TABLE OF CONTENTS
TABLE OF CONTENTS
LIST OF TABLE AND FIGURES
CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW1
1.2 LITERATURE REVIEW
1.2.1: The skin and the normal skin microbiota
1.2.2: Burden, prevalence and epidemiology of Staphylococcus aureus infections
1.2.3: Clinical manifestations and pathogenesis of Staphylococcus aureus Infections
1.2.4: Resistance of Staphylococcus aureus to antibacterial agents
1.2.5: Prevention, control and treatment of Staphylococcus aureus infections
1.2.6: Medical relevance of medicated (antibacterial) soap
1.3 STATEMENT OF THE PROBLEM
1.5.1: General objectives
1.5.2: Specific objectives
1.6 LIMITATIONS OF STUDY
CHAPTER 2: MATERIALS AND METHODS
2.1.1: Materials and instruments
2.1.2: Preparation of 70% alcohol
2.1.3: Preparation of soap solutions
2.1.4: Preparation of soap impregnated discs
2.1.5: Preparation of McFarland standard
2.1.6: Media preparation.
184.108.40.206: Preparation of Mannitol salt agar
220.127.116.11: Preparation of Muller Hinton agar
2.2.1: Sample collection and isolation of microorganisms
2.2.2: Identification and confirmation of isolates
2.2.3: Antimicrobial susceptibility testing
CHAPTER 3: RESULTS
3.1: Prevalence of Staphylococcus aureus on skin of subjects
3.2: Antimicrobial patterns
CHAPTER 4: DISCUSSION
LIST OF TABLES AND FIGURES
Table 1: colonial morphology of test microorganism
Table 2: S. aureus distribution on the different parts of the skin
Table 3: Zone diameter of inhibition (mm)
Figure 1: Histogram relating the zone diameters at different dilutions of medicated soaps
Table 4: Diameter of zone of inhibition for positive control (antibiotics discs) and negative control (physiological saline solution)
Table 5: ANOVA table verifying significance for zone diameter of inhibition
Figure 2: Coagulase positive slide test showing coagulation, for the identification of Staphylococcus aureus
Figure 3: 0.5 McFarland standard used for adjusting the turbidity of bacterial suspension
Figure 4: Catalase test positive result for the identification of Staphylococci
Figure 5: Yellow, circular colonies fermenting mannitol by Staphylococcus aureus on mannitol salt agar
Figure 6: Muller Hinton agar plates in which Antimicrobial susceptibility testing was done
I wish to express my sincere gratitudes to my supervisor Prof. Roland Ndip Ndip for giving me the opportunity to work with him, for accepting the topic and for the scientific supervision of this work. I equally thank; Prof. Samuel Wandji and all lecturers and Staff of Department of Microbiology and Parasitology, Faculty of Science, University of Buea (UB) for the skills they have imparted in me for the past three academic years, permitting me to do this work. I also wish to thank Mr. Mbaabe Felix for technical assistance.
I will be forever grateful to my parents, Mr. Akaolisa Joel and Mrs. Akaolisa Maryann for all they had to endure to provide the finances for my studies, as well as for all the pieces of advice and immense contribution they chipped in to steer my imagination in the right direction. A million thanks to my siblings, Laura, Verine, MacDonald and Treasure as well as to every other member of the family and also friends like Pelagie, Julius, Luke and Ewane who put in one thing or another to assist me in this quest.
And above all, I give all the glory to God Almighty for seeing me through.
Following invasion of the human body by bacteria, most bacteria produce substances that are toxic to the human body. In the fight against these infections, the production of antibacterial substances (synthetic compounds of different forms) has been a major step in solving this problem. In this study, three medicated soaps: Dettol, Pharmapur and Tetmosol were investigated for their antibacterial activity at different concentrations against Staphylococcus aureus using the disc diffusion method. Saline was used as a negative control and a readymade antibiotic impregnated disc (Ciprofloxacin) was used as a positive control. Bacterial identification was by standard microbiological techniques which included: Colonial examination, Gram staining and biochemical testing. Dettol medicated soap had the highest antibacterial activity (26mm at 1/10 soap dilution) while Tetmosol showed the least antibacterial activity (6mm at 1/103 soap dilution). S. aureus was found on 4 of the 6 samples that were analysed giving a prevalence of 66.67%. All the medicated soaps showed antibacterial activity which depended on the concentration (dilution) of soap sample on S. aureus; hence, the use of antibacterial soaps is recommended as a means of reducing risks of transmission and infection by bacteria.
1.0 INTRODUCTION AND LITERATURE REVIEW
The human skin makes contact with thousands of microbes that are pathogenic to man. When proper hygiene is not practiced, these microbes replicate, produce toxic substances and eventually cause disease. Some of these microbes cause diseases on the skin while others become pathogenic only when they migrate to mucous membranes and other vital organs.
Staphylococcus aureus has been known to be one of the many dangerous and versatile pathogens with high death rates in humans as a consequence of community-acquired and hospital-acquired infections. It has shown little change in mortality rates and a steadily increasing morbidity. S. aureus is a member of the Micrococcaceae family; a group of pyogenic cocci known to cause various suppurative or pus forming diseases in humans and other animals. It is a non-motile and non-spore forming facultative anaerobic organism that grows by aerobic respiration or fermentation. On microscopical examination, the organisms appear as Gram-positive cocci in clusters. The distinction of S. aureus from other staphylococcal species is very important in clinical microbiology laboratories because this pathogen serves as an important source of nosocomial infections; this is done on the basis of the yellow pigmentation of colonies and positive results of coagulase, mannitol fermentation and deoxyribonuclease test (Lowy, 1998; Murray et al, 2003). S. aureus ’ ability to synthesize the enzyme coagulase makes it the most pathogenic of the Staphylococci. Prevention and treatment of infections caused by this pathogen is very important to public health especially with the emergence of multi drug resistant strains which has led to the search for new antibiotics or the modification of existing ones (Lyon and Skurray, 1987).
Soaps play an important role in the prevention and treatment of infections because they have the ability to remove and kill pathogens and this ability is well exploited in skin hygiene (Riaz et al, 2009). Skin hygiene, particularly of hands, is considered one of the primary mechanisms to reduce risk of transmission of infectious agents by both the contact and faecal-oral routes. The washing of hands with soaps and water is a routine practice which was established many generations before now as a means to ensure personal hygiene and over the decades, bathing, scrubbing and washing traditions have become established within the health care setting (Larson, 1999; Bhat et al, 2011). The importance of hand washing with soap is more crucial when it is associated to health care workers because of possible cross contamination of bacteria and other pathogens that may be pathogenic or opportunistic (Richards et al, 1999). Soaps, detergents and hand sanitizers used for hygiene and sanitation are expected to effectively remove infectious agents. It is for this purpose that medicated soaps which possess chemical agents/compounds that are antimicrobial are produced. These soaps produced by different manufacturers have different antibacterial strengths which is due to difference in the concentration and/or antimicrobial content of the chemicals/compounds found in them. The analysis of the antimicrobial activity of these medicated soaps is therefore necessary as a major step to maintaining proper hygiene in our communities.
1.2 LITERATURE REVIEW
1.2.1 The skin and the normal skin microbiota
The 1.8m2 human skin has folds, invaginations and specialized niches. This ecosystem inhabits diverse groups of microorganism providing them with shelter and nutrients (Grice and serge, 2011). It is part of the human innate immune system and its primary role is to serve as a physical barrier, protecting our bodies from potential assault by foreign organisms or toxic substances. It is the first line of defense against invasion by infectious agents. The epithelial surfaces of the skin are very impermeable to most infectious agents and desquamation of the skin epithelium also help to remove bacteria and other infectious agents that have adhered to epithelial surfaces thus protecting the human body. However, when the skin is breached, disease can occur due to infection. The skin is also an interface with the outer environment and, as such, is colonized by a diverse collection of microorganisms which may be symbiotes, commensals or parasites. These include; bacteria, fungi and viruses as well as mites. Many of these microorganisms are harmless (symbiotes and commensals) and the symbiotes may in some cases provide vital functions that the human genome has not evolved. Symbiotic microorganisms occupy a wide range of skin niches and protect against invasion by more pathogenic or harmful organisms by competing with them for nutrients and adhesions proteins on the skin or produce substances toxic to them and may also play a role in educating the billions of T cells that are found in the skin, priming them to respond to similarly marked pathogenic cousins (Grice and Serge, 2011). These microorganisms can be categorized either as “resident flora” or contaminants (“transient flora”) (Rotter, 1966). According to Nobel and Somerville (1974), the normal flora (“resident flora”) of the skin composed primarily of Gram-positive cocci and diphtheroids which may represent a selective barrier against proliferation of potentially pathogenic organisms. In some individuals, small numbers of Gram-negative organisms or yeast may also comprise this normal flora, but their proliferation may be influenced by the normal ecological balance (Aly and Maibach, 1976).
1.2.2 Burden, prevalence and Epidemiology of Staphylococcus aureus infections
The diversity of bacteria is seen in their presence and wide distribution all over the surface of the earth. They are found in soil, water, sewage, standing water as free-living organisms and can become pathogenic upon contact with the human body. (Bhat et al, 2011; Johnson et al, 2002). Infection by a bacterium does not necessarily result in a disease state. Disease occurs when (1) The bolus of infection is high, (2) when the immune system is compromised, (3) when the virulence of the invading organism is great. Transient bacteria are known to be the cause of most bacterial infections. Bacterial infectious diseases represent an important cause of mortality and morbidity worldwide. The rates of diseases resulting in deaths is even worse in developing countries where sanitary conditions are poor, health care services are limited and worse-still few people can afford the health care services presented to them.
Staphylococcus aureus bacteraemia (SAB) represents a significant burden on health care systems. According to an analysis of a large US database, SAB was associated with a longer median duration of hospital stay, higher median total treatment cost, and greater risk of mortality, compared with bacteraemia caused by any other pathogen (Shorr et al; 2006). In U.S hospitals in the national nosocomial infections surveillance system, S. aureus accounted for up to 13% of isolates recovered from patients with nosocomial infections from 1979 through 1995 and the percentage has increased in recent years. Community-acquired infections with S. aureus are also common (Eiff et al, 2001). S. aureus is the most frequent bacteria pathogen among clinical isolates from hospital in-patients in the United States and is the second most prevalent bacterial pathogen among clinical isolates from out-patients. According to the SENTRY Antimicrobial surveillance program, which examined more than 81,000 isolates during the period of 1997-2002, S. aureus was the most common cause of nosocomial bacteraemia in North America (prevalence, 26.0%) and Latin America (prevalence, 21.6%) and the second most common cause of bacteraemia in Europe (prevalence, 19.5%). The incidence of nosocomial MRSA infections has also greatly increased in recent years in the Unites states and Europe (Naber, 2009).
1.2.3 Clinical manifestations and pathogenesis of Staphylococcus aureus
The versatility of S. aureus is seen in a variety of clinical conditions which include: septicaemia, pneumonia, wound sepsis, septic abortion, osteomyelitis, septic arthritis, post-surgical infections and toxic shock syndrome (Nkwelang et al, 2009). These results in high rates of morbidity and mortality in humans worldwide (Murray et al, 2004).
Approximately 30% of healthy individuals can asymptomatically carry S. aureus on mucous membranes for weeks or months but is only transiently carried on the intact skin. Five stages are seen in the pathogenesis of S. aureus. These include: (1) colonization, (2) local infection, (3) systemic dissemination and/or sepsis, (4) metastatic infection, and (5) toxinosis. Once in blood, the organism spreads widely to peripheral sites in distant organs and septic shock can occur. Without specific therapy, the mortality rate resulting from spread of infection is high. Specific infections result from haematogenous dissemination, these include: endocarditis, osteomyelitis, renal carbuncle, septic arthritis, or epidural abscess. Even when the organism itself does not invade the bloodstream, specific syndromes such as toxic shock syndrome, scalded skin syndrome and food borne gastroenteritis result from the local or systemic effects of specific toxins (Archer, 1998).
1.2.4 Resistance of Staphylococcus aureus to antibacterial agents.
The excessive use of antibiotics has led to resistance. Resistance to antibiotics in most cases, is coded for by gene carried on plasmids, accounting for the rapid spread of resistant bacteria (Harris et al, 2002). S. aureus like many other human pathogens has evolved resistance to commonly used antimicrobial agents. Multidrug-resistant S. aureus strains have been reported all over the world with increasing frequencies. These include isolates that are resistant to methicillin, lincosamides, macrolides, aminoglycosides, fluoroquinolones or a combinations of these antibiotics. Methicillin resistant strains of S. aureus (MRSA) , have emerged intermediate resistance to glycopeptides which are the main drugs with reliable activity against MRSA (Eiff et al, 2001). This is a major problem to the pharmaceutical industry and has led to the search for new antimicrobials or the modification of existing antimicrobials to increase their spectra of activity.
Methicillin resistance is due to the acquisition of a new penicillin-binding protein, PBP2a. This protein has a low affinity for most β -lactam antibiotics and, therefore, mediates cross resistance to all these compounds. This high level of resistance not only impedes successful therapy for infections but also allows the organisms to persist in the hospital, expanding its reservoir. (Archer, 1998).
1.2.5 Prevention, control and treatment of Staphylococcus aureus infections.
Person to person contact transmission is a mode of transmission of infections by certain pathogens. Cleaning of hands immediately after using the toilet, treatment of suspected cases and even regular cleaning and disinfection of hands to eliminate bacteria picked up by exchanging greetings, touching money and even contact with tables in classrooms and the working surfaces of offices are a few of the many methods used to prevent and control infections by pathogens transmitted by this route. S. aureus is transmitted by this route and successful transmission can lead to skin infections like abscesses, scalded skin syndrome and even food borne gastroenteritis. According to Kimel (1996), scrubbing the body or hands particularly with soaps is the first defense against bacteria and other pathogens that cause such infections hence these methods can be implemented in the control and prevention of S. aureus infections (Kimel, 1996).
Antimicrobial agents found in some medicated soaps have the ability to destroy some pathogens like most Staphylococci and methicillin resistant S. aureus (MRSA). These agents include: Triclosan, chloroxylenol and Trichlorocarbanilide. Chemotherapy for S. aureus is becoming increasingly difficult. Some antibiotics like ampicillin, erythromycin and tetracycline were used for treatment of Staphylococcal infections but with the emergence of multi-drug resistant strains, the use of these drugs for treatment diminishes. The pharmaceutical industry is responding either by modifying existing compounds to broaden their spectra or by developing novel compounds. The result of this response includes oxazolidinones and a combination drug consisting of semisynthetic derivatives of streptogramin A (dalfropristin) with streptogamin B (quinipristin). Vancomycin is often the only effective agent available for therapy especially for the treatment of MRSA infections, but its extensive use may help to promote colonization and infection with Vancomycin-resistant enterococci. Attempts to find other unique compounds that may attack novel or new bacterial targets are also on the way (Archer, 1998; Anstead et al, 2013).
1.2.6 Medical relevance of medicated (antibacterial) soap.
The medical importance of medicated soaps depends on their ability to either kill or inhibit the growth of microorganisms. Some medicated soaps have a broad spectrum of antimicrobial activity on all types of microorganisms (bacteria, viruses, and fungi etc.) while other medicated soaps have activity against a limited group of microorganisms (narrow spectrum of activity). For a soap to be effectively used for the medical purposes, it should be able to ensure protection against a wide range of microorganisms. Soaps are water-soluble or insoluble cleansing agents made from animal and vegetable fats, oils and greases; or chemically, the sodium or potassium salt of a fatty acid formed by the saponification interaction of fats and oils with alkali. Based on the formulation and functional chemical constituents, soaps could be categorized as toilet soaps, antiseptic soaps or medicated soaps. The antiseptic and medicated soaps are made to fight pathogenic microbes and other germs due to special chemical additives in them while the toilet ones are made for conventional cleaning purposes (Ogunnowo et al, 2010).
Antibacterial soaps can remove 65 to 85% of bacteria from the human skin with Triclosan, Trichlorocarbanilide, and P-Chloro-in-xylenol (PCMX/Xylenol) being commonly used antibacterials in medicated soaps which elicit this effect (Osborne and Grube, 1982; Larson et al, 1989).
Although many people consider that an antimicrobial portion of soaps is effective at preventing communicable disease, recent studies have proven that too much of it can have an opposite effect spreading disease/infection instead of preventing them (Poole, 2002). According to Bettley (1960), some antibacterial soaps can cause nummular eczema, eczematous dermatitis, and eczematous eruption (Bettley, 1960).
1.3 STATEMENT OF THE PROBLEM
Bacterial infection is a public health problem (Johnson et al, 2002). The best method for killing or inhibiting their growth is of great importance to man; as such confirmation of the efficient antibacterial activity of Dettol, Pharmapur and Tetmosol which are commonly used medicated soaps is a milestone to achieving this goal.
Medicated soaps have different antibacterial strengths.
1.5.1 General objectives
- To investigate the antibacterial effect of three different brands or types of medicated soaps (Dettol, Pharmapur and Tetmososol) on Staphylococcus aureus.
1.5.2 Specific objectives
- To determine the prevalence of Staphylococcus aureus from the skin.
- To determine the antimicrobial susceptibility pattern of different concentrations of soap on Staphylococcus aureus.
1.6 LIMITATIONS OF STUDY
This study was limited by a number of factors; these included:
Ø Absence of an oven for sterilizing and drying of the filter paper discs before inoculation following storage. To reduce this, discs were dried in sterile (Autoclaved), well labelled glass petri dishes.
Ø The working laboratory was available for short periods of time. Autoclaved filter papers which were supposed to be dipped in soap solution for an hour to ensure complete saturation of discs with soaps solutions. This was mitigated by dipping the filter papers for 25mins.
- Absence of a Bunsen burner (flame) for the practice of aseptic techniques. This was mitigated by using an Alcohol lamp.
- The study was limited to S. aureus which is just one of the many pathogens of concern to Public health.
2.0 MATERIALS AND METHODS
This study was carried in the Life Science Laboratory of the Faculty of Science, University of Buea, Cameroon.
2.1.1 Materials and instruments
Mannitol salt agar (MSA), Muller Hinton agar (MHA), Medicated soap solutions (Dettol, Pharmapur, Tetmosol), Petri dishes, 70% alcohol, 95% alcohol Gloves, cotton, Aluminium foil, Swab sticks, Test tubes, Alcohol lamp, McCartney bottles, Forceps, Graduated Syringe, Micropipette, pipette tips, electronic balance, 0.5 McFarland standard, Saline (0.85% NaCl), beaker, conical flask, inoculating loop, spreader, ruler, sterile blade, filter papers, medicated soap impregnated discs, Antibiotic discs, Safranin, Crystal violet, iodine, H2O2, plasma, distilled water.
2.1.2 Preparation of 70% alcohol
70% alcohol was made by diluting 95% alcohol with water.
Volume (V1) of 95% alcohol to be diluted= 80mL
Volume (V2) of water needed=?
Concentration= [from 95% (C1) to 70% (C2)]
Therefore, for 108.57-80=28.57 hence, 28.57mL of water was added to 80mL of 95% alcohol to give 70% alcohol.
2.1.3 Preparation of soap solutions
One hundred millilitres of water was autoclaved in a bottle and allowed to cool to room temperature. Using an electronic balance and a sterile blade, 1g of soap was scraped from the tablet of each soap (Dettol, Pharmapur, Tetmosol). Using a sterile graduated syringe, 9mL each of sterile water was put in 9 McCartney bottles (3 for the 1st dilution, the next 3 for the 2nd dilution and the last 3 for the 3rd dilution). One gram of each soap was then put in each of the 3 bottles for the 1st dilution, capped with the lids and then labelled. After it had dissolved, serial dilution was then done for the 2nd and 3rd dilution by transferring 1mL soap solution; resulting in different concentrations of each soap solution ranging from 111.1mg/ml to 1.37mg/ml (Obi, 2014).
2.1.4 Preparation of soap impregnated discs
Using a disc borer, a filter paper was bored into 8mm discs and transferred into a closed bottle, then autoclaved. Using sterile forceps, the discs were then immersed into the soap solutions for 25mins after which they were removed and placed in well labelled sterile glass petri dishes and allowed to dry at room temperature.
2.1.5 Preparation of McFarland standard
McFarland standard (0.5) was prepared by using 1% w/v barium chloride (BaCl2) and 1.175% v/v sulphuric acid (H2SO4). 0.5mL of 1% w/v BaCl2 was added to 99.5mL of 1.175% v/v H2SO4 and mixed.
2.1.6 Media preparation
Assuming that each petri dish should contain 20mL of medium, the media were prepared according to the manufacturer’s instructions. 12 plates of Mannitol salt agar and 3 plates of Muller Hinton agar were needed for the study.
18.104.22.168 Preparation of Mannitol salt agar (MSA)
111g → 1000mL
Xg → 240mL
Mannitol salt agar (26.64g) was measured using an electronic balance and placed in 240mL of water in a beaker and then heated to dissolve completely. The beaker was then covered and the medium was autoclaved at 121Abbildung in dieser Leseprobe nicht enthalten for 15mins. It was then removed from the autoclave and allowed to cool after which approximately equal amounts were aseptically poured into the 12 petri dishes and allowed at room temperature to solidify. The media-containing petri dishes were then stored in the refrigerator at 4Abbildung in dieser Leseprobe nicht enthalten until used.
22.214.171.124 Preparation of Muller Hinton agar
38g → 1000mL
Xg → 60mL
Muller Hinton agar (2.28g) was measured using an electronic balance and placed in 60mL of water in a beaker and then heated to dissolve completely. The beaker was then covered and the medium was autoclaved at 121Abbildung in dieser Leseprobe nicht enthalten for 15mins. It was then removed from the autoclave and allowed to cool after which equal amounts were aseptically poured into the 3 petri dishes and allowed at room temperature to solidify. The media-containing petri dishes were then stored in the refrigerator at 4Abbildung in dieser Leseprobe nicht enthalten until used.
2.2.1 Sample collection and isolation of microorganisms
The medicated soap samples were purchased from a pharmacy in Buea. Using sterile swab sticks, samples were collected from the anterior nares of the nose, hand and the armpit of 2 human subjects. Six Mannitol salt agar plates were removed from the refrigerator allowed to cool to room temperature. The swab sticks containing the samples were then streaked (streak plate technique) on the plates and labelled. The plates were then incubated at 37Abbildung in dieser Leseprobe nicht enthalten for 24hrs. After 24hrs suspected colonies were sub-cultured on the remaining 6 mannitol salt agar plates to obtain pure cultures.
2.2.2 Identification and confirmation of isolates
This was done for each of the 6 samples by Gram staining followed by examination under the microscope (oil immersion). Further confirmation was by biochemical testing using catalase and coagulase (Cheesbrough, 2005).
Gram staining: A colony was picked with a sterile (flamed) inoculating loop and emulsified on a slide to make a smear. It was then air dried and heat fixed by passing over the flame. The slide was then floated with 5 drops of crystal violet and allowed for 1min and then washed off. Five drops of iodine was placed on the slide and allowed for 1min and then washed. Next it was decolorized by tilting in alcohol for 5secs. After these, 5 drops of safranin were placed on the slide and then washed off gently. The slide was allowed to dry and examined under the microscope (x1000).
Catalase test: Catalase test was done by putting 2ml of H2O2 in a test tube then, a colony was placed in it to observe for effervescence.
Coagulase test: Coagulase test was done by collecting 5ml of venous blood and placed in an EDTA tube and then centrifuged to obtain plasma. A drop of distilled water was dropped on a slide using a dropper and using a flamed wire loop, a colony of the organism that has been checked for positive results of Gram stain was emulsified to make a thick suspension. A loopful of plasma was then added to the suspension and mixed gently (Cheesbrough, 2005).
2.2.3 Antimicrobial susceptibility testing
This was done using the Kirby-Bauer disc diffusion technique (Cheesbrough, 2005).The working bench was sterilized with 70% alcohol. Three Muller Hinton agar (MHA) plates that were stored in the refrigerator were removed and placed on the bench for their temperature to adjust to room temperature. Eight millimetre discs were placed in a little amount of saline and allowed for 25mins (This served as the negative control discs).
Four millilitres of saline solution was measured accurately and transferred into a sterile McCartney bottle. An inoculating loop was then flamed and when it had cooled was used to pick colonies of then test organism (Staphylococcus aureus) and transferred into the bottle containing saline solution. This mixture was then adjusted to the turbidity of McFarland standard. This produced a bacterial suspension of approximately same turbidity with that of 0.5 McFarland standard. The 3 plates of MHA were then labelled 10-1, 10-2 and 10-3 for the 1st, 2nd and 3rd dilutions respectively. Using a micropipette, 100µL of the bacterial suspension was then transferred into each of the agar plates aseptically and a sterile spreader was used to spread suspension. The plates were inverted and allowed for 5mins. Using sterile forceps, the discs impregnated with different medicated soaps were then placed at different positions in the appropriate plate (discs were placed in the plate labelled with the concentration of soap found in them) 25mm apart from each other and 15mm away from the petri dish wall. The antibiotic control discs (Ciprofloxacin) that severed as positive control discs was placed at the center of the 3 different discs and the saline impregnated discs were placed in each plate. The plates were then incubated in inverted positions at 37Abbildung in dieser Leseprobe nicht enthalten for 24hrs. Results were then obtained in millimetres (mm). Statistical analysis was then done using Analysis of variance (ANOVA).