The bulk of reported instances of foodborne unwellness in the UK originate from the place.
This is due to unhygienic patterns when handling, fixing or devouring nutrient. Kitchen sponges can move as reservoirs and vehicles for foodborne pathogens leting cross-contamination onto surfaces and human custodies during cleansing. Air drying is n’t sufficient plenty to kill the micro-organisms held within them so kitchen sponges should be disposed of or disinfected after their usage in order to forestall foodborne unwellness. Microwaves have been used in several scenes for sterilization and disinfection so this survey was conducted to see what effects a domestic microwave would hold on the endurance of microorganisms common to kitchen sponges when they are heated for short periods of clip. The micro-organisms used in this probe were Bacillus subtilis, Escherichia coli, Salmonella typhimurium or Staphylococcus aureus where all but the former are foodborne pathogens. Bacillus subtilis was used as an optimal index for microwave sterilization.
Kitchen sponges were cut into little rectangles, ( 80 millimeter x 20 millimeters x 10 millimeter ) and inoculated with one of the four micro-organisms. They were subjected to different lengths of microwave intervention, ( 15 seconds, 30 seconds and 60 seconds ) utilizing a domestic microwave with an end product energy of 455 W. A consecutive dilution from 10-1 to 10-4 was made utilizing the alimentary stock from the screw-capped glass bottles the sponges were microwaved in and each dilution was plated onto selective media. The experiment was repeated three times for all of the micro-organisms in order to obtain an norm and a control was used in each where the inoculated sponge received no microwave intervention.The Gram-negative bacteriums, E.
coli and Salmonella typhimurium were found to be the most effected by microwave warming as Salmonella typhimurium was wholly eliminated and E. coli had a 3.39-log decrease after 60 seconds of intervention at the 10-1 dilution. The Gram-positive bacteriums, Bacillus subtilis and Staphylococcus aureus were the most immune as no consequences could be obtained for the former due to inordinate growing on the home bases and Staphylococcus aureus merely had a 0.15-log decrease at the 10-1 dilution after 60 seconds of microwave warming.This survey showed that micro-cooking a kitchen sponge is effectual against microorganisms common to kitchen sponges, particularly those that are Gram-negative.
Therefore microwave warming should be used for disinfection as it is fast and cost-efficient so it could easy be adopted as modus operandi in families so the spread of foodborne pathogens can be prevented and the incidence of foodborne disease lowered.
As people live life styles where they work longer hours and are unable to concentrate on kitchen hygiene it has enabled the incidence of foodborne unwellness to increase. Communal kitchens, ( such as those in student adjustment and inns ) have besides allowed the prevalence of nutrient toxic condition to increase due to there being a deficiency of duty or cognition for kitchen hygiene between those who are sharing the kitchen, ( Ojima et al. , 2002 ; Sharp and Walker, 2003 ) . In the twelvemonth 2000, in England and Wales, there were 86,528 reported instances of foodborne unwellness, ( Sharp and Walker, 2003 ) nevertheless at that place could hold been more as many instances of foodborne unwellness are non reported.
It has been estimated that between 50-80 % of these instances originate from the place, ( Redmon and Griffith, 2005 ; Sharp and Walker, 2003 ) due to incorrect patterns when handling, fixing and devouring nutrient, ( Mattick et al. , 2003 ; Sharma et al. , 2009 ) . Topographic points within kitchens that are often touched with human custodies or countries that are damp are found to be the most contaminated with ‘high Numberss of fecal coliforms, coliforms and heterotrophic bacteriums than other countries in the kitchen ‘ , ( Sharma et al. , 2009 ) .
As assorted foodborne pathogens such as E. coli and Salmonella spp. can last on custodies, surfaces, utensils and kitchen fabrics or sponges for hours upon initial contact there is possible for foodborne disease, ( Kusumaningrum et al. , 2003 ; Rusin et al.
, 2002 ) . If a contaminated object or contaminated custodies come into contact with a individual ‘s oral cavity they can acquire nutrient poisoning so in order to forestall foodborne unwellness improved hygienic methods in the kitchen should be employed, ( Carrasco, et al. , 2008 ; Rusin et al. , 2002 ) .Foodborne unwellness can be caused by devouring nutrient or drink which is contaminated with micro-organisms or toxins produced by them. Escherichia coli is a comparatively harmless micro-organism as it is present in the enteric piece of land of worlds and is involved in our metamorphosis by synthesizing vitamins such as vitamin K, ( Farthing, 2004 ) . However, infective strains of E. coli, such as E.
coli 0157: H7, can bring forth enterotoxins which are exotoxins that are secreted from the cells. These enterotoxins act on the little bowels where they cause them to secret big sums of fluid into the enteric lms which consequences in purging and diarrhea. ( Madigan, et al. , 2005 ) .
The genus Salmonella, like E. coli, includes Gram-negative, bacillar bacteriums which are besides involved in foodborne unwellness. In contrast to E. coli, Salmonella spp. do non bring forth toxins but big sums of Salmonella which have been ingested colonise the big and little bowels which consequences in common foodborne unwellness symptoms such as purging and diarrhea. ( Madigan, et al. , 2005 ) . Even though Staphylococcus aureus is a micro-organism which normally colonises the tegument, it can do foodborne unwellness where it produces enterotoxins like E.
coli. However some enterotoxins produced by S. aureus act as superantigens where they induce big Numberss of lymph cells which leads to intestinal and systemic redness, Madigan, et al.
, 2005 ) . Other micro-organisms which cause foodborne unwellness include Campylobacter jejuni, Clostridium perfringens, Listeria monocytogenes.Kitchen sponges are normally used during washing-up, to pass over sinks and to pass over kitchen surfaces. They can move as reservoirs for foodborne pathogens as they remain wet, enabling the micro-organism to last. As air drying is n’t sufficient plenty to kill these pathogens so other agencies of disinfection are needed to make so to forestall the spread of these micro-organisms when the sponges are in usage, ( Sharma et al. , 2009 ) . A survey by Mattick et al. , ( 2003 ) showed that kitchen sponges which were used during the rinsing up procedure had transferred E.
coli 0157: H7 to surfaces every bit good as Salmonella spp. but the former was transferred more often. Josephson, Rubino and Pepper, ( 1997 ) , showed that 33 % of kitchen sponges from 10 kitchens in the US were contaminated with E. coli and 67 % were contaminated with fecal coliforms. Other surveies affecting kitchens from the US found that there were 15.4 % of sponges that were contaminated with Salmonella spp.
, ( Enriquez, Enriquez-Gordillo and Gerba, 1997 ) and 4 % were contaminated with Staphylococcus aureus harmonizing to Hilton and Austin, ( 2000 ) . They besides demonstrated that kitchen sponges had higher bacterial counts when compared to dishrags and this was confirmed by Josephson, Rubino and Pepper, ( 1997 ) . All of these surveies indicate that disinfection of kitchen sponges is needed following their usage in order to forestall the spread of foodborne pathogens.Surveies have shown that the most effectual ways to demobilize micro-organisms in kitchen sponges is by utilizing microwave irradiation or a dish washer as opposed to chemical agencies, ( Mattick et al. , 2003 ; Sharma et al. , 2009 ) . One of the earliest surveies by Olsen, ( 1965 ) used microwave energy to extinguish the presence of micro-organisms in staff of life and found that it reduced the Numberss of spores produced by Aspergillus Niger, Penicillium spp. , every bit good as Rhizopus nigricans following two proceedingss of heating at 5 KW.
Microwaves have been used for ‘many industrial applications such as annealing, dissolving, blanching, cookery, desiccation, sterilization and pasteurization ‘ , ( Yaghmaee and Durance, 2005 ) . They have been employed in many scenes such as in infirmaries where they are used for the sterilization of hospital waste and sterilizing medical utensils, ( Kim et al. , 2009 ) . Other utilizations include the decomposition of organic stuff every bit good as for the riddance of pathogens present in carnal manures, nutrient and dirt, ( Banik et al. , 2003 ; Hong, et al.
, 2004 ; Kim et al. , 2009 ; Tonuci et al. , 2008 ) .Microwave warming has been known to kill many micro-organisms within short exposure periods such as Bacillus Cereus, Campylobacter jejuni, Clostridium perfringens, Escherichia coli, Enterococcus, Listeria monocytogenes, Proteus Mirabilis, Pseudomonas aeruginose, Salmonella spp. , Staphylococcus aureus, Streptococcus faecalis, and Listeria spp.
, ( Woo et al. , 2000 ; Yaghmaee and Durance, 2005 ) . However for sterilization of spore-forming micro-organisms longer periods of exposure are needed ( Woo et al. , 2000 ) , but no micro-organism has been found to be microwave resistant, ( Yaghmaee and Durance, 2005 ) .A microwave works by breathing non-ionising radiation and can do different ‘biological effects depending upon field strength, frequences, wave signifiers, transition and continuance of exposure ‘ , ( Banik et al.
, 2003 ) . Within the electromagnetic spectrum, microwaves are ‘between millimetre moving ridges, ( 0.01 m ) and radiowaves, ( 1 m ) , matching to frequences between 30 and 0.3 GHz ‘ , ( Hong et al. , 2004 ) .
Microwave are able to disinfect stuffs by dielectric warming where heat is generated by polar molecules, ( such as H2O and methyl alcohol ) absorbing the high frequence radiation which causes them to vibrate and aline with each other with the hovering electrical field, ( Banik et al. , 2003 ; Hong et al. , 2004 ; Kim et al. , 2009 ; Tonuci et al.
, 2008 ) . The agitation of the dipolar molecules causes environing molecules to vibrate excessively so the generated heat and spread throughout the non-metallic stuff, ( Banik, et al. , 2003 ; Tonuci et al.
, 2008 )But how does a microwave eliminate micro-organisms? This is n’t presently known as there is contention between whether microbic decease is due to thermic effects caused by the microwave or by non-thermal effects due to ‘difficulty in maintaining the temperature invariable during the microwave irradiation ‘ , ( Banik et al. , 2003 ) . Thermal effects induce the doing ‘the denaturation of nucleic acids, proteins and membrane break ‘ ( Yaghmaee and Durance, 2005 ) and non-thermal effects result in DNA escape, the release of proteins from the cells and cell surface harm, ( supported by the riddance of certain micro-organisms below their thermic devastation point ) , ( Woo et al. , 2000 ) . Papadopoulou et al. , ( 1995 ) , was the first to describe that the riddance of micro-organisms could be due to differences between thermic and non-thermal effects of microwaves when detecting the effects microwaves had on enteric bacterias.In this survey the effects of a microwave oven at 455 W on four different species of bacteriums which were inoculated onto pieces of kitchen sponge were observed. The micro-organisms used were Bacillus subtilis, Escherichia spiral, Salmonella typhimurium and Staphylococcus aureus and they were each subjected to different lengths of microwave warming, ( 15 seconds, 30 seconds and 60 seconds ) .
These micro-organisms were chosen as they are involved in foodborne illness apart from Bacillus subtilis which was chosen to move as ‘an optimal index bacteria for microwave sterilization ‘ , ( Kim et al. , 2009 ) . There were controls used for each species which were non heated within the microwave. This survey was conducted to find if microwave warming is sufficient plenty to extinguish the bulk of micro-organisms. This would show whether or non utilizing a microwave would supply a fast and effectual method of disinfection which could be employed in domestic families to suit in with our busy life styles.
2. Materials and Methods
The micro-organisms used in this experiment were vegetive Bacillus subtilis, Escherichia coli, Salmonella typhimurium and Staphylococcus aureus.
The micro-organisms were provided already grown on alimentary agar home bases and were each subcultured onto extra food agar plates to guarantee their viability.
A battalion of four field cellulose sponges, ( with no scouring tablets ) mensurating 160 mm x 110 mm x 10 mm each were purchased from Sainsbury ‘s.
The 900 W domestic microwave used throughout the experiment was from the trade name Sharp, ( model R-353 ) . The power scene to which it was set was unknown as the screen of the microwave was n’t working. So to decide this, the microwave was calibrated utilizing an equation designed by Thomas and Webb, which is described below, ( Webb, et al. , and Mima, et al.
) . This revealed that the microwave was working at 455 W.
2.4 Calibration of the Microwave
To graduate the microwave, 1 L of H2O, ( at room temperature ) , was heated for 60 seconds in the microwave. The difference between the concluding temperature and the initial temperature was calculated and multiplied by 70. This reveals the electrical power end product of the microwave, ( Webb, et al. , 1998 ; Mima, et al. , 2008 ) .
2.5 Preparation of Cultures
A civilization for each micro-organism was prepared by taking settlements from its alimentary agar home base utilizing a unfertile vaccinating cringle. This cringle was so suspended into a Sterilin® incorporating 15 milliliter of alimentary stock. The civilizations were incubated for two hours at room temperature before being used in the experiment.
2.6 Preparation of Sponges
A sponge was cut, while still in its original packaging to maintain it every bit unfertile as possible, with a unfertile razor blade. The sponge was cut into 80 millimeters x 20 millimeters x 10 millimeter pieces and these pieces were so wrapped in aluminum foil.
2.7 Inoculation of Sponges
1 milliliter of the civilization was pipetted utilizing a P1000 Gilson pipette onto a piece of sponge, which was taken from the Sn foil and held utilizing unfertile forceps. Once inoculated the piece of sponge was so put into a screw-capped bottle incorporating 100 milliliter of alimentary stock, utilizing the unfertile forceps.
2.8 Exposing Sponges to Microwave Radiation
Once all of the sponge pieces were inoculated and put into screw-capped bottles, these bottles were so labelled either ‘control ‘ , ‘15 seconds ‘ , ’30 seconds ‘ or ‘1 minute ‘ and were so put into the microwave and heated for their designated clip periods.
2.9 Extracting Microorganisms from Sponges
After the exposure to micro-cook radiation, a tenfold consecutive dilution was prepared utilizing 1 % peptone H2O.
For each piece of sponge, 4.5 milliliter of 1 % peptone H2O was put into four Sterilins® designated either ’10-1 ‘ , ’10-2 ‘ , ’10-3 ‘ or ’10-4 ‘ utilizing a P5000 Gilson pipette. Then 0.
5 milliliter of the alimentary stock inside a screw-capped bottle was taken and pipetted into the ’10-1 ‘ Sterilin® utilizing a P1000 Gilson pipette. This Sterilin® was assorted and so 0.5 milliliter of the solution was removed from it and pipetted into the ’10-2 ‘ Sterilin® and this was repeated for the ’10-3 ‘ and ’10-4 ‘ Sterilins® .
2.10 Selective Plating
100 µl was taken from each Sterilin® and pipetted onto an agar home base, utilizing a P200 Gilson pipette. The inoculant was spread utilizing the spread home base method with a glass rod which was sterilized utilizing intoxicant and a Bunsen burner. The home bases were so incubated at 37 oC for 48 hours. After 48 hours the figure of settlements on the home bases were counted and recorded. The experiment was repeated three times for each micro-organism.
2.11 Statistical Analysis
A one-way ANOVA statistically trial was conducted utilizing Microsoft Excel 2003 to compare the average figure of bacteriums extracted from the kitchen sponges, ( log10 CFU/mL per sponge ) and see if they were significantly different.
The trial assumes that the information is uninterrupted every bit good as about distributed and that the informations sets have discrepancies which are homogeneous. The void hypothesis was that the informations sets had the same mean and the samples were considered significantly different if P & lt ; 0.05.As the ANOVA shows that at least one of the braces in inquiry is significantly different but does n’t bespeak which brace ( s ) so a station hoc trial is needed in order to make so. The station hoc trial chosen was the Tukey ‘s HSD, ( candidly important differences ) station hoc trial and it identifies which of the braces are significantly different and those which are non. This trial could merely be conducted if the void hypothesis for the one-way ANOVA could be rejected. The difference between the braces of agencies were calculated and so compared to the HSD value. If the differences were larger than the HSD value, they were significantly different.
The Tukey ‘s station hoc trial will be explained in farther item in the appendix subdivision on page 37.Besides a step of consequence size was conducted in order to cipher the proportion of discrepancy that the independent variable, ( the microwave intervention ) could account for in the dependant variable, ( cell endurance ) . The consequences showed the per centum of discrepancy that was due to the microwave intervention in which the micro-organism had received.
3.1 Calibration of the Microwave
1 L of H2O was heated in the microwave for 60 seconds in order to graduate the microwave so its electrical power end product could be identified.
The difference between the initial temperature, ( at room temperature ) and the temperature after heating was measured so multiplied by 70. The initial temperature was 18.5 oC and the concluding temperature was 24 oC so the difference, which was 6.5 oC, was multiplied by 70 therefore the electrical power end product for the microwave was 455 W.
3. 2 The Decrease of the Microorganisms Following Microwave Treatment
No consequences could be obtained for Bacillus subtilis due to overgrowth on the tryptone glucose infusion agar, ( TGEA ) plates demoing that it was n’t sufficiently affected by the microwave interventions. Even after 60 seconds of microwave intervention, ( 445 W ) the home bases were still uncountable which can be seen in figure 3 where a home base of Bacillus subtilis following 60 seconds of intervention is compared to a control home base which received no intervention.
Bacillus subtilis has the ability of teeming where the cells migrate in groups along the surface of the agar which enables it to organize biofilms, ( Connelly, Young and Sloma, 2004 and Hamze et al. , 2009 ) so this may explicate why the agar home bases were indecipherable.
The consequences of experimental microwave exposure for E.
coli are shown in Table 1. When kitchen sponges incorporating the bacteria were exposed for 15, 30 and 60 seconds, the figure of feasible cells declined at 30 and 60 seconds, with fewer than half lasting at the latter exposure, compared with unmicrowaved controls. A one-way analysis of discrepancy ( ANOVA ) statistical trial was calculated for the endurance of E. coli following the microwave interventions at the 10-1 dilution to find if there are important statistical differences between them.
The analysis was found to be important, ( F ( 3.8 ) = 982.02, P = 1.30-10 ) , as the P value was less than 0.05. As the ANOVA statistical trial does n’t place which groups are significantly different from each other, a Tukey ‘s LSD station hoc trial was conducted in order to make so. This showed that the 60-second intervention was significantly, ( P & lt ; 0.05 ) more effectual than the other microwave interventions as merely 2.
10 log CFU/mL had survived the intervention. Significantly lower Numberss of E. coli survived on sponges which were exposed to 30 seconds of intervention than those which were exposed to 15 seconds of intervention because merely 4.56 log CFU/mL survived following the former as opposed to 5.
38 log CFU/mL. No statistically important difference was found between the 15-second intervention and the sponge which had received no intervention, ( the control ) . The step of consequence size was calculated utilizing omega-squared, ( ?2 ) to demo the strength of the relationship between the independent variable ( micro-cook intervention ) and the dependant variable, ( cell endurance ) . It showed for E. coli that the microwave intervention in which the bacteria received could account for 99.
60 % of the consequence obtained. E. coli was found to be the most immune out of the two Gram-negative bacteriums as Salmonella typhimurium was eliminated after 60 seconds of intervention whereas there was still some endurance of E.
Table 2 shows the consequences of microwave exposure on the endurance of Salmonella typhimurium on contaminated kitchen sponges. Following each intervention the figure of feasible cells declined in turn, until after 60 seconds Salmonella typhimurium was wholly eliminated. Therefore the 60-second intervention was the most significantly effectual in cut downing the counts of Salmonella typhimurium when compared to the other interventions, which is supported by the ANOVA statistical trial, ( F ( 3,8 ) = 3048.61, P = 1.44-12 ) and Tukey ‘s LSD station hoc trial. The endurance of S. typhimurium was found to be significantly lower after 30 seconds of intervention ( 4.
87 log CFU/mL ) when compared to the endurance found after 15 seconds of intervention, ( 5.12 log CFU/mL ) . There were significantly lowered counts of S. typhimurium following the 15-second intervention when compared to the counts from the sponges which received no microwave intervention, ( 5.44 log CFU/mL ) . There were no interventions which were non found to be significantly different from each other. The deliberate step of consequence size utilizing omega squared, ( ?2 ) showed that 99.
90 % of the microwave intervention could account for the sum of cell endurance for Salmonella typhimurium.
The consequences demoing the diminution of Staphylococcus aureus following the microwave interventions are shown in Table 3. It can be seen that the sum of the bacteria did n’t worsen dramatically as endurance was merely reduced by 0.58 log CFU/mL following 60 seconds of microwave intervention when compared to the control home base. Yet the ANOVA trial, ( F ( 3,8 ) = 99.49, P = 1.13-6 ) and Tukey ‘s LSD station hoc trial showed that the 60-second intervention was found to be the most significantly effectual, ( P & lt ; 0.
05 ) in cut downing the counts of Staphylococcus aureus, ( 4.19 log CFU/mL ) when compared to the other interventions. The endurance of S. aureus was non significantly lowered following the 30-second intervention, ( 4.
68 log CFU/mL ) when compared to the 15-second intervention, ( 4.70 log CFU/mL ) . This was besides the instance between the 15-second intervention and the control, ( 4.77 log CFU/mL ) and no important difference was found when comparing the control sponges to those which underwent 30 seconds of microwave intervention. This may be due to the counts of Staphylococcus aureus following each intervention being so similar to each other. The step of consequence size calculated utilizing Z squared, ( ?2 ) showed that 96.
10 % of the consequence obtained for Staphylococcus aureus was due to the microwave intervention in which the cells had received.
4.1 What enables micro-organisms to last exposure to micro-cook warming?
After each microwave intervention at 455 W the feasible counts of the micro-organisms decreased in turn with fewer cells lasting following longer periods of microwave exposure. This was due to the temperature of the sponge and environing medium increasing to even higher temperatures following longer lengths of intervention. The riddance of the micro-organism followed the Arrhenius relationship where the ‘number of feasible cells reduces exponentially with clip of exposure to a deadly temperature ‘ , ( Yaghmaee and Durance, 2004 ) . One of the jobs with microwave warming is that there can be ‘uneven heating due to inconsistent microwave field distributions and the physical and electrical nature of the sample ‘ , ( Yaghmaee and Durance, 2004 ) . Therefore uneven heating throughout the kitchen sponges may hold enabled micro-organisms to last, ( Evans, Parry and Ribeiro, 1995 ; Gessner and Beller, 1994 ) .
One survey showed the effects of uneven warming by comparing nine poulets which were cooked conventionally with nine microwave heated poulets which were contaminated with Salmonella typhimurium. Five of the nine poulets which were microwaved were still contaminated with S. typhimurium whereas none of the poulets which were conventionally cooked had S. typhimurium nowadays, ( Irwin et al.
, 1993 ) . In order to sterilize stuffs decently this depends on keeping deadly temperatures for sufficient periods of clip in order to extinguish the micro-organism. However microwaves heat stuffs quickly so they can merely make deadly temperatures for short periods of clip which may non be sufficient plenty to extinguish all of the micro-organisms, ( Gessner and Beller, 1994 ) . So this could besides explicate why bacteriums survived following the short lengths of microwave exposure in this experiment.Bacterias are able to originate a heat daze response when they are put under terrible emphasis due to a sudden alteration in the temperature of the environing environment.
Signals from the environment originate the usage of alternate sigma factors which control which booster sequences the RNA polymerase can adhere to for written text. These alternate sigma factors promote the look of operons that encode heat daze proteins, ( HSPs ) . These heat daze proteins can be split into two groups, those that are cytoplasmatic, ( such as chaperones and peptidases ) and those that are membraneous. The heat daze proteins which are peptidases degrade proteins which have been damaged by the heat so they can be removed. ( Cherepkova et al. , 2006 ; Chung, et al. , 2006 ) .Heat daze proteins which act as chaperones are able to brace heat-damaged or freshly synthesised proteins when they bind to them.
Some chaperones are able to forestall the proteins from misfolding and promote refolding, ( such as HSP70 and GroEL, Cherepkova et al. , 2006 ) which promotes the proteins to be in their right conformation, ( Chung, et al. , 2006 ; Guisbert, et al. , 2008 ) . Besides by adhering misfolded proteins where they are hydrophobic, chaperones are able to forestall their collection which can be toxic to cells as protein sums can impair the cells from working usually, ( Nakamoto and Vigh, 2007 ) . Heat daze proteins which are associated with cell membranes to command their construction when the cell is under heat emphasis which can be done by guaranting the stableness of the lipid bilayer for illustration, ( Nakamoto, et al.
, 2007 ) .An addition of temperature can be sensed by the membrane of the cell as it is in direct contact with the external environment and can take to alterations in the membranes fluidness, ( Nakamoto, et al. , 2007 ) . This could trip signalling tracts which lead to the look of the alternate sigma factors so the synthesis of heat daze proteins can increase. These heat daze proteins take or brace heat damaged proteins so the environment within the cell can accommodate to that of the external temperature. In order for the cells to last they need sufficient sums of heat daze proteins so they can get the better of the alteration in the temperature every bit shortly as possible.
As microwaves are able to heat stuffs to high temperature quickly this could hold given the micro-organism within the kitchen sponges deficient clip to bring forth heat daze proteins. This may hold been why Salmonella typhimurium was wholly eliminated following 60 seconds of intervention and E. coli was significantly reduced. As cells were able to last following the 15 and 30 2nd interventions, the addition of temperature may hold non been sufficient plenty to do terrible emphasis to the cells so any heat damaged proteins could hold been dealt with rapidly by heat daze proteins already present.
4.2 Why were Gram-positive bacteriums more immune to micro-cook heating than Gram-negative bacteriums?
As Salmonella typhimurium was wholly eliminated and Escherichia coli was significantly reduced following 60 seconds of microwave intervention but big Numberss of Bacillus subtilis and Staphylococcus aureus still remained, this probe showed that the Gram-positive bacteriums, ( Bacillus subtilis and Staphylococcus aureus ) were more immune to micro-cook heating than Gram-negative bacteriums.
This may be due to the differences in cell wall composing where Gram-positive bacteriums have the bulk of their cell wall comprised of peptidoglycan whereas Gram-negative bacteriums merely have a little sum of peptidoglycan in their cell walls. This could enable them to be more immune to certain emphasiss like heat but factors like the heat daze response or procedures like monogenesis could besides be involved.Woo et al. , ( 2000 ) demonstrated how microwave warming can impact Gram-positive and Gram-negative bacteriums otherwise utilizing Bacillus subtilis and E. coli, where they measured feasible counts every bit good as the sum of nucleic acids and protein in the suspensions. They found that the cell surface of E. coli was more sensitive to micro-cook intervention than Bacillus subtilis as E. coli exhibited terrible cell surface harm but Bacillus subtilis appeared to hold no cell surface harm at all.
They treated microwave-damaged cells with Na dodecyl sulfate ( SDS ) and found that E. coli cells were sensitive to it whereas Bacillus subtilis was n’t affected at all. This indicates that Bacillus subtilis is more immune than E. coli to it due to the composing of its cell wall which supports the result of this probe.
4.3 Why did Escherichia coli show more opposition to micro-cook warming than Salmonella typhimurium?
E. coli was found to be more immune than Salmonella typhimurium when heated by a microwave at 455 W as Salmonella typhimurium was eliminated following 60 seconds of microwave intervention but a few cells of E.
coli survived. Elben, Annous and Sapers, ( 2005 ) showed that several infective and non-pathogenic E. coli 0157: H7 strains were more thermally immune than strains of Salmonella when cell civilizations were heated to 60 oC. Another survey supports this happening farther as E. coli 0157: H7 was found to be more heat resistant than Salmonella and Listeria monocytogenes when heated in a H2O bath at temperatures runing from 55-70 oC, ( Osaili, et al. , 2007 ) . So what could do E. coli more immune than Salmonella?These two Gram-negative bacteriums portion the same alternate sigma factors used to bring forth a heat daze response such as ?E and ?H.
The anti-sigma factor RseA which controls the look of the ?E operon is found in both of the bacteriums but one survey has indicated that E. coli controls the activity of this anti-sigma factor in an alternate manner opposed to Salmonella typhimurium, ( Klein, et al. , 2003 ) .
This alternate control step involves a kinase and phosphatase and no homologues for these have been found in the genome sequence of Salmonella typhimurium which indicates that there may be differences in how these two bacteriums control their sigma factors needed for the heat daze response, ( Bang, et al. , 2005 ) . Therefore this could explicate why E. coli showed more opposition to micro-cook warming than Salmonella typhimurium.
It has been suggested that thermic opposition in bacteriums can be due to a assortment of factors such as pH and H2O activity plus factors sing experimental technique can impact the heat opposition of Salmonella, ( Jin, Zhang and Boyd, 2008 ) . Besides the growing stage of the cells influences their heat opposition as those which are in stationary stage are more immune to heat every bit good as other emphasiss than those which are in the exponential stage, ( Buchanan and Edelson, 1999 ) . Blackburn, et al. , ( 1997 ) suggest that heat opposition could besides be due to differences in the physiology and biochemistry of the bacteriums but it could besides depend on the strains of E. coli and Salmonella typhimurium used. So there is a whole scope of factors which could explicate why E.
coli showed higher opposition.
4.4 Why was Bacillus subtilis more immune so Staphylococcus aureus?
Both of the Gram-positive micro-organisms used in this survey showed a high degree of opposition to micro-cook warming at 455 W as no consequences could be recorded for Bacillus subtilis due to overgrowth on the agar home bases and Staphylococcus aureus was merely reduced by 0.
58 log CFU/mL following 60 seconds of intervention. As no consequences could be recorded for the former, it can be assumed that the bacteria was more immune than Staphylococcus aureus.The heat daze response initiated in both of the Gram-positive bacteriums depends on the alternate sigma factor ?B but this sigma factor controls the look of over 100 cistrons in response to emphasize in Bacillus subtilis and merely 30 cistrons for Staphylococcus aureus, ( Giachino, Engelmann and Bischoff, 2001 ) . ?B is encoded by the sigB operon which comprises of eight cistrons in Bacillus subtilis but consists of merely four cistrons in Staphylococcus aureus as the latter lacks the cistrons rsbR, rsbS, rbsT and rsbX and no orthologs for these cistrons have been found, ( Pane-Farre , et al. , 2006 ) .
This may intend that the heat daze response of Bacillus subtilis could be activated easier than in Staphylococcus aureus as the operon in the former has been shown to be more sensitive to energy emphasis which does n’t trip the look of the SigB operon in Staphylococcus aureus to bring on the look of ?B, ( Pane-Farre , et al. , 2006 ) .Another ground as to why Bacillus subtilis exhibited more opposition to micro-cook warming may be due to it being a sporulating being whereas Staphylococcus aureus is non. Bacteria undergoing monogenesis become immune to rough environments, such as utmost temperatures, by undergoing asymmetric division with ultimately consequences in the formation of a prespore and a female parent cell. The latter engulfs the prespore and synthesises constituents needed for the prespores differentiation into a spore finally ensuing in a spore with a dehydrated cytol and spore coat which is largely comprised of peptidoglycan, ( Driks, and Losick 1991 ; Gilmore, et al. , 2004 ; Piggot and Hibert, 1991 ) One survey showed that if Bacillus subtilis was in the procedure of monogenesis while it underwent heat daze so the spores produced would exhibit a greater opposition to heat, ( Movahedi and Waites, 2000 ) .
Mendez et al. , ( 2004 ) generated SigB omission mutations and showed that monogenesis is linked to the heat daze response as the mutations were thermosensitive and had defects in monogenesis, ( every bit good as in other emphasis responses ) hence demoing that the two tracts are linked together which would enable it to be become even more immune to any emphasis it was undergoing. As monogenesis is promoted by the bacteria being starved of cardinal foods such as glucose, phosphoric, C or N during growing, ( Errington, 2003 ; Piggot, and Hilbert, 2004 ) . This may hold occurred with the bacteria in this survey as it was grown on a alimentary agar home base prior to the probe and resulted in a bacterial lawn so foods may hold become limited over clip taking to the bacteria sporulating.As stated earlier, Bacillus subtilis has the ability of teeming where cells form microcolonies and are able to migrate together across the agar, ( or other surfaces ) for rapid colonization, ( Connelly et al. , 2004 ; Hamze et al. , 2009 ) . This procedure has been linked to the protein ClpP which is induced under conditions of thermic emphasis as it acts as a peptidase which degrades heat-damaged proteins within Gram-positive bacteriums, ( Frees, et al.
, 2007 ) . This protein is involved in procedures such as cell division, heat opposition, motility and monogenesis, ( Msadek, et al. , 1998 ) .
Mutants of clpP are found to be non-motile and thermosensitive screening that these procedures are linked together, ( Msadek, et al. , 1998 ) . Therefore Bacillus subtilis has several procedures which enable it to go immune to terrible changes in its surrounding environment which is really advantageous for this dirt bacteria where rapid alterations could happen at any clip, ( Mendez et al. , 2004 ) . For that ground Bacillus subtilis was more immune than Staphylococcus aureus when they were exposed to micro-cook warming.
4.5 How could the experiment be improved?
This experiment could hold been improved in several ways.
First the temperature should hold been measured prior to and following each intervention in order to find at which temperature the micro-organisms are eliminated but as this was n’t done in order to maintain the solution the kitchen sponges were in every bit unfertile as possible, an alternate method may necessitate to be come up with or the asepsis compromised. Second, the experiments for each micro-organism should hold been repeated further in order to obtain a more accurate mean of consequences. Besides, as merely the medium power scene was used in the experiment, ( due to the inability to alter the power puting on the microwave in usage as it did non work decently ) , the experiment could hold been conducted utilizing full power as several surveies have shown that the full power scene was the most sufficient to extinguish micro-organisms. One survey showed that E. coli could be wholly eliminated following 30 seconds of microwave intervention, ( Park, Bitton and Melker, 2006 ) . Alternatively a figure of microwave power scenes could hold been used so that their effects on the micro-organism could hold been compared with each other. Furthermore if the length of exposure was increased in extra microwave interventions so the sum of clip needed to wholly extinguish the micro-organisms could be identified.
Several other micro-organisms which are involved in foodborne unwellness, ( such as Listeria spp. ) could hold been used to see how microwave warming can restrict their endurance on kitchen sponges. Besides the consequence of microwave warming on other micro-organisms like barms and casts or even viruses could hold been investigated to happen out the length of microwave exposure needed to extinguish them. Finally, as scientists are non certain as to whether it is the thermic effects or non-thermal effects generated by microwaves that cut down the figure of micro-organisms, the harm inflicted to the microbic cells in this survey should hold been looked at in order to find what type of effects are extinguishing them and to see if there are any differences between Gram-positive and Gram-negative bacteriums.
This survey showed that one minute of microwave warming at 455 W is effectual against fecal coliforms, ( E. coli and Salmonella spp.
) but for Gram-positive and sporulating micro-organisms longer periods of microwave warming or a higher end product power are needed. However this signifier of disinfection would supply a speedy and easy manner of disinfecting kitchen sponges within the place environment to restrict the spread of foodborne pathogens. It could cut down the figure of instances of foodborne disease arising from the place if this method of disinfection was made modus operandi in families. A survey will be needed in the hereafter to look at the long term effects of micro-cooking kitchen sponges in domestic families in order to see if it does supply an effectual manner of disinfection.
I would wish to thank Professor Bignell for his aid and encouragement, Steve for learning me techniques in the research lab, my household and Chris for their support.
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7.1 Growth Media
1 % Peptone Water:1 % of the entire sum of H2O was suspendedBrilliant Green Agar, ( BGA ) :52g was suspended per 1 liter of H2OEosin Methylene Blue Agar, ( EMB ) :37.5g was suspended per 1 liter of H2OMannitol Salt Agar, ( MSA ) :111g was suspended per 1 liter of H2OAlimentary Agar:28g was suspended per 1 liter of H2OAlimentary Broth:25g was suspended per 1 liter of H2OTryptone Glucose Extract Agar:Formula g/litre‘Lab-Lemco ‘ pulverization 3.0Tryptone 5.0Glucose 1.0Agar 15.0pH 7.0 ± 0.2All solutions were autoclaved at 121oC for 15 proceedingss, ( apart from Brilliant Green Agar which was microwaved until it was merely boiled )
7.2 Result Tables
7.3 Statistical Analysis
E. coli ( for 10-1 dilution )
Tables for One-way ANOVA, ( Analysis of Variance )
As there were 12 consequences, ( three for each of the four microwave interventions ) the grades of freedom was 11 ( n-1 ) . As the P value is less than 0.05 and the F value is larger than the F critical value the consequences are significantly different and the void hypothesis saying that the agencies are the same can be rejected.
Tukey ‘s HSD station hoc trial
As the braces of consequences which are significantly different harmonizing to ANOVA necessitate to be identified, Tukey ‘s HSD station hoc trial was conducted to make so. For this trial the candidly important difference, ( HSD ) value needs to be identified as this is what the difference of the mated agencies from the consequences will be compared to. To cipher the HSD value this expression is used:* ‘MS within ‘ is the value taken from the ANOVA tabular array.* ‘n ‘ is the figure of groups* ‘q ‘ is the studentised scope statistic which was taken from the studentised scope statistic tabular array. The value ‘q ‘ is obtained by placing the figure of values in each of the groups on the top of the tabular array and the grades of freedom on the side and so the value for P & lt ; 0.001 or P & lt ; 0.05 can be chosen. In this instance the latter value was chosen.For E. coli ‘q ‘ is 4.26 as the grades of freedom were 11 and the figure of values in each of groups was three. So the HSD value was calculated by:Next the difference between the group means need to be calculated:So all of the mean differences are found to be significantly different apart from the difference between the mean of the control and the 15-second intervention.
Measure of Consequence
The expression for the step of consequence is:* The ‘SS Between ‘ , ‘SS Total ‘ and ‘MS Within ‘ are taken from the ANOVA tabular array.* ‘k ‘ is the figure of groups.‘k ‘ in this instance is 4 so:So 99.6 % of the discrepancy for E. coli cell endurance was due to the microwave intervention in which it received.Salmonella typhimurium ( for 10-1 dilution )As there were 12 consequences, ( three for each of the four microwave interventions ) the grades of freedom was 11 ( n-1 ) . As the P value is less than 0.05 and the F value is larger than the F critical value the consequences are significantly different and the void hypothesis saying that the agencies are the same can be rejected.
Tukey ‘s HSD station hoc trial
To cipher the HSD value this expression is used:‘q ‘ = 4.26All of the mean differences are found to be significantly different.
Measure of Consequence
The expression for the step of consequence is:‘k ‘ in this instance is 4 so:So 99.9 % of the discrepancy for Salmonella typhimurium cell endurance was due to the microwave intervention in which it received.Staphylococcus aureus ( for 10-1 dilution )As there were 12 consequences, ( three for each of the four microwave interventions ) the grades of freedom was 11 ( n-1 ) . As the P value is less than 0.05 and the F value is larger than the F critical value the consequences are significantly different and the void hypothesis saying that the agencies are the same can be rejected.
Tukey ‘s HSD station hoc trial
To cipher the HSD value this expression is used:‘q ‘ = 4.26All of the mean differences are found to be significantly different apart from the difference between the control and the 15-second intervention, the control and the 30-second intervention and the 15-second intervention and the 30-second intervention.
Measure of Consequence
The expression for the step of consequence is:‘k ‘ in this instance is 4 so:So 96.1 % of the discrepancy for Staphylococcus aureus cell endurance was due to the microwave intervention in which it received.