Comparative Study Of Disinfectant Efficiency Of Ethanol, Bleach And Phenolics Against Pseudomonas Aeruginosa And Staphylococcus Aureus

Project and Seminar Material for Microbiology

Comparative Study Of Disinfectant Efficiency Of Ethanol, Bleach And Phenolics Against Pseudomonas Aeruginosa And Staphylococcus Aureus


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.

Differences 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.

Table Of Contents

Preliminary Page(s)

  • Title Page
  • Certification
  • Dedication
  • Acknowledgements
  • Table of Contents
  • List of Tables
  • List of Figures
  • List of Plates
  • Abstract

Chapter One

  • 1.0 Introduction
  • 1.1 Aims and Objectives

Chapter Two

  • 2.0 Literature Review
  • 2.1 History of Disinfectants
  • 2.2 About Disinfectants
  • 2.3 Sources of Contamination of Surfaces
  • 2.4 Types of Disinfectants
  • 2.5 Properties of A Disinfectant
  • 2.6 General Features of Disinfectant
  • 2.7 General Features of the Test Organisms
  • 2.9 Mechanism of Actions of Disinfectants against Bacteria
  • 2.10 Resistant Action of Bacteria
  • 2.11 Advantages and Disadvantages of Disinfectants
  • 2.12 General Guidelines in the Use of Disinfectants

Chapter Three

  • 3.0 Material and Method
  • 3.1 Isolation of Bacteria
  • 3.2 Identification of Isolates
  • 3.3 Preparation of Disinfectants
  • 3.4 Antimicrobial Susceptibility Testing (Using Kirby Bauer Diffusion Assay Well Method)

Chapter Four

  • 4.0 Results

Chapter Five

  • 5.0 Discussion
  • 5.1 Recommendations
  • 5.2 Conclusion
  • References
  • Appendix 1
  • Appendix II
  • Appendix III

List Of Tables

  • Table 1: Results of diameter of zone of inhibition of Ethanol, Phenolics and Bleach for Staphylococcus aureus
  • Table 2: Results of Diameter of Zone Inhibition of Ethanol, Phenolics, Bleach for Pseudomonas aeruginosa
  • Table 3: Pseudomonas aeruginosa response to Ethanol, Phenolics and Bleach
  • Table 4: Staphylococcus aureus response to Ethanol, Phenolics and Bleach

List Of Figures


  • Fig 1: Pseudomonas aeruginosa disinfectants A test result
  • Fig 2: Pseudomonas aeruginosa disinfectants B test result
  • Fig 3: Pseudomonas aeruginosa disinfectants C test result
  • Fig 4: Staphylococcus aureus disinfectants A test result
  • Fig 5: Staphylococcus aureus disinfectants B test result
  • Fig 6: Staphylococcus aureus disinfectants C test result


  • Fig 7: Disinfectant A on Pseudomonas aeruginosa
  • Fig 8: Disinfectant B on Pseudomonas aeruginosa
  • Fig 9: Disinfectant C on Pseudomonas aeruginosa
  • Fig 10: Disinfectant A on Staphylococcus aureus
  • Fig 11: Disinfectant B on Staphylococcus aureus
  • Fig 12: Disinfectant C on Staphylococcus aureus

List Of Plates

  • 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

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 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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