DISINFECTING ACTIONS OF ETHANOL, BLEACH (SODIUM HYPOCHLORITE) AND PHENOLICS (ISOL) ON SOME BACTERIAL SPECIES

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 ﾿