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EFFICACY OF NEWLY EMERGING DETERGENTS AGAINST BACTERIA AND MOLDS ISOLATED FROM HOME KITCHEN SURFACES AND FLOORS

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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.
Dilute the detergents to the appropriate effective concentration as over diluting it may
reduce its effectiveness hence it loses its antimicrobial properties.
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3)
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of Disinfectants Against the Bacteria Salmonella
4)
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survey of used cellulose sponges and cotton dish clothes from domestic
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