for 2 h with 300 ml of 1N sodium

hydroxide (10 g in 250ml). The mixture was centrifuged at 3000 rpm for 3

min. The pellet obtained after centrifugation was washed thrice with de-

35

ionised water and twice with standard ethanol. The cleansed pellet was

heated overnight at 80°C. It was ground to a fine powder and weighed.

3.4.4. Method- III using different capping agents (Sulabha, Singh et

al., 2009)

Method –IIIa. Using Thioglycerol as capping agent

Zinc oxide of were prepared by suspending 0.2 M zinc acetate in 20 ml of

Dimethyl sulfoxide. It was stirred for about 30 min. 1.2 M of potassium

hydroxide prepared in 10 ml of ethanol was added drop wise to zinc acetate

suspended in dimethyl sulfoxide. After stirring for 5 min, 0.12 ml of

thioglycerol was added and stirring continued for an hour till the solution

turned milky. The particles were then washed with methanol thrice and

were later dispersed in methanol. The absorption spectrum using UV-VIS

spectrophotometer was measured (Shailaja et al., 2013).

Method - IIIb using Triethanol amine as capping agent

The same procedure as above was followed but 0.12ml of Triethanol amine

was used as a capping agent.

Method –IIIc.Using Oleic acid as capping agent

The same procedure as above was followed but 0.12ml of Oleic acid was

used as a capping agent.

36

3.5. CHARACTERIZATION OF ZNO NANOPARTICLES

3.5.1. UV-VIS Spectrophotometer

In a UV-VIS spectroscopic measurement light absorption as a function of

λ provides information about electronic transitions occurring in the

material. The fraction of light transmitted is described by Beer-Lamberts

law, which states that the fraction of the light measured after interaction

with the sample or reflectance versus the incident intensity is dependent

on the path length of light through the sample, absorption cross section of

the transition and the difference in the population of the initial state.

The characterization of the nanoparticles was carried out using Systronics

2201 in the wavelength range 300 to 400 nm using a double beam UV-VIS

spectrophotometer with DMSO as the solvent (reference std) in which the

nanoparticles were dispensed for the analytical study of the ZnO

nanoparticles.

3.5.2. X-ray diffraction (XRD)

X-ray diffraction is a versatile, non-destructive analytical method for

identification and quantitative determination of various crystalline forms,

known as ‘phases’ of compound present in powder and solid samples.

X-rays correspond to electromagnetic radiation in the wave length range

of 1Ao. The wave length range is below that of UV light and above that of

gamma rays. This radiation is produced when charged particles

decelerated by metals, thus producing a continuum called

37

Bremsstrahlung radiation. X-rays are generally produced when the

electrons of several thousands of electron volts are decelerated or stopped

by the metals. This will produce a white radiation up to a threshold

frequency corresponding to the kinetic energy of the particle. (Pradeep,

2007). Three kinds of radiations are generally used for diffraction: X-rays,

electrons and neutrons. The characteristic X- ray used for diffraction is

the copper Kα radiation at 1.5418 Ao wave length. Two approaches are

generally used for the analysis of X -ray diffraction data. These are the

Laue equations and the Bragg’s law.

In the Laue equation diffraction from a one-dimensional crystal may be

treated in the same way as the diffraction by an optical grating. Upon

projection. The grating is like an array of points similar to a crystal. The

diffraction condition is nλ=dsinθ. (Pradeep,2007).

In 1912, W. L. Bragg recognized a predictable relationship among several

factors.

Bragg’s equation

nλ =2dsinθ,

λ- Wavelength of X-ray d- interplanar spacing,

θ- diffraction angle n- 0,1,2,3

The size of the particle can be determined by Scherrer’s formula (Singh et

al., 2009) using FWHM of XRD patterns

38

size of the particles is D =0.9λ

βCos θ

Whereλ is wave length of X-ray source, β is full-width at half- maximum

in radians, θ is Bragg’s diffraction angle.

Structural properties of the ZnO nanoparticles synthesized using wet

chemical method were recorded on Rigaku X-ray powder diffractometer

(Cu radiation, λ = 0.1546 nm) running at 40 kV and 40 mA (Tokyo, Japan).

3.5.3. Electron Microscopy

“Electron Microscopy can be defined as a specialized field of science that

employs the electron microscope as a tool and uses a beam of electrons to

form an image of a specimen(Stadtländer, 2007).”

3.5.3.1. Transmission electron microscopy (TEM)

“TEM is a microscopy technique in which a beam of electrons is

transmitted through an ultra-thin specimen, interacting with the

specimen as it passes through it. An image is formed from the interaction

of the electrons transmitted through the specimen; the image is magnified

and focused onto an imaging device, such as a fluorescent screen, on a

layer of photographic film, or to be detected by a sensor such as a charge-

coupled device.”

39

TEM images were observed on TECNAI FE12 TEM instrument operating at

120 kV using SIS imaging software. The particles were dispersed in

methanol and a drop of it was placed on formvar-coated copper grid

followed by air drying.

3.5.3.2.Selected area diffraction

“Selected area (electron) diffraction (abbreviated as SAD or SAED), is a

crystallographic experimental technique that can be performed inside a

transmission electron microscope (TEM).”It was used to identify the crystal

structure and examine the defects.

3.5.3.3. Scanning Electron Microscope (SEM)

“The scanning electron microscope (SEM) uses a focused beam of high-

energy electrons to generate a variety of signals at the surface of solid

specimens. The signals that derive from electron sample interactions

reveal information about the sample including external morphology”

40

Figure 3.1. SEM lay out and function(www.ammrf.org.au)

3.5.3.4. Energy-dispersive X-ray microanalysis (EDX)

“EDX is complementary to SEM. It was done to determine the composition

of the features in the SEM image.”

SEM (FEI Quanta 200 FEG with EDS) was used for morphology

assessment of the synthesized Zinc Oxide nanoparticles. The sample was

collected on a round cover glass (1.2 cm), washed with deionized water and

dried in a desiccator at Room Temperature. The cover glass was then

mounted on a SEM stub and coated with gold for SEM analysis.

41

3.5.4. FTIR

“FT-IR stands for Fourier Transform InfraRed, the preferred method of

infrared spectroscopy. In infrared spectroscopy, IR radiation is passed

through a sample. Some of the infrared radiation is absorbed by the

sample and some of it is passed through (transmitted). The resulting

spectrum represents the molecular absorption and transmission, creating

a molecular fingerprint of the sample. Like a fingerprint no two unique

molecular structures produce the same infrared spectrum. This makes

infrared spectroscopy useful for several types of analysis.”

FT-IR spectra were recorded on Thermo Nicolet Nexus (Washington, USA)

670 spectrophotometer.

3.5.5. Thermogravimetric analysis (TGA): (Coats, AW et al., 1963;

Tikhonov N A et al., 2009)

“Thermogravimetric analysis or thermal gravimetric analysis (TGA) is a

method of thermal analysis in which changes in physical and chemical

properties of materials are measured as a function of increasing

temperature (with constant heating rate), or as a function of time (with

constant temperature and/or constant mass loss)”.

Thermal decomposition profile was recorded on Mettler Toledo TGA

851einstrument in the temperature range 25 - 800 °C with a heating rate

of 10 °C.

42

3.6. ANTIBACTERIAL ACTIVITY OF ZNO NANOPARTICLES

(Hewitt, 2012)

Antibacterial activity of ZnO nanoparticles against acne causing

organisms isolated in the present study, standard cultures P. acnes MTCC

1951, S.aureus MTCC 7443 and S.epidermidis MTCC 435 procured from

IMTECH, Chandigarh were tested by agar diffusion method (well method).

Agar diffusion method is a means of measuring the effect of an

antibacterial agent against bacteria. The cultures were inoculated over the

dried surface of BHI and nutrient agar, respectively. Wells bored in the

agar were impregnated with ZnO nanoparticles dispersed in

methanol/DMSO and methanol/DMSO were used as controls. The

compound diffuses from the well into the agar. The concentration of the

compound will be highest near the well and will decrease as distance from

the well increases. If the compound is effective against bacteria at a certain

concentration, no colonies will grow where the concentration in the agar

is greater than or equal to the effective concentration creating a zone of

inhibition. Thus, the size of the zone of inhibition is a measure of the

compound's effectiveness: the larger the clear area around the well, the

more effective is the compound.

The BHI agar plates inoculated with the cultures P. acnes and the wells

impregnated with ZnO nanoparticles were incubated at 37oC in anaerobic

chamber for 4 days and the agar plates inoculated with S. epidermidis and

S. aureus and the wells incorporated with the ZnO nanoparticles were

43

placed in the incubator and incubated for 48h and 24h respectively. The

zones of inhibition were measured.

3.6.1. Stability of ZnO nanoparticles at different temperatures

To test the stability of nano sized Zinc Oxide at different temperatures, the

ZnO nanoparticles were placed at room temperature (RT), in oven at a

temperature of 45o C, in refrigerator at a temperature of 4 oC for 24h and

their antibacterial activity was tested against Propionibacterium acnes

culture.

P. acnes was cultured on BHI agar and the wells were incorporated with

the ZnO nanoparticles placed at different temperatures. The plates were

placed in Anaero gas pak and incubated for 4 days and the antibacterial

activity was determined by measuring the zone of inhibition.

3.6.2. Stability of ZnO nanoparticles with time

The antibacterial activity of the ZnO nanoparticles against the acne

causing organisms was carried out for about 6 months.

3.6.3. Antibacterial activity of commercial ZnO nanoparticles

Commercial nano sized Zinc oxide were procured from Nano labs and the

antibacterial activity was tested by agar well diffusion method.

44

3.7.MINIMUM INHIBITORY CONCENTRATION- MIC

3.7.1. Minimum Inhibitory Concentration of ZnO Nanoparticles

against P.acnes.

Two sets of 5 tubes in each set with 5 ml of BHI broth were taken.

To one set of BHI broth ZnO nanoparticles of different

concentrations (0.5mg-2mg) were added. One tube with only BHI

broth was used as blank.

Second set of 5 tubes with BHI broth were inoculated with P. acnes

and ZnO nanoparticles of different concentrations (but same as set

I) were added and the tubes were incubated in Anaerobic chamber

for 4 days.

BHI broth inoculated with P. acnes culture was used as control

3.8.PREPARATION OF COSMETIC SKIN CARE PRODUCTS WITH ZNO

NANOPARTICLES

3.8.1. Preparation of cold cream with ZnO nanoparticles

Stearic acid and liquid paraffin were taken in a clean sterilised beaker and

heated to melt the stearic acid. In another beaker water was taken and

heated till the appearance of the first bubble. It was removed from the

flame and was mixed well after the addition of borax powder. To the

mixture of Stearic acid and liquid paraffin, borax water mixture was added

45

slowly, stirred vigorously to get a creamy consistency. Few drops of

perfume was added and stirred. It was then stored in an air tight container.

An emulsion of ZnO nanoparticles and cold cream was prepared and left

over night to test for stability and its antibacterial activity against

Propionibacterium acnes.

3.8.2. Preparation of Vanishing cream I with ZnO nanoparticles

Stearic Acid, stearyl alcohol, cetyl alcohol were taken into a clean sterile

beaker and melted at 75 oC. In another beaker 0.9g of potassium hydroxide

is dissolved at 75oC in purified water. 0.1g of methyl paraben and 0.05g

of propyl paraben were added to the above mixture, followed by the

addition of 0.1% of ZnO nanoparticles (prepared using thioglycerol as a

capping agent). This above mixture was added to the stearic acid mixture

and stirred slowly and continuously until a smooth cream is formed at RT.

3.8.3. Vanishing cream II with ZnO nanoparticles

Oily phase (A) is obtained by mixing Stearic acid, Lanolin, Propylene

glycol, propyl paraben were mixed and heated to 70 oC.

Aqueous mixture of TEA, methyl paraben and water were mixed and

heated at 70oC. This forms the Aqueous phase (B)

These two were mixed with continuous stirring till homogenous

emulsion is formed. It was cooled to RT and then mixed with 0.1 %

of ZnO nanoparticles.

46

3.8.4. Preparation of Calamine lotion using ZnO nanoparticles:

The wax was heated and dissolved gently in arachis oil and 45ml of

purified water at the same temperature and cooled to get the cream.

Calamine was mixed with ZnO nanoparticles in water and then added to

the cream and mixed uniformly.

3.8.5. Preparation of scrub with ZnO nanoparticle

The scrub was prepared using orange peel, oat meal, Liquid paraffin,

stearyl alcohol, cetyl alcohol, methyl paraben, propyl paraben, SDS, ZnO

nanoparticles, Almond oil, Disodium EDTA (pinch) and Isopropanol (2-3

drops)

3.8.6. Preparation of Face pack

Face pack was prepared using Multani mitti. ZnO nanoparticles were

added to Multani mitti at a concentration of 1 mg/mg and Rose Water.

3.8.7. Addition of ZnO nanoparticles to commercial products

ZnO nanoparticles were added to commercially available Neem face wash,

walnut scrub and face wash used for acne and the antibacterial activity of

these products against P. acnes was tested by agar well diffusion method.

This was done to compare the antibacterial effect of these commercial

products with and without the addition of ZnO nanoparticles.

47

3.8.8. Preparation of the plant extracts

The plant samples collected were washed thoroughly under running tap

water to remove dust and were then dried.

Extracts were prepared using Tulsi (leaves), Neem (leaves), Mint (leaves),

Sandalwood (bark) and Aloe vera (leaves).

The plant extracts were prepared using water. 1g of the samples and

homogenized with 10 ml of water. The crude preparations were left

overnight at Room Temperature (RT) and then centrifugation was carried

out at 4000 rpm for 5 min. The supernatants containing the plant extracts

were transferred to test tubes and stored at 4°C.

3.8.9. Addition of ZnO nanoparticles to the plant extracts

The ZnO nanoparticles were added to the extracts at a concentration of

1mg of Zinc Oxide nanoparticle for 1ml of the extract.

3.8.10. Antibacterial activity of the cosmetic skin care products

prepared using ZnO nanoparticles

Antibacterial activity of the creams, scrubs prepared with ZnO

nanoparticles was tested by standard agar well diffusion method and the

cosmetic skin care products prepared without the nanoparticles were used

as control.

48

3.9.Invitro Cytotoxicity tests

Measurement of cell viability and proliferation forms the basis for

numerous in vitro assays of a cell population’s response to external

factors. The MTT Cell Proliferation Assay measures the cell proliferation

rate and conversely, when metabolic events lead to apoptosis or necrosis,

the reduction in cell viability.

To carry out the test DMEM (Dulbecco's modified Eagles medium), MTT [3-

(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], trypsin,

EDTA Phosphate Buffered Saline (PBS) and were purchased from Sigma

Chemicals Co. (St. Louis, MO) and Fetal Bovine Serum (FBS) were

purchased from Gibco. 25 cm 2 and 75 cm2 flask and 96 well plated

purchased from Eppendorf India.

3.9.1. Maintenance of cell line:

The MCF 10A epidermal cell line were purchased from NCCS, Pune and

the cells were maintained in DMEM supplemented with including 10%

FBS and the antibiotics penicillin/streptomycin (0.5 mL-1), in atmosphere

of 5% CO 2 /95% air at 37 0 C.

3.9.2. MCF 10A cell viability by MTT Assay:

MTT Assay is a colorimetric assay that measures the reduction of yellow

3-(4, 5-dimethythiazol- 2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) to

dark purple colored formazan by mitochondrial succinate dehydrogenase.

The assay depends both on the number of cells present and on the

49

assumption that dead cells or their products do not reduce tetrazolium.

The MTT enters the cells and passes into the mitochondria where it is

reduced to an insoluble, dark purple coloured formazan crystals. The cells

are then solubilized with DMSO and the released, solubilized formazan

reagent is measured spectrophotometrically at 570 nm.

Cell viability was evaluated by the MTT Assay with three

independent experiments with six concentrations of compounds in

triplicates.

MCF 10 A cells were trypsinized to perform the tryphan blue assay

to know viable cells in cell suspension.

Cells were counted by haemocytometer and seeded at density of

5.0 X 10 3 cells / well in 100 μl media in 96 good plate culture

medium and incubated overnight at 37 0 C.

After incubation, remove the old media and add fresh media 100 µl

with different concentrations of test compound in representative

wells in 96 plate.

After 48 hrs., the drug solution was discarded and the fresh media

with MTT solution (0.5 mg / mL-1) was added to each well and plates

were incubated at 37 0 C for 3 h. At the end of incubation time,

precipitates were formed as a result of the reduction of the MTT salt

to chromophore formazan crystals by the cells with metabolically

active mitochondria.

50

The optical density of solubilized crystals in DMSO were measured

at 570 nm on a microplate reader. The percentage growth inhibition

was calculated using the following formula and concentration of test

drug needed to inhibit cell growth by 50 % values is generated from

the dose-response curves for each cell line using with origin

software.

3.10.Anti-Inflammatory effect of the Cream on Wistar Albino Mice

infected with P. acnes

To evaluate the degree of inflammation by P. acnes, male Wistar

albino mice of 25 g weight were selected and acclimatized. They are

grouped into 3 groups of 6 animals per group namely:

Group 1: These animals were treated only with the cream for a

period of 1 month to study the toxicity of the cream.

Group 2: These animals received intradermal injection of

only P. acnes to study the degree of inflammation.

Group 3: These animals were injected with P. acnes suspension, and

after 24h cream was applied.

Changes in inflammation were observed and recorded from day 1 to

last day of work- day 6.

51

P. acnes suspensions were prepared at concentrations of 107 CFU/

ml. Culture suspended in PBS (Phosphate buffer solution) were

injected in 20 μL aliquots intradermally in the right ears of the rat.

Left ears received only PBS injection.

After 24h of P. acnes injection, significant cutaneous erythema,

formation of lesions were observed in the right ears of groups 2 & 3.

Group 2 was left untreated and group 3 was used for the study of

Anti – Inflammatory effect of the cream. 2 mg cream was applied on

the right ears of mice and the decrease in thickness of the ear was

measured using a microcaliper.

Group 1 received only cream for 1 month

52

CHAPTER 4.

RESULTS AND DISCUSSION

4. An Overview of results obtained during the present study

I. Isolation and characterization of acne causing organisms

Isolation of acne causing microorganisms

The acne causing organisms were isolated from acne present on the skin surface of patients

And then noculated into tryptic soy broth, brain heart infusion broth and

thioglycollate broth

The organisms were cultured onto Brain heart infusion agar , Casmans and Clostridial agar and incubated anaerobically

Identification of the organism

The organisms were identified based on Colony morphology Gram staining and Biochemical tests

Gram positive bacilli and cocci were present

The cultures obtained were compared with the standsard cultures from Microbial Type Culture Collection centre

Gram positive bacilli which were predominantly present were identified

based on Fatty Acid Methyl Ester analysis and 16 S r RNA gene

sequencing

The acne causing organism was identified to

be Propionibacterium acnes

53

II. Synthesis and Characterization of ZnO nanoparticles

Synthesis of ZnO nanoparticles

ZnO nanoparticles were prepared using wet chemical method

Different capping agents like thioglycerol, triethanolamine and oleic acid

were used to prevent agglomeration

Characterization of the ZnOnanoparticles

ZnO nanoparticles were characterized using UV-VIS, XRD, SEM –EDX,

TEM, SAED, TGA and FTIR

The ZnO nanoparticles were synthesized and well characterized.

ZnO nanoparticles capped with thioglycerol are well separated and

have a spherical shape with a particle size of 40-50nm

Antibacterial activity of ZnO nanoparticles

Antibacterial activity of ZnO nanoparticles against acne causing

organisms was studied invitro using agar well diffusion method and the

zone of inhibition was measured

Minimum Inhibitory Concentration of ZnO nanoparticles against

Propionibacterium acnes was measured using broth culture and

Absorbance was measured

Stability of the nanoparticles was tested with temperature and time

These results obtained were compared with the antibacterial activity of

ZnO nanoparticles procured from Nano labs and antibiotic tetracycline

against acne causing organisms

End Result:

The ZnO nanoparticles were effective against the organisms

causing acne and the Minimum inhibitory concentration

against P. acnes was found to be 0.3mg ml-1

54

III. Preparation of cosmetic (skin care) products with ZnO

nanoparticles

Preparation of different cosmetic products like creams, lotions, scrubs,

face wash and face pack with ZnO nanoparticles

Tested the antibacterial activity of these products against acne causing

organisms invitro by agar well diffusion method. The Zone of inhibition

was measured.

Cytotoxicity of ZnO nanoparticles was determined using 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and

MCF 10A (epidermal cell line).

Effect of cream prepared with ZnO nanoparticles on P. acnes in vivo and

P. acnes induced inflammation in mice was studied

End Result:

ZnO nanoparticles are effective agents against P. acnes in vitro

and its effect in vivo was observed in mouse ears induced with P.

acnes inflammation.

The nanoparticles were not toxic to the epidermal cell lines up to

a concentration of 100 µg ml-1

55

4.1. ISOLATION OF ACNE CAUSING ORGANISM:

According to the Global Burden of Disease (GBD) study, acne affects

approximately 85% of young adults aged 12–25 years. Acne consistently

represents the top three most prevalent skin conditions in the general

population, in various countries throughout the world. Acne vulgaris-

associated disease burden exhibits global distribution and has continued

to grow in prevalence over time within this population. This continued

growth suggests an unmet dermatologic need worldwide for this disorder

and potential opportunities for improved treatment.

The present treatments available for treating acne have side effects. ZnO

nanoparticles were selected in the present study to test their potential to

be used as an alternative treatment for acne because they are proved to

be effective antibacterial agents and are biocompatible. No studies were

carried out to test the antibacterial activity of ZnO nanoparticles against

acne.

To achieve this objective, the acne causing organisms were isolated from

acne present on the skin surface of patients. The organisms were

inoculated into BHI (Brain Heart Infusion) broth, Tryptic soy broth and

thioglycollate broth. The samples (50) were labelled as S1, S2, S3, S4, S5,

Pa1, Pa2, Pa3, Pa4, Pa5……………………….Pa45 and incubated in an

anaerobic jar with a disposable carbondioxide generator envelope. After

56

incubation the cultures were plated onto BHI, Casmans and Clostridial

agar and incubated anaerobically for 7 days. About 20 different colony

types were morphologically identified and subcultured for further analysis.

From each sample, one colony of each different type was isolated in pure

culture and incubated anaerobically. Further studies were carried out

using 9 cultures which showed good growth.

Standard Propionibacterium acnes cultures, P. acnes MTCC 1951, P. acnes

MTCC 3297 procured from IMTECH were cultured in BHI and tryptic soy

broth and were incubated anaerobically. After incubation the standard

cultures were subcultured on BHI agar plates.

Staphylococcus epidermidis MTCC 435 was cultured into BHI broth and

incubated for 48h under anaerobic conditions and were later inoculated

onto BHI agar and maintained.

Staphylococcus aureus MTCC 7443 was cultured on Nutrient agar medium

and incubated at 37oC under aerobic conditions for 24h.

4.2. IDENTIFICATION OF THE ORGANISM

The identification of the organisms was done on the basis of its gram

nature, colony morphology, microscopic observation, biochemical tests

such as testing for the production of Indole, nitrate reduction, catalase

test, carbohydrate fermentation tests, ability to hydrolyze gelatin, FAME

analysis and 16S r RNA gene sequencing.

57

4.2.1. Identification of the organism based on the colony morphology

and gram staining

The colony morphology and microscopic observation of the 9 isolates is

tabulated in Table 4.1. from the observation we can infer that both gram

positive bacilli and cocci were isolated from acne but gram positive bacilli

were predominant. The results obtained were compared with that of the

colony morphology and microscopic observation of the standard MTCC

cultures (Microbial Type Culture Collection, IMTECH) tabulated in Table

4.2.

From Table 4.1 which shows the colony morphology of the isolates on BHI

agar we can infer that the isolates Pa1 and Pa6 are gram positive cocci

hence identified as Staphylococcus epidermidis and S. aureus as the

cultures were grown under anaerobic conditions. The identification was

confirmed by performing biochemical tests, growth on MSA plates and

biofilm formation which is one of the virulence factors and cause of its

growth on medical devices and nosocomial infections.

Pa2, Pa3, Pa4, Pa5, Pa7, S3 and S5 have shown similar colony morphology

and were all gram positive bacilli as tabulated in Table 4.1.Colony

morphology and microscopic view on gram staining of the isolate Pa3 can

be seen in Fig. 4.1a and b. The further identification was done based on

the conventional biochemical tests, FAME analysis and 16S r RNA

sequencing.

58

To characterize the gram positive bacilli molecular methods were employed

as the literature shows that the causative organism of acne is a gram

positive anaerobic bacilli (Kirschbaum et al., 1963).

The individual colony characteristics and gram nature of the standard

MTCC cultures P. acnes MTCC 1951, P. acnes MTCC 3297, S. epidermidis

MTCC 435 and S. aureus MTCC 7443 is tabulated in Table 4.2 and the

same observation can be noted from Fig. 4.2a, 4.2b.,4.2c. The gram

positive bacilli isolated during the present study showed a similar

morphology with that of the standard MTCC cultures P.acnes MTCC 1951

and P.acnes MTCC 3297 so the isolates are identified as Propionibacterium

species.

59

Table 4.1. Colony morphology of the isolates

S.No Sample Colony morphology Gram stain

1. Pa1 Round, slimy, opaque,

small colonies

Gram positive

cocci

2. Pa2 Small, circular, raised,

creamy, mucoid colonies.

Gram positive

bacilli

3. Pa3 Circular, opaque, creamy

pin head colonies.

Gram positive

bacilli

4. Pa4 Small, Round, cream

coloured pin head

colonies

Gram positive

bacilli

5. Pa5 Slimy, round, cream

colored colonies.

Gram positive

bacilli

6. Pa6 Small, round, opaque,

mucoid colonies

Gram positive

cocci

7 Pa7 Big, round, elevated,

creamy colonies

Gram positive

bacilli

8. S3 Round, opaque, raised,

creamy colonies

Gram positive

bacilli

9. S5 Round, opaque, raised,

creamy colonies

Gram positive

bacilli

60

(a) (b)

Figure 4.1. Colony morphology of Pa3 isolate on BHI agar (a) and

Microscopic view on gram staining (b)

61

Table 4.2. Colony characteristics of standard cultures (IMTECH)

S. No Culture Colony morphology Gram stain

1. MTCC 1951 Round, opaque ,raised,

creamy pinhead colonies

Gram positive

bacilli

2. MTCC 3297 Small, round, opaque,

regular, creamy colonies.

Gram positive

bacilli

3. MTCC 435 White raised cohesive

colonies

Gram positive

cocci

4. MTCC 7443 Slightly raised, creamy

yellow colonies

Gram positive

cocci in

clusters

MTCC 1951 and 3297 -Propionibacterium acnes

MTCC 435- Staphylococcus epidermidis;

MTCC 7443- Staphylococcus aureus

62

a.

b. c.

Figure 4.2. Colony morphology of P. acnes MTCC 1951 on BHI (a)

Microscopic view on gram staining (b)

Microscopic view (Phase contrast) of P.acnes MTCC 1951 (c )

63

4.2.2. Characterization of the gram positive bacilli using

biochemical methods

The isolates which were gram positive anaerobic rods were tested for sugar

fermentation, TSI, catalase test, hydrolysis of gelatin, nitrate reduction

and Indole production, the results are tabulated in Table 4.3, 4.4 and 4.5.

From the observations we can interpret that the isolates Pa3, Pa4 showed

positive reaction for catalase, nitrate reduction test (Cummins, 1986). Pa3

and Pa4 showed positive for Indole, Adonitole and did not ferment esculin

which are the characteristic features of P.acnes (Mc Gliney et al., 1978).

The observations are similar to those published by Shinkafi et al., 2013

they isolated acne causing organisms from face, neck and back. The

further identification of the isolates Pa3 and Pa4 is done based on the

hemolysis, FAME analysis and 16 SrRNA gene sequencing. To characterize

the gram positive bacilli molecular methods were employed as the

literature shows that the causative organism of acne is a gram positive

anaerobic bacilli (Kirschbaum et al., 1963).

4.2.2.1. Hemolysis

Hemolysis of the defibrinolysed sheep blood agar was shown by the

organisms Pa3, Pa4 and P.acnes MTCC 1951 and MTCC 3297 strains due

to the production of hemolysins, the virulence factors. Hemolysis of blood

agar by Pa3 is shown in the Fig 4.3.

64

Table 4.3 – Sugar fermentation tests for the gram positive anaerobic rods

Identification Glucose Maltose Sucrose Esculin* Adonitole

Pa2 - - - - -

Pa3 + - - - +

Pa4 + - - - +

Pa5 - - - - -

Pa7 + + + - -

S3 + - + - -

S4 + - + - -

MTCC1951 + - - - +

MTCC3297 + - - - +

in the case of Esculin + indicates a positive ferric citrate test. The pH in

these tubes did not change significantly hence -. Fermentation of

Carbohydrates in question is indicated by +

65

Table 4.4. Biochemical tests- TSI, catalase, gelatinase and nitrate

reductase

+ indicates production of catalase, reduction of NO3- to NO2

- and

liquefaction of Gelatin respectively

S.No Sample

name

Triple sugar

iron test

Gelatinase

test

Nitrate

reduction

test

Catalas

e test

1. Pa2 Acidic + + +

2. Pa3 Acidic + + +

3. Pa4 Acidic + + +

4. Pa5 Acidic - - +

5. Pa7 Alkaline slant/

acidic butt

+ + -

6. S3 Alkaline slant/

acidic butt

+ + +

7. S5 Acidic

slant/acidic

butt

- - -

8. 1951 Acidic + + +

9. 3297 Acidic - + -

66

Table 4.5- Identification of cultures based on IMViC reactions

S.no Sample Indole MR VP Citrate

1. Pa2 + - - +

2. Pa3 + - + -

3. Pa4 + - + +

4. Pa5 + + - -

5. Pa7 - - - +

8. S3 + - - +

9. S5 + - - +

10. 1951 + + - -

11. 3297 + + - -

67

Figure 4.3. Hemolysis of the blood by Pa 3

4.3. Characterization of the isolates based on FAME analysis and

16 S r RNA gene sequencing

The biochemical characterization of the isolates Pa3 and Pa4 correlated

with that of the MTCC strains P. acnes MTCC 1951 and P. acnes MTCC

3297. The cultures were characterized based on the FAME analysis and

16 S r RNA typing.

4.3.1. FAME Analysis

The results of FAME Analysis done using Agilent GC 6850, ANAER 6

method are tabulated in Table 4.6. The fatty acids identified through FAME

analysis of the isolate Pa3 are shown in Fig. 4.4.The ANAER 6 method

using MIDI Sherlock Microbial identification system identified the

organism to be Propioniobacterium acnes. The major fatty acids C12: 0, C14:

0, C15:0, C15: 0, C16: 0, C17: 0, C18:0 were identified.

68

“SI is a numerical value, which determines the close relation between the

fatty acid composition of an unknown and compares it with the mean fatty

acid composition of the strains used to create the library entry listed as its

match. The SI is an expression of the relative distance of the unknown

sample from the population mean. It is not a “probability” or percentage.

Samples with an SI of 0.500 or higher and with a separation of 0.100

between the first and second choice are considered good library

comparisons. If the SI is between 0.300 and 0.500 and well separated from

the second choice (>0.100 separation), it may be a good match, but an

atypical strain (it would fall very far away from the mean on the normal

distribution curve).” The isolate Pa4 did not find any match in the BHIBLA

anaerobic library and the SIM Index was found to be 0.0 as seen in Fig.

4.5, Table 4.7, 4.8,4.9.

69

S.No Sample Id Analysis Method

Used

Result

1 Pa-3 FAME ANAER6 Propionibacterium acnes

Figure 4.4. Chromatogram of isolate Pa3 showing the peaks representing

the FA by Agilent GC 6850

min2.5 5 7.5 10 12.5 15 17.5 20

pA

28

30

32

34

36

38

40

42

44

FID1 A, (E14522.560\A0031067.D)

0.1

82

0.4

18

0.6

46

0.8

69

1.0

86

1.3

08

1.4

98

1.5

94

1.7

44

1.9

78

2.0

12

2.0

88

2.1

33

2.2

47

2.3

92

2.6

27

2.7

21

2.9

41

3.0

56

3.3

44

3.4

93

3.6

70

3.8

62

3.9

48

4.0

90

4.2

41

4.4

10

4.6

32

4.7

86

4.9

39

5.1

52

5.3

10

5.4

63

5.5

89

5.6

62

6.0

82

6.2

15

7.2

08

7.5

22

8.5

02

8.6

40

10.4

65

10.7

80

11.8

86

12.0

46

13.8

20

13.8

99

14.3

07

15.5

54

70

Table 4.6. FAME Analysis of Pa3

RT Response

Ar/Ht

RFact ECL Peak Name Percen

t

Comment1

Comment2 0.182 3278 0.0

62 ---- 4.010 ---- < min rt

0.418 5367 0.086

---- 4.466 ---- < min rt 0.646 5884 0.0

73 ---- 4.907 ---- < min rt

0.869 4030 0.066

---- 5.338 ---- < min rt 1.086 7714 0.1

27 ---- 5.756 ---- < min rt

1.308 6673 0.083

---- 6.185 ---- < min rt 1.498 3702 0.0

68 ---- 6.553 ---- < min rt

1.594 4728 0.062

---- 6.739 ---- < min rt 1.744 3.84E+

8 0.029

---- 7.029 SOLVENT PEAK

---- < min rt 1.978 10811 0.0

23 ---- 7.482 ---- < min rt

2.012 10837 0.0

29

---- 7.547 ---- < min rt 2.088 2451 0.0

22 ---- 7.693 ---- < min rt

2.133 395 0.027

---- 7.780 ---- < min rt 2.247 1965 0.0

44 ---- 8.000 ---- < min rt

2.392 2788 0.060

---- 8.281 ---- < min rt 2.627 1812 0.0

75 ---- 8.736 ---- < min rt

2.721 1553 0.049

---- 8.917 ---- < min rt 2.941 3263 0.0

87 ---- 9.342 ----

3.056 1983 0.051

---- 9.564 ---- 3.344 3114 0.0

93 ---- 10.08

7 ----

3.493 2612 0.076

---- 10.295

---- 3.670 1703 0.0

54 ---- 10.54

8 ----

3.862 581 0.037

---- 10.817

---- 3.948 655 0.0

37 ---- 10.93

7 ----

4.090 3179 0.071

---- 11.103

---- 4.241 1780 0.0

43 ---- 11.26

3 ----

4.410 3846 0.117

---- 11.441

---- 4.632 2711 0.0

79 ---- 11.67

5 ----

4.786 2381 0.055

---- 11.838

---- 4.939 2345 0.0

55 1.041 12.00

0 12:0 FAME 2.1

4 ECL deviates 0.000

Reference -0.002 5.152 3252 0.1

00 ---- 12.18

1 ----

5.310 1305 0.067

---- 12.314

---- 5.463 1699 0.0

63 ---- 12.44

4 ----

5.589 911 0.047

---- 12.551

---- 5.662 115 0.0

09 1.019 12.61

2 13:0 ISO FAME

---- < min ar/ht

6.082 799 0.0

50 ---- 12.96

7 ----

6.215 1754 0.054

---- 13.068

---- 7.208 997 0.0

51 ---- 13.77

7 ----

7.522 1383 0.038

0.980 14.000

14:0 FAME 1.19

ECL deviates 0.000

Reference -0.001 8.502 83305 0.0

39 0.967 14.62

5 15:0 ISO FAME

70.45

ECL deviates 0.002

Reference 0.000 8.640 9575 0.0

39 0.965 14.71

3 15:0 ANTEISO FAME

8.08

ECL deviates -0.001

Reference -0.003 10.46

5 334 0.0

27 0.950 15.81

3 16:1 CIS 9 FAME

0.28

ECL deviates -0.005

10.78

0 7011 0.0

42 0.948 15.99

8 16:0 FAME 5.8

1 ECL deviates -0.002

Reference -0.004 11.88

6 8198 0.0

43 0.942 16.63

1 17:0 ISO FAME

6.76

ECL deviates 0.001

Reference -0.003 12.04

6 1465 0.0

44 0.942 16.72

2 17:0 ANTEISO

FAME

1.21

ECL deviates -

0.001

13.82

0

988 0.0

53

0.937 17.72

5

18:2 CIS 9,12

FAME

0.8

1

ECL

deviates 0.002

13.89

9 1500 0.0

48 0.937 17.77

0 18:1 CIS 9 FAME

1.23

ECL deviates -0.001

14.30

7 1057 0.0

48 0.936 18.00

0 18:0 FAME 0.8

7 ECL deviates 0.000

Reference -0.006 15.55

4 1463 0.0

59 0.936 18.70

8 22:0 NHC 1.2

0 ECL deviates -0.004

71

Figure 4.5. Chromatogram of bacterial sample Pa4 showing the fatty acid

peaks through Agilent GC 6850

min2.5 5 7.5 10 12.5 15 17.5 20

pA

28

30

32

34

36

38

40

42

44

FID1 A, (E14522.560\A0051069.D)

0.0

66

0.3

05

0.5

42

0.7

65

0.9

13

0.9

89

1.2

11

1.5

07

1.6

04

1.7

45

1.9

78

2.0

11

2.0

88

2.1

60

2.2

52

2.3

15

2.6

53

2.7

29

2.8

15

2.9

83

3.2

80

3.4

28

3.6

14

3.7

97

3.8

82

4.0

16

4.1

74 4

.394

4.5

78

4.7

31

4.9

40

5.2

65

5.3

90

5.5

41

5.7

54

5.8

62

6.1

84

6.3

67

6.6

44

6.7

59

6.9

05

7.5

18

8.5

00

8.6

44

9.0

91

10.4

72

10.5

33

10.7

83

11.8

88

12.3

37

13.9

99

14.3

12

15.9

04

17.1

50

Sample Id Analysis Method Used Result

Pa-4 FAME ANAER6 No Match

72

Table 4.7. FAME Analysis of Pa4

RT Response

Ar/Ht

RFact ECL Peak Name

Percent Comment1

Comment2 0.066 2339 0.04

8 ---- 3.789 ---- < min rt

0.305 4908 0.078

---- 4.252 ---- < min rt 0.542 6263 0.07

6 ---- 4.709 ---- < min rt

0.765 3961 0.064

---- 5.140 ---- < min rt 0.913 3352 0.06

2 ---- 5.427 ---- < min rt

0.989 4866 0.082

---- 5.574 ---- < min rt 1.211 6964 0.07

7 ---- 6.003 ---- < min rt

1.507 8506 0.117

---- 6.574 ---- < min rt 1.604 4011 0.06

4 ---- 6.761 ---- < min rt

1.745 3.849E+8

0.029

---- 7.033 SOLVENT PEAK

---- < min rt 1.978 10801 0.02

4 ---- 7.484 ---- < min rt

2.011 9633 0.028

---- 7.548 ---- < min rt 2.088 2072 0.02

1 ---- 7.696 ---- < min rt

2.160 1249 0.045

---- 7.837 ---- < min rt 2.252 273 0.02

1 ---- 8.014 ---- < min rt

2.315 1915 0.054

---- 8.136 ---- < min rt 2.653 3807 0.09

0 ---- 8.789 ---- < min rt

2.729 88 0.014

---- 8.935 ---- < min rt 2.815 3190 0.07

8 ---- 9.102 ----

2.983 2727 0.057

---- 9.425 ---- 3.280 4219 0.08

4 1.141 9.999 10:0

FAME 2.03 ECL

deviates -0.001

Reference -0.002 3.428 1153 0.04

7 ---- 10.207 ----

3.614 1834 0.057

---- 10.468 ---- 3.797 451 0.03

0 ---- 10.724 ----

3.882 545 0.029

---- 10.842 ---- 4.016 3102 0.06

7 ---- 11.023 ----

4.174 1856 0.048

---- 11.191 ---- 4.394 8819 0.04

2 1.066 11.423 10:0 3OH

FAME 3.96 ECL

deviates 0.001

4.578 2421 0.07

0 1.057 11.618 12:0 ISO

FAME 1.08 ECL

deviates 0.010

Reference 0.009 4.731 1981 0.05

5 ---- 11.779 ----

4.940 17624 0.033

1.041 12.000 12:0 FAME

7.73 ECL deviates 0.000

Reference -0.001 5.265 859 0.04

6 ---- 12.276 ----

5.390 1866 0.068

---- 12.382 ---- 5.541 1340 0.05

5 ---- 12.510 ----

5.754 1854 0.084

1.016 12.690 13:0 ANTEISO FAME

0.79 ECL deviates -0.013

Reference -0.014 5.862 816 0.05

0 ---- 12.781 ----

6.184 1395 0.056

---- 13.046 ---- 6.367 469 0.03

6 ---- 13.177 ----

6.644 1244 0.053

---- 13.374 ---- 6.759 7762 0.03

8 0.993 13.457 Sum In

Feature 2 3.25 ECL

deviates 0.001

12:0 3OH FAME 6.905 1343 0.06

7 0.990 13.561 Sum In

Feature 3 0.56 ECL

deviates -0.001

15:0 ISO ALDE ?? 7.518 3192 0.03

8 0.980 13.998 14:0

FAME 1.32 ECL

deviates -0.002

Reference -0.003 8.500 5145 0.04

2 0.967 14.624 15:0 ISO

FAME 2.10 ECL

deviates 0.001

Reference -0.001 8.644 743 0.04

3 0.965 14.716 15:0

ANTEISO FAME

0.30 ECL deviates 0.002

Reference 0.000 9.091 953 0.03

4 0.961 15.001 15:0

FAME 0.39 ECL

deviates 0.001

Reference -0.001 10.472 17821 0.04

3 0.950 15.817 16:1 CIS

9 FAME 7.13 ECL

deviates -0.001

10.533 14824 0.04

2 ---- 15.853 ----

10.783 80763 0.041

0.948 16.001 16:0 FAME

32.24 ECL deviates 0.001

Reference -0.002 11.888 2393 0.04

6 0.942 16.632 17:0 ISO

FAME 0.95 ECL

deviates 0.002

Reference -0.002 12.337 12933 0.04

5 0.941 16.888 17:0 CYC

FAME 5.12 ECL

deviates -0.001

13.999 59643 0.04

7 0.937 17.824 Sum In

Feature 10

23.53 ECL deviates 0.000

18:1c11/t9/t6 FAME

14.312 3602 0.048

0.936 18.000 18:0 FAME

1.42 ECL deviates 0.000

Reference -0.004 15.904 15514 0.05

0 0.935 18.903 19 CYC

11,12/:1FAME

6.11 ECL deviates -0.001

Reference -0.006 17.150 1540 0.08

1 ---- 19.616 ----

---- 7762 --- ---- ---- Summed Feature 2

3.25 12:0 3OH FAME

13:0 DMA ---- 1343 --- ---- ---- Summed

Feature 3 0.56 15:0 ISO

ALDE ?? UN 13.570 ---- 59643 --- ---- ---- Summed

Feature 10

23.53 18:1c11/t9/t6 FAME

UN 17.834

73

Table 4.8. Comparison with Streptococcus mitis

0 10 20 30 40 50

10:0 FAME

10:0 3OH FAME

12:0 ISO FAME

12:0 FAME

13:0 ANTEISO FAME

14:0 FAME

15:0 ISO FAME

15:0 ANTEISO FAME

15:0 FAME

16:1 CIS 7 FAME

16:1 CIS 9 FAME

16:1 CIS 11 FAME

16:0 FAME

17:0 ISO FAME

17:0 CYC FAME

17:0 FAME

18:2 CIS 9,12 FAME

18:1 CIS 9 FAME

18:0 FAME

19 CYC 11,12/:1FAME

Summed Feature 2

Summed Feature 3

Summed Feature 10

[BHIBLA] Streptococcus-mitis

Sim Index: 0.000 (Distance: 18.116)

74

Table 4.9. Comparison with Wolinella succinogenes

0 10 20 30 40 50 60 70 80 90 100

10:0 FAME

10:0 3OH FAME

12:0 ISO FAME

12:0 FAME

13:0 ANTEISO FAME

14:0 FAME

15:0 ISO FAME

15:0 ANTEISO FAME

16:0 ALDE

15:0 FAME

15:0 ISO DMA

15:0 ANTEISO DMA

16:1 CIS 9 FAME

16:0 FAME

16:0 DMA

17:0 ISO FAME

17:0 ANTEISO FAME

17:1 CIS 11 FAME

17:0 CYC FAME

17:0 FAME

UN 17.103 17:0i DMA

17:0 ANTEISO DMA

18:2 CIS 9,12 FAME

18:1 CIS 9 FAME

18:0 FAME

19 CYC 11,12/:1FAME

Summed Feature 2

Summed Feature 3

Summed Feature 5

Summed Feature 10

[BHIBLA] Wolinella-succinogenes

Sim Index: 0.000 (Distance: 22.032)

75

The identification of Pa3 which was characterized as Propionibacterium

acnes based on biochemical tests and FAME analysis was confirmed by

16S r RNA gene sequencing.

4.3.2. 16 S r RNA gene sequencing:

The 16S r RNA gene was sequenced and after the completion of BLAST

analysis using the Ez Taxon database as shown in Table 4.10, organism

was identified to be Propionibacterium acnes with 100% identity match

with Propionibacterium acnes strain: DSM 1897(T) , Accession number

AWZZ01000008 (EZ TAXON database). On comparing the results of

sequencing with that of FAME we can infer that both the methods correlate

with each other in confirmation of the identity of the organism isolated

from acne.

The biochemical tests, FAME and 16S r RNA sequencing results confirm

the identity of Pa3 to be Propionibacterium acnes. The isolate was positive

for indole and nitrate reduction characteristic of P. acnes but not P.

granulosumor P. avidum(Holt et al., 1994).16 S r RNA sequencing analysis

provides phylogenetically useful information and is a better and faster way

of identification of the fastidious organisms as the conventional methods

take more than 48 h. The organism is thus identified as

Propionibacterium acnes DSM 1897(T) using BLAST similarity search and

Ez Taxon database

76

Table. 4.10. Blast Similarity Search Results for the isolate Pa3 using

Ez Taxon database

Accession Description PWS

(%)

Different

/ Total

Comp

letene

ss (%)

AWZZ01000008 Propionibacterium acnes

strain: DSM 1897(T)

100 0/1398 100

AFAM01000003 Propionibacterium

humerusii strain: P08(T)

98.26 24/1397 100

AGBA01000019 Propionibacterium avidum

strain: ATCC 25577(T)

96.44 49/1385 100

JQ283460 Propionibacterium olivae

strain: IGBL1 (T)

94.4 76/1358 94.73

JQ283461 Propionibacterium

damnosum strain:

IGBL13(T)

94.36 77/1365 95.7

AOSS01000369 Propionibacterium

granulosum strain: DSM

20700(T)

94.32 78/1374 100

AJ704569 Propionibacterium

acidopropionici strain:

NCFB 563 (T)

94.24 79/1372 98.85

AF234623 Propionibacterium

microaerophilum strain:

M5(T)

93.74 86/1373 100

AUDD01000055 Propionibacterium jensenii

strain: DSM 20535(T)

93.74 86/1373 100

AJ704572 Propionibacterium thoenii

strain : NCFB 568 (T)

92.79 99/1373 99.46

77

>Consensus _Pa3 sequence

TTACACATGCAAGTCGAACGGAAGGCCCTGCTTTTGTGGGGTGCTCGAGT

GGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCTTGACTTTGGGATAA

CTTCAGGAAACTGGGGCTAATACCGGATAGGAGCTCCTGCTGCATGGTG

GGGGTTGGAAAGTTTCGGCGGTTGGGGATGGACTCGCGGCTTATCAGCT

TGTTGGTGGGGTAGTGGCTTACCAAGGCTTTGACGGGTAGCCGGCCTGA

GAGGGTGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGG

GAGGCAGCAGTGGGGAATATTGCACAATGGGCGGAAGCCTGATGCAGCA

ACGCCGCGTGCGGGATGACGGCCTTCGGGTTGTAAACCGCTTTCGCCTG

TGACGAAGCGTGAGTGACGGTAATGGGTAAAGAAGCACCGGCTAACTAC

GTGCCAGCAGCCGCGGTGATACGTAGGGTGCGAGCGTTGTCCGGATTTA

TTGGGCGTAAAGGGCTCGTAGGTGGTTGATCGCGTCGGAAGTGTAATCTT

GGGGCTTAACCCTGAGCGTGCTTTCGATACGGGTTGACTTGAGGAAGGTA

GGGGAGAATGGAATTCCTGGTGGAGCGGTGGAATGCGCAGATATCAGGA

GGAACACCAGTGGCGAAGGCGGTTCTCTGGGCCTTTCCTGACGCTGAGG

AGCGAAAGCGTGGGGAGCGAACAGGCTTAGATACCCTGGTAGTCCACGC

TGTAAACGGTGGGTACTAGGTGTTGGGGTCCATTCCACGGGTTCCGTGCC

GTAGCTAACGCTTTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTA

AAACTCAAAGGAATTGACGGGGCCCCGCACAAGCGGCGGAGCATGCGGA

TTAATTCGATGCAACGCGTAGAACCTTACCTGGGTTTGACATGGATCGGG

AGTGCTCAGAGATGGGTGTGCCTCTTTTGGGGTCGGTTCACAGGCTGGT

GCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAA

CGAGCGCAACCCTTGTTCACTGTTGCCAGCAGGTTATGGTGGGGACTCAG

TGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGT

CATCATGCCCCTTATGTCCAGGGCTTCACGCATGCTACAATGGCTGGTAC

AGAGAGTGGCGACCTGTGAGGGTGAGCGAATCTCGGAAAGCCGGTCTCA

GTTCGGATTGGGGTCTGCAACTCGACCTCATGAAGTCGGAGTCGCTAGTA

ATCGCAGATCAGCAAGGCTGCGGTGAATACGTTCCCGGGGCTTGTACACA

CCGCCCGTCAACTCATGAAAGTTGGTAACACCCGAAGCCGGTGGCCTAAC

CGTTGTGGGGAGCCGTCGAATGG

78

4.4. Characterization of the gram positive cocci using biochemical

methods

The isolates Pa1 and Pa6 were identified to be gram positive cocci and the

biochemical tests were performed to characterize the bacteria and the

results tabulated in Table 4.10, 4.11 and Table 4.12 were compared with

the standard MTCC 435 and MTCC 7443 strains of S. epidermidis

(Fig.4.6a, 4.6b)and S. aureus respectively. Typical reaction patterns for

IMViC, catalase, sugar fermentation tests were reported by

Shinkafi et al., 2013 who isolated Staphylococci from face, neck and back.

Table 4.11. Sugar fermentation test

S.No Isolate Glucose Maltose Lactose Esculin

1. Pa1 + + + -

2. Pa6 + + + -

3. MTCC 435 + + + -

4. MTCC 7443 + + + -

Fermentation of Carbohydrates in question is indicated by +

79

Table 4.12. Typical reaction patterns of the isolates

S.No Isolate TSI Nitrate

test

Gelatin

hydrolysis

Catalase

1. Pa1 - + - +

2. Pa6 - + - +

3. MTCC 435 - + - +

4. MTCC 7443 - Nd - +

Nd- Not determined

80

(a) (b)

Figure 4.6. Colony morphology (a)

Microscopic view on gram staining (b)

81

Table 4.13. IMViC reaction patterns of the isolates

S.No Isolate Indole MR VP Citrate

1. Pa1 - + - +

2. Pa6 - + - +

3. MTCC 435 - + - +

4. MTCC 7443 - Nd - +

Nd- Not determined

The cultures Pa 1 and Pa6 did not show any hemolysis on the blood agar

plates. The cultures were plated on to Mannitol Salt Agar (MSA) to identify

the bacteria based on their ability to ferment Mannitol.Mannitol salt

agar or MSA is a commonly used selective and differential media,

containing carbohydrate mannitol and phenol red as a pH indicator for

detecting acid produced by mannitol-fermenting Staphylococci.

S.aureus produce yellow colonies with yellow zones, where as

other Staphylococci like S. epidermidis produce small pink or red colonies

with no color change to the medium (organism capable of fermenting

mannitol, an acidic byproduct is formed that will cause the phenol red in

the agar to turn yellow).

From Fig 4.7a, we can analyze that the culture Pa6 is

S. epidermidis as it is not able to ferment Mannitol and Pa1 as

82

S. aureus as it showed yellow colored colonies on MSA as shown in

Fig.4.7b.

4.5. Biofilm formation by isolate Pa 6 and S. epidermidis 435

Catheters and other medical devices are generally contaminated by the

Biofilms. The major virulence factor for S. epidermidis is the ability to form

biofilms. It produces an extracellular material made up of sulfated

polysaccharides known as Polysaccharide Intercellular Adhesion (PIA). It

allows other bacteria to bind to the already existing biofilm, creating a

multilayer biofilm. Fig. 4.8a and 4.8b, shows black colonies with a dry

crystalline consistency indicating the biofilm formation by Pa6 and

S. epidermidis MTCC 435.

Based on the biochemical tests, ability to ferment mannitol and formation

of biofilm formation, the organism Pa1 is identified as Staphylococcus

aureus and Pa6 is identified as Staphylococcus epidermidis.

83

(a) (b)

Figure 4.7. Colony morphology on MSA plateof Pa6 isolate (a)

Pa1 isolate (b)

Figure 4.8. BHI plates showing biofilm formation by

isolate Pa6 (a) and S. epidermidis MTCC 435 (b)

84

4.6. SYNTHESIS OF ZnO NANOPARTICLES:

The ZnO nanoparticles were synthesized using different methods. ZnO

nanoparticles are synthesized by wet chemical method, which is a simple

method and the synthesis can be carried out under ambient atmosphere

at RT. Different methods of synthesis and capping agents were employed

for the synthesis. Capping agents like Tri ethanolamine (TEA), Thio

glycerol and Oleic acid have been used for the synthesis of the ZnO

nanoparticles using Zinc acetate, KOH, ethanol and DMSO. It has been

found that Thioglycerol is more effective capping agent as compared to

oleic acid and TEA with less particle size and more antibacterial activity

(Singh et al., 2009).

The ZnO nanoparticles synthesized using thioglycerol as capping agent

were very effective antibacterial agents against P. acnes compared to TEA,

OA or starch as inferred from Table 4.19.Thioglycerol capped ZnO

nanoparticles were well characterized by UV-VIS spectroscopy, X-ray

diffraction (XRD), Scanning electron microscopy-Energy Dispersive X-Ray

Analyser (SEM-EDX), Transmission electron microscopy (TEM), Fourier

Transform Infrared spectroscopy (FTIR) and Thermo gravimetric analysis

(TGA).

85

CHARACTERIZATION OF ZNO NANOPARTICLES

4.7.1. Characterization using UV-VIS spectrophotometer

UV-VIS absorption spectrum of the as-synthesized ZnO sample was

recorded in the wavelength range 300 to 400 nm using a double beam UV-

VIS spectrophotometer with DMSO as the solvent (reference) in which the

nanoparticles were dispensed for the analytical study of ZnO

nanoparticles. Fig. 4.9 shows the spectrum recorded at room temperature.

A peak or absorption band was observed at 334 nm that is in accordance

with reported in literature and published in Principles of Nanotechnology

by Sulabha Kulakarni ( 2007 ).

86

Figure. 4.9. UV-VIS absorption spectrum of as-synthesized ZnO

nanoparticles dispersed in DMSO

4.7.1.1. Calculation of the band gap energy

Band Gap Energy (E) = h*c/λ (Hoffman et al., 1995)

h = Planck’s constant = 6.626 x 10-34 joules sec

c = Speed of light = 3.0 x 108 meter/sec

λ = Cut off wavelength = 334 x 10-9 meters

Table 4.14. Calculations for band gap

h C λ E eV

6.626 x 10-34

joules sec

3.0 x 108

meter/sec

334

nm

5.8 x 10-19

joules

3.625

where 1eV = 1.6 X 10-19 Joules (conversion factor)

The band gap is calculated and the values tabulated in Table 4.14. In the

bulk material the bands are actually comprised of adjacent energy levels

of a huge number of atoms and molecules (Juhi Soniet al., 2015). As the

particle size reaches the nanoscale, the number of overlapping of orbitals

or energy levels decreases and the band width becomes narrow and there

is an increase in energy gap between valence band and conduction band.

The energy gap of nanoparticles is more than the corresponding bulk

material. There is a shift of absorption spectrum toward blue region

exhibiting lower electrical conductivity of the nanomaterial (Juhi Soni et.

al., 2015). The band gap energy calculated in the present study is similar

to published in literature by Juhi Soni et. al., 2015, whose reports suggest

87

that the increase in concentration of the capping agents increases the

band gap.

4.7.2. X-Ray Diffraction studies

ZnO nanoparticles were characterized by using XRD in the scanning

scanning range of 2 - 80 o (2θ) using copper Kα radiation with a wavelength

of 1.5406 Ao. Fig. 4.10 shows the X-ray diffraction pattern of ZnO

nanoparticles. The XRD powder diffraction pattern of the ZnO

nanoparticles obtained by sol-gel synthesis shows characteristic peaks of

ZnO, zinc acetate and ZHA. The broadening of the X-ray diffraction lines

reflects the nanoparticle and ultra-fine nature of the crystallites. The

peaks obtained for ZnO-NPs at 2θ = 31.7o, 32o, 34.5o, 36.12o, 56.5o, 62.5o

are characteristic hexagonal wurtzite structure of ZnO nanoparticles. The

other peaks are due to the presence of acetate and other intermediates.

Zn5(OH)8 (CH3COO)2.2H2O ( Zinc hydroxide acetate) can be considered as

an important intermediate product and its formation and transformation

into ZnO can be represented by the following reactions: (Luković Golić et.

al., 2011)

5Zn(CH3COO)2.2H2O Zn5(OH)8(CH3COO)2.2H2O + 8CH3COOH

Zn5(OH)8(CH3COO)2 .2H2O 5ZnO + 2CH3COOH + 5H2O

ZHA (Zinc Hydroxide Acetate) is an intermediate product of the hydrolysis

reaction, preferentially formed in the presence of H2O and OH- ions. It can

be easily transformed into ZnO at higher temperatures and with prolonged

refluxing.

88

Table 4.15 shows that the calculated d-values that are in good agreement

with those taken from the JCPDS- Joint Committee of Powder Diffraction

Standards card file data for ZnO powder. Debye Scherrer formula was used

to calculate the particle size D and the values are tabulated in Table 4.15.

The average particle size is calculated using D = Kλ/β cos θ.

λ = 0.154 nm, the X-ray radiation used, θ is the Bragg diffraction angle of

the XRD peak and β is the measured broadening of the diffraction line

peak at an angle of 2θ, at half its maximum intensity (FWHM) in radian.

The XRD peak can be widened by internal stress and defects, so the

average size calculated by this method is normally smaller than the actual

value (Raoufi et al., 2013). The average particle size calculated using Debye

Scherrer equation is 11.12 nm.

Table 4.15. XRD parameters 2θ and d-value

ZnO prepared in

this work

JCPDS 36-1451

2θ d-value 2θ d-value

31 2.897 31.770 2.814

32 2.843 34.44 2.603

34.5 2.620 36.25 2.475

36.12 2.491 47.53 1.911

56.5 1.632 56.60 1.624

62.5 1.484 62.86 1.477

89

Figure 4.10. XRD pattern of ZnO nanoparticles

Zn5 (OH)8 (CH3COO)2 2H2O ZnO

Zn3 (OH)4(CH3COO)2 Zn (CH3COO)

90

4.7.3. SEM-EDX analysis

Fig. 4.11, shows the SEM images of ZnO capped with 0.12 ml of

thioglycerol. Though the nanoparticles are not observed clearly in SEM

images, but are seen in TEM Fig. 4.13. Fig. 4.12 and Table 4.16, shows

EDX analysis results. It shows zinc and oxygen are in stoichiometric ratio.

Presence of sulphur is due to the capping agent thioglycerol.

Figure 4.11. SEM of ZnO nanoparticles

91

Figure 4.12. EDX of ZnO nanoparticles

Table 4.16. Quantification of EDX data

Element Weight %

Zn 67.75

Oxygen 15

Potassium 15

Sulphur 1.25

Carbon 1.00

92

4.7.4. TEM Analysis of ZnO nanoparticles

Figure 4.13.TEM Analysis (a), SAED pattern of ZnO nanoparticles (b)

TEM images of ZnO nanoparticles are shown in the Figure 4.13a. Results

of Transmission Electron Microscope shows that the ZnO nanoparticles

capped with thioglycerol are well separated. TEM analysis shows that the

particle size is around 10nm. From the Fig. 4.13b selected area electron

diffraction (SAED) patterns of ZnO nanocrystals it can be inferred that as

observed in XRD, SAED also shows characteristic patterns that are due

to ZnO, zinc acetate and Zn5 (OH)8 (CH3COO)2 2H2O , an intermediate

compound observed during the synthesis.

93

4.7.5. TGA analysis of ZnO nanoparticles

The thermo gravimetric analysis of the nanoparticles as shown in Fig. 4.14

shows three step decomposition. The first step between room temperature

and 130 °C could be due the evolved solvent molecules that are present

during the synthesis process. Steps between 200 and 350 °C and 350

and 475 °C are due the decomposition of zinc acetate and

Zn5(OH)8(CH3COO)2.2H2O, an intermediate compound observed during

the synthesis and these observations correlate with the observation by

XRD.

Figure 4.14. TG/DTA Analysis of ZnO nanoparticles

94

4.7.6. FTIR Analysis of ZnO nanoparticles:

Figure 4.15. FTIR Analysis of ZnO nanoparticles

FTIR analysis was carried out to analyze the ZnO nanoparticles. From the

Fig. 4.15 and Table 4.17, we can interpret that the peaks observed at

3387 cm-1 and 1020 cm-1 correspond to the O-H stretching. The peaks

observed at 1573cm-1, 651 cm-1 and 450 cm-1 can be assigned to Zn-O

stretching. The bands in the range from 900-1300 cm-1 and

1300- 1500 cm-1 are associated with C-O and C-H vibrations. (Vanaja and

Rao, 2016., Ghule et.al., 2006).

95

Table 4.17. FTIR interpretation of ZnO nanoparticles

Wave number (cm-1 ) Assignment

3400-3700 Alcohol/phenol O-H stretch

3387 and 1020 O-H stretching

900-1300 C-O

1300-1500 C-H

1700- 1630 C=O stretch

1573, 651 and 450 ZnO

From these results we can infer that the as-synthesized zinc oxide

nanoparticles have been well characterized and were used to prepare

cosmetic skin care products that can be used against acne causing

organisms.

4.8. ANTIBACTERIAL ACTIVITY OF ZNO NANOPARTICLES

The nanoparticles were synthesized using different methods and various

capping agents. The antibacterial activity of these nanoparticles was tested

against the standard MTCC cultures procured from IMTECH, Chandigarh,

P. acnes MTCC 1951, S. epidermidis MTCC 435, S. aureus and Pa3 by

agar well diffusion method. The ZnO nanoparticles synthesized using three

different methods were labelled as ZnO-I, ZnO-II, ZnO-III. The stability of

the nanoparticles with time was tested for about three weeks against the

96

organisms and the results were tabulated. From the Table 4.18, 4.19,4.20

and Fig.4.16,4.17,4.18, we can infer that the nanoparticles were effective

against the organisms and out of the three methods employed the

nanoparticle synthesized using method three that is ZnO –III is more

effective against the organisms P. acnes and S. epidermidis. The ZnO

nanoparticles were dispersed in methanol and DMSO and the solvents

methanol and DMSO were used as controls. The activity of the

nanoparticles increased when DMSO was used as solvent. DMSO used as

control did not show any activity. The increase in activity of the ZnO

nanoparticles in DMSO is because of better dissolution of the

nanoparticles in DMSO when compared to methanol. The nanoparticles

were found to be stable and they showed good antibacterial activity till the

third week of synthesis, thus making them potential antibacterial agents

against acne causing organisms. ZnO –III was synthesized using

thioglycerol, oleic acid and triethanolamine as capping agents. Out of the

three capping agents used, thioglycerol capped ZnO nanoparticles have

shown to be better antibacterial agents against P. acnes, S. epidermidis

and S. aureus. This may be due to the effect of the capping agent. The

thioglycerol capped particles are much smaller than the TEA and Oleic

acid capped ZnO nanoparticles. This is because of the amine group of

triethanol amine, which is much greater in comparison to the ‘S’ of

thioglycerol which is less hindered. The large size of Sulphur makes it a

good capping agent than TEA. Oleic acid also is an effective capping agent

97

but at a higher concentration. Similar results have been reported by

Singh et al, Ghosh et al and Tan et al. “Low molecular weight thiols or their

esters such as 2-mercaptoethanol, 1-thioglycerol and dithiothreitol have

been used as agents to denature proteins by reducing the disulfide

linkages leading to the tautomerization and breaking up of quaternary

protein structure. They have been studied as anticancer agents by

damaging the DNA of the cancer cells (Mueller et al., 2008; Yoon et al.,

2007).

Table 4.18. Antibacterial activity of ZnO nanoparticles tested for 3 weeks

I, II, III represent the nanoparticles synthesized using three methods and

1st,2nd, 3rd represent weeks

ZnO nanoparticles I I

I

II

II

II

III

III

III

Week 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd

Organisms Zone of Inhibition in cm

P. acnes MTCC

3297

1.8 2.0 2.0 1.8 1.8 1.8 2.0 2.0 2.0

S. aureus 2.0 2.0 2.0 2.0 2.1 2.1 1.9 1.9 2.0

S. epidermidis 1.8 1.9 1.9 1.8 1.8 1.8 2.0 2.0 2.0

98

Figure. 4.16. Graphical illustration of antibacterial activity of ZnO

nanoparticles against S. epidermidis

1.81.9 1.9

1.8 1.8 1.8

2 2 2

0

0.5

1

1.5

2

2.5

1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd

I I I II II II III III III

zon

e o

f in

hib

itio

n

Antibacterial activity of ZnO nanoparticles against S.epidermidis

ZnO I ZnO II ZnO III

99

a. b.

Figure 4.17. Antibacterial activity of ZnO nanoparticles against isolates

S3(a) , S5 (b).

a.b.

Figure 4.18. Antibacterial activity of ZnO nanoparticles against and Isolate

Pa3 (a), P.acnes MTCC 1951 (b).

100

a. b.

Figure 4.19. Antibacterial activity of ZnO nanoparticles I, II and III against

Propionibacterium acnes 3297

Figure 4.20. Antibacterial activity of ZnO nanoparticles I, II and III against

S. aureus

101

Figure 4.21. Effect of temperature on the antibacterial activity of ZnO

nanoparticles against P. acnes MTCC 1951

102

Table 4.19. Effect of different capping agents on antibacterial activity of

ZnO nanoparticles

S.No Organism Zone of Inhibition cm

TG TEA OA

1. P.acnes

MTCC 1951

2.8 2.3 2.0

2. P.acnes

MTCC 3297

2.0 1.2 1.5

3. Isolate Pa3 2.5 2.0 2.0

4. S.epidermidis

MTCC 435

1.8 1.7 1.2

5. S.aureus

MTCC 7443

2.0 1.7 1.2

103

Table 4. 20. Antibacterial activity of ZnO nanoparticles

S.No Organism Zone of inhibition

ZnO NP –III (in cm)

1. Pa2 1.8

2. Pa3 2.5

3. Pa4 1.5

4. Pa5 1.7

5. S3 2.4

6. S5 2.5

7. P.acnes 1951 2.8

8. P.acnes 3297 2.0

104

Table 4.21. Stability of ZnO nanoparticles when placed at different

temperatures

S.No. Sample name

ZoI (Cm)of ZnO np placed at

RT

ZoI (Cm) of ZnO np placed in

oven

ZoI (Cm) of ZnO np placed in

Fridge

1. P.acnes

MTCC1951

2.8 0.5 1.5

2. P.acnes

MTCC3297

2.0 1.4 -

3. Pa4 1.5 - -

4. Pa3 2.5 1.5 1.5

5. Pa2 1.5 - -

6. Pa5 1.7 - -

7. S.epidermidis

MTCC 435

1.8 - -

8. S.aureus

MTCC 7443

1.8 - -

105

The nanoparticles were placed at RT, in Oven and in Refrigerator for 24 h

and the antibacterial activity of these nanoparticles was tested against the

different isolates and standard MTCC cultures by agar well diffusion

method and the zone of inhibition was measured after incubation.

Table 4.22. MIC of ZnO nanoparticles against P. acnes

Flask

No

BHI broth

/np/culture

OD at 540 nm

1. 0.5mg 0.0

2. 1mg 0.00

3. 1.5mg 0.01

4. 2mg 0.02

5. 0.5mg+culture 0.25

6. 1 mg+ culture 0.1

7. 1.5 mg+ culture 0.01

8. 2mg+culture 0.02

9. Culture 0.28

106

The results clearly indicate that the ZnO nanoparticles are effective

antibacterial agents. To study the least concentration effective against the

organism, different concentrations of the nanoparticles and the broth

cultures of P.acnes were taken and incubated at

37oC for 4 days and absorbance was recorded (Table 4.18). The

nanoparticles were effective at a concentration of 1.5 mg/5 ml which is

0.3mg in 1 ml. The stability of the nanoparticles at different temperatures

was tested by placing the nanoparticles at RT (RT), in oven at a

temperature of 45o C, in Refrigerator at a temperature of 4 o C for 24h. The

observation of P. acnes plates (Fig. 4.21) shows that the nanoparticles are

stable at RT and the antibacterial activity of the nanoparticles against the

acne causing organism P. acnes decreased when the nanoparticles were

placed at low temperatures. The nano particles bring about a change in

the levels of proteins inside the cell. This might be due to the increase in

the cell permeability and cellular internalization of the nanoparticles.

Nanoparticles possess greater surface area which results in more

interaction with the cell making them better antibacterial agents than the

bulk particles. On entry into the bacterial cells, the zinc nano-particles

react with the proteins (enzymes, cellular proteins) and nucleic acids

(especially DNA), and denature them, thus inhibiting the replication. One

of the main advantages of employing zinc oxide nano-particles as anti-

bacterial agents is that there are very few chances of the bacteria

developing resistance, since their activity is nonspecific. However, the

107

inhibitory effect of zinc nanoparticles depends upon the concentration of

nanoparticles used, the number of bacteria in the sample and the type of

bacteria in the sample. It has also been found that zinc oxide nano-

particles prevent the attachment of bacteria to the host cell surface and

the formation of bio-films. ZnO is the current focus of the Nanotechnology

Safety Initiative under National Institute of Environmental Health and

Safety. These features of zinc oxide nano-particles suggest that they would

make excellent anti- microbial agents for treating acne.

4.9. ANTIBACTERIAL ACTIVITY OF ZNO NANOPARTICLES

(COMMERCIAL) AND TETRACYCLINE AGAINST P. acnes

The antibacterial activity of the nanoparticles synthesized was compared

with the standard nanoparticles purchased from nano labs. The size of the

commercial nanoparticle was in the range of 50-75nm. The activity of the

nanoparticle was around 2.5 cm which was similar to that of the activity

shown by the nanoparticles synthesized using the wet chemical method.

The method of synthesis employed by the nano labs is not known.

The antibacterial activity of the Tetracycline antibiotics which are used for

topical application and also taken orally is tested by agar well diffusion

method. The in vitro activity of the antibiotic is similar to that shown by

the nanoparticles (Fig. 4.22). Though the antibiotic is effective against the

organism, the organism generally develops resistance towards the

antibiotic (Oprica et al., 2006).

108

4.10.PREPARATION OF COSMETIC SKIN CARE PRODUCTS USING

ZnO NANOPARTICLES

Different face products like creams, scrubs, face packs, natural and

commercial products were prepared using ZnO nanoparticles and their

antibacterial activity was tested in vitro.

Figure 4.22. Antibacterial activity of Tetracycline antibiotic against

isolates S3 and S5

Figure 4.23. Antibacterial activity of commercial ZnO nanoparticles

against isolate Pa3.

109

a. b.

c.

Figure 4.24. Antibacterial activity of vanishing cream I (a) and II (b)

against P. acnes 1951 and calamine (c )prepared using ZnO nanoparticle

against Pa3.

110

Figure 4.25. Antibacterial activity of cold cream prepared with ZnO

nanoparticles against the isolates

Figure 4.26. Effect of temperature on antibacterial activity of cream

against P.acnes MTCC 1951

111

Table 4.23. Antibacterial activity of cream prepared with ZnO

nanoparticles against acne causing organisms

Vanishing cream I and II, calamine and cold cream prepared with ZnO

nanoparticles as one of the components were very effective against the

acne causing organism. Cold cream though effective in vitro (Fig. 4.25) is

not suitable for application during all seasons as cold cream is a seasonal

cream, which is an emulsion of fats and water that is used to soften the

skin. It acts as a cleansing cream and is generally applied after removal of

makeup keep it hydrated and glowing. Cold creams are famous during

S.NO Sample name ZoI (in Cm) of

cream I

1. 1951 2.8

2. 3297 1.8

3. Pa4 1.5

4. Pa3 3.0

5. Pa2 1.5

6. Pa5 1.7

7. S. epidermidis 1.1

8. S. aureus 1.7

112

winters and its application is avoided during the summers due to the hot

and humid climate. Calamine lotion prepared without the addition of ZnO

nanoparticles used as control was effective against the acne causing

organisms. The addition of ZnO nanoparticles increased the activity of the

lotion (Fig. 4.24 c). Though it is an effective agent in treating acne, side

effects like the interaction of calamine with the drugs/ medicines have

been reported, hence it has to be used under strict monitoring of a Medical

practitioner and is to be applied in the doses specified by the Physician.

Vanishing creams act as moisturizing agents and do not give greasy feeling

as they vanish quickly on absorbance by the skin. Moisturizer an

emulsifying cream, also known as 'emollient' is used to control the

moisture content and help in reducing the dryness of the skin. It is a

complex mixture of chemical agents, especially designed to make the outer

layer of the skin (epidermis) softer and more pliable. The basic function of

a moisturizer is to increase the skin’s hydration, or water content, by

reducing the rate of evaporation. Vanishing creams incorporated with ZnO

nanoparticles thus can be used as good topical agents against acne as they

can be used in all seasons, keep the skin moisturized, non-greasy with no

side effects (Table 4.23, Fig. 4.24. a, b, c).

The ZnO nanoparticles were also used in the preparation of mild scrubs

which were prepared with natural ingredients like orange peel and oat

meal, as commercially available scrubs are little harsh on the skin and

113

might be painful. Scrub basically helps in removing the dead cells and

hardened sebum thus clearing the skin. Scrubs were prepared using a

combination of orange peel (sun dried and powdered), oat meal (powdered),

ZnO nanoparticle, SDS, EDTA, methyl and propyl paraben in trace

amounts. Scrubs were also prepared with only orange peel and all other

ingredients without oat meal, all ingredients and oat meal without the

orange peel to see the effect of orange peel and oat meal separately and in

combination. Orange peel and oat meal scrubs were prepared and the

activity was tested with and without SDS to show the specific effect of

nanoparticles on the acne causing organisms. From the results as shown

in Table 4.16 and Fig. 4.27, 4.28, 4.29, 4.30, we can infer that the scrub

incorporated with the ZnO nanoparticles showed very good activity. Scrub

prepared using combination of oat meal and orange peel showed better

activity followed by scrub made using only oat meal and other ingredients

compared to that of scrub prepared using only orange peel. The ZnO

nanoparticles were added to the commercial walnut scrub and the scrub

without nanoparticles was used as control. From Table 4.15 and Fig. 4.28

we can infer that the nanoparticle incorporated scrub was very effective

against the organism.

Face packs were made of multani mitti or the fullers’ earth and ZnO

nanoparticles were added to it. Multani mitti, also known as the Fuller’s

earth is a clay substance which is very rich in magnesium chloride. It can

be used as a cleanser, toner and most importantly as multani mitti facial

114

packs. Multani mitti, a dry powder is incorporated with the nanoparticles.

1g of clay was mixed with 1g of the nanoparticles and constituted in either

Rose water or water just before testing its effect as an antibacterial agent

against P. acnes. Multani mitti without the nanoparticles was used as

control. The activity of multani mitti against P. acnes increased when

mixed with the nanoparticles (Table 4.24, Fig. 4.29, 4.30).

The antibacterial activity of the ZnO nanoparticles incorporated into

commercial walnut scrub and Neem face wash was tested by using Agar

well diffusion method. From the results as shown in Fig.4.31, it can be

analyzed that the nanoparticle incorporated face products were effective

against acne causing organism in vitro. The stability of the activity of the

nanoparticle incorporated creams, scrubs was tested for a period of six

months and the activity of the nanoparticles was stable in the face

products (Table 4.24). Tulsi, Neem, Aloe Vera and Mint are known to be

natural products which are generally used against acne as home remedies

and most of the commercial face washes recommended for acne contain

these extracts as one of the core ingredients. The plant extracts were mixed

with ZnO nanoparticles at a concentration of 1mg/ml and the antibacterial

activity was tested against acne causing organism, P. acnes. Mint and

Neem were effective against the organism but their activity was enhanced

on mixing with the nanoparticles. Tulsi and Sandalwood extracts were

effective against P. acnes only when mixed with ZnO nanoparticles(Fig.

4.32), ZnO nanoparticles were effective against P. acnes in vitro and the

115

skin care products prepared using nanoparticles were stable with time and

temperature. Cytotoxicity of ZnO nanoparticles was determined using MTT

Assay and MCF 10A cell lines (epidermal cell line). In vivo studies of

vanishing cream prepared using ZnO nanoparticles were tested in Mice.

Table 4.24. Antibacterial activity of cosmetic skin care products prepared

using ZnO nanoparticles against isolate Pa3

Sample 1stweek

(Zone diameter –cm)

3 Months

Zone diameter –cm)

6 Months

(Zone diameter –cm)

Control

(without ZnO NP)

Scrub 2.5 2.5 2.4 -

Oat meal scrub

2.0 2.0 2.0 -

Orange peel scrub

1.2 1.2 1.1 -

Scrub (without

SDS)

3.5 - -

Face pack (MRW)

3.5 3.5 3.0 -

Facepack

(MW)

3.5 3.0 3.0 -

Wallnut scrub

3.0 Nd Nd -

Facewash –

Neem

2.5 Nd Nd Nd

Face Wash (C )

2.0 Nd Nd Nd

Nd- Not determined; C- Commercial

116

Figure. 4.27. Antibacterial activity of orange peel scrub with nanoparticles

Figure. 4.28. Antibacterial activity of Scrub with orange peel, oat meal

and ZnO nanoparticles indicated as 22.

Scrub with only oat meal and ZnO nanoparticles indicated as 23

117

Figure. 4.29. Antibacterial activity of Multani mitti- ZnO nanoparticles in

water and control.

Figure. 4.30. Antibacterial activity of Multani mitti ZnO nanoparticles in

rose water and control

118

Figure. 4.31. Antibacterial activity of Commercial scrub with

nanoparticles; control without nanoparticles

Figure. 4.32. Antibacterial activity of Plant extracts mixed with ZnO

nanoparticles

119

4.11. Cytotoxicity effect of the sample ZnO nanoparticles on MCF

10 A Cell line (human epidermal cell line)

Measurement of cell viability and proliferation forms the basis for

numerous in vitro assays of a cell population’s response to external

factors. The MTT assay, one of the rapid colorimetric methods to determine

the cell viability, was used to assess the cytotoxicity of ZnO nanoparticles.

The human epidermal cell lines MCF 10 A were selected to study the

cytotoxic effect of ZnO nanoparticles and the results obtained were

tabulated. The reports prove that ZnO nanoparticles are not toxic to

human epidermal cell lines and the 90 % cell viability was seen when

treated with the ZnO nanoparticles up to a concentration of 100 µg/ml.

IC50 was not determined as the nanoparticles were not toxic to the cell

lines.

Sl. No Compound in MCF10 A IC50 (µg/ml)

1 ZnO nanoparticles ND

ND- not determined

120

Concentration of cells µg/ml

cells

4.33. Graphical illustration showing the cytotoxicity effect of the ZnO

nanoparticles on MCF 10 A Cell line

Table 4.25. Cytotoxicity of ZnO nanoparticle against MCF 10 A cell line

Con.

µg/ml

Absorbance at

570nm

% cell

Viability Avg

%

Cytotoxicity

100 1.544 1.666 1.544 90.45 89.98 90.63 90.35 9.65

75 1.358 1.452 1.398 91.54 91.49 91.47 91.5 8.5

50 1.358 1.552 1.398 91.74 91.76 91.75 91.75 8.25

25 1.817 1.899 1.888 100 100 100 100 0

10 1.958 1.952 1.998 100 100 100 100 0

5 2.544 2.666 2.544 100 100 100 100 0

Con. – concentration

Avg- Average

% o

f In

hib

itio

n

121

a b

4.34. Photomicrographs showing the MCF 10A epidermal cell lines a.

control cells (untreated) and b. cells treated with 100 µg/ml ZnO

nanoparticles. There was no change in the morphology of the cells.

4.12. Effect of cream prepared with ZnO nanoparticles on P.acnes

MTCC 1951 in vivo and P.acnes induced inflammation in mice

P. acnes suspensions were prepared at concentrations of 107 CFU/ ml.

P. acnes suspended in PBS were injected in 20 μL aliquots intra dermally

in the right ears of the mice of group III. Left ears received only PBS

injection. After 24 h of P. acnes injection, significant cutaneous erythema,

ear swelling and granulomatous response were observed in the right ears

of groups 2 & 3. Group 2 was left untreated and group 3 was used for the

study of Anti – Inflammatory effect of the cream. 2 mg cream was applied

on the right ears of mice and the decrease in thickness of the ear was

measured using a micro caliper. Cutaneous erythema and swelling

reduced in the ears of Mice with P. acnes infection when cream prepared

122

with the nanoparticle was applied topically. P. acnes induced ear swelling

significantly reduced from day 4 and the ear was normal by day 6 as

observed in Fig.4.35 and Table 4.27. Whereas the sole application of cream

for a month (without pre injection of P. acnes) in group 1 did not induce

inflammation and there was no change in the morphology of the ear.

Inflammation and ear swelling persisted in Group 3 even after a month as

it was left untreated. From the results of in vitro and in vivo studies in

mice, we can infer that the ZnO nanoparticles are effective antibacterial

agents against P. acnes as the present treatments available have side

effects or the organism develops resistance to the agents.

ZnO nanoparticles on the other hand are mild and the key findings of

Premanathan et al., 2011, that the ZnO nanoparticles induce toxicity in a

cell specific and proliferation dependent manner. HL 60 and normal

PBMC’s were assessed for the toxicity of ZnO nanoparticles by MTT assay.

ZnO nanoparticles displayed greater toxicity towards cancer cells than the

normal cells.

Figure 4.35. Left normal ear and right P. acnes induced ear of mice

123

Table 4.26. Group I of mice- application of only cream on the right ears

S.NO Mice

1

Mice

2

Mice

3

Mice

4

Mice

5

Mice

6

Mean STD

V

Day

1

R-ear 2.8 2.7 2.8 2.7 2.8 2.8 2.766 0.05

Day

0

L-ear 2.8 2.7 2.8 2.7 2.8 2.8 2.766 0.05

Day

2

R-ear 2.9 2.8 2.9 2.8 3 3 2.9 0.08

Day

1

L-ear 2.8 2.7 2.8 2.7 2.8 2.8 2.766 0.05

Day

3

R-ear 2.8 2.7 2.8 2.7 2.8 2.8 2.766 0.05

Day

2

L-ear 2.8 2.7 2.8 2.7 2.8 2.8 2.766 0.05

Day

4

R-ear 2.8 2.7 2.8 2.7 2.8 2.8 2.766 0.05

Day

3

L-ear 2.8 2.7 2.8 2.7 2.8 2.8 2.766 0.05

Day

5

R-ear 2.8 2.7 2.8 2.7 2.8 2.8 2.766 0.05

Day

4

L-ear 2.8 2.7 2.8 2.7 2.8 2.8 2.766 0.05

Day

6

R-ear 2.8 2.7 2.8 2.7 2.8 2.8 2.766 0.05

Day

5

L-ear 2.8 2.7 2.8 2.7 2.8 2.8 2.766 0.05

Day

7

R-ear 2.8 2.7 2.8 2.7 2.8 2.8 2.766 0.05

Day

6

L-ear 2.8 2.7 2.8 2.7 2.8 2.8 2.766 0.05

*R- Right , L- Left

124

Table 4.27.Group II of mice- right ears injected with P.acnes and left

untreated

S.NO Mice

1

Mice

2

Mice

3

Mice

4

Mice

5

Mice

6

Mean STDV

Day

0

R-ear 3 2.9 3 2.9 3.1 3.2 3.02 0.12

L-ear 3 2.9 3 2.9 3.1 3.2 3.017 0.117

Day

1

R-ear 3.2 3.1 3.1 3.2 3.3 3.3 3.2 0.09

L-ear 3.1 3 3 2.9 3.2 3.2 3.07 0.12

Day

2

R-ear 3.6 3.4 3.4 3.3 3.5 3.4 3.4 0.1

L-ear 3 2.9 3 2.9 3.1 3.2 3.02 0.12

Day

3

R-ear 3.5 3.5 3.4 3.4 3.5 3.4 3.45 0.05

L-ear 2.9 2.8 2.8 3 2.9 2.8 2.87 0.08

Day

4

R-ear 3.5 3.5 3.4 3.4 3.5 3.4 3.45 0.05

L-ear 2.9 2.8 2.8 3 2.9 2.8 2.87 0.08

Day

5

R-ear 3.5 3.5 3.4 3.4 3.5 3.4 3.45 0.05

L-ear 2.9 2.8 2.8 3 2.9 2.8 2.87 0.08

Day

6

R-ear 3.5 3.5 3.4 3.4 3.5 3.4 3.45 0.05

L-ear 2.9 2.8 2.8 3 2.9 2.8 2.87 0.08

125

Table- 4.28. Group III of mice- right ears injected with P.acnes and

treated with cream

S.NO

Mice

1

Mice

2

Mice

3

Mice

4

Mice

5

Mice

6

Mean STDV

Day

0

R-ear 3 2.9 3 2.9 3.1 3.2 3.02 0.12

L-ear 3 2.9 3 2.9 3.1 3.2 3.017 0.117

Day

1

R-ear 3.2 3.1 3.1 3.2 3.3 3.3 3.2 0.09

L-ear 3.1 3 3 2.9 3.2 3.2 3.07 0.12

Day

2

R-ear 3.6 3.4 3.4 3.3 3.5 3.4 3.4 0.1

L-ear 3 2.9 3 2.9 3.1 3.2 3.02 0.12

Day

3

R-ear 3.5 3.5 3.4 3.4 3.5 3.4 3.45 0.05

L-ear 3 2.9 3 2.9 3.1 3.2 3.0167 0.1169

Day

4

R-ear 3.4 3.3 3.4 3.3 3.3 3.3 3.333 0.052

L-ear 3 2.9 3 2.9 3.1 3.2 3.016 0.116

Day

5

R-ear 3.3 3.2 3.2 3.3 3.3 3.3 3.267 0.052

L-ear 3 2.9 3 2.9 3.1 3.2 3.016 0.1169

Day

6

R-ear 3 2.9 3 2.9 3.1 3.2 3.02 0.12

L-ear 3 2.9 3 2.9 3.1 3.2 3.016 0.116

126

The resistance of organisms to various antibiotics and a possibility of

recurrence of the disease pose a threat to the public health. Both gram

positive and gram negative bacteria have shown resistance to major

antibiotics and thus an alternate therapeutic treatment has to be

employed in order to eradicate the infectious diseases in both community

and hospital environment (Lowy,1998)

Nanotechnology, particularly the nanoparticles are the new antibacterial

agents that can be used effectively in the treatment of infectious diseases,

due to their small size and more surface area they prove to be potentially

useful agents for treating the infections.

Preliminary studies carried out by Ameer Azam et al, 2012, shows that

ZnO inhibits the growth of organisms. When compared to CuO, Fe2O3

metal oxides, bulk ZnO has been used as a major component in the

preparation of cream, lotions and ointments used for dermatological

application, same has been reported by Sawai in 2003. The antibacterial

effect of the nanoparticles is more because of their small particle size and

larger surface area which helps in efficient interaction of the nanoparticle

with the organism and hence impart cytotoxicity to the microorganisms.

This is in accordance with the reports of Wani et al., 2012, and Qilin et al.,

2008, who have reported the enhancing effect of ZnO nanoparticles against

S.aureus and for disinfection of water.

127

The present study focused on the antibacterial activity of ZnO

nanoparticles on acne causing organisms- P. acnes, S. aureus and

S. epidermidis. ZnO nanoparticles were effective at a concentration of

0.3 mg ml-1 and was not toxic to the epidermal cell lines up to a

concentration of 0.1mg ml-1

ZnO nanoparticles which have good antibacterial effect cause the

disorganization of membranes in gram positive organisms and results in

the changes in the level of proteins due to the internalization of these

nanoparticles which is a result of modification of ZnO nanoparticles.

Due to the advantages of ZnO nanoparticles, the focus of the research is

to evaluate the antibacterial activity of ZnO nanoparticles against acne

causing organisms.

Acne vulgaris is a major skin disease which effects the pilosebaceous

glands. Many treatments are such as use of antibiotics retinoids, BPO,

plant extracts have been used for treating acne.

Neem is an effective antibacterial agent against acne, but the duration of

treatment is long. Margosa or neem oil is most widely used as a medical

remedy but has a disagreeable small and bitter taste. National Reye’s

syndrome foundation have reported though rare, that the prolonged use

of this will result in metabolic acidosis, hepatic and organ failure. Addition

of ZnO nanoparticles have enhanced the activity and thus the treatment

of acne can be faster with less concentration of neem and this helps to

128

overcome the side effects of neem. Reports by Maria Jose et al., 2013 on

antifungal activity have shown orange peel to be effective at a MIC of 8000

mg for vapour contact.

Antibacterial activity of Orange peel was carried out against food borne

pathogens like S. aureus, E.coli, Salmonella species have been reported by

Hasija et al.,. Their studies reported the Zone of Inhibition by orange peel

to be around 10mm. The addition of ZnO nanoparticles to the orange peel

in the present study resulted in increase in the activity by 3 times against

P. acnes. Literature does not reveal use of oat meal as an effective

antibacterial agent. Oatmeal in the present study is employed as a means

of formulation for the application of ZnO nanoparticles on to face for acne

treatment.

ZnO nanoparticles can thus be used as potential antibacterial agents to

treat acne and various cosmetics prepared with the nanoparticles can be

used against acne as ZnO nanoparticles are effective against

Propionibacterium acnes, the acne causing organism.

129

CHAPTER 5.

SUMMARY AND CONCLUSION

Propionibacterium acnes isolated from acne is a slow growing, aero

tolerant, anaerobic gram positive bacterium that colonizes sebum rich

follicles, causes acne vulgaris commonly called as acne, a skin disease that

is most common during adolescence, afflicting more than 85% of

teenagers and over 40 million people in US alone (Darren et al., 2009, Fried

et al., 2006, Taglietti et al., 2008).

Antibiotics like tetracycline and antimicrobial agents such as benzoyl

Peroxide BPO have been used epicutaneously to treat acne. The oxidizing

agent BPO (Benzoyl peroxide), one of the most frequently used

epicutaneous medication has several side effects such as erythema,

scaling, burning and flare (Taglietti et al., 2009, Castro et al.,2008). Until

the late 1970s, P. acnes were universally susceptible to oral and topical

formulations of clindamycin, tetracycline, and other antimicrobial agents

(Eady et al., 2003). However, since the 1980s, numerous reports of

increasing Propionibacterium species resistance worldwide have been

published (Eady et al., 2003, Haider et al., 2004, Ross et al., 2003).

Resistance rates in 50% to 90% of isolates in Spain and Hungary have

been associated with the widespread therapeutic use of various agents

(Ross et al., 2003). On the other hand, Propionibacterium acnes have

shown resistance to tetracycline. And its sensitivity to azithromycin has

130

been reported to have several side effects such as accumulation in lungs

post antibiotic usage.

Growing resistance of microorganisms to potent antibiotics has renewed a

great interest towards investigating bactericidal properties of

nanoparticles and their nanocomposites as an alternative treatment

method.

Research related to the antibacterial activity of nanoparticles will enable

new developments in the field of nano medicine. Targeted drug delivery,

diagnostics, magnetic resonance, imaging etc., are possible because of the

biological cell interactions of the nanoparticles. The applications of

nanotechnology are countless. Quantum dots can be used in display

technology and biological imaging. DNA nanotechnology helps us

construct well-defined structures out of DNA and other nucleic acids. The

antimicrobial activity of nanoparticles has been studied with human

pathogenic bacteria. Several natural and engineered nano materials have

demonstrated strong antimicrobial properties through diverse

mechanisms including photo catalytic production of reactive oxygen

species that damage the cell components of bacteria and viruses (e.g.:

TiO2, ZnO), and also the bacterial cell envelope e.g.,ZnO and Ag nano

particles( Shailaja et al., 2013).

ZnO nanoparticles exhibit strong antibacterial activities on a broad

spectrum of bacteria and do not induce any cytotoxicity. Jones et

alobserved that smaller zinc oxide particles were more toxic to the

131

Microorganisms than the bulk particles. Zinc oxide nano-particles activity

increases with a reduction in the particle size (Qilin et al., 2008).

Zinc oxide nanomaterial are used in the preparation of substances

possessing medically useful properties. Due to its antibacterial properties,

zinc oxide is applied on the skin, in the form of powders, antiseptic creams,

surgical tapes and shampoos, to relieve skin irritation, diaper rash, dry

skin and blisters. Zinc oxide nano-particles possess excellent durability

and heat resistance (Qilin Li, 2008).Zinc oxide is more commonly used

than titanium dioxide nano-particles in sunscreens, since it is a better

blocker of ultraviolet light, and also causes lesser side effects. Zinc oxide

nano-particles also have good biocompatibility to human cells.

Research related to antibacterial activity of ZnO nanoparticles was carried

out against Staphylococcus aureus, E.coli, Klebsiella spp, Pseudomonas

spp, Proteus spp.,etc., No work has been published related to the

antimicrobial activity of ZnO nanoparticles against acne causing

organisms.

132

In the present study -

Acne causing organism is isolated using different media and

identified as Propionibacterium acnes based on biochemical

characters, FAME analysis and 16s rRNA typing.

Staphylococcus epidermidis and Staphylococcus aureus, associated

with acne were also isolated and characterized based on the

biochemical tests.

ZnO nanoparticles were synthesized by using wet chemical method

and were well characterized by XRD, TEM, SEM, EDX, FTIR, TGA,

UV-VIS. The characterization revealed that the zinc oxide

nanoparticles synthesized were of nanosize and pure.

ZnO nanoparticles synthesized by all three methods showed activity

against all the isolates. ZnO nanoparticles prepared using

thioglycerol as capping agent was more effective against the acne

causing organisms.

The nanoparticles were found to be stable exhibiting more or less

same activity for six months, thus making them potential

antibacterial agents. The activity of the nanoparticles was more

when placed at RT.

Creams, scrubs, face wash, lotions, face packs, dermato -cosmetic

herbal formulations prepared with ZnO nanoparticles as a major

component and the antibacterial activity of these products against

acne proved that these products can be used for treating acne.

133

In vivo studies carried out in mice showed good antibacterial activity

of ZnO nanoparticles against induced P. acnes infection and the

nanoparticles were effective in reducing the P. acnes induced

inflammation.

Evaluation of antibacterial activity of ZnO nanoparticles in vitro and

in vivo against Propionibacterium acnes concludes that an emulsion

of ZnO nanoparticles and vanishing cream, scrub, face packs,

lotions and cold cream can be used for treating acne.

134

5.1. Achievements

Objective Achievement

To Isolate and characterize the

acne causing organism.

Acne causing organisms were isolated and identified as

Propionibacterium acnes.

To synthesize and Characterize the

ZnO nanoparticles

Zinc oxide nanoparticles were

synthesized using different

methods and capping

agents.

Stability of ZnO nanoparticles with time and temperature

ZnO nanoparticles were stable at

Room Temperature and they were

stable upto 6 months.

To test the antibacterial activity of

ZnO nanoparticles against acne

causing organisms

The ZnO nanoparticles were found

to be effective against the

microorganisms.

To prepare cosmetic products with

nanoparticles

Cosmetic products (for skin care)

with nanoparticles were prepared.

To Study the Antibacterial activity

of the cosmetic products with nanoparticles using standard procedures

The nanoparticles were effective

against the organisms and MIC

against P. acnes is 0.3 mg ml-1 .

To test the stability of creams and

emulsions with time

The stability of the products was

checked for about 6 months and

they were found to be stable.

To test the toxic effects of zinc oxide nanoparticles in Mice in which P. acnes infection is induced

In vivo studies showed good antibacterial activity of ZnO nanoparticles against induced

P.acnes infection and the nanoparticles were effective in

reducing the P.acnes induced inflammation.

Testing the Cytotoxicity of zinc oxide nanoparticles in epidermal

cell lines

Cytotoxicity properties of sample ZnO nanoparticles against MCF 10 A cell line.

The nanoparticles were not toxic to the epidermal cell lines up to a concentration of 100 µg ml-1

135

5.2. Conclusion

The study concludes that the ZnO nanoparticles are effective agents

against P.acnes in vitro and its effect in vivo was observed in mouse ears

induced with P. acnes inflammation. The results obtained highlight the

potential of using ZnO nanoparticles as an alternative treatment option to

the antibiotic therapy of acne vulgaris as significant antibiotic resistance

and resistance to many drugs have been identified for P. acnes strains

from acne patients with long-term antibiotic treatments.

5.3. FUTURE PERSPECTIVES

Assessment of Quality of the products prepared using ZnO

nanoparticles.

Formulation or standardization of dose for therapy and

commercialization of cream, face packs and scrubs.

Clinical trials and statistical analysis.

136

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7. APPENDIX

I. Preparation of Cosmetic skin care products

1. Preparation of Vanishing cream

Composition

Components g

Stearic acid 13.0

Stearyl alcohol 1.0

Cetyl alcohol 1.0

Methyl paraben 0.1

Propyl paraben 0.05

KOH 0.90

Purified water 100ml

ZnO Nano Particles 0.1%

Stearic acid , stearyl alcohol ,cetyl alcohol – melted at 75 oC in a conical

flask

a) KOH is dissolved in purified H2O at 75 oC, in another conical flask

b) Preservative - methyl paraben, propyl paraben & ZnO Nanoparticles

are added to it

Contents of ‘b’ are added into ‘a’, at 75 OC slowly with continuous stirring,

the mixture is stirred slowly until a smooth cream is formed at RT.

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2. Vanishing cream II

Composition

Components g

Stearic acid 25.00

Triethanolamine 1.35

Lanolin 4.00

Propylene glycol 5.00

Methyl Paraben 0.18

Propyl paraben 0.02

ZnO nanoparticles 0.1%

Water 100ml

Stearic acid, Lanolin, Propylene glycol and propyl paraben are mixed and

heated to 70 oC (A) -Oily phase. Triethanol amine, methyl paraben, water

are mixed and heated at 70 oC (B) – Aqueous phase. Contents of B are

transferred to A in small portions with continuous stirring till homogenous

emulsion is formed. Then the emulsion is mixed with ZnO nanoparticles.

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3.Preparation of Calamine Lotion

Composition

Component g

Calamine 4.0

ZnOnanoparticles 0.1%

Arachis oil 30

Emulsifying wax 6

Water 69.9 ml

Wax is heated gently and dissolved in arachis oil, 45 ml of purified water

is added at the same temperature, stirred and cooled. Calamine and ZnO

are titrated with remaining quantity of pure water. This mixture is added

to the cream and mixed uniformly.

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4.Preparation of Scrub

Composition

Components g

Stearyl alcohol 0.5

Cetyl alcohol 0.5

Methyl paraben 0.05

Propyl paraben 0.05

SDS 0.2

ZnO nanoparticles 0.1%

Orange peel 1.0

Oat meal 1.0

Disodium EDTA pinch

Liquid paraffin 3 ml

Isopropanol 2-3 drops

Stearyl alcohol, Cetyl alcohol, stearic acid melted at 75 oC in a conical

flask. Methyl paraben, propyl paraben, SDS, ZnO nanoparticles, Orange

peel, oat meal, EDTA, Liquid paraffin, Isopropanol are added to it.

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II. Media

Formula Per Litre Purified Water

1. Brain Heart Infusion ( BHI) Agar

Components g/L

Brain Heart, Infusion from (Solids) 8.0

Peptic Digest of Animal Tissue 5.0

Pancreatic Digest of Casein 16.0

Sodium Chloride 5.0

Glucose 2.0

Disodium Hydrogen Phosphate 2.5

Agar 13.5

2. Nutrient Agar

Components g/L

Peptic digest of animal tissue 5.000

Sodium chloride 5.000

Beef extract 1.500

Yeast extract 1.500

Agar 15.000

Final pH (at 25°C) 7.4 ± 0.2

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3. Nutrient Broth

Components g/ L

Peptic digest of animal tissue 5.000

Sodium chloride 5.000

Beef extract 1.500

Yeast extract 1.500

Final pH (at 25°C) 7.4±0.2

4. Peptone water

Components g/L

Peptic digest of animal tissue 5.000

Sodium chloride 5.000

Final pH (at 25°C) 7.2±0.2

5. Simmons Citrate Agar:

Components g/L

Magnesium sulphate 0.20

Ammonium dihydrogen phosphate 1.00

Dipotassium phosphate 1.00

Sodium citrate 2.00

Sodium chloride 5.00

Bromo thymol blue 0.08

Agar 15.00

pH 6.8± 0.2

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6. MR-VP Medium:

Ingredients g/L

Buffered peptone 7.00

Dextrose 5.00

Dipotassium phosphate 5.00

pH 6.9± 0.2

7.Mannitol Salt Agar Base:

Ingredients g/L

Proteose peptone 10.00

Beef extract 1.00

Sodium chloride 75.00

D-Mannitol 10.00

Phenol red 0.025

Agar 15.00

pH 7.4± 0.2

III.Reagents

1. Reagent for testing nitrate reduction

Reagent A: 8 g of sulphanilic acid is dissolved in 1 L of 5 N acetic

acid

Reagent B: 0.6 g of N-N- Dimethyl Naphthyl amine is dissolved in

0.1 L of 5 N acetic acid

Reagent C: Zn dust