ABSTRACT
The study of minimum inhibitory concentration, minimum bactericidal concentration and the killing time of ethanol, sodium hypochlorite and hydrogen peroxide on Staphylococcus aureus was carried out. The test organism, Staphylococcus aureus was isolated from the nasal cavity of humans. The media used was nutrient agar, streak plate method was employed. Colonial morphology, Gram staining and biochemical tests were used in the identification of the test organism, Staphylococcus aureus. Ethanol, Sodium hypochlorite and Hydrogen peroxide were utilized as disinfectants in this study. The study of the minimum inhibitory concentration, minimum bactericidal concentration and the killing time of these three disinfectants was conducted on Staphylococcus aureus using the tube dilution method (Quantitative method) under the time intervals of 3, 7 and 10 minutes respectively. The killing time of ethanol at concentration of 70%, 80% and 95% was at 10 minutes. For sodium hypochlorite, at concentrations of 1% and 2%, the killing time was 10 minutes while at 3% concentration, the killing time ranged from 7 and 10 minutes. For Hydrogen peroxide, at 5% concentration, the killing time ranged from 7 and 10 minute while at concentration 10% and 15%, the killing time ranged from 3, 7 and 10 minutes. At the different concentrations of ethanol used, the minimal bactericidal concentration and minimal inhibitory concentrations were 1000µl/ml and 250µl/ml respectively. At 15% concentration, hydrogen peroxide showed the least values of minimum bactericidal concentration and minimum inhibitory concentration which were 500µl/ml and 62.5µl/ml respectively. At 5% concentration, hydrogen peroxide showed the highest values of minimum bactericidal concentration and minimum inhibitory concentration at 2000µl/ml and 250µl/ml respectively. The minimum bactericidal concentration and minimum inhibitory concentration of 1% Sodium hypochlorite were at 2000µl/ml while 2% and 3% had similar effect in terms of the minimum inhibitory concentration and minimum bactericidal concentration which occurred at 2000µl/ml and 1000µl/ml respectively. The results showed that 15% Hydrogen peroxide had the highest efficacy against Staphylococcus aureus, both during the determination of the killing time and in the determination of the minimum inhibitory concentration and minimum bactericidal concentration while 1% Sodium hypochlorite which showed both minimum inhibitory concentration and minimum bactericidal concentration at 2000µl/ml had the least efficacy against Staphylococcus aureus.
TABLE OF CONTENTS
Title Page i
Certification ii
Dedication iii
Acknowledgements iv
Table of Contents v
List of Tables vi
List of Figures vii
Abstract viii
CHAPTER ONE
1.0 Introduction 1
1.1 Justification of the study 5
1.2 General aim of the study 5
1.3 Objectives of the study 6
CHAPTER TWO
2.0 Literature Review 7
2.1 History of Disinfectants 8
2.2 About Disinfectants 9
2.3 Sources of Contamination of Surfaces 9
2.4 Types of Disinfectants 10
2.4.1 Non-oxidizing disinfectants 10
2.4.2 Alchohols 11
2.4.3 Aldehyde 12
2.4.4 Phenol 12
2.4.5 Quaternary ammonium compounds 13
2.4.6 Oxidizing agents 13
2.4.7 Sodium hypochlorite 14
2.4.8 Chlorine dioxide 14
2.4.9 Hydrogen peroxide 14
2.4.10 Iodine 15
2.4.11 Peracetic acid 15
2.4.12 Home disinfectants 15
2.5 Properties of a Disinfectant 17
2.6 General Features of Disinfectant 17
2.7 Factors influencing the efficacy of
disinfectant 20
2.8 General Features of the Test Organisms 21
2.9 Mechanism of
Actions of Disinfectants against Bacteria 22
2.10 Resistant Action of Bacteria 23
2.10.1 Staphylococcus
aureus sensitivity 24
2.11 Advantages and Disadvantages of
Disinfectants 24
2.11.1 Advantages 24
2.11.2 Disadvantages 26
2.12 General Guidelines in the Use of
Disinfectants 27
CHAPTER THREE
3.0 Material and Method 29
3.1 Study area 29
3.2 Sources and concentration of
disinfectants used 29
3.3 Preparation of different concentration of
disinfectants 29
3.4 Media used 31
3.5 Collection of test sample and isolation 31
3.6 Biochemical identification of bacteria
isolate 31
3.6.1 Gram staining 32
3.6.2 Catalase test 32
3.6.3 Coagulase test 32
3.7 Materials 33
3.8 Killing time of different dilutions of
the disinfectant used in this study
using suspension test 33
3.9 Minimium inhibitory concentration 34
3.10 Minimium bactericidal concentration 34
CHAPTER FOUR
4.0 Results 35
CHAPTER FIVE
5.0 Discussion 41
5.1 Conclusion 42
5.2 Recommendations 43
References
Appendix
LIST OF TABLES
TABLE TITLE PAGES
4.1 Results of the killing
time of the different dilutions of the disinfectants used 36
4.2 Shows the minimum inhibitory concentration and
minimum
bactericidal concentration of different
dilutions
of the disinfectants used. 37
LIST OF FIGURES
FIGURES TITLE PAGES
4.1: The minimum inhibitory
concentration and minimum bactericidal
concentration
of ethanol. 38
4.2: The minimum inhibitory concentration and minimum
bactericidal
concentration
of hydrogen peroxide. 39
4.3: The minimum
inhibitory concentration and minimum bactericidal
concentration of sodium hypochlorite. 40
CHAPTER ONE
1.0 INTRODUCTION
Bacteria are a major cause
of disease and even human death. Disinfectant as an effective agent to kill or
eliminate bacteria is widely used in varies ways, especially in microbial
laboratory. Disinfectants can be mainly divided into five agents: alkylating,
sulfhydryl combining, oxidizing, dehydrating and permeable. The most commonly
used disinfectants in lab are ethanol, sodium hypochlorite and sometimes
hydrogen peroxide. The main constituent of sodium hypochlorite acts by
oxidizing the cell of microorganisms and attacking essential cell components
including lipid, protein and DNA (Ho-Hyuk Jang et al., 2008).
Ethanol as a dehydrating
agent causes the cell membrane damage, rapid denaturalization of proteins with
subsequent metabolism interference and cell lyses (Larson and Morton, 1991).
Hydrogen peroxide as a daily
used disinfectant, normally works by producing destructive hydroxyl free
radicals that can attack membrane lipids, DNA and other essential cell
components. Catalase produced by aerobic organisms and facultative anaerobes
that possess cytochrome systems, can protect cells from metabolically produced
hydrogen peroxide by degrading hydrogen peroxide to water and oxygen. Many
studies have been done on comparison of disinfectant efficiency; ethanol and
sodium hypochlorite are believed to have immediate effect against most
organisms (Carly N. Jordan, et al., 2006). For bacterial strains, E.coli has
been used widely in disinfectant test as a pathogen indicator.
In this study, a
disinfectant experiment was conducted using different concentration of
laboratory ethanol, commercial bleach (sodium hypochlorite) and hydrogen
peroxide against pure Staphylococcus aureus. The efficiency of disinfectant
was compared under three different contact times. 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 die in
devastating epidemics in which the cause was not understood. Entire families died because vaccination and
antibiotics were not available to fight infection (Johnson and Case, 1995).
Disinfectants
used in the food processing industry include oxidizing agents, e.g.,
hypochlorite, hydrogen peroxide (H2O2), ozone
and peracetic acid, denaturing agents, e.g., alcohol-based products,
non-oxidizing and surface tension diminishing agents, and enzyme-based
compounds (Wirtanen et al., 2001).
These disinfectants must be effective, safe and easy to use, and easily rinsed
off from surfaces, leaving no toxic residues that could affect the health
properties and sensory values of the final products (Wirtanen et al., 2000). Thus the appropriate
concentration of a disinfectant should be applied in the factory in order to
avoid propagation of food-borne diseases. Usually an effective cleaning and
sanitation step is a part of a program to inactivate microorganisms, preventing
the accumulation of microbial cells and particulates on the surfaces of
equipment as well as biofilm formation (Peng et al., 2002). In the processes of cleaning and disinfection of the
food industry, a concentration that is too low increases the risk of
acquisition of resistance to the disinfectant, and a concentration that is too
high increases the cost and the environmental burden (Luppens et al., 2002). Before application, each
suitable disinfectant should be tested against different microbial strains and
under working conditions representative of the food industry. The design of
food-processing equipment is also important to achieve better “cleanability” of
the food contact surface once bacterial adhesion has occurred. Comparative
cleaning studies carried out on materials like stainless steel, glass, nylon
and polyvinyl compounds showed no significant differences in cleanability when
the surfaces were new (Le Clercq-Perlat and Lalande, 1994).
Hydrogen peroxide contains properties, germicidal
effectiveness, and potential uses for stabilized hydrogen peroxide in the
health-care setting. It ascribes good germicidal activity to hydrogen peroxide
and attest to its bactericidal, virucidal, sporicidal, and fungicidal
properties (Rutala et al., 1993).Hydrogen
peroxide works by producing destructive hydroxyl free radicals that can attack
membrane lipids, DNA and other essential cell components. Catalase, produced by
aerobic organisms and facultative anaerobes that possess cytochrome systems,
can protect cells from metabolically produced hydrogen peroxide by degrading
hydrogen peroxide to water and oxygen. This defense is overwhelmed by the
concentrations used for disinfection (Omidbakhsh
and Sattar, 2006).
Sodium
hypochlorite has traditionally been used as a disinfectant because of its effectiveness
in killing a wide range of organisms, However, NaOCl use has many limitations,
including the severe corrosive effect on metals and some other materials, the
release of toxic chlorine gas when mixed with ammonia or acidic body fluids,
and the mutagenic and carcinogenic materials that may result from the
interaction between the organic matter and chlorine in many forms. These side
effects represent the main limitation of the use of chlorine and limit its use
to being a popular cleaner for domestic and housekeeping purposes (Yilmaz et al., 2005).
Ethanol in the healthcare setting refers to chemical
compounds ethanol that have generally underrated germicidal characteristics.
The ethanol is rapidly bactericidal rather than bacteriostatic against vegetative
forms of bacteria; they also are tuberculocidal, fungicidal, and virucidal but
do not destroy bacterial spores. Their cidal activity drops sharply when
diluted below 50% concentration, and the optimum bactericidal concentration is
60%–90% solutions in water (volume/volume) (Ali et al., 2001).The most feasible explanation for the
antimicrobial action of ethanol is denaturation of proteins. This mechanism is
supported by the observation that absolute ethyl alcohol, a dehydrating agent,
is less bactericidal than mixtures of alcohol and water because proteins are
denatured more quickly in the presence of water (Ali et al., 2001).The bacteriostatic action was believed
caused by inhibition of the production of metabolites essential for rapid cell
division.
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).
Staphylococcus aureus is an invasive, toxigenic
organisms is called disinin patients with abnormal host deficiencies. Staphylococcus 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).
1.1 JUSTIFICATION OF THE STUDY
It is important to establish the concentration of disinfectants
that can be inhibitory and bacteriostatic on bacteria and also the duration of
time for their effectiveness. In microbiology, the minimum inhibitory
concentration (MIC) is the lowest concentration of an antimicrobial that will
inhibit the visible growth of a microorganism after overnight incubation.
Minimum inhibitory concentrations are important in diagnostic laboratories to
confirm resistance of microorganisms to an antimicrobial agent and also to
determine the potency of new antimicrobial agents. MIC is generally regarded as the most basic
laboratory measurement of the activity of an antimicrobial, the minimum
bactericidal concentration (MBC) is the lowest concentration of an
antibacterial agent required to kill a particular bacterium. It can be
determined from broth dilution minimum inhibitory concentration (MIC) tests by
sub culturing to agar plates that do not contain the test agent. The MBC is
identified by determining the lowest concentration of antibacterial agent that
reduces the viability of the initial bacterial inoculum by ≥99.9%.
1.2 GENERAL AIM OF THE STUDY
1.
To determine
the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration
(MBC) and killing time of ethanol, hydrogen peroxide and sodium hypochlorite on
Staphylococcus aureus.
1.3 OBJECTIVES OF THE
STUDY
1. To determine the duration of time for
the effectiveness of ethanol, sodium hypochlorite and hydrogen peroxide on Staphylococcus aureus.
2. To determine the minimal inhibitory
concentration and minimal bactericidal concentration of ethanol on Staphylococcus aureus.
3. To determine the minimal inhibitory
concentration and minimal bactericidal concentration of Hydrogen peroxide on Staphylococcus aureus.
4. To determine the minimal inhibitory
concentration and minimal bactericidal concentration of Sodium hypochlorite on Staphylococcus aureus.
5. To find out the concentration of
disinfectants that will be effective in Gram positive Staphylococcus aureus.
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