Formulations Containing Eucalyptus Oil For Delivery Of Chlorhexidine Biology Essay


Chlorhexidine belongs to a group of antiseptics/ anti-infectives, antibacterial/ antimicrobials, it is having a vast application and is mainly used to prevent nosocomial infections as 50% of nosocomial infections are related to medical devices and Chlorhexidine is used as anti-infective either alone or in combination with other anti-bacterial. In this study Eucalyptus oil has been proposed to be used as it acts as a penetration enhancer for Chlorhexidine and some studies has proven its antimicrobial action. Main organism causing medical device related nosocomial infections are S. epidermidis, S. aureus, P.aerogenosa, Candida albicans etc and of these strains some of them are associated with biofilm which is made up glcocalyx to which bacteria attaches themselves and hence this leads to antibacterial resistance. Chlorhexidine and eucalyptus oil show synergistic effect against strains of S. epidermidis which is the main causative organism of them, but this combination still needs to be proven for higher penetration into biofilm though some studies has shown it penetrates biofilm but their result shows minimal activity. Hence to improve the penetration of Chlorhexidine and eucalyptus oil into biofim; microsphere technology for drug delivery can be used as in recent past it has been shown that PLA (Poly-Lactic acid) microsphere helps antimicrobial drug to penetrate biofilm and hence helps in eradication of bacteria and prevention of antimicrobial resistance. PLA microsphere of eucalyptus oil and Chlorhexidine can be prepared by coacervation and solvent evaporation method respectively; here two different methods are used because there is not enough literature present which proves formation of PLA microsphere of Chlorhexidine with coacervation method.

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To determine the efficacy of PLA microsphere of Chlorhexidine and eucalyptus oil chemostat method can be used, release kinetics for Chlorhexidine and eucalyptus oil is considered as a function of drug loading and amount of microsphere is related with extent of killing bacteria biofilm, bacterial strains are grown on silicon tubing and vortex mixing is done after this they are inoculated in on agar plate and biofilm cells is maintained at 108 CFU per cm of tubing after which microspheres are added and antimicrobial free solution is pushed into tubing. To evaluate concentration of Chlorhexidine spectrophotometry method can be used and different values can be obtained from standard curve. Though this chemostat system method has not been established for Chlorhexidine as this design has been referred from formulation of Ciprofloxacin PLA microsphere so other method like HPLC determination can be used.


Antiseptics are the compounds consist of biocides (chemical component) which destroy or inhibit the growth of micro-organism in or on living tissue. These are used in preventing nosocomial infection and are generally present in many topical formulations.

Chlorhexidine (C22H30Cl2N102C6H12O7), is a cationic bisbiguanide. It is one of the most widely used biocides used in antiseptics because it shows broad spectrum of activity and low irritancy profile.

Mcdonnell. G & Russel.A.D (1999)

Chlorhexidine binds to the mucous membrane and on the cell surface it reacts with negatively charged group and this leads to loss of cytoplasmic components, membrane damage and enzyme inhibition and at higher concentration it causes coagulation of proteins and extensive damage to the cell

Hope.C.K & Wilson.M(2004)

Chlorhexidine is bactericidal at higher concentration; it has been found out that its uptake by E.coli and S. aureus increases at higher concentration and pH. It acts by crossing the cell wall mainly by passive diffusion and attacks bacterial cytoplasmic membrane, this leads disruption of semipermeable membrane and hence leakage of intracellular component. This leakage stops at higher concentration of chlorhexidine because of coagulation of intracellular component. It shows portioning effect in yeast in cell wall, plasma membrane and cytoplasm of cells. Increase in concentration of biguanide shows lysis effect on Saccharomyces cerevisiae protoplasts, this effect reduces at higher concentration.

Chlorhexidine does not effect mycobacterium as this bacterium shows resistance.

Mcdonnell. G & Russel.A.D (1999)

Eucalyptus oil belongs to the class of terpenes, terpenes consist of carbon, hydrogen and oxygen atom. I t has been found that eucalyptus oil acts as penetration enhancer for many drugs and in study it has been demonstrated that it increases permeation coefficient of drug by 34 fold

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Williams. A.C & Barry.B .W(2003)

1,8- cineole is a major chemical component of eucalyptus oil, to assess the interaction of eucalyptus oil with human skin Raman spectroscopy method was used in which excised full thickness human skin was taken and result of investigation showed alteration lipid domain order-disordered equilibrium in stratum corneum and hence increasing the permeability of drug.

Aquil. M et. al (2007)

Eucalyptus oil has been shown to produce antibacterial effect on gram positive as well as gram negative bacteria,on bacteria strains like S.epidermidis, S.aureus, Candida albicans, E.coli etc

Current Problems

Many germicides has been detected with associated contact urticaria and Chlorhexidine is one of them. Taylor.J.S mentioned in the report that so far two studies has been done to assess the severity of Chlorhexidine associated contact urticaria, first study has been carried on a man, 66 years old suffering with chronic hand eczema and stasis dermatitis encounter localized urticarial reactions shortly after surgical procedure latest one occurring after application of Hibiscrub (4% Chlorhexidine) and Citanest gel (0.05% chlorhexidine), though open test on forearm showed negative result. Second study was done on 37 year old atopic women who suffered with urticaria, for procedure for copper IUCD when prepared with 0.05% Chlorhexidine, a prick test has been performed and it shows that 0.5% Chlorhexidine digluconate and acetate (0.5%) produces urticaria after 20 min

Warner. M.R. et. al (1997)

Karpanen. T.J. et. al proposed a studies which defines penetration of 2% Chlorhexidine gluconate (CHG) into full thickness of human skin. Penetration of CHG into full thickness excised human skin has been estimated with the use of Franz cell at different intervals of 2 min, 30 min, and 24 hours. Study was designed to investigate penetration of CHG up to thickness of 1500 µm using microtome and analysis has been done HPLC method. Result of the investigation showed that penetration of Chlorhexidine was poor at 2min and 30 min interval and also its concentration below 300µm skin depth was very low though concentration of Chlorhexidine was higher after 24 hours of exposure on upper layer that is at 100µm, hence this study confirms that penetration of aqueous CHG into full length skin tissue was poor and therefore this study laid the foundation to select the antiseptic agent to treat deep tissue bacterial infection other than Chlorhexidine or to work on strategies on how to increase the penetration of aqueous Chlorhexidine.

Karpanen. T.J. et. al (2008)

Anaphylaxis reaction with Chlorhexidine has been reported but are rare, in this study 3 cases has been reported with use of Instillagel, which is used as uretheral lubricant and it contains Chlorhexidine as one of the component. To confirm the presence of Chlorhexidine Skin prick test(SPT) has been performed, This test took place Manchester Royal Infirmary.

Case 1:

A 33 year old patient under gone elective urethrotomy and is on general anaesthesia, patient history revealed conjunctivitis on exposure to cats and irregular urticaria which is in dormant phase. General anaesthesia was given with propofol and remifentanil to insert laryngeal mask airway, during the procedure tramadol, gentamycin ketorolac was given and at the end of the procedure urinary catheter was inserted using Instillagel, patient became unconscious after 5 min of his transferred into room and signs of low blood pressure and low oxygen has been noted with marked cutaneous flushing, hence case of anaphylaxis has been suspected therefore patient was resuscitated and patient was supplied with 100% oxygen and 400µg Epinephrine and Gelofusine 1000 ml given through i.v. route, marked improvement in health of patient noted with in 10 min of treatment. To confirm the Anaphylaxis reaction mast cell tryptase (MCT) has been taken at 1, 4 and 16 hours interval, were noticeably elevated at 1 and 4 h intervals and at 16 h are decreased with lowest limit. At Anaesthetic Reaction Clinic (ARC) patient underwent SPTs for hydrochloride, Chlorhexidine 0.05%, Chlorhexidine 0.25% and it has been concluded that cardiovascular collapse was due to presence of chlorhexidine.

Harper.N. et. al (2008)

Previous Scientific Literature

Ringer M et. al carried out prospective randomised trial on three known antiseptics that is 10% povidone-iodine,70% alcohol and 2% Chlorhexidine to assess their antiseptic effect to prevent infection associated with arterial catheters. In these study catheters has been randomised to these three antiseptics and it has been noted that Chlorhexidine was most effective as the infection related to catheter insertion and post operative site infection found out to be greatly reduced.

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Alvarado .C.J. (1991)

George. N et. al carried out randomised trial to explain the importance of use of antiseptics or water for periuretheral cleaning before uretheral catherization. It has been found that 0.1% chlorhexidine gluconate is most readily used and is applied onto the mucous membrane. In this study 436 patients were involved of which 219 treated with water and 217 treated with 0.1% Chlorhexidine gluconate, 38 of which was having urinary tract bacteriuria, after 24 hours urine sample was analysed and it has been find out that periuretheral cleaning by antiseptic does not reduces the risk of production of bacteriuria.

Hood.R.H. et. al (2001)

Bergan. T et. al carried out a study on pregnant women to determine the efficacy of Chlorhexidine in preventing the vaginal transmission of bacteria such as Streptococcus agalactiae from mother to child. In this study vaginal douching method was employed to apply chlorhexidine and mothers has been monitored in reference phase (first 4 months) for vaginal flora and in next 5 months a randomized double blinded trial has been carried out with vaginal douching with either 0.2% Chlorhexidine or sterile saline and it has been find out the transmission of microbes has been greatly reduced within patient treated with 0.2% Chlorhexidine 35% to 18%. In a parallel study it has been shown 0.2% Chlorhxidine reduces risk of UTI infection to 3.5% as compared to 6.2% in patient treated with saline. Hence it has been proved mothers treated with chlorhexidine have very less chances of transmitting microbes to neonates as compared to mothers treated with saline.

Bergan. T. et. al. (1999)

Hall. J et. al studied the effectiveness of Chlorhexidine powder over hexachlorophane against Staphylococcus aureus in neonates. Previously studies has been carried out by phage type surveillance techniques, to study the epidemic of methicillin-susceptible S.aureus (MSSA, PT 53,85) on 202 babies and certain measures has been taken to overcome this problem like washing hand and using hexachlorophane powder as antiseptic. At one this hexachlorophane was unavaible which were used for prophalaxis and it has been replaced by 1% Chlorhexidine for umbilical decontamination and it has been found that number of monthly MSSA strain was decreased and the therapy with 1% Chlorhexidine was continued instead of hexachlorophane in neonates.

Hall. J. et. al (2004)

Recent Literature

Nosocomial infections and resistant to antimicrobials used against methicillin resistant Staphyloccocus aureus and medical conditions such soft tissue and bone or implant infections proved to be a major point of concern.

Antimicrobials effects of several essential oils such as eucalyptus oil, lemon grass, Tea tree, Thyme etc has been identified against many bacteria such as Staphyloccocus strains (including MRSA), Candida strains and Streptococcus strains causing nosocomial infections, in this study anti infective such as Chlorhexidine, povidone iodine, hydrogen peroxide and other oils such as paraffin and olive oil served as controls. To assess the activity of different antimicrobial oils Agar diffusion test has been carried out and result shows that of oil used in the study Thyme oil shows the highest activity and eucalyptus and Tea tree oil shows almost similar activity even against MRSA resistant bacterias

Sherry. E. et. al. (2009)

It has been proved that triclosan topical application acts as antistaphylogenic antiseptic in case of atopic dermatitis. Use of 2% triclosan has been proved to cause exacerbation of disease as it leads to secondary reactions. Chlorhexidine exhibits same activity as that of triclosan against Staphylococcal organisms, so to overcome the side effects associated to triclosan a combination of these antibiotics has been prepared which contains 2% of triclosan and 0.34% of Chlorhexidine dihydrochloride and their antibacterial activity has been evaluated preclinically by comparing with reference compound such fusidic acid and the result after investigation showed that combination was inferior to reference but produces same degree of reduction in pathogen in atopic dermatitis when low concentration of triclosan and Chlorhexidine combination used.

Jost.G. et. al. (2007)

A study has been carried out to assess the efficacy of eucalyptus oil as a antimicrobial agent and its derivative 1,8- cineol which is a recognised penetration enhancer alone or in combination of Chlorhexidine gluconate(CHG) against micro-organisms grown on planktonic and biofilms culture. In this study Minimum inhibitory concentration for each bacteria has been estimated by using micro broth dilution method and bactericidal effect has been estimated by use of bioluminescence and to keep a check on effect of interaction between 1,8- cineol, eucalyptus oil and CHG a chequerboard assays has been used. It has been noted, result obtained demonstrated that CHG was more active against planktonic bacteria and synergistic effects has been seen with both eucalyptus oil and 1 ,8- cineol with CHG against strains of Staphylococcus aureus, MRSA, candida, and Pseudomonas,this synergistic effect on biofilm culture is still under research and have not been achieved the desired results. Combination of CHG with Eucalyptus oil proved to be more efficacious then with1,8- cineol. (2009)

Bacterial resistance to many antimicrobial has been seen in the past, use of essential oil has been proved to be beneficial to overcome this resistance, as many essential oils such a thymol oil, eucalyptus oil etc has been proved to show antimicrobial activity against many strains of bacteria residing onto the skin. Combination of Chlorhexidine (gluconate) with these essential oil specifically with eucalyptus oil has been shown to produce finest synergistic antimicrobial effect and better penetration of drug compound though maximum antimicrobial activity has been achieved with thymol oil. However the base for synergistic effect of eucalyptus oil and Chlorhexidine gluconate is not known as it is believe that both compound having same site of action on bacteria and eucalyptus oil helps changing the physiochemical properties of extracellular matrix of bacteria and hence increasing action of chlorhexidine, thought more of research is required to prove this point. Another main point of in which further research work is needed is the synergistic effect of Chlorhexidine with essential oil against S. epidermidis , Chlorhexidine alone is not susceptible to S. epidermidis as it produces resistance and at sub inhibitory concentration of Chlorhexidine, biofilm mode of growth of bacteria increases.

Karpanen.T.J. (2008)

Intended Design

This study is based on synergistic effect of Chlorhexidine and eucalyptus oil in treating major cause of nosocomial infections such as catheter venous infections. Synergistic effect of eucalyptus oil and Chlorhexidine can be used to penetrate the biofilm of bacteria, biofilm consists of microbes which attaches to the surface of catheter, this leads to production of glycocalyx matrix where micro colonies of microbes grows, aged biofilm offers advantage to bacteria to grow rapidly as it remains ineffective to host immune response

Anwar.H. (1999)

Most of the nosocomial infections has been associated the medical device related infections such as central venous catheters, urological medical devices, dental implants etc. Microbial infection caused by different strains of bacteria and some of them are biofilm associated bacteria, in which bacteria attaches itself to the cell surface or cell to cell adhesion or microorganism pluristratifaction and metamorphosis on the surfaces of medical devices. Medical device related infections occurs in post-operative period, another form of device related infections linked to biofilm mineralization which causes encrustration majorly in case of urinary tract infection, hence these infections needs to treated as the morbidity, mortality cases associated with these infections are on a high. Hence to overcome this problems some steps has been taken such as coating of medical devices with antimicrobial, anti-infective drugs or employing bacterial surface modification techniques.

Ludwig. A. Et. al. (2008)

Biofilms are the small microbial ecosystems with different strains of microbes which aggregates, co-operate with each other for their survival. Bacteria in these biofilms grow in dense matrix having nutrient channels and water; bacteria adhere with matrix which is made up of polymeric compounds known as extracellular polymer substances (EPS). Resistance to many antimicrobial has been seen in the presence of these bacterial biofilms, due to structural heterogenecity or changes in physiology, metabolic or genetic composition in bacteria itself renders resistance to many anti-microbial.

Sihorkar. V. & Vyas. S.P. (2001)

In recent years, for eradicating bacterial biofilm on medical device associated nosocomial infection, lipid/ polymer-based drug delivery carrier/ microspheres has been employed which carry active drug molecule and also helps in better penetration of antimicrobial agent across biofilm or accumulation of carrier onto the biofilm surface or by preventing biofilm formation.

Ludwig. A. Et. al. (2008)

Antimicrobial spectrum of combination of eucalyptus oil and Chlorhexidine digluconate[CHG] (more soluble form when combined with gluconic acid) against planktonic and biofilm cultures of S. epidermidis has been proved earlier by Karpanen et. al

Method of Investigation

This study is based on work done by Rogers.J. et al. In which ciprofloxacin microspheres has been used in eradication of biofilm cells and planktonic cells of P. aeruginosa and S. aureus , though according to another study done by Paul. S. et. al. to compare the efficacy of Chlorhexidine with that of ciprofloxacin and tobramycin against Staphylococcus aureus and Pseudomonas aeruginosa related infections, in this study rabbit eye cornea was used to inject S. aureus and P. aeruginosa and MIC values for each antibiotic has been determined, for Chlorhexidine MIC was ≤40 μg/mL against both bacterial strains and Colony forming unit (CFU) of S. aureus and P. aeruginosa for Chlorhexidine was 1.1 Ã- 103 when compared to other two antibiotics which was 4.7 Ã- 104, 1.9 Ã- 105 respectively. Hence it can be interpreted from the study that Chlorhexidine is more active against P. aeruginosa and S. aureus.

Paul. S. et. al. (2007)

In this study Poly-lactic acid (PLA) can used to prepare microsphere of Chlorhexidine as PLA has proved to be histocompatible and biodegradable in case of surgical sutures, prosthetic devices etc.

Materials and Methods:

Encapsulation of Eucalyptus oil in PLA microsphere:

To explain this part reference of preparation of thyme oil (also an essential oil and blend well with eucalyptus oil)[] can be considered in encapsulation of eucalyptus oil into PLA microsphere using same method as used for thyme microsphere encapsulation.


Poly(DL-lactide) used as wall forming agent, dimethylformamide (DMF)- PLA solvent, eucalyptus oil - core material, surface active agent such as Tween 20, Octamethylcyclotetrasiloxane(OCMTS) - hardening agent.


To Prepare PLA microspheres of eucalyptus oil coacervation method can be used.PLA is an organic solvent and is water insoluble, it is used to prepare microspheres of drugs, oil etc by using double emulsion technique (o/w/o). Eucalyptus oil is water in- soluble therefore to incorporate this oil an o/w emulsion should be prepared. PLA is to be dissolved in DMF, as DMF has high water solubility; this is dissolved in water which leads to precipitation of PLA around eucalyptus oil.

Characterization Techniques

Microcapsule size distribution: This can be determined by laser dispersion using laser diffraction particle size analyzer.

Morphology Determination: This can be determined by using optical microscopy or cryogenic electron microscopy.

Quantification of Eucalyptus oil: This can be done using Gas chromatography technique.

Encapsulation efficiency of microsphere can be calculated mathematically by using following formula:

where mtotal = amount of loaded essential oil (g) and mout = amount of non-encapsulated essential oil (g).

Martins. I.M. et. al. (2009)

Encapsulation of Chlorhexidine in PLA microsphere

Microencapsulation of Chlorhexidine can be done by using oil/water emulsion solvent evaporation method as it also shows hydrophobic characteristics In this method PLA(100000 g/mol) and Chlorhexidine is dissolved in dichloromethane, then solution is added with Poly-vinyl alcohol(surfactant) then this system is emulsified using homogeniser (13000 rpm) for 1 min. Continuous stirring of solution is done until all of dichloromethane evaporates, centrifugation of particle is done then they are washed and freeze dried.

Fay.F. et. al. (2008)

Characterization Techniques:

Encapsulation efficiency: Microsphere encapsulation of Chlorhexidine can be calculated by carrying out assay of uncapsulated free Chlorhexidine by reverse phase high performance liquid chromatography, hence parameters to calculate efficiency by the mathematically formula given above can be obtained by this technique.

Open chemostat system can be used to describe this study, in this organism to be tested are grown in soy broth up to mid- exponential phase into which 2,2- dipyridyl(chelating agent) is added, 100 pieces of silicon tubing to be added to the chemostat and autoclaving should be done at 121°C for 20 min, this system should be maintained at 37°C for 7 days so as to allow biofilm formation, after this bacterial solution prepared earlier should be transferred to chemostat and each chemostat volume should be maintained at 100 ml. On 7th day of inoculation planktonic cells should be determined from nutrient agar plates and biofilm cells counts which can be obtained from silicon tubing should be done by washing tubing with phosphate buffer (PBS), after this vortex mixing done for about 3 min, serially diluted samples should be prepared and inoculation of nutrient agar plate at 37°C is done to count the number of cells, efficiency of removing biofilms can be checked by repeating the procedure until biofilm cell remain constant, it has been noted 99% of cells is removed during first vortex- mixing and number of biofilm cells should be maintained at 108 CFU per cm of tubing.

After viable count determination, microspheres should be added to nylon screen and it is to be placed in chemostat, then antibiotic free medium should be pushed into chemostat, samples to be removed and viable counts for planktonic or biofilms is to be performed on agar plate.

Chlorhexidine concentration in chemostat can be determined by centrifugation at different time intervals, sample analysis should be carried out Spectrophotometically. Standard curve for Chlorhexidine can be plotted at different concentration. Microspheres should be added at a rate of 1/day for 4 days, when all the bacteria gets killed then again replace the sample to check possible re-growth.

Anwar.H. (1999)


Aquil. M., Ahad. A., Sultana. Y., Ali. A (2007) Status of Terpenes as Skin Penetration Enhancer. Drug Discovery Today:12, page no.:1061-1067

This reference provides insight information about the main constituent of Eucalyptus oil, 1,8-cineol and its ability to be used as Penetration enhancer

Bergan. T., Stray-Pedersen.B., Hafstad.A., Normann.E., Grøgaard.J., Vangdal.M. (1999). International Journal of Antimicrobial Agents:12,page no. 245-251

This reference explains about the study which is performed to check the efficacy of Chlorhexidine in preventing transmission of microbes from mother to Child.

Harper.N., Parkes.A.W., Herwadkar.A., Pumphrey.R (2008) Anaphylaxis to the chlorhexidine component of Instillagel: a case series. British Journal of Anaesthesia: 102, page no.: 65-68

This reference provides one more example of complication associated with use of Chlorhexidine formulation by quoting different case studies

Hendry.E.R.,Worthington.T., Conway.B.R., Lambert.P.A.(2009) Antimicrobial efficacy of eucalyptus oil and 1,8-cineole alone and in combination with chlorhexidine digluconate against microorganisms grown in planktonic and biofilm cultures. Journal of Antimicrobial Chemotherapy:64, page no.:1219-1225.

This reference explained the efficacy of eucalyptus oil and 1,8-cineol as penetration enhancer and in combination with Chlorhexidine as antimicrobial agent against planktonic and biofilm cultures of S. epidermidis.

Hope.C.K & Wilson.M(2004) Analysis of the Effects of Chlorhexidine on Oral Biofilm Vitality and Structure Based on Viability Profiling and an Indicator of Membrane Integrity. Antimicrobial Agents and Chemotherapy: 48, page no.:1461-1468

This reference provides insight into drug mechanism of action.

Karpanen. T.J., Worthington.T., Conway. B.R., Hilton. A.C., Elliott.T.S.J., Lambert. P.A. (2008) Penetration of Chlorhexidine into Human Skin. Antimicrobial agents and Chemothereapy:52, page no.: 3633-3636

This reference gives insight into Penetration of Chlorhexidine into human skin at different concentration and ways to improve its penetration.

Karpanen.T.J., Worthington.T., Hendry.E.R., Conway.B.R., Lambert.P.A.(2008) Antimicrobial efficacy of chlorhexidine digluconate alone and in combination with eucalyptus oil, tea tree oil and thymol against planktonic and biofilm cultures of Staphylococcus epidermidis Journal of Antimicrobial Chemotherapy:62, page no.:1031-1036

This reference explains about use of essential oil in overcoming bacterial resistance and synergistic effect of Chlorhexidine with eucalyptus oil against strains of S, epidermidis

Maki. D.G., Ringer. M., Alvarado .C.J.(1991) Prospective randomised trial of povidone-iodine, alcohol, and chlorhexidine for prevention of infection associated with central venous and arterial catheters. Lancet: 338, page no.: 339-343

This reference gives comparison of three antibiotics to prove the efficacy of Chlorhexidine.

Mcdonnell. G & Russel.A.D. (1999) Antiseptics and Disinfectants: Activity, Action, and Resistance. Clinical Microbiology reviews: 12, page no.:147-179

This reference provide general introduction about the Antiseptics and Chlorhexidine and how do Chlorhexidine acts on bacteria's

Owusu-Ababio .G., Rogers. J., Anwar .H (1999) Effectiveness of ciprofloxacin microspheres in eradicating bacterial biofilm . Journal of Controlled Release:57, page no.: 151-159

This reference provides the basis to describe the basis of intended design of this proposal as it explains the application of antimicrobial microspheres in penetrating biofilm of bacteria and it also explain the basis of method which can be used to assess the effectiveness of the intended microspheres

Sihorkar. V. & Vyas. S.P. (2001) Biofilm Consortia on Biomedical and Biological Surfaces: Delivery and Targeting Strategies. Pharmaceutical Research: 18, page no.: 1247-1254

This reference provides further general information related to biofilm

Tamilvanan. S., Venkateshan. N., Ludwig. A.(2008) The potential of lipid- and polymer-based drug delivery carriers for eradicating biofilm consortia on device-related nosocomial infections. Journal of Controlled Release:128, page no.: 2-22

This reference helps in explaining the effect of biofilm on medical devices and how biofilm formation takes place on medical devices which causes antibacterial resistance and other related phenomenon.

Warnke. P.H., Becker. S.T., Podschun. R., Sivananthan .S., Springer. I.N., Russo. P.A., Wiltfang. J., Fickenscher. H., Sherry. E.(2009) The battle against multi-resistant strains: Renaissance of antimicrobial essential oils as a promising force to fight hospital-acquired infections. Journal of Cranio-Maxillofacial Surgery: 37, page no.: 392-397

This reference provides information regarding use of essential oil as an antibacterial agent in case of nosocomial infections, keeping some anti-infective as control.

Warner. M.R., Taylor. S.J., Leow. Y.H. (1997) Agents Causing Contact Urticaria. Clinics in Dermatology:15, page no.: 623-635

This reference provides insight into Medical conditions such as urticaria produced by Chlorhexidine at certain concentration in some patients

Webster.J., Hood.R.H., Burridge.C.A., Doidge.M.L, Phillips.M.K., George.N (2001) Water or antiseptic for periurethral cleaning before urinary catheterization: A randomized controlled trial.AJIC:29, page no., 389-394.

This reference provides information about importance of Periuretheral wash with antiseptics before surgery and explains efficacy of Chlorhexidine

Wilcox.M.H., Hall.J., Gill.A.B., Fawley.W.N., Parnell.P., Verity.P (2004) Effectiveness of topical chlorhexidine powder as an alternative to hexachlorophane for the control of Staphylococcus aureus in neonates. Journal of Hospital Infection:56, page no.: 156-159

This reference provides information about the effectiveness of Chlorhexidine powder over hexachlorophane in neonates against certain stains of bacteria

Williams. A.C & Barry.B .W(2003) Penetration enhancers. Advanced Drug Delivery Reviews:56, page no.:603-618

This reference provides introduction of Eucalyptus oil and its antimicrobial efficiency.

Wohlrab. J., Jost .G., Abeck. D. (2007) Antiseptic efficacy of a low-dosed topical triclosan/chlorhexidine combination therapy in atopic dermatitis. Skin Pharmacology and Physiology:20, page no.: 71-76

This reference explains the side effects related with triclosan and Chlorhexidine when they are used alone and their beneficial physiological effects when used in combination.