EFFICACY OF NEWLY EMERGING DETERGENTS AGAINST BACTERIA AND MOLDS ISOLATED FROM HOME KITCHEN SURFACES AND FLOORS Fredrick Omondi Odongo1* Sharon Tanui2, Miriam Jumba3, Oludare Temitope Osuntokun4 1&3 Faculty of Biological and physical Science, Department of Microbiology and Biotechnology University of Nairobi, P.O.Box.30197-00100, Nairobi, Kenya 2 Faculty of Health Science, Department of Nursing, University of Nairobi, P.O.Box.30197-00100, Nairobi, Kenya Department of Microbiology, 4 Faculty of Science, Adekunle Ajasin University, Akungba-Akoko, Ondo state Nigeria ABSTRACT In this study the efficacy of various newly emerging detergents used in these kitchens which are purported to clean the kitchen surfaces and other kitchen equipment was investigated. Reflecting the increase in infections by pathogens that emerged in recent times, the effectiveness of measures for preventing cross-contamination should be continuously evaluated. Hygienic codes that have been developed need also to be continuously updated and justified if necessary, to minimize the bacterial transmission in the domestic environment by any newly identified or previously known pathogen. Any effective measures to control or reduce the microorganism in the home would reduce the public health concerns related to their exposure. Microorganisms are ubiquitous in nature. One might think that one is safe from these microorganisms at home, but they can be found almost everywhere especially in areas where food is prepared. It is common practice to clean food and food products prior to consumption. However, it is equally important to sanitize the places where food is stored and prepared and also to sanitize the locations and items commonly used within a typical house. As we embark on our day to day lives eating is a normal occurrence in our lives as it gives us strength and good health. A part of our existence, we usually fall sick and to prevent that hygiene has become a major concentration of our lives. In this same effort we take great care so that the food we eat is hygienic. The emerging detergents on the other hand were considered as most of the time they are the same ones that are used to clean these kitchen surfaces. Kitchen swabs were collected from two kitchens and the bacteria isolated through plating on nutrient agar plates. Three samples of detergents were also considered. Whole concentrate of the detergents were used to see if any of them had antimicrobial properties. Sterilized discs were dipped into the detergents and seeded with the bacteria during plating. After incubation, the detergents found with antimicrobial properties were serial diluted to get the most effective concentration of the detergent. Clorox floral detergent was found to be most effective with a minimum inhibitory concentration (MIC) of 10-3. Bio-clean was found to be the least effective with a minimum inhibitory concentration of 10-1. Gram positive bacteria were the more inhibited than Gram negative bacteria as indicated by the mean inhibition zones of 20.67 and 18.33 respectively for the three tested detergents. Bacteriawere the most inhibited than mold however the difference in zone of inhibition is minimal. This is shown from the control done with a bacterium E. coli with an inhibition zone of 15 mm as compared to mold species having a zone of inhibition of 13 mm. The results obtained further showed that; isolates from poorly unhygienic or rather poor background kitchen surfaces had many growth and many bacteria obtained from the cultured plates. However isolates from hygienic clean environment had less growth and few bacteria isolated. Keywords: Emerging Detergents, Home Kitchen Surfaces 1.0 INTRODUCTION Kitchen is probably the most crucial area that harbors and transmits infection in most of our home settings. Microbes are prevalent everywhere in the kitchen, in sponges, countertops, cutting boards, kitchen utensils, refrigerators, towels and even stove tops. Growth of undesirable contaminating bacteria not only causes deterioration in the sensory and organoleptic properties of food but can also cause illnesses. Most pathogenic microorganisms in food products are intestinal in origin, however some are found in nasal passages, in throat, on hair and on skin. Thus food handlers are often a main source of contamination and cross-contamination. Salmonella spp. and Campylobacter spp. are easily transferred from kitchen surfaces, utensils, hands and other food items (1). Kitchen towels and sponges have been greatly ignored and these have led to emergence of new detergents that have been proposed to be very effective for eliminating these microorganisms. The former had been found to harbor numerous microorganisms such as Camphylobacter species Bacillus cereus, Escherichia coli, Salmonella species, Staphylococcus aureus, yeast and moldswhich are responsible for causing food poisoning and infections that can lead to severe dysentery. (2) The focus on personal hygiene brought about a new definition of “clean”: Rather than being washed free of dirt and other substances, sites must be free of microorganisms. (Levy, 2001). In this study concerning kitchen hygiene and cleanliness it was found that cleaning with detergents resulted in reduction of bacterial populations on kitchen surface. In recent years, there has been increasing concern about indoor air quality in kitchen, living rooms and surrounding areas. The family home can sometimes become a source of microbial contamination, where molds and bacteria proliferate. Inadequate ventilation and high moisture levels resulting from water damage episode or excess humidity often cause the proliferation of molds and bacteria on visible surfaces or hidden inside structure (3). Early studies on bacterial contamination in the kitchen were conducted in the late 1960s investigating bacterial load of hand towels, kitchen surfaces and dishcloth towels. Such cloths were heavily contaminated with bacteria and suspected as one of the main vectors for spreading and dissemination of the bacteria in the kitchen (4). However, no clear studies have ever been carried out to investigate bacterial loads on some vectors found in some of our kitchen, the cockroaches. These insects appear to act as reservoirs that harbor and encourage the growth of potential pathogens since they crawl on the surface pick up food substances and bacteria on the kitchen surfaces. Also several kitchen sites particularly wet area and sink drain areas appear to act as reservoirs that harbor and encourage the growth of these pathogens. Although food particles are usually cleaned from the surface when good hygienic practices are carried, bacteria attached to these surfaces are not visible to the eye and may therefore be left to multiply and increase in numbers. If the bacteria attached are found on the kitchen surfaces preparing of food on the surface may lead to illness if food comes into contact with these surfaces. Several studies have shown that cross contamination from raw products via vectors and kitchen cloths or sponges and utensils to food not subjected to further cooking contributed to occurrence of outbreak of food borne Salmonellosis. (5). The risk of cross contamination during regular domestic cleaning is important since kitchens were found to be potential vehicles of pathogens in domestic kitchen (5) and pathogens were able to survive in kitchen surfaces and kitchen sponges for at least weeks. (6). 2.0 MATERIALS AND METHODOLOGY 2.1 SOURCES OF THE SAMPLES 2.2.1. SOURCES OF KITCHEN SURFACE SWABS For this study two kitchen swabs samples were obtained. The two swabs were from home kitchen areas. Sampling methods used tried to encompass a wide economic and social class level Sites of collection of the samples included: Common house hold kitchen swab samples. From two household; One from a less privileged house hold (Kangemi slum) and the other from a suburb (Westland home). Transportation of the samples from the point of collection to the laboratory was done by use of sterile containers which were autoclaved before use. 2.2.2. SOURCE OF DISINFECTANTS OR DETERGENTS USED In the selection of the disinfectants samples, the criteria used took into consideration the old disinfectants that are believed to have been there for over ten years and the newly emerging detergents and disinfectants that are recently taking over the market. For this study only the newly emerging detergents were selected for use since most probably the old ones had been subjected for the same study. The disinfectants used are found in the local Kenyan supermarkets. The disinfectants used in this study include: i. Clorox Floral Bleach ii. Pride Washing liquid iii. Bio-clean Lemon Fresh FIGURE 1: Detergents used for this study; pride washing liquid, Clorox and Bio-clean 2.3. MEDIA PREPARATION 2.3.1. NUTRIENT AGAR MEDIA To make the nutrient agar plates, 28 grams nutrient agar powder was first weighed using an electric weighing scale into a small weighing boat. After the agar powder was weighed, it was transferred into a conical flask containing 200ml of distilled water. The conical flask was then swirled while warming to ensure homogenous mixing of the nutrient agar powder with water. As the powder was being mixed with water, the 200ml of distilled water was topped up with another 800ml of distilled water. The mixture was then placed into an autoclave and autoclaved at a pressure of 15 atmospheres for 15 minutes at 1210C. After autoclaving, the agar was cooled to 550C or a temperature conducive enough for holding the flask but not below 450C before pouring into the Petri dishes(7). The agar is cooled; a surface was wiped with alcohol to sterilize the surface on which the Petri Dishes are placed. Approximately 15ml of the agar solution was poured into the bottom of the petri dishes. Each dish was cooled and upon solidification of the agar, the dishes were then wrapped and placed in the refrigerator for storage. 2.4. ISOLATION OF MICROORGANISMS FROM SAMPLES To obtain microorganisms from kitchen swabs; fill a small test tube partly full of sterilized water. Dipped bacteria laden swab into the water. This transferred some of bacteria collected into the water. To ensure maximum representation of the microorganisms in the samples during culturing and to obtain good results, a test run is carried out with the concentrated extract and the raw kitchen swab. Inoculate the dish by pouring the water into the dish and spreading for the kitchen swab extract and for the raw kitchen swab it is touched on the surface of nutrient agar medium(8). 2.5. CULTURING AND SERIAL DILUTION PROCESS The method used to inoculate the extract is by use of the spread plate method. The technique is carried out for each of the four kitchen swab sample extracts by inoculating 100µl on to the dry agar plate which were then seeded with sterile disks containing the whole concentrate of the selected disinfectant and are then incubated at room temperature and results obtained after 24 hours. This is followed by measuring zone of inhibition so as to identify the most effective detergent that is found to have antimicrobial properties. After the first culture with whole concentrate, the detergent found to have the greatest antimicrobial properties was serial diluted to the power of 10-1-10-4 as follows; 9ml of distilled water is first sterilized by autoclaving for an hour, which is then followed by placing the tubes into a micro flow chamber which was sterilized with 70% alcohol so as to avoid any contamination. 1ml of each detergent is obtained by use of a micropipette from the whole concentrate and added into tube containing 9ml of sterile water. This procedure is repeated two more times until the last dilution of 10-4. After serial dilution the sterile discs are then added to the tube for each detergent and left to soak for fifteen minutes so as to avoid any form of contamination. Culturing began by first sterilizing the micro flow chamber bench with 70% alcohol. After sterilization, the agar plates are labeled on the lower surface for easy identification. The plates are then partitioned using marker so as to provide each detergent with a potion into which seeding of the disc containing the detergents will be done. After labeling the plates, culturing commenced by first sterilizing the L-shaped glass rod by dipping it into absolute alcohol which is then flamed and let to cool. 100µl of each sample was obtained and transferred onto the plate and using the sterile rod, the sample is spread evenly over the surface discs containing the specific dilutions are seeded onto the plate containing the cultures and are then incubated for 24 hours after which the zones of inhibition are measured and the results obtained(9). 2.6. CHARACTERIZATION OF THE OBTAINED BACTERIAL ISOLATES After the culturing process the cultured plate were observed for growth after 24 hours. Those that were found to contain microbial growth are characterized on their basic of forms, sizes, cellular morphologies and Gram reactions 2.6.1. GRAM STAINING Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell constituents. The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet. This procedure began by obtaining clean glass slides then placing a drop of water on the slide. Using an inoculating loop it was first sterilized by flaming it over a flame. After cooling the inoculating loop, a loop full of bacteria from the bacterial culture on the plates was spread on the slide to make a smear. The smear was then air dried then heat fixed by passing the slide over the flame three times to avoid washing off the bacterial smear. After heat fixing the slide was placed on a staining rack and flooded with crystal violet stain for one minute. The crystal violet was then washed off gently using running water while holding the slide at an angle. After washing the slide was flooded with iodine solution for another one minute after which it was rinsed off using running water. The slide was then decolorized using absolute alcohol but caution was taken so as not to over decolorize. The excess decolorizing agent was washed off using tap water. The slides was then flooded with Safranin for 30 seconds as a counter stain. The Safranin was then washed off using tap running water with held at an angle. The slide was then air dried and the bottom was dried using blotting paper. The slide was then observed under a compound microscope using oil emulsion lens at a magnification of X100 (10). 3.0. RESULTS 3.1. RESULTS FOR HOME 1 AND HOME 2 AND THE RESPECTIVE EFFICACY The results obtained from Home 1 and Home 2 after the cultures grew indicated that the three of the newly emerging detergents had antimicrobial properties. After seeding the agar plates with whole concentrate of the detergents the three detergents were serial diluted to find the most effective concentration. The cultures grown were also gram stained to determine the morphology of the various colonies which grew from the isolates of the kitchen surface swabs. The results obtained are all summarized in the table below. The bacterial colonies were characterized according to their physical manifestation on the agar plates and the gram staining characteristic observed. Also included were the tables showing the inhibition zones of the seeded detergents. TABLE 1: Table below shows summarized colony characteristics of the bacteria cultured from the two homes floor and kitchen surfaces. No. of colonies Bacterial Colony characteristics Gram reaction shape/morphology Home 1 Uncountable Rods and cocci (slum) Home White in colour, Gram positive mucoid, smooth margin 2 Uncountable Rod shaped Cream, round margin, Gram negative (middle class raised, shiny, pinpoint, suburb) mucoid 3.1.1. HOME 1 (SLUM REIDENTIAL HOME) Plate 1 shows inhibition zones of the seeded detergents. Plate 1 shows that Clorox floral, pride washing liquid and bio-clean had inhibition zones. Clorox floral had the highest inhibition zone followed by pride and finally bio-clean. Plate 1 Plate 2 shows bacterial colonies control for home 1 without seeded detergents, i.e.directly touched the swabs on the surface of the agar. Plate 3 is an indication of the gram stain of home one isolates. Plate 2; colony without seeded detergent plate 3; gram stain magnification X1000 TABLE 2: Table showing inhibition zones of concentrated detergents for home 1 kitchen surface swab extract. Detergents Inhibition zone measurement (mm) Bio-clean 10 Clorox Floral Bleach 27 Pride washing liquid 25 3.1.2. HOME 2 (SUBURB WELL PRIVILEDGED HOME) Plate 4, showing cultures from home 2 seeded using whole concentrate of the different detergents indicating the formed inhibition zones. Plate 4 Plate 6 Magnification X1000 Plate 5 Plate 5 shows bacterial colonies control for home 2 without seeded detergents, directly touched the swabs on the surface of the agar. Plate 6 is an indication of the gram stain of the colony. TABLE 3: Showing inhibition zones from home 2 kitchen surface swab extract and seeded detergents Detergents Inhibition zone measurement (mm) Bio-clean 5 Clorox Floral Bleach 30 Pride washing liquid 20 30 20 HOME 1 10 HOME 2 HOME 2 0 CLOROX HOME 1 PRIDE BIO-CLEAN FIGURE 2: Graph showing the inhibition zones which were measured of the various detergents used in the study and the general trend of their efficacy. y-axis is diameter of inhibition (mm) and x-axis are the detergents used Plate 7 Clorox seeded on a bacteria E. coli Plate 8Clorox seeded on a mold species (control) 3.2. SERIAL DILUTIONS The below tables show results of the three effective detergents which have been serial diluted to various concentrations and seeded with the bacterial cultures from the collected samples. TABLE 4:Below shows serial dilutions for detergents seeded on kitchen surface swab cultures from home 1 Detergents 10-1 10-2 10-3 10-4 Bio-clean 2 mm 0 mm 0 mm 0 mm Clorox Floral Bleach 20 mm 14 mm 8 mm 0 mm Pride washing liquid 12 mm 7 mm 0 mm 0 mm TABLE 5: Below table shows serial dilutions for detergents seeded on kitchen surface swab cultures from Home 2 and respective inhibition zones. Detergents 10-1 10-2 10-3 10-4 Bio-clean 0 mm 0 mm 0 mm 0 mm Clorox Floral Bleach 18 mm 12 mm 3 mm 0 mm Pride washing liquid 12 mm 1 mm 0 mm 0 mm PLATES 9-13: Showing the inhibition zones of plates seeded with the most effective detergents, Clorox; whole concentrate and the ones after serial dilution. Colony isolates were from home 2 Plate 9: whole concentrate Plate 11: dilution to 10-2 Plate 10: dilution to 10-1 Plate 12: serial diluted to 10-3 no inhibition zone Plate 13: serial diluted to 10-4, no inhibition zone 30 25 20 15 HOME 1 10 HOME 2 5 0 HOME 2 HOME 1 100 100 10-1 10-2 10-3 10-4 10 FIGURE 3: Graph showing inhibition zones of seeded Clorox at different serial dilutions. X- Axis represents the dilution factors while y- axis represents the diameter of inhibition (mm) TABLE 6: Zone of inhibition for Bioclean, Clorox and Pride detergents from Homes 1 & 2 Detergent Zone Of Inhibition Home 1 Home 2 Bioclean 10 5 Clorox 27 30 Pride 25 20 TABLE 7: Summary of variables analysed with means and variances SUMMARY Groups Count Sum Average Variance Home 1 3 62 20.66667 86.33333 Home 2 3 55 18.33333 158.3333 TABLE 8:Results of statistical test ANOVA Source of Variation SS df MS F P-value F crit Between Groups 8.166667 1 8.166667 0.066757 0.808868 7.708647 Within Groups 489.3333 4 122.3333 Total 497.5 5 4.0. DISCUSSION In the study the less common new brands of detergents used in each home were assessed to determine their effect on bacteria that was proved to contaminate the tested site. The two samples that were obtained were first cultured two of which were control without any seeded detergents, then with whole concentrate of the different detergents and results were obtained showed that out of the three detergents used on the two cultured samples; Clorox Floral Bleach and pride washing liquid proved to be the most effective followed lastly by Bio-clean of which serial dilutions were done and culturing of the samples was done using the diluted detergents and the results obtained showed that; • Home 1 sample, Clorox floral was the most effective showing the highest zone of inhibition, followed by pride washing liquid, then bio-clean. • Home 2 sample, again Clorox floral bleach proved to be the most effective with the highest inhibition zone, followed by pride and then bio-clean. The above results indicate that Clorox floral bleach was the most effective detergent among all the three samples as it exhibited the highest inhibition zone in all the four dilutions followed by pride washing liquid and finally bio-clean was the least effective detergent that exhibited the lowest zones of inhibition at all the four dilutions on the two samples. These results were in agreement with another study by (11,12) who examined the use of dishwashing detergents as an aid to kill pathogens commonly found in the kitchen. Clorox floral detergent was found to be most effective with a minimum inhibitory concentration (MIC) of 10 -3. Bio-clean was found to be the least effective with a minimum inhibitory concentration of 10-1. Gram positive bacteria were the more inhibited than Gram negative bacteria as indicated by the mean inhibition zones of 20.67 and 18.33 respectively for the three tested detergents. Bacteria were the most inhibited than mold however the difference in zone of inhibition is minimal. This is shown from the control done with a bacterium E. coli with an inhibition zone of 15 mm as compared to mold species having a zone of inhibition of 13 mm. We accept the hypothesis since the F (observed value) is less than < FCV. In this study 0.066757<7.708647, so we accept the hypothesis. Interpretation Since I accepted the hypothesis, I am 95% confident that the newly emerging detergents (Bioclean, Clorox and Pride) are effective against the bacteria and molds isolated from Homes 1 & 2 kitchen surfaces and floors. Exposure of strains of bacteria of molds to lethal doses of hypo chloric acid causes a decrease in ATP production. Chlorine dioxide acts on permeability of the external membrane of the bacteria e.g. E. coli through a primary lethal phenomenon that consists in a substantial leakage of K┼ ion; such leakage does not occur for macromolecules. Sub lethal doses inhibit cellular respiration due to a nonspecific oxidizing effect, as for the case of seeding the plates after serial dilution Home characteristics, particularly those associated with increased moisture levels, have been linked to respiratory symptoms due to the growth of microbial flora of bacteria and molds. (13,14) reported that in the first 7 year of a house becoming damp, the increased level of exposure to microorganisms especially fungal and bacteria may increase the risk of allergic rhinitis. This study revealed varied microbial loading from routinely used sites. Outbreaks of food poisoning frequently occur as a result of improper food preparation in which cross-contamination in combination within adequate storage or cooking was implicated in many instances (15,16). Microorganisms, including some potentially pathogenic species, are commonly found in the entire home environment which includes wet sites such as kitchen surfaces and floor areas. Other wet sites, such as sink, dishcloths and similar cleaning utensils, after lab test were found to be frequently and heavily contaminated due to constant human contact and failure to properly clean the surfaces(17,18,19). These results suggest that, in the kitchen, although raw food is usually the main source of contamination, the sink, dustbin and surrounding areas can also act as a source of reservoir that may harbor and encourage establishment of free-living bacterial and fungal populations. The results obtained are also indication of poor hygiene this is reflected on the samples from slum area showing clear growth of many bacterial cultures and fungal populations(20). CONCLUSION It can be concluded that the project was successful as the goal set out in the general and specific observations were met. Microorganisms were isolated and cultured as proof of contamination. The microorganisms were collected through swabbing from two different homes this would bring out the effects of hygiene on the bacterial population and type. The sample detergents were also found to be effective as zones of inhibition were measured. In conclusion Clorox floral was the most effective followed by Pride washing liquid with the least effective being Bio clean dishwashing liquid. Clorox floral bleach had an active ingredient of Sodium Hypochlorite 4.06% m/v when packed. Pride washing liquid was composed of Anionic surfactants, sodium chloride, preservatives, colorants, and perfume and di-ethanol amide. For bio-clean its components were not indicated by the manufacturers proving its low effectiveness in terms of the low or no inhibition zone. RECOMMENDATION I. Always disinfect the kitchen surfaces (e.g. using bleach such as Clorox floral) or wash them using hot clean water regularly. Also does help to keep numbers down by keeping the kitchen surface and floors dry regularly as dry surfaces discourage growth and survival of bacteria. II. Wash raw foods with clean water before putting them on the kitchen surfaces and floor for preparation. III. Use of detergents as has a beneficial effect on the numbers of bacteria on the home floor and kitchen surfaces; however, one should vary the detergents used as the microorganisms may develop resistance against a specific kind of detergent. IV. Always clean the surfaces first before disinfecting to expose the bacteria to the disinfectants as some of the bacteria may be covered by biofilm or dust particles V. Use cloths which are impregnated with disinfectant or ant-bacterial agents for cleaning the surfaces. Although this method is not as effective as the detergent or antimicrobial agent wear off with time. VI. 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