Hygiene; foodborne illness

Published: Last Edited:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.


The majority of reported cases of foodborne illness in the UK originate from the home. This is due to unhygienic practices when handling, preparing or consuming food. Kitchen sponges can act as reservoirs and vehicles for foodborne pathogens allowing cross-contamination onto surfaces and human hands during cleaning. Air drying isn't sufficient enough to kill the microorganisms held within them so kitchen sponges should be disposed of or disinfected after their use in order to prevent foodborne illness. Microwaves have been used in several settings for sterilisation and disinfection so this study was conducted to see what effects a domestic microwave would have on the survival of microorganisms common to kitchen sponges when they are heated for short periods of time. The microorganisms used in this investigation were Bacillus subtilis, Escherichia coli, Salmonella typhimurium or Staphylococcus aureus where all but the former are foodborne pathogens. Bacillus subtilis was used as an optimum indicator for microwave sterilisation.

Kitchen sponges were cut into small rectangles, (80 mm x 20 mm x 10 mm) and inoculated with one of the four microorganisms. They were subjected to different lengths of microwave treatment, (15 seconds, 30 seconds and 60 seconds) using a domestic microwave with an output energy of 455 W. A serial dilution from 10-1 to 10-4 was made using the nutrient broth 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 microorganisms in order to obtain an average and a control was used in each where the inoculated sponge received no microwave treatment.

The Gram-negative bacteria, E. coli and Salmonella typhimurium were found to be the most effected by microwave heating as Salmonella typhimurium was completely eliminated and E. coli had a 3.39-log reduction after 60 seconds of treatment at the 10-1 dilution. The Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus were the most resistant as no results could be obtained for the former due to excessive growth on the plates and Staphylococcus aureus only had a 0.15-log reduction at the 10-1 dilution after 60 seconds of microwave heating.

This study showed that microwaving a kitchen sponge is effective against microorganisms common to kitchen sponges, especially those that are Gram-negative. Therefore microwave heating should be used for disinfection as it is fast and cost-effective so it could easily be adopted as routine in households so the spread of foodborne pathogens can be prevented and the incidence of foodborne disease lowered.

1. Introduction

As people live lifestyles where they work longer hours and are unable to focus on kitchen hygiene it has enabled the incidence of foodborne illness to increase. Communal kitchens, (such as those in student accommodation and hostels) have also allowed the prevalence of food poisoning to increase due to there being a lack of responsibility or knowledge for kitchen hygiene between those who are sharing the kitchen, (Ojima et al., 2002; Sharp and Walker, 2003). In the year 2000, in England and Wales, there were 86,528 reported cases of foodborne illness, (Sharp and Walker, 2003) however there could have been more as many cases of foodborne illness are not reported. It has been estimated that between 50-80% of these cases originate from the home, (Redmon and Griffith, 2005; Sharp and Walker, 2003) due to incorrect practices when handling, preparing and consuming food, (Mattick et al., 2003; Sharma et al., 2009). Places within kitchens that are frequently touched with human hands or areas that are moist are found to be the most contaminated with ‘high numbers of faecal coliforms, coliforms and heterotrophic bacteria than other areas in the kitchen', (Sharma et al., 2009). As various foodborne pathogens such as E. coli and Salmonella spp. can survive on hands, surfaces, utensils and kitchen cloths or sponges for hours upon initial contact there is potential for foodborne disease, (Kusumaningrum et al., 2003; Rusin et al., 2002). If a contaminated object or contaminated hands come into contact with a person's mouth they can get food poisoning so in order to prevent foodborne illness improved hygienic methods in the kitchen should be employed, (Carrasco, et al., 2008; Rusin et al., 2002).

Foodborne illness can be caused by consuming food or drink which is contaminated with microorganisms or toxins produced by them. Escherichia coli is a relatively harmless microorganism as it is present in the intestinal tract of humans and is involved in our metabolism by synthesising vitamins such as vitamin K, (Farthing, 2004). However, pathogenic strains of E. coli, such as E. coli 0157:H7, can produce enterotoxins which are exotoxins that are secreted from the cells. These enterotoxins act on the small intestines where they cause them to secret large amounts of fluid into the intestinal lumen which results in vomiting and diarrhoea. (Madigan, et al., 2005). The genus Salmonella, like E. coli, includes Gram-negative, rod-shaped bacteria which are also involved in foodborne illness. In contrast to E. coli, Salmonella spp. do not produce toxins but large amounts of Salmonella which have been ingested colonise the large and small intestines which results in common foodborne illness symptoms such as vomiting and diarrhoea. (Madigan, et al., 2005). Even though Staphylococcus aureus is a microorganism which usually colonises the skin, it can cause foodborne illness where it produces enterotoxins like E. coli. However some enterotoxins produced by S. aureus act as superantigens where they induce large numbers of lymphocytes which leads to intestinal and systemic inflammation, Madigan, et al., 2005). Other microorganisms which cause foodborne illness include Campylobacter jejuni, Clostridium perfringens, Listeria monocytogenes.

Kitchen sponges are commonly used during washing-up, to wipe sinks and to wipe kitchen surfaces. They can act as reservoirs for foodborne pathogens as they remain wet, enabling the microorganisms to survive. As air drying isn't sufficient enough to kill these pathogens then other means of disinfection are needed to do so to prevent the spread of these microorganisms when the sponges are in use, (Sharma et al., 2009). A study by Mattick et al., (2003) showed that kitchen sponges which were used during the washing up process had transferred E. coli 0157:H7 to surfaces as well as Salmonella spp. but the former was transferred more frequently. Josephson, Rubino and Pepper, (1997), showed that 33 % of kitchen sponges from ten kitchens in the US were contaminated with E. coli and 67 % were contaminated with faecal coliforms. Other studies involving 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 according to Hilton and Austin, (2000). They also demonstrated that kitchen sponges had higher bacterial counts when compared to dishcloths and this was confirmed by Josephson, Rubino and Pepper, (1997). All of these studies indicate that disinfection of kitchen sponges is needed following their use in order to prevent the spread of foodborne pathogens.

Studies have shown that the most effective ways to inactivate microorganisms in kitchen sponges is by using microwave irradiation or a dishwasher as opposed to chemical means, (Mattick et al., 2003; Sharma et al., 2009). One of the earliest studies by Olsen, (1965) used microwave energy to eliminate the presence of microorganisms in bread and found that it reduced the numbers of spores produced by Aspergillus niger, Penicillium spp., as well as Rhizopus nigricans following two minutes of heating at 5 KW.

Microwaves have been used for ‘many industrial applications such as tempering, thawing, blanching, cooking, dehydration, sterilisation and pasteurisation', (Yaghmaee and Durance, 2005). They have been employed in many settings such as in hospitals where they are used for the sterilisation of hospital waste and sterilising medical utensils, (Kim et al., 2009). Other uses include the decomposition of organic material as well as for the elimination of pathogens present in animal manures, food and soil, (Banik et al., 2003; Hong, et al., 2004; Kim et al., 2009; Tonuci et al., 2008).

Microwave heating has been known to kill many microorganisms 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 sterilisation of spore-forming microorganisms longer periods of exposure are needed (Woo et al., 2000), but no microorganism has been found to be microwave resistant, (Yaghmaee and Durance, 2005).

A microwave works by emitting non-ionising radiation and can cause different ‘biological effects depending upon field strength, frequencies, wave forms, modulation and duration of exposure', (Banik et al., 2003). Within the electromagnetic spectrum, microwaves are ‘between millimetre waves, (0.01 m) and radiowaves, (1 m), corresponding to frequencies between 30 and 0.3 GHz', (Hong et al., 2004). Microwave are able to disinfect materials by dielectric heating where heat is generated by polar molecules, (such as water and methanol) absorbing the high frequency radiation which causes them to vibrate and align with each other with the oscillating 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 surrounding molecules to vibrate too so the generated heat and spread throughout the non-metallic material, (Banik, et al., 2003; Tonuci et al., 2008)

But how does a microwave eliminate microorganisms? This isn't currently known as there is controversy between whether microbial death is due to thermal effects caused by the microwave or by non-thermal effects due to ‘difficulty in keeping the temperature constant during the microwave irradiation', (Banik et al., 2003). Thermal effects induce the causing ‘the denaturation of nucleic acids, proteins and membrane disruption' (Yaghmaee and Durance, 2005) and non-thermal effects result in DNA leakage, the release of proteins from the cells and cell surface damage, (supported by the elimination of certain microorganisms below their thermal destruction point), (Woo et al., 2000). Papadopoulou et al., (1995), was the first to report that the elimination of microorganisms could be due to differences between thermal and non-thermal effects of microwaves when observing the effects microwaves had on enterobacteria.

In this study the effects of a microwave oven at 455 W on four different species of bacteria which were inoculated onto pieces of kitchen sponge were observed. The microorganisms used were Bacillus subtilis, Escherichia coil, Salmonella typhimurium and Staphylococcus aureus and they were each subjected to different lengths of microwave heating, (15 seconds, 30 seconds and 60 seconds). These microorganisms were chosen as they are involved in foodborne illness apart from Bacillus subtilis which was chosen to act as ‘an optimum indicator bacterium for microwave sterilisation', (Kim et al., 2009). There were controls used for each species which were not heated within the microwave. This study was conducted to determine if microwave heating is sufficient enough to eliminate the majority of microorganisms. This would demonstrate whether or not using a microwave would provide a fast and effective method of disinfection which could be employed in domestic households to fit in with our busy lifestyles.

2. Materials and Methods

2.1 Cultures

The microorganisms used in this experiment were vegetative Bacillus subtilis, Escherichia coli, Salmonella typhimurium and Staphylococcus aureus. The microorganisms were provided already grown on nutrient agar plates and were each subcultured onto additional nutrient agar plates to ensure their viability.

2.2 Sponges

A pack of four plain cellulose sponges, (with no scrubbing pads) measuring 160 mm x 110 mm x 10 mm each were purchased from Sainsbury's.

2.3 Microwave

The 900 W domestic microwave used throughout the experiment was from the brand Sharp, (model R-353). The power setting to which it was set was unknown as the screen of the microwave wasn't working. So to resolve this, the microwave was calibrated using 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 calibrate the microwave, 1 L of water, (at room temperature), was heated for 60 seconds in the microwave. The difference between the final temperature and the initial temperature was calculated and multiplied by 70. This reveals the wattage output of the microwave, (Webb, et al., 1998; Mima, et al., 2008).

2.5 Preparation of Cultures

A culture for each microorganism was prepared by removing colonies from its nutrient agar plate using a sterile inoculating loop. This loop was then suspended into a Sterilin® containing 15 ml of nutrient broth. The cultures 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 keep it as sterile as possible, with a sterile razor blade. The sponge was cut into 80 mm x 20 mm x 10 mm pieces and these pieces were then wrapped in aluminium foil.

2.7 Inoculation of Sponges

1 ml of the culture was pipetted using a P1000 Gilson pipette onto a piece of sponge, which was taken from the tin foil and held using sterile forceps. Once inoculated the piece of sponge was then put into a screw-capped bottle containing 100 ml of nutrient broth, using the sterile 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 then labelled either ‘control', ‘15 seconds', '30 seconds' or ‘1 minute' and were then put into the microwave and heated for their designated time periods.

2.9 Extracting Microorganisms from Sponges

After the exposure to microwave radiation, a ten-fold serial dilution was prepared using 1% peptone water. For each piece of sponge, 4.5 ml of 1% peptone water was put into four Sterilins® designated either '10-1', '10-2', '10-3' or '10-4' using a P5000 Gilson pipette. Then 0.5 ml of the nutrient broth inside a screw-capped bottle was taken and pipetted into the '10-1' Sterilin® using a P1000 Gilson pipette. This Sterilin® was mixed and then 0.5 ml 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 plate, using a P200 Gilson pipette. The inoculum was spread using the spread plate method with a glass rod which was sterilized using alcohol and a Bunsen burner. The plates were then incubated at 37 oC for 48 hours. After 48 hours the number of colonies on the plates were counted and recorded. The experiment was repeated three times for each microorganism.

2.11 Statistical Analysis

A one-way ANOVA statistically test was conducted using Microsoft Excel 2003 to compare the mean number of bacteria extracted from the kitchen sponges, (log10 CFU/mL per sponge) and see if they were significantly different. The test assumes that the data is continuous as well as approximately distributed and that the data sets have variances which are homogenous. The null hypothesis was that the data sets had the same mean and the samples were considered significantly different if P < 0.05.

As the ANOVA shows that at least one of the pairs in question is significantly different but doesn't indicate which pair(s) then a post hoc test is needed in order to do so. The post hoc test chosen was the Tukey's HSD, (honestly significant differences) post hoc test and it identifies which of the pairs are significantly different and those which are not. This test could only be conducted if the null hypothesis for the one-way ANOVA could be rejected. The difference between the pairs of means were calculated and then compared to the HSD value. If the differences were larger than the HSD value, they were significantly different. The Tukey's post hoc test will be explained in further detail in the appendix section on page 37.

Also a measure of effect size was conducted in order to calculate the proportion of variance that the independent variable, (the microwave treatment) could account for in the dependent variable, (cell survival). The results showed the percentage of variance that was due to the microwave treatment in which the microorganisms had received.

3. Results

3.1 Calibration of the Microwave

1 L of water was heated in the microwave for 60 seconds in order to calibrate the microwave so its wattage output could be identified. The difference between the initial temperature, (at room temperature) and the temperature after heating was measured then multiplied by 70. The initial temperature was 18.5 oC and the final temperature was 24 oC so the difference, which was 6.5 oC, was multiplied by 70 therefore the wattage output for the microwave was 455 W.

3. 2 The Reduction of the Microorganisms Following Microwave Treatment

Bacillus subtilis

No results could be obtained for Bacillus subtilis due to overgrowth on the tryptone glucose extract agar, (TGEA) plates showing that it wasn't sufficiently affected by the microwave treatments. Even after 60 seconds of microwave treatment, (445 W) the plates were still uncountable which can be seen in figure 3 where a plate of Bacillus subtilis following 60 seconds of treatment is compared to a control plate which received no treatment. Bacillus subtilis has the ability of swarming where the cells migrate in groups along the surface of the agar which enables it to form biofilms, (Connelly, Young and Sloma, 2004 and Hamze et al., 2009) so this may explain why the agar plates were unreadable.

Escherichia coli

The results of experimental microwave exposure for E. coli are shown in Table 1. When kitchen sponges containing the bacterium were exposed for 15, 30 and 60 seconds, the number of viable cells declined at 30 and 60 seconds, with fewer than half surviving at the latter exposure, compared with unmicrowaved controls. A one-way analysis of variance (ANOVA) statistical test was calculated for the survival of E. coli following the microwave treatments at the 10-1 dilution to determine if there are significant statistical differences between them. The analysis was found to be significant, (F(3.8) = 982.02, P = 1.30-10), as the P value was less than 0.05. As the ANOVA statistical test doesn't identify which groups are significantly different from each other, a Tukey's LSD post hoc test was conducted in order to do so. This showed that the 60-second treatment was significantly, (P < 0.05) more effective than the other microwave treatments as only 2.10 log CFU/mL had survived the treatment. Significantly lower numbers of E. coli survived on sponges which were exposed to 30 seconds of treatment than those which were exposed to 15 seconds of treatment because only 4.56 log CFU/mL survived following the former as opposed to 5.38 log CFU/mL. No statistically significant difference was found between the 15-second treatment and the sponge which had received no treatment, (the control). The measure of effect size was calculated using omega-squared, (ω2) to show the strength of the relationship between the independent variable (microwave treatment) and the dependent variable, (cell survival). It showed for E. coli that the microwave treatment in which the bacterium received could account for 99.60 % of the result obtained. E. coli was found to be the most resistant out of the two Gram-negative bacteria as Salmonella typhimurium was eliminated after 60 seconds of treatment whereas there was still some survival of E. coli cells.

Salmonella typhimurium

Table 2 shows the results of microwave exposure on the survival of Salmonella typhimurium on contaminated kitchen sponges. Following each treatment the number of viable cells declined successively, until after 60 seconds Salmonella typhimurium was completely eliminated. Therefore the 60-second treatment was the most significantly effective in reducing the counts of Salmonella typhimurium when compared to the other treatments, which is supported by the ANOVA statistical test, (F(3,8) = 3048.61, P = 1.44-12) and Tukey's LSD post hoc test. The survival of S. typhimurium was found to be significantly lower after 30 seconds of treatment (4.87 log CFU/mL) when compared to the survival found after 15 seconds of treatment, (5.12 log CFU/mL). There were significantly lowered counts of S. typhimurium following the 15-second treatment when compared to the counts from the sponges which received no microwave treatment, (5.44 log CFU/mL). There were no treatments which were not found to be significantly different from each other. The calculated measure of effect size using omega squared, (ω2) showed that 99.90 % of the microwave treatment could account for the amount of cell survival for Salmonella typhimurium.

Staphylococcus aureus

The results showing the decline of Staphylococcus aureus following the microwave treatments are shown in Table 3. It can be seen that the amount of the bacterium didn't decline dramatically as survival was only reduced by 0.58 log CFU/mL following 60 seconds of microwave treatment when compared to the control plate. Yet the ANOVA test, (F(3,8) = 99.49, P = 1.13-6) and Tukey's LSD post hoc test showed that the 60-second treatment was found to be the most significantly effective, (P < 0.05) in reducing the counts of Staphylococcus aureus, (4.19 log CFU/mL) when compared to the other treatments. The survival of S. aureus was not significantly lowered following the 30-second treatment, (4.68 log CFU/mL) when compared to the 15-second treatment, (4.70 log CFU/mL). This was also the case between the 15-second treatment and the control, (4.77 log CFU/mL) and no significant difference was found when comparing the control sponges to those which underwent 30 seconds of microwave treatment. This may be due to the counts of Staphylococcus aureus following each treatment being so similar to each other. The measure of effect size calculated using omega squared, (ω2) showed that 96.10 % of the result obtained for Staphylococcus aureus was due to the microwave treatment in which the cells had received.

4. Discussion

4.1 What enables microorganisms to survive exposure to microwave heating?

After each microwave treatment at 455 W the viable counts of the microorganisms decreased successively with fewer cells surviving following longer periods of microwave exposure. This was due to the temperature of the sponge and surrounding medium increasing to even higher temperatures following longer lengths of treatment. The elimination of the microorganisms followed the Arrhenius relationship where the ‘number of viable cells reduces exponentially with time of exposure to a lethal temperature', (Yaghmaee and Durance, 2004). One of the problems with microwave heating 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 have enabled microorganisms to survive, (Evans, Parry and Ribeiro, 1995; Gessner and Beller, 1994). One study showed the effects of uneven heating by comparing nine chickens which were cooked conventionally with nine microwave heated chickens which were contaminated with Salmonella typhimurium. Five of the nine chickens which were microwaved were still contaminated with S. typhimurium whereas none of the chickens which were conventionally cooked had S. typhimurium present, (Irwin et al., 1993). In order to sterilise materials properly this depends on maintaining lethal temperatures for sufficient periods of time in order to eliminate the microorganisms. However microwaves heat materials rapidly so they can only reach lethal temperatures for short periods of time which may not be sufficient enough to eliminate all of the microorganisms, (Gessner and Beller, 1994). So this could also explain why bacteria survived following the short lengths of microwave exposure in this experiment.

Bacteria are able to initiate a heat shock response when they are put under severe stress due to a sudden change in the temperature of the surrounding environment. Signals from the environment initiate the use of alternative sigma factors which control which promoter sequences the RNA polymerase can bind to for transcription. These alternative sigma factors promote the expression of operons that encode heat shock proteins, (HSPs). These heat shock proteins can be split into two groups, those that are cytoplasmic, (such as chaperones and proteases) and those that are membraneous. The heat shock proteins which are proteases degrade proteins which have been damaged by the heat so they can be removed. (Cherepkova et al., 2006; Chung, et al., 2006).

Heat shock proteins which act as chaperones are able to stabilise heat-damaged or newly synthesised proteins when they bind to them. Some chaperones are able to prevent the proteins from misfolding and encourage refolding, (such as HSP70 and GroEL, Cherepkova et al., 2006) which promotes the proteins to be in their correct conformation, (Chung, et al., 2006; Guisbert, et al., 2008). Also by binding misfolded proteins where they are hydrophobic, chaperones are able to prevent their aggregation which can be toxic to cells as protein aggregates can impair the cells from functioning normally, (Nakamoto and Vigh, 2007). Heat shock proteins which are associated with cell membranes to control their structure when the cell is under heat stress which can be done by ensuring the stability of the lipid bilayer for example, (Nakamoto, et al., 2007).

An increase of temperature can be sensed by the membrane of the cell as it is in direct contact with the external environment and can lead to changes in the membranes fluidity, (Nakamoto, et al., 2007). This could trigger signalling pathways which lead to the expression of the alternative sigma factors so the synthesis of heat shock proteins can increase. These heat shock proteins remove or stabilise heat damaged proteins so the environment within the cell can adapt to that of the external temperature. In order for the cells to survive they need sufficient amounts of heat shock proteins so they can overcome the change in the temperature as soon as possible. As microwaves are able to heat materials to high temperature rapidly this could have given the microorganisms within the kitchen sponges insufficient time to produce heat shock proteins. This may have been why Salmonella typhimurium was completely eliminated following 60 seconds of treatment and E. coli was significantly reduced. As cells were able to survive following the 15 and 30 second treatments, the increase of temperature may have not been sufficient enough to cause severe stress to the cells so any heat damaged proteins could have been dealt with quickly by heat shock proteins already present.

4.2 Why were Gram-positive bacteria more resistant to microwave heating than Gram-negative bacteria?

As Salmonella typhimurium was completely eliminated and Escherichia coli was significantly reduced following 60 seconds of microwave treatment but large numbers of Bacillus subtilis and Staphylococcus aureus still remained, this investigation showed that the Gram-positive bacteria, (Bacillus subtilis and Staphylococcus aureus) were more resistant to microwave heating than Gram-negative bacteria. This may be due to the differences in cell wall composition where Gram-positive bacteria have the majority of their cell wall comprised of peptidoglycan whereas Gram-negative bacteria only have a small amount of peptidoglycan in their cell walls. This could enable them to be more resistant to certain stresses like heat but factors like the heat shock response or processes like sporulation could also be involved.

Woo et al., (2000) demonstrated how microwave heating can affect Gram-positive and Gram-negative bacteria differently using Bacillus subtilis and E. coli, where they measured viable counts as well as the amount of nucleic acids and protein in the suspensions. They found that the cell surface of E. coli was more sensitive to microwave treatment than Bacillus subtilis as E. coli exhibited severe cell surface damage but Bacillus subtilis appeared to have no cell surface damage at all. They treated microwave-damaged cells with sodium dodecyl sulphate (SDS) and found that E. coli cells were sensitive to it whereas Bacillus subtilis wasn't affected at all. This indicates that Bacillus subtilis is more resistant than E. coli to it due to the composition of its cell wall which supports the outcome of this investigation.

4.3 Why did Escherichia coli show more resistance to microwave heating than Salmonella typhimurium?

E. coli was found to be more resistant than Salmonella typhimurium when heated by a microwave at 455 W as Salmonella typhimurium was eliminated following 60 seconds of microwave treatment but a few cells of E. coli survived. Elben, Annous and Sapers, (2005) showed that several pathogenic and non-pathogenic E. coli 0157:H7 strains were more thermally resistant than strains of Salmonella when cell cultures were heated to 60 oC. Another study supports this finding further as E. coli 0157:H7 was found to be more heat resistant than Salmonella and Listeria monocytogenes when heated in a water bath at temperatures ranging from 55-70 oC, (Osaili, et al., 2007). So what could make E. coli more resistant than Salmonella?

These two Gram-negative bacteria share the same alternative sigma factors used to generate a heat shock response such as σE and σH. The anti-sigma factor RseA which controls the expression of the σE operon is found in both of the bacteria but one study has indicated that E. coli controls the activity of this anti-sigma factor in an alternative way opposed to Salmonella typhimurium, (Klein, et al., 2003). This alternative control measure 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 bacteria control their sigma factors needed for the heat shock response, (Bang, et al., 2005). Therefore this could explain why E. coli showed more resistance to microwave heating than Salmonella typhimurium.

It has been suggested that thermal resistance in bacteria can be due to a variety of factors such as pH and water activity plus factors regarding experimental technique can affect the heat resistance of Salmonella, (Jin, Zhang and Boyd, 2008). Also the growth phase of the cells influences their heat resistance as those which are in stationary phase are more resistant to heat as well as other stresses than those which are in the exponential phase, (Buchanan and Edelson, 1999). Blackburn, et al., (1997) suggest that heat resistance could also be due to differences in the physiology and biochemistry of the bacteria but it could also depend on the strains of E. coli and Salmonella typhimurium used. So there is a whole range of factors which could explain why E. coli showed higher resistance.

4.4 Why was Bacillus subtilis more resistant then Staphylococcus aureus?

Both of the Gram-positive microorganisms used in this study showed a high level of resistance to microwave heating at 455 W as no results could be recorded for Bacillus subtilis due to overgrowth on the agar plates and Staphylococcus aureus was only reduced by 0.58 log CFU/mL following 60 seconds of treatment. As no results could be recorded for the former, it can be assumed that the bacterium was more resistant than Staphylococcus aureus.

The heat shock response initiated in both of the Gram-positive bacteria depends on the alternative sigma factor σB but this sigma factor controls the expression of over 100 genes in response to stress in Bacillus subtilis and only 30 genes for Staphylococcus aureus, (Giachino, Engelmann and Bischoff, 2001). σB is encoded by the sigB operon which comprises of eight genes in Bacillus subtilis but consists of only four genes in Staphylococcus aureus as the latter lacks the genes rsbR, rsbS, rbsT and rsbX and no orthologs for these genes have been found, (Pané-Farré, et al., 2006). This may mean that the heat shock 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 stress which doesn't activate the expression of the SigB operon in Staphylococcus aureus to induce the expression of σB, (Pané-Farré, et al., 2006).

Another reason as to why Bacillus subtilis exhibited more resistance to microwave heating may be due to it being a sporulating organism whereas Staphylococcus aureus is not. Bacteria undergoing sporulation become resistant to harsh environments, such as extreme temperatures, by undergoing asymmetric division with ultimately results in the formation of a prespore and a mother cell. The latter engulfs the prespore and synthesises components needed for the prespores differentiation into a spore ultimately resulting in a spore with a dehydrated cytoplasm and spore coat which is mostly comprised of peptidoglycan, (Driks, and Losick 1991; Gilmore, et al., 2004; Piggot and Hibert, 1991) One study showed that if Bacillus subtilis was in the process of sporulation while it underwent heat shock then the spores produced would exhibit a greater resistance to heat, (Movahedi and Waites, 2000). Mendez et al., (2004) generated SigB deletion mutants and showed that sporulation is linked to the heat shock response as the mutants were thermosensitive and had defects in sporulation, (as well as in other stress responses) therefore showing that the two pathways are linked together which would enable it to be become even more resistant to any stress it was undergoing. As sporulation is promoted by the bacterium being starved of key nutrients such as glucose, phosphorous, carbon or nitrogen during growth, (Errington, 2003; Piggot, and Hilbert, 2004). This may have occurred with the bacterium in this study as it was grown on a nutrient agar plate prior to the investigation and resulted in a bacterial lawn so nutrients may have become limited over time leading to the bacterium sporulating.

As stated earlier, Bacillus subtilis has the ability of swarming where cells form microcolonies and are able to migrate together across the agar, (or other surfaces) for rapid colonisation, (Connelly et al., 2004; Hamze et al., 2009). This process has been linked to the protein ClpP which is induced under conditions of thermal stress as it acts as a protease which degrades heat-damaged proteins within Gram-positive bacteria, (Frees, et al., 2007). This protein is involved in processes such as cell division, heat resistance, motility and sporulation, (Msadek, et al., 1998). Mutants of clpP are found to be non-motile and thermosensitive showing that these processes are linked together, (Msadek, et al., 1998). Therefore Bacillus subtilis has several processes which enable it to become resistant to severe alterations in its surrounding environment which is very advantageous for this soil bacterium where rapid changes could occur at any time, (Mendez et al., 2004). For that reason Bacillus subtilis was more resistant than Staphylococcus aureus when they were exposed to microwave heating.

4.5 How could the experiment be improved?

This experiment could have been improved in several ways. Firstly the temperature should have been measured prior to and following each treatment in order to determine at which temperature the microorganisms are eliminated but as this wasn't done in order to keep the solution the kitchen sponges were in as sterile as possible, an alternative method may need to be come up with or the sterility compromised. Secondly, the experiments for each microorganism should have been repeated further in order to obtain a more accurate mean of results. Also, as only the medium power setting was used in the experiment, (due to the inability to change the power setting on the microwave in use as it did not work properly), the experiment could have been conducted using full power as several studies have shown that the full power setting was the most sufficient to eliminate microorganisms. One study showed that E. coli could be completely eliminated following thirty seconds of microwave treatment, (Park, Bitton and Melker, 2006). Alternatively a number of microwave power settings could have been used so that their effects on the microorganisms could have been compared with each other. Furthermore if the length of exposure was increased in additional microwave treatments then the amount of time needed to completely eliminate the microorganisms could be identified. Several other microorganisms which are involved in foodborne illness, (such as Listeria spp.) could have been used to see how microwave heating can limit their survival on kitchen sponges. Also the effect of microwave heating on other microorganisms like yeasts and molds or even viruses could have been investigated to find out the length of microwave exposure needed to eliminate them. Finally, as scientists are not sure as to whether it is the thermal effects or non-thermal effects generated by microwaves that reduce the number of microorganisms, the damage inflicted to the microbial cells in this study should have been looked at in order to determine what type of effects are eliminating them and to see if there are any differences between Gram-positive and Gram-negative bacteria.

4.6 Conclusion

This study showed that one minute of microwave heating at 455 W is effective against faecal coliforms, (E. coli and Salmonella spp.) but for Gram-positive and sporulating microorganisms longer periods of microwave heating or a higher output power are needed. However this form of disinfection would provide a quick and easy way of disinfecting kitchen sponges within the home environment to limit the spread of foodborne pathogens. It could reduce the number of cases of foodborne disease originating from the home if this method of disinfection was made routine in households. A study will be needed in the future to look at the long term effects of microwaving kitchen sponges in domestic households in order to see if it does provide an effective way of disinfection.

5. Acknowledgement

I would like to thank Professor Bignell for his help and encouragement, Steve for teaching me techniques in the laboratory, my family and Chris for their support.

6. References

1. Bang, L.S., Frye, J.G., McClelland, M., et al., (2005) Alternative sigma factor interactions in Salmonella σE and σH promote antioxidant defences by enhancing σS levels. Molecular Microbiology, 56, (3), pages 811-823

2. Banik, S., Bandyopadhyay, S., & Ganguly, S., (2003) Bioeffects of Microwave - a Brief Review. Bioresource Technology, 87, (2), pages 155-159

3. Blackburn, C.D., Curtis, L.M., Humpheson, L., et al., (1997) Development of thermal inactivation models for Salmonella enteritidis and Escherichia coli O157:H7 with temperature, pH and NaCl as controlling factors. International Journal of Food Microbiology, 38, (1), pages 31-44

4. Buchanan, R.L., & Edelson, S.G., (1999) Effect of pH-dependent, stationary phase acid resistance on the thermal tolerance of Escherichia coli O157:H7. Food Microbiology, 16, pages 447-458

5. Carrasco, L., Mena, K.D., Mota L.C., et al., (2008) Occurrence of faecal contamination in households along the US-Mexico border. Letters in Applied Microbiology, 46, (6), pages 682-687

6. Cherepkova, O.A., Lyutova, E.M., Eronina, T.B., et al., (2006) Accelerated Protein Aggregation Induced by Macrophage Migration Inhibitory Factor under Heat Stress Conditions. Biochemistry (Moscow), 71, (2), pages 140-145

7. Chung, H.J., Bang, W., & Drake, M.A., (2006) Stress response of E. coli. Comprehensive Reviews in Food Science and Food Safety, 5, (3), pages 52-64

8. Connelly, M.B., Young, C.B., & Sloma, S., (2004) Extracellular proteolytic activity plays a central role in swarming motility in Bacillus subtilis. Journal of Bacteriology, 186, (13), pages 4159-4167

9. De Muth, J.E., (2006) Basic Statistics and Pharmaceutical Statistical Applications, 2nd edition, Chapman and Hall/CRC, pages 451-453. ISBN: 0849337992

10. Driks, A., & Losick R., (1991) Compartmentalised expression of a gene under the control of sporulation transcription factor σE in Bacillus subtilis. Proceedings of the National Academy of Sciences, 88, (22), pages 9934-9938

11. Dytham, C., (2003) Choosing and Using Statistics: a Biologist's Guide, 2nd edition, Oxford: Blackwell Publishing, pages 94-102. ISBN: 1405102438

12. Eblen, D.R., Annous, B.A., & Sapers, G.M., (2005) Studies to select appropriate nonpathogenic surrogate Escherichia coli strains for potential use in place of Escherichia coli O157:H7 and Salmonella in pilot plant studies. Journal of Food Protection, 68, (2), pages 282-291

13. Enriquez, C., Enriquez-Gordillo, R. & Gerba, C., (1997). Bacteriological survey of used cellulose sponges and cotton dishcloths from domestic kitchens. Dairy Food and Environmental Sanitation, 17, (1), pages 20-24

14. Errington, J., (2003) Bacillus subtilis Sporulation: Regulation of Gene Expression and Control of Morphogenesis. Microbiological Reviews, 57, (1) pages 1-33

15. Evans, M.R., Parry, S.M., & Ribeiro, C.D., (1995) Salmonella outbreak from microwave cooked food. Epidemiology and Infection, 115, (2), pages 227-230

16. Farthing, M.J.G., (2004) Bugs and the gut: an unstable marriage Best Practice & Research Clinical Gastroenterology, 18, (2), pages 233-239

17. Frees, D., Savijoki, K., Varmanen, P., et al., (2007) Clp ATPases and ClpC proteolytic complexes regulate vital biological processes in low GC, Gram-positive bacteria. Molecular Microbiology, 63, (5), pages 1285-1295

18. Gessner, B.D., & Beller, M., (1994) Protective Effect of Conventional Cooking versus Use of Microwave Ovens in an Outbreak of Salmonellosis, American Journal of Epidemiology, 139, (9), pages 903-909

19. Giachino, P., Engelman, S., & Bischoff, M., (2001) σB activity depends on RsbU in Staphylococcus aureus. Journal of Bacteriology, 183, (6), pages 1843-1852

20. Gilmore, M.E., Bandyopadhyay, D., Dean, A.M., et al., (2004) Production of muramic δ-lactam in Bacillus subtilis spore peptidoglycan. Journal of Bacteriology, 186, (1), pages 80-89

21. Gravetter, F.J., & Wallnau, L.B., (2009) Statistics for the Behavioural Sciences, 8th edition, Wadsworth, pages 426-428. ISBN: 0495602949

22. Guisbert, E., Yura, T., Rhodius, V.A., et al., (2008) Convergence of molecular, modelling, and systems approaches for an understanding of the Escherichia coil heat shock response. Microbiology and Molecular Biology Reviews, 72, (3), pages 545-554

23. Hamze, K., Julkowska, D., Autret, S., et al., (2009) Identification of genes required for different stages of dendritic swarming in Bacillus subtilis, with a novel role for phrC. Microbiology, 155, pages 398-412

24. Hilton, A.C., & Austin, E., (2000) The kitchen dishcloth as a source and vehicle for foodborne pathogens in a domestic setting. International Journal of Environmental Health Research, 10, (3), pages 257-261

25. Hong, S.M., Park, J.K., & Lee Y.O, (2004) Mechanisms of Microwave Irradiation involved in the Destruction of Faecal Coliforms from Biosolids. Water Research, 38, (6), pages 1615 - 1625

26. Irwin, D.J., Rao, M., Barham, D.W., et al., (1993) An outbreak of infection with Salmonella enteritidis phage type 4 associated with the use of raw shell eggs. Communicable disease report, CDR review, 3, (13), pages R179-R183

27. Jin, T., Zhang, H., & Boyd, G., et al., (2008) Thermal resistance of Salmonella enteritidis and Escherichia coli K12 in liquid egg determined by thermal-death-time disks. Journal of Food Engineering, 84, pages 608-614

28. Josephson, K.L., Rubino, J.R., & Pepper, I.L., (1997) Characterization and quantification of bacterial pathogens and indicator organisms in household kitchens with and without the use of a disinfectant cleaner. Journal of Applied Microbiology, 83, (6), pages 737-750

29. Kim, S.Y., Shin, S.J., Song, C.H., et al., (2009) Destruction of Bacillus licheniformis Spores by Microwave Irradiation. Journal of Applied Microbiology, 106, (3), pages 877-885

30. Klein, G., Dartigalongue, C., and Raina, S., (2003) Phosphorylation-mediated regulation of heat shock response in Escherichia coli. Molecular Microbiology 48, (1), pages 269-285.

31. Kusumaningrum, H.D., Riboldi, G., Hazeleger, W.C, et al., (2003) Survival of Foodborne Pathogens on Stainless Steel Surfaces and Cross-Contamination to Foods. International Journal of Food Microbiology, 85, (3), pages 227-236

32. Madigan, M.T., Martinko, J.M., Dunlap, P.V., et al., (2005) Brock Biology of Microorganisms, 11th edition, Prentice Hall, United States of America, ISBN: 0132017849, pages 934-938.

33. Mattick, K., Durham, K., Domingue, G., et al., (2003) The Survival of Foodborne Pathogens during Domestic Washing-up and Subsequent Transfer onto Washing-up Sponges, Kitchen Surfaces and Food. International Journal of Food Microbiology, 85, (3), pages 213-226

34. Mendez, M.B., Orsaria, L.M., Philippe, V., et al., (2004) Novel roles of the master transcription factors Spo0A and sigma(B) for survival and sporulation of Bacillus subtilis at low growth temperature. Journal of Bacteriology, 186, (4), pages 989-1000

35. Mima, E.G., Pavarina, A.C., Neppelenbroek, K.H., et al., (2008) Effect of Different Exposure Times on Microwave Irradiation on the Disinfection of a Hard Chairside Reline Resin. Journal of Prosthodontics, 17, (4), pages 312-317

36. Movahedi, S., & Waites, W., (2000) A two-dimensional Ppotein gel electrophoresis study of the heat stress response of Bacillus subtilis cells during sporulation. Journal of Bacteriology, 182, (17), Pages 4758-4763

37. Msadek, T., Dartois, V., Kunst, F., et al., (1998) ClpP of Bacillus subtilis is required for competence development, motility, degradative enzymes synthesis, growth at high temperatures and sporulation. Molecular Microbiology, 27, (5), pages 899-914

38. Nakamoto, H., & Vigh, L., (2007) The small heat shock proteins and their clients. Cellular and Molecular Life Sciences, 64, (3), pages 294-306

39. Ojima, M., Toshima, Y., Koya, E., et al., (2002) Hygiene Measures Considering Actual Distributions of Microorganisms in Japanese Households. Journal of Applied Microbiology, 93, (5), pages 800-809

40. Olsen, C.M., (1965) Microwaves inhibit bread mold. Food Engineering, 37, (7), pages 51-53

41. Osaili, T.M., Griffis, C.L., Martin, E.M., et al., (2007) Thermal Inactivation of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in Breaded Pork Patties. Journal of Food Science, 72, (1), pages M56-M61

42. Pané-Farré, J., Jonas, B., Förstner, K., et al., (2006) The σB regulon in Staphylococcus aureus and its regulation. International Journal of Medical Microbiology, 296, pages 237-258

43. Papadopoulou, C., Demetriou, D., & Panagiou, A., et al., (1995) Survival of enterobacteria in liquid cultures during microwave radiation and conventional heating, Microbiological Research, 150, (3), pages 305-309

44. Park, D.K., Bitton, G., & Melker, R., (2006) Microbial Inactivation by Microwave Radiation in the Home Environment. Journal of Environmental Health, 69, (5), pages 17-24

45. Piggot, P.J, & Hilbert, D.W., (2004) Sporulation of Bacillus subtilis. Current Opinion in Microbiology, 7, (6), pages 579-586

46. Redmon, E.C., & Griffith, C.J., (2005) Consumer Food Handling in the Home: A Review of Food Safety Studies. Journal of Food Protection, 66, (1), pages 130-161

47. Sharma, M., Eastridge, J., & Mudd, C., (2009) Effective Household Disinfection Methods of Kitchen Sponges. Food Control, 20, (3), pages 310-313

48. Tonuci, L.R.S., Paschoalatto, C.F.P.R., & Pisani, R., (2008) Microwave Inactivation of Escherichia coli in Healthcare Waste. Waste Management, 28, (5), pages 840-848

49. Webb, B.C., Thomas, C.J., Harty, D.W.S, et al., (1998) Effectiveness of Two Methods of Denture Sterilization. Journal of Oral Rehabilitation, 25, (6), pages 416-423

50. Woo, I., Rhee, I., & Park, H., (2000) Differential Damage in Bacterial Cells by Microwave Radiation on the Basis of Cell Wall Structure. Applied and Environmental Microbiology, 66, (5), pages 2243-2247

51. Yaghmaee, P., & Durance, T.D., (2005) Destruction and Injury of Escherichia coli during Microwave Heating Under Vacuum. Journal of Applied Microbiology, 98, (2), pages 498-506

7. Appendix

7.1 Growth Media

1% Peptone Water:

1% of the total amount of water was suspended

Brilliant Green Agar, (BGA):

52g was suspended per 1 litre of water

Eosin Methylene Blue Agar, (EMB):

37.5g was suspended per 1 litre of water

Mannitol Salt Agar, (MSA):

111g was suspended per 1 litre of water

Nutrient Agar:

28g was suspended per 1 litre of water

Nutrient Broth:

25g was suspended per 1 litre of water

Tryptone Glucose Extract Agar:

Formula g/litre

‘Lab-Lemco' powder 3.0

Tryptone 5.0

Glucose 1.0

Agar 15.0

pH 7.0 ± 0.2

All solutions were autoclaved at 121oC for 15 minutes, (apart from Brilliant Green Agar which was microwaved until it was just 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 twelve results, (three for each of the four microwave treatments) the degrees 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 results are significantly different and the null hypothesis stating that the means are the same can be rejected.

Tukey's HSD post hoc test

As the pairs of results which are significantly different according to ANOVA need to be identified, Tukey's HSD post hoc test was conducted to do so. For this test the honestly significant difference, (HSD) value needs to be identified as this is what the difference of the paired means from the results will be compared to. To calculate the HSD value this formula is used:

* ‘MS within' is the value taken from the ANOVA table.

* ‘n' is the number of groups

* ‘q' is the studentised range statistic which was taken from the studentised range statistic table. The value ‘q' is obtained by identifying the number of values in each of the groups on the top of the table and the degrees of freedom on the side and then the value for P < 0.001 or P < 0.05 can be chosen. In this case the latter value was chosen.

For E. coli ‘q' is 4.26 as the degrees of freedom were 11 and the number 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 treatment.

Measure of Effect

The formula for the measure of effect is:

* The ‘SS Between', ‘SS Total' and ‘MS Within' are taken from the ANOVA table.

* ‘k' is the number of groups.

‘k' in this case is 4 so:

So 99.6 % of the variance for E. coli cell survival was due to the microwave treatment in which it received.

Salmonella typhimurium (for 10-1 dilution)

As there were twelve results, (three for each of the four microwave treatments) the degrees 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 results are significantly different and the null hypothesis stating that the means are the same can be rejected.

Tukey's HSD post hoc test

To calculate the HSD value this formula is used:

‘q' = 4.26

All of the mean differences are found to be significantly different.

Measure of Effect

The formula for the measure of effect is:

‘k' in this case is 4 so:

So 99.9 % of the variance for Salmonella typhimurium cell survival was due to the microwave treatment in which it received.

Staphylococcus aureus (for 10-1 dilution)

As there were twelve results, (three for each of the four microwave treatments) the degrees 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 results are significantly different and the null hypothesis stating that the means are the same can be rejected.

Tukey's HSD post hoc test

To calculate the HSD value this formula is used:

‘q' = 4.26

All of the mean differences are found to be significantly different apart from the difference between the control and the 15-second treatment, the control and the 30-second treatment and the 15-second treatment and the 30-second treatment.

Measure of Effect

The formula for the measure of effect is:

‘k' in this case is 4 so:

So 96.1 % of the variance for Staphylococcus aureus cell survival was due to the microwave treatment in which it received.