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

Certification ii

Dedication iii

Acknowledgements iv

Table of Contents List of Tables List of Figures x

Abstract

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

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

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