ABSTRACT
This disinfecting actions of three disinfectants (ethanol, bleach and phenolics on two selected organisms, Staphylococcus aureus and Pseudomonas aeruginosa were studied. The bacterial species employed for the study were pure isolates from the stock cultures of Microbiology laboratory, Michael Okpara University of Agriculture Umudike. Their identities were confirmed using a three-way analysis including cultural examination, microscopic examination and biochemical tests. The methods used for the investigation included the Disc Diffusion method and Kirby Bauer Diffusion Assay Well method. Different concentrations of bleach (1%, 2%, 3%, 4% and 6%) were used on both test organisms. Also (50%, 60%, 80%, 85% and 95%) of ethanol as well as (5%, 10%, 20%, 25%, and 30%) phenolics were used. Differences in concentrations tested were because the original concentrations of the disinfectants differ. After 24 hours of incubation at 37°C, the results showed that all the disinfectants inhibited the growth of the test organisms in their concentrated forms with the exception of ethanol which was effective at concentrations above 90%. In both methods, the diameters of the zones of inhibition were measured using a ruler calibrated in millimeters, using different concentrations, and their efficacies varied. In the Disc Diffusion method, 30% Phenolics was found to exhibit the highest activity on Staphylococcus aureus with the diameter of inhibition of 37mm while 1% bleach showed the least with the diameter of inhibition of 0mm, while using the Kirby Bauer Assay Well Method, 30% Phenolics exhibited the highest activities on Pseudomonas aeruginosa with the diameter of inhibition of 31mm while 95% Ethanol and 1% Bleach exhibited the least inhibition activities.
TABLE OF CONTENTS
Title Page
Title Page i
Certification ii
Dedication iii
Acknowledgements iv
Table of Contents v
List of Tables ix
List of Figures x
Abstract xi
CHAPTER ONE
INTRODUCTION
1.0. Introduction 1
1.1. Aims and Objectives 2
CHAPTER TWO
LITERATURE
REVIEW
2.1. History of Disinfectants 3
2.2. About Disinfectants 6
2.3. Division of Disinfectants 6
2.4. Classes and Types of Disinfectants 7
2.5. Properties of a Disinfectant 8
2.6. Factors Affecting the Efficacy of Disinfectants 9
2.7. Mechanism of Actions of Disinfectants against
Bacteria 13
2.8. General Features of the Test Organisms 13
2.9.0. Confirmation/Identification and
Characterization of Test Organisms 14
2.9.1. Gram staining 14
2.9.2. Motility test 14
2.9.3. Catalase test 15
2.9.4. Coagulase test 15
2.9.5. Methyl red test 15
2.9.6. Voges-Proskaeur test 15
2.9.7. Indole test 15
2.9.8. Citrate test 16
2.9.9. Oxidase test 16
2.10.1. Resistant Action of Bacteria 16
2.10.2. Role of Biofilm in Reduced Sensitivity to
Disinfecting Agents 16
2.10.3. Mechanism of Plasmid Mediated Resistance 17
2.10.4. Mutational Resistance 17
2.11. Advantages and Disadvantages of Disinfectants 17
2.12. General Guidelines in the Use of Disinfectants 18
2.13. Disinfectant Tests 18
2.13.1. Carrier Tests 19
2.13.1.1. The AOAC (American association of Official
Analytical Chemists Use Dilution Test 19
2.13.2. Suspension Tests 19
2.13.2.0. Types of Suspension Test 20
2.13.2.1. Qualitative Suspension Test 20
2.13.2.2. Quantitative Suspension Test 20
2.13.3.1. Phenol Coefficient 20
2.13.3.2. Rideal Walker Method 20
2.13.3.3. The Chick Martin Test 21
2.14. Capacity Test 21
2.14.1. Kelsey-Syke Test 21
2.15. Test for Stability and Long Term Effectiveness 21
2.16. Practical Tests 22
2.16.1. Surface Disinfection Tests 22
2.16.2. Surface Time Kill Test 22
2.17. In Use Test 23
2.18. Testing Schemes 23
2.19. Bactericidal Tests 23
2.20. Disc Diffusion Method 24
2.21. Agar Well Diffusion Method 24
2.22. Kirby Bauer Diffusion Assay Well Method 25
2.23. Neutralizers 25
CHAPTER
THREE
MATERIALS
AND METHOD
3.1. Collection of Disinfectant Product 27
3.2.0. Sample Collection 27
3.2.1. Normal Saline Preparation 27
3.2.2. Media Preparation 27
3.2.3. Sub-culturing 27
3.2.4. Preparation of Disinfectants 27
3.3.0. Confirmation of Bacterial Cultures Used in the
Study 28
3.3.1. Gram Staining Techniques 28
3.3.2. Motility Test 29
3.3.3. Catalase Test 29
3.3.4. Coagulase Test 29
3.3.5. Methyl Red Test 29
3.3.6. Voges-Proskaeur Test 30
3.3.7. Indole Test 30
3.3.8. Citrate Test 30
3.3.9. Oxidase Test 30
3.4.1. Determination of Microbicidal Activity of Each
Disinfectant
by subjecting it to
Bactericidal Test 31
3.4.2. Paper Disc Diffusion Method 31
3.4.3. Impregnation of the Discs 31
3.4.4. Inoculation of Impregnated Disc 31
3.5.0. Determination of the Disinfectant Concentration
that is Bactericidal
(Using Kirby Bauer
Diffusion Assay Well Method) 32
3.6. Showing Patterns of the Antimicrobial Efficacies
of Varying 32
Concentration of the
Disinfectants on the Test Organisms
3.7. Determination of Susceptibility of the Test Gram
Positive
and Gram Negative Bacteria
to the Test Disinfectant 32
CHAPTER FOUR
RESULTS
4.1.
Results on Confirmation of Bacteria Isolates 33
4.2. The Microbicidal Activity
of the Disinfectants. (Using Disc Diffusion Method) 33
4.3. Disinfectant
Concentrations that appeared to be Bactericidal
(Using Kirby Bauer Diffusion Assay Well
Method) 34
4.4. Efficacies of Varying
Concentrations of Test Disinfectants
on the Test Organisms ` 34
4.5. Susceptibility of the
test Gram Positive and Gram Negative bacteria
to the experimental
disinfectants 35
CHAPTER FIVE
DISCUSSION,
CONCLUSION AND RECOMMENDATION
5.1. Discussion 44
5.2. Conclusion 45
5.3. Recommendation 45
REFERENCES 46
LIST OF TABLES
Table Title Page
4.1. Confirmation of
Bacterial Isolates
37
LIST OF FIGURES
Figure Title Page
4.1.1. Susceptibility
pattern of Staphylococcus aureus to
disinfectant A (ethanol),
(using the Disc Diffusion Method) 38
4.1.2. Susceptibility pattern of Staphylococcus aureus to disinfectant B (bleach),
(using the Disc Diffusion Method 38
4.1.3. Susceptibility pattern of Staphylococcus aureus to disinfectant C (phenolics),
(using the Disc Diffusion Method) 39
4.1.4. Susceptibility pattern of Pseudomonas aeruginosa to disinfectant A (ethanol),
(using the Disc Diffusion Method) 39
4.1.5. Susceptibility pattern of Pseudomonas aeruginosa to disinfectant B (bleach),
(using the Disc Diffusion Method) 40
4.1.6. Susceptibility pattern of Pseudomonas aeruginosa to disinfectant C (phenolics),
(using the Disc Diffusion method) 40
4.2.1. Susceptibility pattern of Staphylococcus aureus to disinfectant A (ethanol),
(using Kirby Bauer Diffusion Assay Well method) 41
4.2.2. Susceptibility pattern of Staphylococcus aureus to disinfectant B (bleach),
(using Kirby Bauer Diffusion Assay Well method) 41
4.2.3. Susceptibility pattern of Staphylococcus aureus to disinfectant C (phenolics),
(using Kirby Bauer Diffusion Assay Well method) 42
4.2.4. Susceptibility pattern of Pseudomonas aeruginosa to disinfectant A (ethanol),
(using Kirby Bauer Diffusion Assay Well method) 42
4.2.5. Susceptibility pattern of Pseudomonas aeruginosa to disinfectant B (bleach),
(using Kirby Bauer Diffusion Assay Well method) 43
4.2.6.
Susceptibility pattern of Pseudomonas
aeruginosa to disinfectant C (phenolics),
(using Kirby Bauer Diffusion Assay Well method) 43
CHAPTER ONE
INTRODUCTION
Microorganisms are minute living things that are
individually too small to be seen with the unaided eyes (Tortora et al., 2007). Though 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
usually harmless but pose a threat to the sick and the 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 only began about 100 years ago. It was Pasteur's work on
microorganisms that led scientists to beliefs that microbes were a possible
cause of diseases and needs to be eliminated or destroyed. Some examples of
these microbes are: Bacteria, fungi, viruses and protozoa etc., (Tortora et al., 2007).
In the mid 1800s, the Hungarian physician Igaz
Semmeliveis and English physician Joseph Lister used these thoughts to develop
some of the first microbial control practices for medical procedures. These
practices include hand washing with microbicidal chloride of lime and use of
techniques such as 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 that
control microbial growth. Contract 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 substance (Bhatia and Icchpujani,
2008).
Bacteria are major causes of disease and even human
death. A disinfectant is one of the diverse group of chemicals which reduces
the number of microorganisms present (normally on an inanimate object). There
are various official definitions of of the process of disinfection and
disinfectant agents. A disinfectant 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). Disinfectants can be mainly divided into five agents: alkylating,
sulfhydry combining, oxidizing, dehydrating and permeable. The most commonly used
disinfectants 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 acting on biological material that is
present on any surface. Their effects are by oxidizing the cell of the
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 surface, 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 and
cell lysis (Larson and Morton, 1991). Another surface disinfectant is the
compound that contains phenol group, a popular commercial brand of Isol, (a
saponated brand of cresol) as Phenolics are intermediate level disinfectants
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 the comparison of disinfectant efficiency and Ethanol
and Bleach are believed to have immediate effect against most organisms (Carly et al., 2006). In this study,
disinfectant experiment was conducted using different concentrations of
laboratory Ethanol, household Bleach (hypo) and Phenolics against Staphylococcus aureus and Pseudomonas aeruginosa.
Staphylococcus aureus is a Gram positive cocci
that occurs in 40%-50% of humans, with hospitalized patients as well as medical
and paramedical staff showing higher incidence of carriage (Bhatia and
Icchpujani, 2008). Pseudomonas aeruginosa
however, is a classical Gram negative opportunistic pathogen with innate
resistant to many antibiotics and disinfectants. It is invasive, toxigenic and
produces infection in patients with abnormal host deficiencies (Stephen et al., 2004).
1.1. AIMS
AND OBJECTIVES OF THIS RESEARCH
1.
To confirm the bacterial cultures that will be used in the
study.
2.
To determine the microbicidal activity of each disinfectant,
by subjecting it to bactericidal tests.
3.
To determine the concentration of disinfectant that is
bactericidal.
4.
To show patterns of the antimicrobial efficacies of varying
concentration of the disinfectant on the test organisms.
5.
To determine the susceptibility of the test Gram positive Staphylococcus aureus and Gram negative Pseudomonas aeruginosa to the test
disinfectants.
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