COMPARATIVE STUDY OF DISINFECTANT EFFICIENCY OF ETHANOL, BLEACH AND PHENOLICS AGAINST Pseudomonas aeruginosa and

COMPARATIVE STUDY OF DISINFECTANT EFFICIENCY OF ETHANOL, BLEACH AND PHENOLICS AGAINST Pseudomonas aeruginosa and Staphylococcus aureus

BY

IKE PEACE .A .

(MB/2007/262)

DEPARTMENT OF MICROBIOLOGY AND BIOTECHNOLOGY

FACULTY OF NATURAL SCIENCE

CARITAS UNIVERSITY, AMORJI – NIKE EMENE, ENUGU

AUGUST 2010

COMPARATIVE STUDY OF DISINFECTANT EFFICIENCY OF ETHANOL, BLEACH AND PHENOLICS AGAINST Pseudomonas aeruginosa and Staphylococcus aureus

BY

IKE PEACE .A.

(MB/2007/262)

A RESEARCH PROJECT PRESENTED TO THE DEPARTMENT OF MICROBIOLOGY AND BIOTECHNOLOGY OF THE FACULTY OF NATURAL SCIENCE

IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF BACHELOR IN SCIENCE (B.SC.) IN MICROBIOLOGY AND BIOTECHNOLOGY,

CARITAS UNIVERSITY, AMORJI – NIKE EMENE, ENUGU

AUGUST 2010

CERTIFICATION

          This is to certify that this project work “Comparative Study of Disinfectant efficiency of Ethanol, Bleach and Phenolics against Pseudomonas aeruginosa and Staphylococcus aureus” was carried out by Ike Peace A. under the supervision of Mrs. Iloghalu B.U.  

         It is found worthy of acceptance in partial fulfillment of the requirement for the award of Bachelor in Science (B.S.c.) Degree in Microbiology and Biotechnology.

_______________                                               _______________

Ike Peace A.                                                                   Date

_______________                                               _______________

Mrs. Iloghalu B.U.                                                         Date

Supervisor

_______________                                               _______________

Mr. Amadi E. C.                                                   Date

Head of Department                        

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Prof. Ogbonna J.C.                                                        Date

External Supervisor

DEDICATION

This work is dedicated to God Almighty

ACKNOWLEDGEMENTS

          I wish to acknowledge with gratitude of heart the immensely effort of my supervisor Mrs. Iloghalu        B.U. for her contributions to see that this project is successful.

          My special thanks goes to my parents and siblings for their encouragements, financial assistant to me during the process of the work.

          My earnest heart of thanks goes to the Head of Department, Mr. Amadi .E.C. and all the lecturers of Microbiology Department, for elevating me to a greater height.

          Thanks so much.

          Also wish to thank all my friends and many others who helped me in the preparation of this project.  All your efforts are highly appreciated.

          God bless you all.

TABLE OF CONTENTS

Title Page                                                                                                                 i

Certification                                                                                                                        ii

Dedication                                                                                                                iii

Acknowledgements                                                                                                            iv

Table of Contents                                                                                                   v

List of Tables                                                                                                           vi

List of Figures                                                                                                         vii

List of Plates                                                                                                            viii

Abstract                                                                                                                    ix

CHAPTER ONE

1.0       Introduction                                                                                                  1

1.1       Aims and Objectives                                                                                   2

CHAPTER TWO

2.0       Literature Review                                                                                        5

2.1       History of Disinfectants                                                                              6

2.2       About Disinfectants                                                                                     7

2.3       Sources of Contamination of Surfaces                                                     8

2.4       Types of Disinfectants                                                                                9

2.5       Properties of A Disinfectant                                                                       18

2.6       General Features of Disinfectant                                                               19

2.7       General Features of the Test Organisms                                                   24

2.9       Mechanism of Actions of Disinfectants against Bacteria                      26

2.10    Resistant Action of Bacteria                                                                      27

2.11    Advantages and Disadvantages of Disinfectants                                                29

2.12    General Guidelines in the Use of Disinfectants                                      33

CHAPTER THREE

3.0       Material and Method                                                                                   36

3.1       Isolation of Bacteria                                                                                    36

3.2       Identification of Isolates                                                                             37

3.3       Preparation of Disinfectants                                                                      40

3.4       Antimicrobial Susceptibility Testing (Using Kirby Bauer

Diffusion Assay Well Method)                                                                  41

CHAPTER FOUR

4.0       RESULTS                                                                                                      43

CHAPTER FIVE

5.0       Discussion                                                                                                     57

5.1       Recommendations                                                                                       60

5.2       Conclusion                                                                                                    51

References                                                                                                                62

Appendix 1                                                                                                               69

Appendix II                                                                                                              71

Appendix III                                                                                                            74

LIST OF TABLES

Table 1:          Results of diameter of zone of inhibition of Ethanol,

Phenolics and Bleach for Staphylococcus aureus                      44

Table 2:          Results of Diameter of Zone Inhibition of Ethanol,

Phenolics, Bleach for Pseudomonas aeruginosa                                    45

Table 3:          Pseudomonas aeruginosa response to Ethanol, Phenolics

and Bleach                                                                                        46

Table 4:          Staphylococcus aureus response to Ethanol, Phenolics

and Bleach                                                                                        47

LIST OF FIGURES

PATTERNS OF THE ANTIMICROBIAL EFFICACIES OF VARYING CONCENTRATIONS OF THE DISINFECTANT ON THE TEST ORGANISMS USING HISTOGRAM

a.                  Fig 1: Pseudomonas aeruginosa disinfectants A test result                48

b.                  Fig 2: Pseudomonas aeruginosa disinfectants B test result                 48

c.                  Fig 3: Pseudomonas aeruginosa disinfectants C test result                 49

d.                  Fig 4: Staphylococcus aureus disinfectants A test result                     49

e.                  Fig 5: Staphylococcus aureus disinfectants B test result                     50

f.                   Fig 6: Staphylococcus aureus disinfectants C test result                     50

PATTERNS OF THE EVALUATION OF THE BACTERIAL PERCENTAGE RESPONSE TO EACH DISINFECTANT USING A PIE CHART

a.                  Fig 7: Disinfectant  A on Pseudomonas aeruginosa                             52

b.                  Fig 8: Disinfectant B on Pseudomonas aeruginosa                              52

c.                  Fig 9: Disinfectant C on Pseudomonas aeruginosa                              53

d.                  Fig 10: Disinfectant A on Staphylococcus aureus                                 53

e.                  Fig 11: Disinfectant B on Staphylococcus aureus                                  54

f.                   Fig 12: Disinfectant C on Staphylococcus aureus                                  54

LIST OF PLATES

Plate 1: Plates showing zones of inhibition                                       74

Plate 2: MacConkey media with colonies of Pseudomonas aeruginosa 74

Plate 3: Some of the used plates                                                       75

Plate 4: Biochemical test for Pseudomonas aeruginosa                              75

ABSTRACT

Ethanol, Bleach and Phenolics are three kinds of disinfectants which have been widely used in common laboratories.  In this study, a compared experiment on these three disinfectants efficiency was conducted against Staphylococcus aureus and Pseudomonas aeruginosa using agar hole diffusion method.  Different concentrations of bleach (1%, 2%, 3%, 4% and 5%) were used on both organisms.  Also (50%, 60%, 70%, 85% and 95%) of ethanol as well as (5%, 10%, 20%, 25%, and 30%) Phenolics were used. Diffrences in  concentrations tested was because, the original concentrations of the disinfectants differs. After 24 hours of incubation at 37⁰C, the results showed that all the disinfectants inhibited the growth of the test organism in their concentrated forms.  The diameter of zone of inhibitions were measured around each well by using a ruler in millimeters, using different concentrations, their efficacies varied.  The results showed that 30% Phenolics had the best efficiency against both test organisms and 5% bleach had a better effect on Staphylococcus aureus than Pseudomonas aeruginosa, while ethanol showed least sensitivity. 70% concentration gave the highest effect on Staphylococcus aureus as compared with Pseudomonas aeruginosa.

           

CHAPTER ONE

1.0       INTRODUCTION

            Microorganisms are minute living things that individually are too small to be seen with the unaided eyes (Tortora et al, 2007).  Though only a minority of microorganisms are pathogenic (disease producing), practical knowledge of microbes is necessary for medicine and related health sciences.  For example hospital workers must be able to protect patients from common microbes that are normally harmless but pose a threat to the sick and injured.  Thousands of people died in devastating epidemics; the cause of which was not understood.  Entire families died because vaccination and antibiotics were not available to fight infection (Johnson and Case, 1995).  This leads to scientific control of microbial growth.  This began only about 100 years ago.  It was Pasteur’s work on microorganism that led scientists to believe that microbes were a possible cause of diseases and need to be eliminated or destroyed.  Some examples off these microbes are; Bacteria, fungi, viruses and protozoa etc (Tortora et al, 2007).

            In the mid 1800s, the Hungarian physician Ignaz Semmeliveis and English physician Joseph Lister used these thoughts to develop some of the first microbial control practice for medical procedures.  These practices include hand washing with microbes killing chloride of lime and use of techniques of aseptic surgery to prevent microbial contamination of surgical wounds (Hamamah, 2004).  Over the last century, scientists have continued to develop a variety of physical methods and chemical agents to control microbial growth.  Control directed at destroying harmful microorganisms is called disinfection.  It usually refers to the destruction of vegetative (non-endospore forming) pathogens example bacteria by using a disinfectant to treat an inert surface or substances (Bhatia and Icchpujani, 2008).

            Bacteria are major causes of disease and even human death.  A disinfectant is one of the diverse groups of chemicals which reduces the number of microorganisms present (normally on an inanimate object).  There are various official definitions of the process of disinfection and disinfectants agents.  It is defined as a chemical that inactivates vegetative microorganism but not necessarily high resistant spores (ISO, 2008).  Cleaning and disinfection of surfaces are essential steps for maintaining the cleanliness of pharmaceutical industries, hospitals and environments (Rollins, 2000).  Disinfectant as effective agents that kill or eliminates bacteria is widely used in various ways; especially in microbial laboratory.  Disinfectant can be mainly divided into five agents; alkylating, sulfhydryl combining, oxidizing, dehydrating and permeable.  The most commonly used disinfectants in laboratories are ethanol, bleach and Isol (Larson and Morton, 1991).  Bleach also known as sodium hypochlorite is a broad spectrum disinfectant, non specific in their action, only action biological material that is present on any surface. They effects by oxidizing the cell of microorganism and attacking essential cell components including lipid, protein and DNA (Ho-Hyuk Jang et al, 2008).    Ethanol, as  a dehydrating agent, lies between the highly specific and broadly based categories.  It is effective against actively growing bacteria and viruses with a lipid based outer surfaces, but is not effective against bacterial spores or viruses that prefer watery environment.  They cause cell membrane damages, rapid denaturalization of proteins with subsequent metabolism interference an cell lyses (Larson and Morton, 1991).  Another surface disinfectant is the compound that contain phenol group, a popular commercial brand of Isol, (a saponated brand of cresol) as a phenolics are intermediate level disinfectant derived from coal tar, that are effective on contaminated surfaces (Bittel and Hughes, 2003).

            However, certain types of viruses and some bacteria are resistant to the killing action of Phenolics compound (ISO, 2008).  Many studies have been done on comparison of disinfectant efficiency, and ethanol and bleach are believed to have immediate effect against most organisms (Carly et al, 2006).  For bacteria species, the effects of ethanol, bleach, phenol on Pseudomonas aeruginosa and Staphylococus aureus are the bedrock of this study.

Pseudomonas aeruginosa is a classical opportunities pathogen with innate resistance to many antibodies and disinfectants.  It is invasive, toxigenic and produces infection in patients with abnormal host deficiencies (Stephen et al, 2004).  Staphylococus aureus occur in 40 – 50% of humans.  Hospitalized patients as well as medical and paramedical staff show higher incidence of carriage of it (Bhatia and Icchpujani, 2008) in this study, disinfectant experiment was conducted using different concentrations of laboratory ethanol as disinfectant A, household bleach (Jik) disinfectant B and saponated brand of cresol (Isol) disinfectant C against Pseudomonas aeruginosa and Staphylococus aureus

1.1       AIMS AND OBJECTIVES

1.         To find out the concentration of disinfectants that will be effective in Gram positive Staphylococcus aureus and Gram negative Pseudomonas areuginosa.

2.         To investigate their differences of sterilizing pattern.

3.         To advise the public on the important of disinfectants and dangers of harmful microorganisms.

CHAPTER TWO

2.0       LITERATURE REVIEW

Chemical agents are used to control the growth of microbes on both living tissue and inanimate objects. Unfortunately, few agents achieve sterility; most of them merely reduce microbial population to safe levels or remove vegetative forms of pathogens from objects. A common problem in disinfection is the selection of an agent. No single disinfectant is appropriate for all circumstances (Tortora, et al, 2007).

Disinfectants can be referred to as chemical antimicrobial agent in contrast to physical antimicrobial agents such as heat and radiation. They are preparations of chemicals (usually liquids) that are intended for application to the surface of non-living material. Many household agents such as ammonia and bleach (hypochlorites) are also disinfectants. Most of them are strong oxidizing agents (Claus, 2009).

         Disinfection and the use of chemical disinfectants is one key strategy of infection control. Disinfection refers to the reduction in the number of living micro-organisms to a number that is considered to be safe to the particular environment. Typically, this entails the destruction of those microbes that are capable of causing diseases (ISO, 2008). The micro-organisms found on surfaces can be bacteria, viruses, fungi and protists. These disease-causing microbes can also be called pathogens, germs and they are responsible for causing infectious diseases. Infectious diseases caused by disease-causing microbes are responsible for more deaths worldwide than any other single cause. These micro-organisms are very good at adjusting to new environment, making it hard to find a way to get rid of them (Lages et al, 2008 ).

Among these micro-organisms, bacterial were grouped by morphology (rod, coccus and helix) staining reactions which classified them under gram positive and gram negatives, presence of endospores and other obvious features. They include all of the pathogenic prokaryotes as well as the nonpathogenic prokaryotes found in soil and water. Actually, relatively few species of bacteria cause disease in humans, animals, plants or any other organisms. Some examples of these bacteria are: Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella choleraesius etc (Tortora, et al, 2007).

2.1       HISTORY OF DISINFECTANTS

       Ancient records show the Egyptians, Chinese and Persians practiced preservation drinking water sanitation, antiseptic for wounds injuries and both physical and chemical methods. Many disinfectants had been in use for 150-200 years and have stood the test of time. Heat was one of the first disinfectants, followed by the use of sulfur dioxide as a fumigant (Omidbakhsh et al, 2006).In the medical field, as early as the 6^th century, surgeons used special vapours to disinfectant operating rooms. Until that time, hospital acquired infections or nosocomial infections were the cause of death in at least 10% of surgical cases, and death of delivering mothers were as high as 25%. Ignorance of microbes was such that during the American civil war, a surgeon might have cleaned his scalpel on his boot sole between incision (Bhatia and Ichhpujani, 2008 ).

            A few centuries later, ethanol was discovered but was not until the mid 1700s as disinfectants to dress wounds. In addition to ethanol, many other chemicals have been used such as, chlorine, vinegar water, hypochlorites, iodine, hydrogen peroxide and phenol. As the chemicals were being introduced, disinfection techniques and reason for disinfecting were becoming understood (Committee on Research standard, 1950).

2.2       ABOUT DISINFECTANTS

            Disinfectants are substances that are applied to non-living objects to destroy microorganisms that are living on the objects (Robertson et al, 2009). Disinfection does not necessarily kill all microorganisms, especially not resistant bacteria spores; it is less effective than sterilization, which is an extreme physical and /or chemical process that kills all types of life. Disinfectants are different from other antimicrobial agents such as antibiotics, which destroy microorganisms in the body, and antiseptics which destroy microorganisms living tissues. Disinfectants are also different from biocides – the latter are intended to destroy all forms of life, not just microorganisms. Sanitizers are substances that simultaneously both clean and disinfect. . Bacteria endospoers are most resistant to disinfectants, but some viruses and bacteria also possess some tolerance (Robertson et al, 2009).

            However, disinfectants have been used to destroy microbes that can cause infections, effectively reducing the risk of disease. They have been used to treat water, making it potable (drinkable) and have also been used in hospitals control the spread of infection by microorganisms. They have also been used in the food industry to keep food free from contaminations that can spread sickness, and destroy microorganisms that can form in human tissues (Sattar et al, 2002).

2.3       SOURCES OF CONTAMINATION OF SURFACES

         Germs are everywhere; Germs or microorganisms that cause illness and disease can grow on many surfaces called fomites (Tortora,2007). According to Agee 2001, he described fomites as any inanimate object that can carry disease causing organisms. Cutting boards, kitchen sink, the change in your pocket and even that pen we keep putting in our mouth are all fomites. Very few things we encounter in our everyday activities, are sterile or microbe free including us (Fisher et al, 2009).

            At births, microbe immediately begin colonize our bodies as they do most every object in the world. They float around until they come in contact with surface that offers food and shelter. Microorganisms especially bacteria are most likely to be found in and on dark moist objects that frequently come in contact with food, dirt or vegetation. Bathroom surfaces, hair brushes, refrigerators, kitchen sink and cutting boards often have lots of microbes on them. But doorknobs and walls have fewer because they are nutrient poor and dry (Van et al, 2005). Most of the microbes on our bodies and other surfaces are harmless, but some are pathogenic or disease –causing. For this reason, number of microorganisms need to be controlled on surrounding objects using various disinfectants (Van et al, 2005).

            

2.4       TYPES OF DISINFECTANTS

            Disinfectants have varying mode of action against microbial cells due to their chemical diversity. Different disinfectants target different sites within the microbial cell. These include the cell wall, the cytoplasmic membrane (where the matrix of the cytoplasm phospholipids and enzyme provide various targets) and the cytoplasm. Some disinfectants, on entering the cell either by disruption of the membrane through diffusion, then proceed to act on intracellular components. These are different approaches to the categorization and sub-division of disinfectants including grouping by chemical nature, mode of activity or by bacteriostatic bactericidal effects on microorganisms (Sandle, 2006).

Some different types of disinfectants are;

Non-Oxidizing Disinfectant: Majority of this group of disinfectants, have specific mode of action against microorganism, but generally they have a narrow spectrum of activity compared to oxidizing disinfectants. This group includes; alcohols, aldehyde, amphoteric (which have both anionic and cationic character, and possess a wide spectrum of activity), phenols and quaternary ammonium compounds (QAC).       

Alcohols: Alcohols usually ethanol or isopropanol, are sometimes use as a disinfectant but more often as an antiseptic (the distinction being that alcohols tend to be used on living tissues rather than non living surfaces). They are non corrosive, but can be a fire hazard. They also have limited residual activity due to evaporation which result in brief contact time, unless the surface is submerged, and have a limited activity in presence of organic material. Alcohols are most effective when combined with purified water to, to facilitate diffusion through cell membrane; 100% alcohol typically denatures only external membrane proteins (Lester, et al, 2005). A mixture of 70% ethanol or isopropanol, diluted in water is effective against a wide range of bacteria, though higher concentrations are often needed to disinfect wet surfaces (Moorer, 2003).

            Additionally, high concentration mixture (such as 80% ethanol+ 5% isopropanol), are required to effectively inactivate lipid-enveloped viruses such as; HIV, hepatitis B and hepatitis C (Van Englenbulg, et al, 2002). Alcohol is at best only partly effective against fungal and bacterial spores (Lages, et al, 2008).

Aldehydes: Aldehydes such as formaldehyde and glutaraldehyde, have a wide microbiocidal activity and are sporocidal and fungicidal. They are partly inactivated by organic matter, and have slight residual activity.

Some bacteria have developed resistance to glutaraldehyde, and it has been found that glutaraldehyde can cause asthma and other health hazards; hence ortho-phthalaldehyde is replacing glutaraldehyde (Omidbakhsh, et al, 2006).

Phenol: Phenolics are active ingredients in some household disinfectants. They are also found in some mouthwashes and in disinfectant soap and hand washes.

-Phenol is probably the oldest known disinfectant as it was first, when it was called carboxylic acid. It is preparations rather corrosive to the skin and sometimes toxic to sensitive people. Impure preparations of phenol were originally made from coal tar. Some phenol cause bacteria cell damage through disruption of proton motive force, while others attack the cell wall, and cause leakage of cellular components and protein denaturation. Those impure preparation of phenol contained low concentration of other aromatic hydrocarbons, including benzene.

-              o-phenyl phenol is often used instead of phenol, since is somewhat corrosive (Weber et al, 2000).

-              chloroxylenol is the principal ingredient in dettol, a household disinfectant and antiseptic (Lester et al, 2005).

-           Hexachlorophene is a phenol that was once used as a germicidal additive to some household products, but was banned due to suspected harmful effects (CRS, 2005)

Quaternary Ammonium Compounds: Quaternary ammonium compounds “quats” such as benzalkonium chloride, are a group of related compounds. Many types of quaternary ammonium compounds are used as mixtures and often in combination with other germicides. Some concentrated formulations have been shown to be effective low level disinfectants (EPA, 2006). Typically, quats do not exhibit efficacy against non-enveloped viruses such as, Norovirus, Rotavirus or polio. Newer synergous, low alcohol formulations, are high effective broad spectrum disinfectants with quick contact times (3-5minutes), against bacteria, enveloped viruses, pathogenic fungi and mycobacterium (Zamani et al, 2007).

     The germicide activity of certain types of quaternary ammonium compounds is considerably reduced organic matter, water hardness and anionic detergents. They have good activity against some vegetative bacteria. These compounds when properly diluted have low odour and are not irritating (Van Engelenburg et al, 2002).

Oxidizing Agents: Oxidizing agent acts by oxidizing thhe cell membrane of microorganisms which resultin a loss of structure and leads to cell lysis and death. A large number of disinfectants operate in this same way. Chlorine and oxygen are strong oxidizers,so their compounds figure heavily here().

Some of them are: sodium hypochlorite (bleach),hydrogen peroxide, iodine, chlorine,pottasium permanganate etc.

Sodium hypochlorite: This very commonly used, common household bleach is a sodium hypochrolite solution and is used in the home to disinfect drains, toilets and other surfaces. In more dilute form, it is used in swimming pools and in still more dilute form, it is used in drinking water. When pools and drinking water are said to be chlorinated, it is actually sodium hypochlorite or related compound, not pure chlorine that is being used. Chlorine partly react with proteinaceous liquids such as blood to form an oxidizing N-chloro compounds, and thus higher concentrations must be used if disinfecting surfaces after blood spills (Weber, et al, 1999).

Other hypochlorites such as calcium hypochlorite are also used especially as in swimming pool additives. Hypochlorite yields an aqeous solution of hypochlorous acid that is the true disinfectant. Hypobromide solutions are also sometimes used (Zamani et al, 2007).   

                                     

Chloramine is often used in drinking water treatment.

Chlorine Dioxide is used as advanced disinfectant for drinking water to reduce water borne diseases. In certain part of the world, it has largely replaced chlorine because it forms fewer by-product. Sodium chlorite, sodium chlorate and potassium chlorate are used as precursors for generating chorine dioxide (Venglarik et al, 2003)

Hydrogen Peroxide: is used in hospitals to disinfect surfaces and is used in solution alone or in combination with other chemicals as a high level disinfectant. Hydrogen peroxide vapour is used as a medical sterilant and as room disinfectant. It has the advantage that it decomposes to form oxygen and water, thus leaving no long term residues. Hydrogen peroxide, as with most other strong oxidants, is hazardous and the solutions are primarily irritants. The vapour is hazardous to the respiratory system and eyes (Lester et al, 2005 ).

Therefore, engineering control, personal protective equipment, as monitoring etc should be employed where high concentration of hydrogen peroxide are used in the work place ( Omidbakhsh, et at, 2006). Hydrogen peroxide is sometimes mixed with colloidal silver. It is often preferred because it causes far fewer allergic reactions than alternative disinfectants. Also used in park aging industries to disinfect foil containers, a 3% solution is also used as an antiseptic (Moorer, 2003 ).

Iodine is usually dissolved in an organic solvent or as lugols iodine solution. It is used in the poultry industry, added to the birds’ drinking water. Although no longer recommended because it increases both scar tissue formation and healing time. Tincture of iodine has also been used as an antiseptic for skin cuts and scrapes (DHQP, 2009)

Ozone: is a gas that can be added to water for sanitation (Moorer, 2003)

Peracetic Acid: is a disinfectant produced by reacting hydrogen peroxide with acetic acid.  It is broadly effective against microorganism and is not deactivated by catalase and peroxidase, the enzymes that breaks down hydrogen peroxide.  It also breakdown to food safe and environmentally friendly residues (acetic acid and hydrogen peroxide) and therefore can be used in non-rinse application.  It can be used over a wide temperature range (0-40⁰C) wide pH range (3.0 – 7.5) in clean-in-place (CIP) processes in hard water conditions; and is not affected by protein residues.

Home Disinfectants: By far, the most cost effective home disinfectant is the commonly used chlorine bleach (a 5% solution of sodium hypochlorite) which is effective against most common pathogens including difficult organisms such as tuberculosis (Mycobacterium tuberclosis), hepatitis B and C, fungi and antibiotic resistant strains of Staphylococcus and Enterococcus).  It even have some disinfectant action against parasitic organisms (Fisher et al, 2009).

            Diluted bleach can be tolerated on the skin for a period of time by most persons, as witnessed by the long exposure to extremely dilute “chlorine” (actually sodium or calcium hyporchlorite) many children get in swimming pools (Venglarik et al,2003).  

            To use chlorine bleach effectively, the surface or item to be disinfected must be clean macroscopically.  In the bathroom or when cleaning after pets, special caution must be taken to wipe up urine first, before applying chlorine to avoid reaction with the ammonia in urine, causing toxic gas by products.  A 1 – 20 solution in water is effective simply by being wiped on and left to dry.  The user should wear rubber gloves and in tight airless spaces, goggles are won.  If parasitic organisms are suspected, it should be applied at 1 to 1 concentrations, or even diluted.  Extreme caution must be taken to avoid contact with eyes and mucous membranes.  Protective goggles and good ventilation are mandatory when applying concentrated bleach.

            Commercial bleach tends to lose strength every time, whenever the container is opened.  Where one does not want to risk the corrosive effort of bleach, alcohol-based disinfectants are reasonably inexpensive and quite safe.  The great draw back to them is their rapid evaporation; sometimes effective disinfection can be obtained only by immersing an object in the alcohol (Moorer , 2003).

            The use of some antimicrobial such as triclosan, particularly in the uncontrolled home environment is controversial because it may lead to the germs becoming resistant.  Chlorine bleach and alcohol do not cause resistance because they are so completely lethal, in a very direct physical way (DHQP, 2009).

2.5       PROPERTIES OF A DISINFECTANT

             A perfect disinfectant would also offer complete and full sterilization without harming other forms of life, be inexpensive and non corrosive.  Unfortunately, ideal disinfectants do not exist (Malik and Goyal, 2006).  Most disinfectants are also, by nature potentially harmful (even toxic) to humans or animals.  Most modern household disinfectants contain bitrex an exceptionally bitter substances which is added to discourage ingestion, as a safety measure (Sandle, 2006).  Those that are used indoors should never be mixed with other cleaning products as chemical reactions can occur.

            The choice of disinfectant to be used depends on the particular situations.  Some disinfectants have a wide spectrum (kill many different types of microorganisms), while others kill a smaller range of disease – causing organisms but are preferred for other properties (they may be non-corrosive, non-toxic, or inexpensive).  There are arguments for creating or maintaining conditions which are not conducive to bacterial survival and multiplication, rather than attempting to kill enables them to evolve rapidly.  Should some bacteria survive a chemical attack, they give rise to new generations composed completely bacteria that have the surviving bacteria qualities in successive generation which are increasingly resistant to the chemical used, and ultimately the chemical is rendered ineffective.  (Huang et al, 2009).

2.6       GENERAL FEATURES OF DISINFECTANT

·                    A disinfectant must have a wide spectrum of activity: This refers to the ability of the disinfectant to kill different types of microorganism which are in different physiological states.

·                    Whether there is a requirement that the disinfectant is sporicidal.  This requirement influences the type of disinfectant purchased.  Sporicidal disinfectant tend to have greater health and safety consideration and some particularly chlorine based disinfectants are aggressive to certain types of surfaces and will cause discolouration and abrasion.

·                    The disinfectant must be rapid in action with an ideal contact time of less than ten minutes. The contact time is the time taken for the disinfectants to band to the microorganism, transverse the cell wall membranes and to reach specific target site.  The longer the contact time, then the longer the surface or article needs to be left prior to use.

·                    The disinfectants selected must have different mode of action 

·                    Some disinfectants require certain temperature and pH ranges in order to function correctly.  One type of disinfectant for example may not be effective in a cold room due to lower temperature.  The reason for this is because the validation standards for disinfectants measure the bacterial activity at 20⁰C and therefore the disinfectant may not be effective at higher or lower temperature.

·                    Prior to the use of disinfectants, it is essential that as much dirt and soil is removed as possible: This requires the application of a detergent.  Some disinfectants are not compatible with certain detergents.  In such circumstances, detergents residues could neutralize the active ingredients in the disinfectant.  Any disinfectant purchased should be compatible with the detergents used.

·                    Other disinfectants leave residue surfaces, whilst thus can mean a continuation of an antimicrobial activity, residues can also lead to sticky surfaces and or the inactivation of other disinfectants.

·                    Different disinfectants are not compatible with all types of surfaces:  The disinfectant must not damage the material to which they are applied to, (although it is recognized that repeated application over several years may cause some corrosion).  For more aggressive disinfectants like alcohol is sometimes necessary in order to remove the residues.  In addition to some disinfectants having a corrosive effects others may be absorbed by fabric, rubber, and so on, which lessens their bactericidal properties (Sandle, 2006)

·                    The disinfectants must meet the requirement of the validation standards, to measure bactericidal, fungicidal and appropriate sporicidal and virucidal activities.

·                    The presentation of the disinfectant is an important choice, whether as a prescribed preparation in a trigger spray or as a ready to use concentrate or an impregnated wipe.

·                    The disinfectnhat must be relatively safe to use in terms of health and safety standards.  Here the main concern is with operator welfare, a related concern is the impact upon the environment.

·                    The cost of the disinfectants is required for use in an aseptic filling area then it will need to be sterile, filtered or supplied sterile in a suitably wrapped container.  Any disinfectant will only be effective if it is used at the correct concentration applied to relatively clean surfaces using appropriate clean room ground mops or cloth and left for the appropriate contact time (Theraud et al., 2004).

            In 1985, the Centre for Diseases Control (CDC’s) guidelines for hand washing and hospital environmental control defined the levels of disinfection, sterilization, high level, intermediate level disinfectant and low level disinfectant.

            Sterilization is the destruction of all microorganism including bacteria spores, high level disinfectant is expected to destroy all microorganisms with the exception of bacteria spores.  The food and drug administration clears these chemicals germicides. 

Intermediates level disinfection in activities Mycobacterium tuberculosis, vegetative bacteria, most viruses and fungi, but does not necessarily kill bacteria spores.

            Low level disinfection inactivates vegetative bacteria, knots viruses and fungi but it cannot be relied upon to kill resistant microorganisms such as tuberclobacilli and bacteria spores (EPA, 2006).

Factors Influencing the Efficacy of Disinfectants

·                    Contraction of the disinfectants and time of effective applications:  Generally, the effectiveness of disinfectants increases with its concentration.  The range of dilution over which a disinfectant is effective varies markedly with different chemicals and some disinfectant becomes quite inactive when diluted only several times beyond the correct dilution for use.  Thus strong enough for long enough principle should be applied.

·                    Number and type of microorganism present:  The velocity of the reaction of disinfectant decreases with increase in the number of organism present.  Also, spore-forming microorganisms are more resistant to disinfectants than non-spore formers.

·                    Effect of pH: Extremes of acidity or alkalinity can effectively limit growth of microorganism.  pH 4.5 to 9, being the limiting range for many organisms.  For many weak bases, such as acridness, the biologic activity has been correlated with the concentration of cation species.  Also, for many weak acids, the antimicrobial activity is primarily or sorely attributed to the undisassociated molecules.

·                    Temperature:  The antimicrobial activity of disinfectants is increased by heat with the limits of the thermo stability of the substance; hence it is preferable to use disinfectant under warm eater than cold condition.

·                    Effects of diluting agent: some diluting agents react with the disinfectant and form complexes with them thereby reducing the availability of the agent.

·                    Hardness of Water: Minerals such as calcium and magnesium can also affect the efficacy of the disinfectants by tying up the active ingredients (Bhatia and Ichhpujani, 2008).

2.7       GENERAL FEATURES OF THE TEST ORGANISMS

Pseudomonas aeruginosa

            This is of the order – Pseudomonales family – Pseudomonceae, and genius pseudomonas.  It is gram negative aerobic rod, non-spring and non-capsulata, usually motile by virtue of a single pollar flagellum.  It is a strict aerobe, but can grow anaerobically in the presence of nitrate.  Most strains produce pigment.  Classically the colony and surrounding medium is greenish-blue due to production of pyocyanin and yellow green fluorescent pigment, flourescin.

            It is oxidase positive and some strains produce dark red brown pigment pyrorubin.  All strains produce diffusible free fluorescent flourescein.  The commonest colonial form is large, low convex with an irregular surface and an edge that is translucent in comparism with the pigment center.

            It is an important nosocomia pathogen, and may be isolated from wide variety of environmental sources e.g. from earth, water, wounds, bathroom walls and sewage.

            Also, it can be infect almost any external site or organ in the body and causes mild and superficial injections resulting to urinary tract infections, infected ulcers, or bed sores, infected burns and eye infections(Tortora, 2007) .

Staphylococcus aureus

            Staphylococcus aureus is coagulase positive which differentiates it from other species.  It is a major pathogen for humans.  Almost every person will have some type of Staphylococcus aureus infection during life time, ranging from poisoning by elaboration of enterotoxin or minor skin infections to life-threatening infections.  Those infections are as follows: superficial infections e.g. skin pustules, boils, impetigo, subcutaneous and submucous abscess e.g. whitlow of finger or palm of hand and breast diseases etc.  Also, Staphylococcial food poisoning, a common cause of vomiting and diarrhoea.

            Morphologically, these are gram positive cocii, mainly joined in grape-like clusters, but some cocci are in single or some in pairs, non-sporingi, non-motile and non-capsulate.  After 24hrs at 37⁰C on nutrients agar or blood agar, the colonies are circular, with a smooth shiny surfaces relatively opaque to transmitted light.

            It is an opportunistic pathogen, but the more pathogenic strains are well endowed with enzymes (coagulase and lipase) and toxins to help establish and protect the organism in host tissue (Bhatia and Ichhpujani, 2008).

2.9       MECHANISM OF ACTIONS OF DISINFECTANTS AGAINST BACTERIA

            Disinfectants vary in their spectrum of activities, mode of action and efficiency.  Some are bacteriostatic where the ability of the bacteria population is halted.  Here the disinfectant can cause selective and reversible changes to cell by interacting with the nucleic acids, inhibiting enzymes or permeating into the cells wall.  Once the disinfectant is removed from contact with the bacteria cells, the surviving bacteria population will potentially grow.  Other disinfectants are bactericidal in that they destroy bacterial cells through different mechanisms including causing structural damage to the cell, autolysis, cell cysis and the leakage in coagulation of cytoplasm (McDonnel and Russels, 2001).

            Within these groupings, the spectrum of activity varies with some disinfectant being effective against vegetative gram positive and gram negative microorganisms only while others are effective against fungi.  Some disinfectants are sporicidal in that they cause the destruction of endospores forming colony, these are the most difficult forms of microorganisms to eliminate from cleaning room surfaces.

            However, a chemical agent does not have to be sporcidal in order to be classed as disinfectant or as a biocide (Namura et al, 1993).  The bacteriostatic bactericidal and sporicidal properties of antibiotic are influenced by many variables not least then active ingredients (Malik and Goyal, 2006).

2.10    RESISTANT ACTION OF BACTERIA

            Disinfectants cause some bacteria to thrive.  To keep sickness at bay, many off us constantly wash hands and disinfect surfaces.  But a new laboratory study shows one pesky bacterium eats cleansers for breakfast.  When disinfectants was applied to laboratory cultures of the bacteria, they adapted to survive not only the disinfectant, but also a common antibiotic.  How they survived was, the bacteria were able to move efficiently, pump out antimicrobial agents.  The adapted bacteria also had a genetic mutation that allowed them to resists (Wisplingloff et al, 2007).

            In principle, this means that residue from incorrectly diluted disinfectant left on hospital surfaces could promote the growth of antimicrobial resistance bacteria (Sad and Gerald, 2010). A major factor is over use or misuse of antimicrobial agents.

            According to Gerald (2010), he said “we need to investigate the effects of using more than one type of disinfection on promoting antibiotic resistant strains.  This will increase the effectiveness of both our first and second line of defense against hospital acquired infection.

            This research is focused on Pseudomonas aeureginosa and Staphylococus aureus bacteria responsible for a range of infections in people with weakened immune system.  In the addition of increase amount of disinfectant to Pseudomonas and Staphylococcus culture, the bacteria adapted to survive.

Pseudomonas Sensitivity to Physical and Chemical Agents

            Temperature of 55⁰C for 60 minutes exerts lethal effect upon P. aeruginosa.  This microbe is resistant to drying.  It survives well in wet environment and can remain alive in water at ambient temperature for months.  It can also multiply in water with minimal nutrients.  To reduce surface contamination due to this organism on instruments, hospitals and drains, disinfectants must be selected with great care. 

            P. aeruginosa is resistant to many disinfectants including quaternary ammonium compounds and hence dettol (Bhatia and IChhpujani, 2008).

Staphylococcus aureus Sensitivity

            Extremely stubborn organism and can survive in adverse environment for a very long time.  Some strains can even withstand temperature of 60⁰C for 30 minutes (Bhatia and Ichhpujani, 2008).  As compared to other bacteria, these are more resistant to the action of disinfectants.  Staphylococcus aureus is very sensitive to aniline dyes and a concentration of 1:5,000,000 of crystal violent can inhibit their growth.  Most strains can grow in the presence of 10% Sodium Chloride (Nacl).  Fatty acid can inhibit the growth of Staphylococcus specie (Bhatia and Ichhpujani, 2008).

2.11    ADVANTAGES AND DISADVANTAGES OF DISINFECTANTS

            Disinfectants are important chemicals used for a variety of purposes.  Within homes, disinfectants are used to kill microorganisms which can grow in places such as the kitchen or the bathroom.  Disinfectants are widely used within the medical establishment, hospitals, health care centre and doctors offices for purposes of killing different types of germs which may cause all kinds of diseases.  As important as they may be, they have both advantages and disadvantages.

Advantages

1.                  One of the most important advantages offered by disinfectants is for the control of diseases.  Disinfectants cam kill germs and microorganism which may not be seen with the naked eyes.  For instance, phenolics types of disinfectant are vary good for killing bacteria.  They are considered as general purpose disinfectant and can kill organic matter and other organism including mycobacterium.  Examples of Phenolics include Benzyl-4 chlorophenol, amyphenol, phenolphenol and Cresol (Sandle, 2006).

2.                  Inexpensive: Disinfectants are largely inexpensive.  They are widely available.  For example, many schools, care facilities and other institutions specify that bleach must be routinely used as a disinfectant because it is effective on a wider range of bacteria and viruses than many other disinfectants and is cheap and accessible (Carly et al, 2006).  Some of the cheapest kinds of disinfectants include halogens.  Halogens are also very effective in killing and controlling viruses.  They are also use for equipment disinfecting; particularly medical equipments contaminate with blood which are soiled with HIV or hepatitis and other diseases.  Bleach with its bleaching potential is used in treating cloths against bacterial and germ contamination.  Examples of halogen disinfectants are hypochloride and ordinary bleach (Zamani et al, 2007).

3.                  Treating Skin wounds: Alcohol is another group of disinfectant used in treating skin and wounds against bacterial infections.  They are used to clean surfaces of wounds on the body; they are also used along with water to maximize their effectiveness.  Ethanol as an example if alcohols do not leave any residue on treated items. A 70% (v/v) aqueous solution of ethanol can be used to soak small pieces of surgical instruments.  A contact time of ten minutes or more is necessary.  Iso propanol is another example of alcohol use for skin disinfection(Stephen et al, 2004).

Disadvantages:

4.                    Disinfectants are hazardous waste.  They contain halogenated compounds.  Most disinfectants contain in excess of environmental safety level of 0.01% of the halogenated compounds.  This makes it hard for disinfectants to be disposed as ordinary waste.  Infact, some disinfectants are considered as not only hazardous but also toxic.  For example, the chlorine in sodium hyhpochlorite (bleach) is chemically bonded, but even under the most stringent quality control, the first stage of bleach production when the most stringent quality control, the first stage of bleach production, when the chlorine gas is produced, result in the creation of toxic by product known as dioxin.  One of a family of organochlorines dioxin has been identified as carcinogen and has been linked to genetic changes and birth defects.  They are known to be toxic to shell fish and other marine and aquatic organisms.  The Nordic Ministers Conference, made up of environmental ministers from Norway, Sweden, and other Nordic countries list bleach as one of a number of substances considered dangerous to the environment (Fraise, 1999).  Bleach sold for household use is usually 5% sodium hypochlorite, whereas industrial bleach is typically 10 – 12% which increases the hazard when it is used in the workplace.  Amylphenol is also an example of a toxic and hazardous disinfectant.  The use of phenol in nurseries is questioned because of toxicity in infants (Carly et al, 2006).

5.                  Irritants: Some disinfectants are irritants and can be harmful to humans particularly in strong concentration.  A saponated brand of cresol (disinfectant C) which contain phenol as the active ingredient is not recommended for semi-critical items because of the lack of validated efficacy data and because the residual disinfectant on porous material may cause tissue irritation even when thoroughly rinsed.  They are generally safe by prolonged exposure to skin may cause irritation.  Household bleach (disinfectant B) at the concentration of 4 – 5% may produce skin and ocular irritations or oropharygeal, esophageal gastric burns.  They are corrosive to metals in high concentration, discoloring or bleaching of fabrics (Gaonkar et al, 2006). 

2.12    GENERAL GUIDELINES IN THE USE OF DISINFECTANTS

            Universal precaution with the appropriate personnel protective equipment should always be used when dealing with contaminated items, during cleaning and decontamination procedure.   Item must be thoroughly cleaned before being disinfected because dirt, blood, mucous and tissue may interfere with the action of the disinfectant.

            The disinfectant in sufficient concentrations at the correct temperature must remain in contact with the surfaces for a specific period of time to allow penetration of all the microbial cell wall and deactivation.

            The concentration, temperature and exposure times are different for each disinfectant and manufacture’s direction for use must be followed carefully.  Chemicals should not be mixed with each other or with detergents since this may inactivate their disinfecting properties or create noxious fumes.  Disinfectant A should never be should used to disinfect hands since it can dry the skin.  Alcohol - based hand rubs, alcohol mixed with emollients, are recommended for the decontamination of lightly soiled hands in situations where proper hand-washing is inconvenient or not possible.

            Alcohols are volatile and flammable and must not be used near open flames.  Working solution should be stores in proper containers to avoid the evaporation of alcohols.

            If air is entrapped under or within an item, the disinfectant cannot completely contact all the surfaces.  Item should be dry to prevent dilution of the disinfectant.  When indicated, it is essential that the disinfectants be thoroughly rinsed from items before the items are used.

            Personnel should take precaution to avoid direct contact with chemical disinfectant and they should always be used in well ventilated areas.

            The Sanitational Products Prevention Program (SP4), a combined initiative of the U.S. Environmental Protection Agency, the state of California and a number of U.S. cities, list bleach among products to use with extreme care or avoid if possible (EPA, 2006).

CHAPTER THREE

3.0       MATERIAL AND METHOD

MATERIALS

The test materials and the media used in this study and the method of their preparation are shown in appendix.

SAMPLES

Disinfectants were bought from main market Enugu (Ethanol disinfectant A, Jik disinfectant B, and Isol disinfectant C).

Bacteria were Isolated from clinical sample and Bathroom.

-              Staphylococcus aureus from wound pus swabs

-              Pseudomonas aeruginosa from female bathrooms in caritas university Enugu.

The samples were aseptically collected using sterile swab stick and brought to the laboratory.

3.1       ISOLATION OF BACTERIA  

Culture media for the organisms ( MacConkey Agar & Blood Agar) were prepared, samples were aseptically inoculated on the media. Incubated at 37^oC for 24hours.

PURIFICATION

After 24hours of incubation, the colonies that appeared to be similar were picked using a sterile wire loop and sub cultured on a nutrient agar in order to get pure colonies of the Isolated organisms (Staphylococcus aureus and Pseudomonas aeruginosa).  

3.2       IDENTIFICATION OF ISOLATES

Pure colonies of all the Isolates were identified using colony morphology on agar plates, Grams stain and Biochemical test.

i           COLONIAL APPEARANCE

            Based on their colonial appearances on the media, used, the test organisms were identified as Staphylococcus aureus and Pseudomonas aeruginosa.

ii          GRAM’S STAINING TECHNIQUE:

            With a sterile wire loop, a colony of each of the test organisms was emulsified on a drop of sterile physiological saline placed on a clean glass slide. The emulsions were allowed to dry, and then fixed over the Bunsen flame briefly. The slides were placed on a staining rack, and then flooded with crystal violet. Stain allowed to stay for 1 minute, after which the stain was washed off with water. The slides were again flooded with the mordant, Lugol’s iodine solution, and allowed to stain for 1 minute, then washed off with water. The slides were decolorized for 5 seconds with alcohol solution, then washed off with water. The slides were finally counterstained with neutral red solution (safranin) for 2 minutes and also washed off with water.  These slides were air-dried and viewed microscopically using 100 x objective (oil immersion) and the Gram’s reaction of the organisms were recorded. The method used was that described by (Cheesebrough,2000).

iii        CATALASE TEST:

            This test is used to differentiate Streptococcus species from Staphylococcus aureus which have similar morphologies.   On a sterile grease free slides, was added 2 drops of hydrogen peroxide solution. A colony of the Staphylococcus aureus was collected using a sterile wire loop and then placed on the solution,bubbles of gas were examined within 10 seconds.   This indicated a catalase positive test, control was also set up using a known colony of Staphylococcus aureus and examined together with the test. the results were read and recorded. Method used was described by (Cheesbrough, 2000) .

iv.        COAGULASE TESTS:

a.         Slide Coagulase Test: This test is used to identify Staphylococcus aureus which produces the enzymes coagulase. A drop of physiological saline was made on 2 separate grease free slides.  A drop of fresh human plasma was added to one of the suspensor (test) and they were mixed. The other suspension was used as negative control. A visible clumping of the test organism with 10 seconds was examined for and result recorded. 

b.         Tube Coagulase Test: This was carried out as a confirmatory test to slide method.  A 1m 10 dilution of fresh human please labeled T for test, P for positive control, and N for the Negative control.  0.2ml or diluted plasmas was pipette into each tube. 0 8ml or the test both culture was added to tube P, and 0.8ml of sterile broth was added to tube No.  The solutions were gently mixed, incubated at 37c and examines for clothing after I hours.  A position result was shown by the presence of fibrin clot Method used was described by (Cheesbrough,2000). 

v.         OXIDASE TEST

            This test is used to differentiate Pseudomonas aeruginosa from other enteric organisms, which are oxidase negative.  A piece of filter paper was paced on a clean Petri dish and 3 drops of freshly prepared oxidase reagent was added.  Using a piece of stick rod, a colony of the test organism suspected to be Pseudomonas aeruginosa was collected and smeared on the fitter paper.  The development of blue – purple colour within 10 seconds indicated a positive result.  The result was read and recorded.  Method applied here was described by (Cheesbough, 2000)     

3.3       PREPARATION OF DISINFECTANTS

Table 1:

è Original concentration in (%) Percent	Disinfectant	Disinfectant	Disinfectant
	95%	5%	30%

            Various concentration of disinfectant A were prepared thus: 95% 85% 70% 60% and 50%

Using this formular     where R = Required concentration

                                                            V = Required volume of water

                                                            O = Original concentration.

If R = 85%, V = 10ml, O = 95%

è           85% X 10ml           = 850ml         = 8.95ml

                        95%                     95

:.  8.95ml of original concentration + 10 – 8.95ml = 1.05ml of water.For 85% concentration of disinfectant A.

Other disinfectants were diluted in the same way:

For each disinfectant, five different disposable tubes were used with disinfectant name, tube number and concentration and labeled thus:

Table 2: Tube Concentration (%)

1	95%
2	85%
3	70%
4	60%
5	50%

3.4       ANTIMICROBIAL SUSCEPTIBILITY TESTING (USING KIRBY BAUER DIFFUSION ASSAY WELL METHOD)

Procedure

-              Obtained twelve sterile disposable Petri dishes and labeled two each for one bacterial and disinfectant.

-              A permanent marker was used to divide each plates into six equal parts and numbered the bottom of the plates according to the concentration for each disinfectant, by writing the names of the disinfectant on the bottom of the plate.  The sixth sector was written water for control. This was done for all the original plate and the replicates.

-              The prepared 25ml nutrient agar media was poured into each of the plates.

-              A loopful of the isolates was  inoculate  uniformly on each of the plates and this was done in all the plates with the test organisms

-           The plates were allowed to dry for few minutes.

-           For the test plates, a sterile cap borer was use to bore well in the six sectors labeled on the plates.

-           1ml each of different concentrations of the three disinfectants was pipette inside the well.  But the centre sector for control 1ml of a sterile water was pipetted.

-           The plates were allowed to stayed for 30minutes before incubation

-           All the plates were incubated overnight at 37oC for 24 hours.  After over night incubation, the plates were examined for zone of inhibitions and was result recorded.Method was described by (Rollins and Joseph, 2000)

CHAPTER FOUR

4.0       RESULTS

RESULTS OF IDENTIFICATION TESTS

1.         Grams Reaction

a.         Staphylococcus aureus: Gram positive cocci of uniform sizes, occurring characteristically in clusters but also singly and in pairs.

b.         Pseudomonas aeruginosa: Gram negative rods

2.         Catalase Test

Control – positive (stock from Ambilin Medical Laboratories)

Test (Staphylococcus aureus) – positive

3.         Coagulase Test

i.          Slide coagulase test - Staphylococcus aureus positive

ii.         Tube coagulase test; Tubes T and P – positive, tube N – negative

4.         Oxidase Test

i.          Pseudomonas aeruginosa – positive

RESULTS OF THE TEST

i.          CONTROLS: The control sectors showed uniform colonies around the well.  No clear zone of inhibition.

ii.         TEST SECTORS: The efficacies of the different disinfectants varied on dilutions.  The result showed that all the disinfections inhibited the growth of the test organisms in their concentrated forms by showing different diameters of zone of inhibitions around each well, which was measured using meter rule in millimeter as shown in tables below:

Table 1: Results of diameter of zone of inhibition of ethanol, phenolics and bleach for Staphylococcus aureus:

Disinfectants	Concentrations (%)	Diameter of zone of inhibitions (mm)
A	95 85 70 60 50	2 7 20 16 14
B	5 4 5 2 1	24 17 14 10 0
C	30 25 20 10 5	38 29 44 21 20

Table 2:

Results of Diameter of zone Inhibitions of ethanol, phenolics, bleach for Pseudomonas aeruginosa

Disinfectants	Concentrations (%)	Diameter of zone of inhibitions (mm)
A	95 85 70 60 50	0 8 16 15 9
B	5 4 5 2 1	18 16 11 7 2
C	30 25 20 10 5	17 16 13 10 7

The tables below evaluated the test organisms response to each compound based on their different concentrations.

Table 3:

Pseudomonas aeruginosa response to Ethanol, Phenolics and Bleach

Disinfectants	Concentrations (%)	Diameter of zone of inhibitions (mm)	Response
A	95 85 70 60 50	0 8 16 15 9	Resistant Resistant Susceptible Intermediate Resistant
B	5 4 5 2 1	18 16 11 7 2	Susceptible Susceptible Intermediate Resistant Resistant
C	30 25 20 10 5	17 16 13 10 7	Susceptible Susceptible Intermediate Resistant Resistant

Table 4:

Staphylococcus aureus response to ethanol, phenolics and bleach

Disinfectants	Concentrations (%)	Diameter of zone of inhibitions (mm)	Response
A	95 85 70 60 50	2 7 20 16 14	Resistant Resistant Susceptible Susceptible Intermediate
B	5 4 5 2 1	24 17 14 10 0	Susceptible Susceptible Intermediate Resistant Resistant
C	30 25 20 10 5	38 29 44 21 20	Susceptible Susceptible Susceptible Susceptible Susceptible

N.B;    Method Source (Johnson and Case, 1995) using this value below as standard:

                                    Diameter of zone of Inhibition (mm)

Resistant                    10 or less

Intermediate              11 - 15

Susceptible                16 or more

PATTERNS OF THE ANTIMICROBIAL EFFICACIES OF VARYING CONCENTRATIONS OF THE DISINFECTANT ON THE TEST ORGANISMS USING HISTOGRAM

Concentration of disinfectant A

Fig 1:   Pseudomonas aeruginosa disinfectants A test result

 

Concentration of disinfectant B

Fig 2:   Pseudomonas aeruginosa disinfectants B test result

 

Concentration of disinfectant C

Fig 3:   Pseudomonas aeruginosa disinfectants C test result

 

Concentration of disinfectant A

Fig 4:   Staphylococcus aureus disinfectants A test result

 

Concentration of disinfectant B

Fig 5:   Staphylococcus aureus disinfectants B test result

 

Concentration of disinfectant C

Fig 6:   Staphylococcus aureus disinfectants C test result

            From the figures 2, 3, 5, 6, it was shown that the diameters of the zones of inhibition decreased as the concentration of disinfectant decreased except in figure 1 and 4 where the higher the concentration, the lower the diameter of zone of inhibition.

PATTERNS OF THE EVALUATION OF THE BACTERIAL PERCENTAGE AND DEGREE OF RESPONSE TO EACH DISINFECTANT USING A PIE CHART

For disinfectant A on Pseudomonas aeruginosa response ewe have:

Degree of response  =          Resistant values       =          17

                                                Intermediate values=           14

                                                Susceptible values   =          16

                                                                                                47

==> Degree of resistant =   17 x 360 = 130.2⁰, % = 17 x 100 = 36.2%

                                                47       1                          47      1

==> Degree of intermediate =   14 x 360 = 107.2⁰, % = 14 x 100 = 29.78%

                                                       47       1                          47      1

==> Degree of susceptible = 16 x 360 = 122.5⁰, % = 16 x 100 = 36.2%

                                                  47       1                          47      1

This process applied for the rest of the disinfectant on each of the test organisms

 

Fig 7: Disinfectant  A on P. aeruginosa

 

Fig 8: Disinfectant B on P. aeruginosa

 

Fig 9: Disinfectant C on P. aeruginosa

 

Fig 10:            Disinfectant A on Staphylococcus aureus

 

Fig 11:            Disinfectant B on Staphylococcus aureus

 

Fig 12:            Disinfectant C on Staphylococcus aureus

            Then to calculate the average and the standard deviation of the diameter of the zone of inhibition for each disinfectant, method used was from (Johnson and Case 1995).

Average for each disinfectant was calculated thus:

            Mean

Where            X = sum of number of diameter of zone of inhibition

                        N = Total number of concentration

For Disinfectant A on P. aeruginosa = 0 + 3 + 16 + 14 + 9 = 8.4

                                                                                    5

Disinfectant B           =          18 + 16 + 11 + 7 + 2            =          10.8

                                                             5   

Disinfectant C           =          17 + 16 + 13 + 10 + 7          =          12.6

                                                             5

Staphylococcus aureus

A         =          2 + 7 + 20 + 16 + 14            =          11.8

                                     5

B         =          24 + 17 + 14 + 10 + 0          =          13

                                     5

C         =          38 + 29 + 24 + 21 + 20        =          26.4

                                     5

            Standard deviation of diameter of the zone of inhibitions for each disinfectant using the formular

                        =         

Where            X = diameter of zone of inhibition for each disinfectant

             = Average mean

            n = Total no of concentration

Solving out for disinfectant A on P. aeruginosa

=>

=>       S.D. for disinfectant A on P. aeruginosa =          6.88

            S.D. for disinfectant B                                 =          11.58

S.D. for disinfectant C                                 =          13.52

Standard deviation of the diameter of zone of inhibition for each disinfectant on Staphylococcus aureus=

S.D. for disinfectant A         =          12.62

            S.D. for disinfectant B         =          13.96

S.D. for disinfectant C         =          25.86

CHAPTER FIVE

5.0       DISCUSSION

From the different diameters of zones of inhibition of the three disinfectants under study, it was discovered that all the disinfectants inhibited the growth of the test organisms in their concentrated forms. On dilutions, their activities varied. Disinfectant C at 30% concentration showed the highest activity on Staphylococcus aureus, whereas Disinfectant. B and A showed the least. The distribution of the activities in decreasing order is as shown phenolics > bleach > ethanol.

Disinfectants B and C showed the highest activities at the concentrations of 5% 30% on Pseudomonas aeruginosa, whereas disinfectant A showed the least on the same organism. The distribution of their activities in decreasing order is as shown, bleach > phenolics > ethanol.

However, on the contrary, disinfectant A has the lowest antimicrobial effect as compared to others on both organisms. From table 6, disinfectant C had the highest inhibitory activity and can be deduced to be highly bactericidal on both organisms.  According to Weber et al, 1999, phenolics which is active ingredient for disinfectant C are active against bacteria (especially gram positive bacteria). This tallies with my findings, a phenolics p[roves highest inhibition against Staphylococcus aureus. Owing to their high activity level, disinfectants C maintain their activities in the presence of organic material ( milk) as they last long on surfaces unlike ethanol which evaporates easily (Weber et al, 1999). Also since the mode of action of phenols in mainly by protein penetration and cell disruption, this extrapolates the bactericidal action of phenols (McDonell and Russel 2001).

Moreover, form the results, it indicated that bleach had an ideal bactericidal effect against both Pseudomonas aeruginosa and Staphylococcus aureus at 55 and 5% Concentrations as seen in tables 3 and 4.   According to Barindra et al 2006, former study, it found that oxidation reactions will occur when bleach is dissolved in water, which can destroy organisms fold structure leading to sterilization. Another study also found similar result that bleach is rapidly bactericidal achieving a 5log10 kill of Pseudomonas aeruginosa and other vegetative organisms in one minute (Fraise, 1999).

The data’s in figures 2, 3, 4 and 5 generally showed that diameters of zone of inhibition decreases as the concentrations of disinfectant decreases, but the observation was stable in disinfectant A. from the results in figures 1 and 4, it was shown that as the concentration of ethanol increased, the diameter decreased. Ethanol are rapidly bactericidal rather than bacteriostatic against vegetative forms of bacteria (gram tve and gram-ve), but their cidal activities drop sharply when diluted below 60% concentration and optimum bactericidal concentration in the range of 60% - 90% solution in water, volume/volume (Moorer,  2003). The result showed that 70% ethanol gave better effect on both test organisms than other ethanol concentrations. According to Moorer 2009, 70% ethanol had been found to be most effective to denature protein thereby killing bacteria, because of its diffusion rate and transportation into the cells organism. It evaporates at a slow rate and less harmful to the hand, this is the reason why it’s been used in the laboratories for disinfection. Below 70% does not denature protein, while 85%-absolute ethanol evaporates fast and leave the protein untouched. They leave traces on the applied surfaces thus, adding unwanted reagents. Also, they are harmful to the skin thereby making it dry and may not be effective.

From this study, it confirmed Carly et al 2006, study which showed similar result that higher concentrations are less effective as the action of denaturing proteins is inhibited without the presence of water. They also evaporate rapidly which makes extended exposure time difficult to achieve unless items are immersed in the ethanol (Carly et al, 2006).

According to Yi Hsing et al, 2002 researches, it also found that some kinds of bacteria cannot be billed easily and have some characteristics of resistance on ethanol. Its sterilization in mainly due to dehydration of protein enzyme deactivation and prevent bacteria growth. Different proteins have different biological characters which cause selectivity in ethanol deactivation of organisms. However, this conforms with Yi Hsing et al, 2002, as Pseudomonas aeruginosa are more resistant to disinfectant A.

In addition, disinfectant C and B are both effective disinfectants for sterilization against pseudomonas aerations and Staphylococcus aureus but disinfection C has the highest inhibitory effect.

Furthermore, the mean value of each of disinfectant described the net effect of the disinfectant on test organisms. From the result in of the mean it can be deduced that queried effectiveness of disinfectant A on test organisms are intermediate and resistant, while that of disinfectant B are resistant and intermediate as to compare with disinfectant C which has the highest average at scriptable and intermediate. Then standard deviation compared how far each value of diameter of zone of inhibition for each disinfectant are away from the mean this showed that result varies in different cases of life.

5.1       RECOMMENDATIONS                              

On further research in the study of the efficacy of disinfectants, I recommend that these antimicrobials used, be tested in the presence of organic substances so as to determine how they work. Also to find out whether the mechanism of action of the agents have effects on the development of resistance by organisms.

On the proper use of these disinfectants, I recommend that individuals, families, hospitals and other laboratories that used these disinfectants to achieve sterility should use them at the correct concentrations and retain adequate activities for example, if disinfectant C which is commonly and commercially used by all at 5% concentration could give the same susceptible response by bacteria with the original concentrations, I suggest at it should be diluted the more instead of using it at the stake concentration for this will reduce economic waste of the agent and help it to last long for the user.

This when done, will help to reduce the rate of worse infection, nosocomial among hospital inhabitants, in our homes and other laboratory  workers, thereby improving health for all.

5.2             CONCLUSION

        The main goal of this study is to compare the efficiency of three disinfectants at five different concentrations. Conclusively, among the three common disinfectants tested in this project, disinfectant C in all its concentration had this best efficiency against both Pseudomonas aeruginosa and Staphylococcus aureus.

      When these antimicrobial agents are used to disinfect sites suspected to be contaminated with gram positive bacteria, they should be used in their concentrated forms. Any dilution above this will only succeed in providing the user with a false sense of security

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APPENDIX 1

A.      ANTIMICROBIAL AGENTS USED

-              Isol (Gongoni Company Limited Nigeria) contain 30% v/v saponated cresol.

-              Jik (Rechik Benchiser Nig. Limited) contains 5% v/v sodium hypochlorite

-              Ethanol (Bulger Pharmaceuticals Enugu Nig. Limited) contain 95% Isopropyl alcohol.

B.      TEST ORGANISMS

Staphylococcus aureus

Pseudomonas aeruginosa

C.      MEDIA

-        Blood agar

-        Nutrient agar medium

-        MacConkey agar medium

D.      REAGENTS

·                    Crystal violet

·                    Safranin

·                    Hydrogen peroxide

·                    Oxidase reagent

·                    Peptone water

·                    iodine

·                    Alcohol

E.      EQUIPMENTS

·                    Disposable Petri dishes

·                    Sterile swabs

·                    Filter paper

·                    Cap borer

·                     Wire loop

·                    Permanent marker

·                    Distilled water

·                    1ml Disposable tubes

·                    Bunsen burner

·                    Pipette

·                    Autoclave

·                    Incubator

·                    Oven

·                    Microscope

·                    Refrigerator

·                    Slides

·                    Conical flask

APPENDIX II

PREPARATION OF MEDIA

Nutrient Agar Medium (Fluka Biochemica)

          This is a general medium that supports the growth of most microbial species.  It was used to enumerate bacteria and to maintain pure cultures of isolates.

Composition

Meat extracts                 1g/l

Yeats extracts                2g/l

Peptone                          5g/l

NaCl                              5g/l

Agar                               15g/l

Final pH                        7.4+ 0.2 at 37⁰C

Procedure

          The media was prepared as directed by the manufacturer.  12.6g of nutrient agar powder was dissolved in 450ml of distilled water in a conical flask which was corked afterwards.  The suspension was to first dissolved completely by heating and then sterilized by autoclaving for 15 minutes at 121⁰C.  The molten medium was allowed to cool at 45⁰C before dispending into a sterile Petri dishes at 25ml each and allowed to solidify.

MacConkey Agar (Fluka Biochemika)

          This is a differential medium, which as used to differentiate lactose fermenter from non-lactose fermenters.

Composition

Peptone                20g/l

Lactose                 10g/l

Bile Salt               5 g/l

NaCl                     3 g/l

Neutral red 0.075g/l

Agar                     12g/l

Final pH               7.4+ 0.2

Procedure

          The powder was prepared according to the manufacturer’s direction.  23.4g of the medium was dissolved in 450mls of distilled water.  It was dissolved completely by heating and then sterilized at 121⁰C for 15 minutes .  The molten medium was allowed to cool to 45⁰C  then poured in a sterile petri dishes at 25ml each and allowed to solidify.

Blood Agar

            2.96g of Nutrient agar powder was weighed out and dissolved in 1ooml of distilled water in a conical flask. It was sterilized by autoclaving at 121oC

 for 15minutes, allowed to cool at 40oC. Then 20ml of blood was added and mixed gently.

Oxidase Reagent

Tetramethyl-p-phenylenediamine dihydrochloride                  0.1g

Distilled water                                                               10ml

Procedure

          Dissolve the chemical in water.  The reagent is not stable.  It is therefore best prepared immediately before used.

Peptone Water

Peptone                          2g/l

Sodium chloride             1g

Distilled water               200ml

pH                                 70.74

Procedure

The peptone and salt were dissolved in the water and dispersed in a Bijou bottle.  The suspension was sterilized by autoclaving at 121⁰C for 15 minuets.

APPENDIX III

Plate 1: Plates showing zones of inhibition

 

Plate 2: MacConkey media with colonies of Pseudomonas aeruginosa

Plate 3: Some of the used plates

Plate 4: Biochemical test for Pseudomonas aeruginosa