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Common bacteria isolated from cellulose based kitchen sponges collected from college campus and effects of microwave exposure on bacterial reduction

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COMMON BACTERIA ISOLATED FROM CELLULOSE BASED KITCHEN
SPONGES COLLECTED FROM COLLEGE CAMPUS AND EFFECTS OF
MICROWAVE EXPOSURE ON BACTERIAL REDUCTION.
By
Andrew J. Kirlis
Quinnipiac University, 2012
A Thesis
Presented to College of Arts and Sciences and Quinnipiac University as Thesis research
August 15, 2012
UMI Number: 1524920
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ABSTRACT:
ANALYSIS OF COMMON BACTERIAL FLORA IN KITCHEN SPONGES
Andrew Kirlis
Master of Molecular and Cellular Biology
College of Arts and Sciences
Quinnipiac University
August 2012
Cellulose based kitchen sponges are a commonly utilized cleaning tool for kitchen
maintenance. Sponges provide a perfect environment for bacterial growth through food
particles and moisture held within. Through analysis of the sponges collected from
college students, common bacteria were isolated on TSA, MSA, XLD, and MacConkey
agars. Effects of microwave radiation exposure on microbial inactivation after 30 and 60
seconds were observed revealing a significant decrease in overall CFU/g of bacteria. TSA
showed a decrease in 89.33% of total colony for units per gram (CFU/g) after 30 seconds
of microwave exposure and 94.73% after 60 seconds. Similar results from MSA plates
showed an overall decrease in CFU/g by 72.11% after 60 seconds, 98.03% decrease of
gram negative rod bacteria on MacConkey agar, and 98.07% decrease on bacteria grown
on XLD agar which included Salmonella. Lactose fermenting bacterial colonies,
commonly opportunistic pathogenic fecal coliform bacteria, were shown to be decreased
by 10.65% and 54.05% after 30 seconds and 60 seconds of exposure respectively. Data
involving microwave radiation exposure showed evidence supporting that overall all
bacteria counts were decreased after microwave exposure, with gram negative rod shaped
bacteria seemingly being the most susceptible to microwave radiation.
2
COMMON BACTERIA ISOLATED FROM CELLULOSE BASED KITCHEN
SPONGES COLLECTED FROM COLLEGE CAMPUS AND EFFECTS OF
MICROWAVE EXPOSURE ON BACTERIAL REDUCTION.
This thesis is approved as creditable and independent investigation by a candidate for the
degree of Master of Molecular and Cellular Biology, and is acceptable as meeting the
thesis requirements for this degree, but without implying that the conclusions reached by
the candidate are necessarily the conclusions of the major department.
Thesis Advisor _________________________________________________________
Lisa A. Cuchara, Ph. D.
Professor, Biomedical Sciences
Quinnipiac University
Quinnipiac University Thesis Committee Member ______________________________
Tom Martin, Ph. D.
Assistant Professor, Biomedical Sciences
Quinnipiac University
Director, Molecular and Cell Biology Graduate Program _________________________
Sarah J. Berke, Ph.D.
Assistant Professor, Biological Sciences
Quinnipiac University
3
ACKNOWLEDMENTS
For contributions in allowing the progression of this research collected I would
like to thank Dr. Lisa Cuchara for all of her contributions, help, and support through this
time. I would also like to thank Lindsay Musgrove and Patrick McInnis for working with
me in collection of the microwave sponge data presented in this thesis. Without their help
this thesis would not be what it is. I would also like to thank Professor Linda Post for
offering support whenever needed and Debbie Greenblatt for continually ordering the
media needed for this research and being around for.
4
TABLE OF CONTENTS
Page
INTRODUCTION……………………………………………………………………….11
Statement of the Project..……….………………………..…………………........11
Literature Review…………………………………………………………….......11
MATERIALS AND METHODS…………………..……………………………..…….. 22
Sample Collection………………………………………………………………..22
Sample Plating and Culture……………………………………………………...22
Media…………………………………………………………………………….23
Initial Test Run ………………………………………………………………….25
Macroscopic and Microscopic Observation……………...……………………...25
16s Ribosomal RNA Sequencing………....…………………………….……….26
RESULTS………………………………………………………………………………..27
DISCUSSION…………………………………………………………………………....74
Data Analysis…………………………………………………………………….74
Overall Conclusions……………………………………………………………...84
Future Studies……………………………………………………………………88
REFERENCES…………………………………………………………………………..90
APPENDICES…………………………………………………………………………...99
5
LIST OF TABLES
Table 1. Sponges collected with corresponding times used …….………………..……..27
by owner and if disinfection had occurred and by what means
Table 2. Trypticase soy agar quantitative plate counts for ………………………...........29
before microwave sponges
Table 3. Mannitol salt agar quantitative plate counts for ………………………….........30
before microwave sponges
Table 4. MacConkey agar quantitative plate counts for……………………...……….....31
before microwave sponges
Table 5. Xylose lysine deoxycholate quantitative plate………………………….….......32
counts for before microwave sponges
Table 6. Average colony forming unit per gram (cfu/g)…………………..………….…33
recorded for sponges before microwave exposure on each media utilized
Table 7. Trypticase soy agar quantitative plate counts…………………….……............35
for sponges subjected to 30 seconds of microwave exposure
Table 8. Mannitol salt agar quantitative plate counts …………………………...…...….36
for sponges subjected to 30 seconds of microwave exposure
Table 9. MacConkey agar plate counts for sponges ……………………………….........37
subjected to 30 seconds of microwave exposure
Table 10. Xylose lysine deoxycholate agar quantitative…….…………………………..38
plate counts for sponges subjected to 30 seconds of microwave exposure
6
LIST OF TABLES CONTINUED
Table 11. Average colony forming unit per gram (cfu/g)…………………………..…...39
recorded for sponges 30 seconds after microwave exposure on each media utilized
Table 12. Trypticase soy agar quantitative plate counts …………………….……….....43
for sponges subjected to 60 seconds of microwave exposure
Table 13. Mannitol salt agar quantitative plate counts ………………………….......…..44
for sponges subjected to 60 seconds of microwave exposure
Table 14. MacConkey agar quantitative plate counts………………………………...…45
for sponges subjected to 60 seconds of microwave exposure
Table 15. Xylose lysine deoxycholate agar quantitative…………………………...……46
plate counts for sponges subjected to 60 seconds of microwave exposure
Table 16. Average colony forming unit per gram (cfu/g) …………………………..…..47
recorded for sponges 60 seconds after microwave exposure on each media utilized
Table 17. Recorded average cfu/g on each media in…………………………………….52
comparison of before, 30 seconds, and 60 seconds of microwave exposure
Table 18. Comparison of average cfu/g decrease for……………………………………58
before microwave to after 30 seconds microwave subjection
Table 19. Comparison of average cfu/g decrease for …………………………………...59
before microwave to after 60 seconds microwave subjection
7
LIST OF TABLES CONTINUED
Table 20. Average of lactose fermenting colonies found………………………………..64
on before microwaving, after 30 seconds, and after 60 seconds plates, with calculated
percentage of bacteria fermenting lactose decrease in comparison to before microwaving
total colonies
Table 21. Mannitol salt agar common colonies………………...………………...……..66
macroscopically and microscopically described
Table 22. MacConkey agar common colonies………………………..…………………68
macroscopically and microscopically described
Table 23. Xylose lysine deoxycholate agar common ………………………………...…72
colonies macroscopically and microscopically described
8
LIST OF FIGURES
Figure 1. Percentage of prior disinfected sponges upon collection……………...……..28
Figure 2. Average log CFU/g on each media before microwaving……………………..34
Figure 3. TSA CFU/g Comparison of before microwave to 30 seconds………………..40
Figure 4. MacConkey CFU/g Comparison of before microwave to 30 seconds………..41
Figure 5. XLD CFU/g Comparison of before microwave to 30 seconds……………….42
Figure 6. TSA CFU/g Comparison of before microwave to 60 seconds………………..48
Figure 7. MSA CFU/g Comparison of before microwave to 60 seconds……………….49
Figure 8. MacConkey CFU/g Comparison of before microwave to 60 seconds………..50
Figure 9. XLD CFU/g Comparison of before microwave to 30 seconds……………….51
Figure 10. Comparison of Average CFU/g of before to 30 seconds on all media……....53
Figure 11. Photograph of TSA Colonies before Microwave Exposure…………………54
Figure 12. Photograph of TSA Colonies after 30 seconds of Microwave Exposure……54
Figure 13. Photograph of MacConkey Colonies before Microwave Exposure…………55
Figure 14. Photograph of MacConkey Colonies after 30 seconds of Microwave
Exposure…………………………………………………………………………………55
Figure 15. Comparison of Average CFU/g of before to 60 seconds on all media………56
Figure 16. Photograph of XLD Colonies before Microwave Exposure………………...57
Figure 17. Photograph of XLD Colonies after 60 seconds of Microwave Exposure…...57
Figure 18. TSA Percent inhibition of CFU/g 30 and 60 seconds compared to before….60
Figure 19. MSA Percent inhibition of CFU/g 30 and 60 seconds compared to before....61
Figure 20. MacConkey Percent inhibition 30 and 60 seconds compared to before……..62
9
LIST OF FIGURES CONTINUED
Figure 21. XLD Percent inhibition of CFU/g 30 and 60 seconds compared to before.....63
Figure 22. Average Lactose fermenting colonies isolated from MacConkey agar ……..65
Figure 23. Mannitol fermenting colonies isolated from MSA…………………………..67
Figure 24. Percent differential lactose fermenting colonies isolated before exposure….69
Figure 25. Percent differential lactose fermenting colonies isolated after 30 seconds….70
Figure 26. Percent differential lactose fermenting colonies isolated after 60 seconds….71
Figure 27. Percent XLD plates positive for Salmonella………………………………...73
10
INTRODUCTION
Research shows the possibility that many pathogenic bacteria associated with
foodborne illness reside in common cellulose kitchen sponges used in everyday cleaning.
Cellulose based kitchen sponges, made primarily out of cellulose wood fibers, are
commonly utilized in the process of cleaning dishes, stoves, and counters in everyday
cleaning and maintenance of the kitchen area, also known as “washing up” (Mattick et
al., 2003). Raw ingredients are commonly contaminated with foodborne pathogens that
spread to sponges, which research states that the process of washing-up is a control point
for preventing cross contamination in this environment (Mattick et al., 2003). Dishcloths
or sponges containing high concentrations of pathogens may be reservoirs and
disseminators of bacterial contamination in the kitchen and have been recognized for the
potential to spread microorganisms (Ikawa and Rossen, 1999).
Various studies have shown that the risk of transfer from contaminated dishes to
food was low, but the contamination of towels and washing-up sponges used to wipe
hands and work surfaces was more of a concern in connection to the spread of foodborne
illness related pathogens (Mattick et al., 2003). Bacteriological surveys conducted for
kitchen dishcloths or sponges have revealed the presence of many enteric pathogens
(Rossen, 1999). Enteric bacteria are gastrointestinal organisms commonly spread through
contamination of food products, such as Escherichia coli, Klebsiella pneumoniae,
Enterobacter cloacae, Salmonella spp., Staphylococcus aureus, and Pseudomonas spp.
with enteric pathogens shown to survive and multiply in wet environments such as
sponges (Rossen, 1999). While kitchen surfaces can grow bacteria necessary for
11
contamination of sponges, moist sponges provided an ideal candidate for harboring
bacteria, in which conditions are optimal for the growth of many genuses of bacteria,
harmless and opportunistic pathogenic.
By providing moisture and food particles as nutrients sponges provide a good
candidate for harboring gram negative bacteria such as Escherichia coli, Salmonella spp.,
and Camplylobacter spp. which are commonly linked to foodborne illness (Erdogrul and
Ferya, 2000; Humphrey et al., 2001). Commonly isolated from cellulose based kitchen
sponges have been bacteria such as Pseudomonas aeruginosa, Klebseilla pneumonia,
Bacillus spp., Dipthreoids spp., Enterobacter cloacae, and Staphylococcus epidermidis,
and Staphylococcus aureus (Alwakeel, 2007; Beumer and Kusuminingrum, 2003). A
study performed in the United Kingdom revealed the isolation of Listeria spp. from
cellulose kitchen sponges, contributing to 19% of foodborne illness related deaths
(Erdogrul and Ferya, 2000; CDC, 2011). According to Beumer et al. (1996), Listeria spp.
was also detected in 101 of 213 houses (47%) with Listeria occurring at all sampling sites
more in particular dishcloths (37%) and surfaces around drain in kitchen and bathrooms
(27%) (Beumer et al., 1996).
In relation to foodborne illness, the Center for Disease Control and Prevention
reported an average of 47.8 million cases a year, with the pathogens resulting in 127,839
hospitalizations and 3,037 deaths per year (CDC, 2011). Enriquez, et al. (1997) revealed
that twenty three species of bacteria have been commonly found in kitchen sponges
comprising 15% Salmonella spp., 36% Pseudomonas spp., and 20% Staphylococcus spp.
(Erdogrul and Ferya, 2000). Also reported in the CDC database was that Salmonella spp.,
commonly associated with kitchen sponges, is reported as being second in the top five
12
pathogens list contributing to the contraction of foodborne illness, revealed to be
involved in 11% of foodborne illness cases, with number one being Norovirus (CDC,
2011). Salmonella spp. alone was reported as the top bacterial species resulting in
foodborne illness hospitalizations, with an estimated number of 19,336 cases a year,
approximately 35% of all foodborne related hospitalizations (CDC, 2011).
A study conducted in England and Wales found that more than 80% of reported
Salmonella and Campylobacter infections were acquired at home (Scott, 1999). It is
estimated that if one person in a household becomes sick with a Salmonella infection,
there is a 60% chance that at least one other family member will also become infected
due to either direct or indirect cross-contamination within the home (Scott, 1999). In
1990, 2766 Salmonella outbreaks were reported in which 86% were classified as
“family” outbreaks, only affecting members of a single family (Scott, 1996).
Campylobacter spp., is shown to be associated with 9% of reported cases of foodborne
illness (Humphrey, 2001; CDC, 2011). In the review by Scott (1995), 1097
Campylobacter outbreaks were recorded, 97% of which were “family outbreaks” (Scott,
1996). In a study conducted by Mattick, et al., (2003), swabs, testing for the most
prominent bacteria, were taken from kitchen sponges and surfaces wiped by the sponges
collected which revealed that Salmonella spp. was shown to grow on a 104 dilution plate,
diluted to 1/10000 original concentration, and found on 3 of 3 swabs collected, and
Escherichia coli shown to grow on 103 plate and found on 5 of 6 swabs; additionally the
sponges were rinsed in broth and plated in an effort to culture all bacteria present in
sponges in which E. coli and Salmonella spp. were found in each sponge (Mattick et al.,
2003).
13
Escherichia coli, being the most commonly reported bacterium in sponges, was
isolated in studies such as Mattick et al. (2003), Simonne (2007), Park et al. (2006),
Carrasco (2006), Humphrey et al. (2001), and Beumer and Kusuminingrum (2003), and
has been reported to being associated with foodborne related hospitalization, comprising
of 4% of all foodborne related hospitalizations (CDC, 2011). A study by Carrasco et al.,
(2008) revealed that 51% of sponges collected from household kitchens from El Paso,
Texas and 48% of those collected from Cuidad Juarez, Mexico were shown to be positive
for fecal coliforms (Carrasco et al., 2008). Fecal contamination is shown through the
presence of fecal coliform bacteria E. coli present in the sponge, with this bacterial
species being normally living in the lower intestines of warm blooded humans and
animals and is known to be an opportunistic pathogenic bacterial species (CDC, 2012).
Noted in the study was that 60% of sponges, 40% of sink knobs, and 28% of countertops
tested positive for fecal coliform contamination (Carrasco et al., 2008).
Pathogenicity is the ability to produce disease in the host organism. The reason
for pathogenicity is that gram negative bacteria, some of which are classified as
pathogenic bacteria, commonly inhabit sponges. Gram negative bacteria are made up of
two layers, the cytoplasmic membrane and a thin peptidoglycan layer, contributing to 5%
to 10% of the gram negative cell (Moen and Gajda, 2006). Between the external surface
of the cytoplasmic membrane and the internal surface of the outer membrane is the
periplasmic
space
containing
lytic
virulence
factors,
such
as
collagenases,
hyaluronidases, proteases, and beta-lactamase, but more notably containing amphipathic
molecule lipopolysaccharide (LPS) (Moen and Gajda, 2006). LPS endotoxin is shown to
be a powerful stimulator of immune responses, in which shedding of this layer activates
14
B-cells and induces macrophage and other cells to release interleukin-I and interleukin-6,
tumor necrosis factor, and other factors in the host resulting in fever and shock (Moen
and Gajda, 2006). Due to the molecule lipopolysaccharide associated with the outer
membrane of gram negative bacteria, these bacteria have been shown to be more readily
associated with pathogenicity than gram-positive bacteria.
Of the bacteria isolated in the studies reviewed, it was shown that most were gram
negative bacterial species, many being opportunistic pathogenic bacteria. Opportunistic
pathogenic bacteria are bacterial species with the potential to cause disease in immunecompromised hosts, due to lack of host defense, which would typically not occur in
healthy individuals (Todar, 2008). One notable bacterial species isolated was
Pseudomonas aeruginosa, an opportunistic pathogenic bacterial species shown to be
linked to urinary tract infections, respiratory system infections, dermatitis, soft tissue
infections, bacteremia, bone and joint infections, and gastrointestinal infections (Todar,
2008). Pseudomonas aeruginosa being the most common bacterium in regards to cross
contamination from kitchen sponges, and a variety of systemic infections in immunesuppressed patients and those with cystic fibrosis (Todar, 2008).
Additional opportunistic bacterial species isolated were Listeria spp. linked to
listeriosis being an infection occurring when an individual ingests food contaminated
with Listeria monocytogenes resulting in conditions such as pneumonia, meningitis,
septicemia, and endocarditis (Listeriosis, 2011). This bacterial species is shown to be
troublesome to individuals with weakened immune systems, elderly, infants, and women
during pregnancy due to affecting immuno-compromised individuals more readily than
healthy counterparts (Listeriosis, 2011).
15
Campylobacter spp. is shown to be commonly linked to gastroenteritis, or
inflammation of the gastrointestinal tract. Staphylococcus aureus, a primary source linked
to food poisoning if swallowing food or water contaminated with this bacterial species,
leading to food poisoning resulting in diarrhea, fever, chills, vomiting through the
absorption of bacterial endotoxins by the gastrointestinal system (Food Poisoning, 2011).
Escherichia coli linked to hemorrhagic colitis, inflammation of the colon resulting
in hemorrhaging, and hemolytic uremic syndrome, induced production of toxic
substances by the digestive system resulting in destroyed red blood cells; Enterobacter
spp. linked to urinary and respiratory tract infections; Salmonella spp. linked to
salmonellosis, food poisoning infection resulting from infection of Salmonella spp.
resulting in muscle pain, vomiting , diarrhea, nausea; and Klebsiella pneumonia linked to
septicemia (Todar, 2008). Staphylococcus aureus has been shown to be the second
common cause of foodborne disease outbreaks behind Salmonella and as prevalent as
Clostridium perfringens (Gorman, 2002). Sponges harboring pathogenic bacteria are a
health risk concern; they are commonly involved in spreading and cross contamination of
microbes around with possible contaminating of people through food. Without proper
methods of disinfection and inactivation of these bacteria many unknowing individuals
could easily infect themselves with pathogenic microbes resulting in possible sickness
which could easily be avoided through proper disinfection.
Common methods of bacterial decontamination have been investigated and
compared in regard to efficiency, in which microwaves and dishwashers are the most
commonly used methods of disinfection in households today. Both of these processes
utilize extreme heat in order to either cook and/or sanitize, which also provide proper
16
conditions in which most bacteria can be inactivated due to the extreme conditions.
Microwave ovens convert high-voltage electricity into electromagnetic energy waves
which targets the moisture in food by vibrating, creating heat which kills off bacteria
subjected to exposure. Other viable remedies for bacterial sanitation have been
introduced, such as lemon juice, bleach, and deionized water, which was shown to reduce
bacterial growth by 37-87% (Durham, 2007).
However, compared to microwave and dishwasher, other methods discussed were
considered not an effective means of disinfection. Disinfection of cloths with a common
household hypochlorite-cleaning product was shown to be effective in reducing or
interrupting this cross-contamination, but not as practical as the microwaving method of
disinfection (Rusin et al., 1998).
Hypochlorite disinfectants have been found to be more effective than phenolic
disinfectants on kitchen and bathroom surfaces. Ammonium compounds were shown to
significantly reduce total bacterial numbers on surfaces (Rusin et al., 1998). Boiling was
also shown to yield greater than 99.9% reduction of bacteria in cellulose based sponges
(Rossen, 1999). Dishwashing method of disinfection was shown to have killed 99.9998%
of bacteria, while the microwave method, microwaving sponge for 1 minute, was shown
to have killed 99.9999% of bacteria initially found on the sponge (Durham, 2007).
Research published mostly state that microwave induced bacterial decontamination
occurs around 1-2 minutes, with a decrease in bacteria by 99.9% (Mattick et al., 2003).
More notably, dishwashing without detergent resulted in adequate reduction of bacteria,
99.9%, in consumer-used sponges (Rossen, 1999).
17
Debate on adequate time for microwave radiation induced inactivation and
decontamination of bacteria has been a topic of concern regarding time for optimal antibactericidal activity. The method of using microwave radiation as a method for bacterial
decontamination has been an effective way of decontamination, with 99% of bacteria
were reduced in 1-2 minutes with total coliform inactivation after 30 seconds of
microwaving (Park et al., 2006). Coliforms are known as lactose fermenting gram
negative bacteria, involving the genuses Citrobacter, Serratia, Hafnia, Enterobacter,
Escherichia and Klebsiella, associated with the normal human flora (CEHS, 2012). After
30 seconds of microwave exposure, no living bacterial cells were found in full size on a
dry sponge, in which it was also noted that two minutes of microwave exposure was
enough to kill most bacteria on a moist sponge (Simonne, 2007). It is thought that the
longer bacteria are subjected to microwave radiation the more bacteria were killed, but on
average one and a half minutes were shown to be the time range for ideal
decontamination (Park et al., 2006). Additionally, yeast and mold growth were shown to
be reduced by 99% after microwave radiation treatment (Durham, 2007). According to
research reviewed, through the use a microwave bacterial load in a cellulose kitchen
sponge will decrease.
Further study into common bacteria found in kitchen sponges and the effects of
microwaving on bacterial growth should be pursued. Studies conducted have identified
some species of bacteria found in kitchen sponges, but no research has been published in
the past few years, with many studies being conducted in other parts of the world. Some
data was collected from studies in Saudi Arabia and the United Kingdom which are still
viable information concerning this research, but different demographic locations harbor
18
different bacterial species which could lead to different bacteria being isolated.
Additionally some sponges were not stated as to which type of population they were
collected from and could be an entirely different demographic, themselves leading to
varying bacteria isolated not commonly associated with kitchen sponges.
16s Ribosomal RNA analysis is a method commonly used for identification of
bacteria in which variable regions of the DNA sequence are utilized for identification. In
the 1980’s, it was proposed that a new method of bacterial species identification could be
developed based on the idea that phylogenetic relationships between species could be
determined by comparing to genetically conservative regions of the genetic code
(Clarridge, 2004). Original candidates for these stable parts were genes coding for the 5s,
16s, and 23s rRNA subunits, with the 16s now being more readily associated in this
phylogenetic taxonomy procedure. This 16s rRNA gene is shown to be comparable with
all bacteria, but notably to the 16s rRNA gene of archeobacteria and 18s rRNA gene of
eukaryotes (Clarridge, 2004). In a study conducted by Dubnau et al., (1965) involving
study into genus Bacillus, it was observed that ribosomal and 16 sRNA genes seem to be
phylogenetically invariant in bacteria, which suggested the existence of a core of stable
genetic material in the genus Bacillus (Dubnau et al., 1965). Utilizing Bacillus spp. it was
noted that conservation of the sRNA and ribosomes were general, in which base
composition, secondary structure, molecular weight, presence of unusual bases, and
functional properties of sRNA are fairly constant in a wide variety of organisms (Dubnau
et al., 1965). Using this information collected Dubnau et al. (1965) adapted the idea that
if the genetic code was universal three bases comprising the anticodon should be the
same in corresponding sRNA molecules from different organisms, concluding that
19
evidence suggests all sRNA sample examined contain a common penta-nucleotide
sequence (Dubnau et al., 1965). Evolutionary conservation of 16s RNA and rRNA was
speculated to be possible due to the genetic code evolved early along with certain
components of translation mechanism and also that rRNA serves additionally to fold in a
specific manner to form ribosomes in which primary sequence efficiently serving both
functions may be limited (Dubnua et al., 1965). Although the absolute rate of change in
the 16s rRNA gene sequence is not known, it does mark evolutionary distance and
relatedness of organisms (Clarridge, 2004).
In observation of the 16s rRNA gene it is shown to be about 1,550 base pairs long
with variable and conserved regions. For analysis of the sequence, primers are chosen
complementary to conserved regions at the beginning of the gene and either at the 540
base pair region or at the end of the sequence, which the variable sequence in-between is
used for phylogenetic taxonomy (Clarridge, 2004). This method of analysis has been
shown to be successful in differentiation between organisms at genus level across all
major phyla of bacteria with classification possible down to species level (Clarridge,
2004). Through using this method of sequencing, highly conserved regions involved with
the 16s rRNA gene can be utilized in primer creation and variable regions in between can
be analyzed for classification down to the species level.
Through this research presented, common bacteria found from a particular
demographic, college students living in dorm rooms built for 5-7 individuals, were
isolated using a series of serial dilutions and various culturing media. This research tested
effects of microwave radiation on bacteria isolated from sponges at intervals of 30 and 60
seconds to determine the effects of various times on the disinfection and inhibition of
20
bacteria contained. Additionally bacteria could be identified using 16s ribosomal RNA
sequencing as to acquire data on the most common bacteria found in sponges.
The purpose of the proposed thesis was to identify common bacterial flora that
inhabit cellulose wood fiber based kitchen sponges and to additionally observe the effects
of 30 and 60 seconds of microwave radiation on the growth and inhibition of bacterial
species contained. Fifty sponges were collected from college students living in an oncampus dorm room of 5-7 individuals and analyzed revealing how many and what types
of bacteria inhabit cellulose based kitchen sponges using quantitative plate counts and
16s ribosomal RNA sequencing. Additionally, the inhibition of bacterial growth on
sponges subjected to varying times of microwave radiation was observed. Bacteria
calculated by colony forming units/gram were compared on sponges collected without
microwave exposure to sponges subjected to microwaving treatment at intervals of 30
seconds and 60 seconds. Each sponge was observed and recorded, and at the end of the
research, bacterial species were identified and the effects of microwave bactericidal
activity analyzed.
21
MATERIALS AND METHODS
Sample Collection:
Common bacterial flora in kitchen sponges of college students were collected,
cultured, and identified. Fifty currently in use kitchen sponges (moist) were collected (six
sponges a week) from students who volunteered their sponges for further analysis.
Length of time of sponge use was initially recorded and only recently used sponges were
accepted for testing, exempting dried kitchen sponges not recently used in everyday
kitchen cleaning activities. Additionally, only sponges used for kitchen based activities,
such as cleaning dishes and counters, were accepted in order for limit bacteria colonizing
to sinks and kitchen areas. Sponges found to have been used in the bathroom were
excluded from further testing or analysis due to different bacteria colonizing these two
different areas of the common household.
Sample Plating and Culture:
Upon collection of six sponges a week, 1 gram of cut sponge was placed in 9
milliliters (mL) of 1.25% phosphate buffer solution in which serial dilutions from 10-1 to
10-6 were made for plating on various selective and differential media. Media selected for
culturing common sponge bacterial flora were Trypticase Soy Agar (TSA), Mannitol Salt
Agar (MSA), MacConkey Agar, and Xylose Lysine Deoxycholate Agar (XLD). Each
agar was chosen carefully in regard for its selective and differential properties, only
allowing specific colonies to grow and differentiation through tests associated with the
media in order for the isolation of various types of bacteria thought to colonize kitchen
22
sponges. Plating was conducted through the process of pipetting 1mL or 0.1mL of 10mL
sponge-phosphate buffer dilution based upon what dilution the plates needed. For 10-1
dilution plate, 1mL of the serial dilution 10-1 was taken and plated using a sanitized glass
rod in order to spread the dilution on the plate. Other plates were plated through the use
of 0.1mL of serial dilution in order to create plate dilutions 1/10 more dilute, ex. 0.1mL
of 10-1 dilution was taken and spread to create plate dilution 10-2 and 1mL of 10-1 dilution
was taken and spread to create a plate dilution of 10-1. Three dilution plates were made
for each sponge by pipetting 0.1mL of 10-1 more concentrated dilution in which dilutions
10-4, 10-5, and 10-6 were first plated on TSA, XLD, and MacConkey agars in which
dilutions were adjusted during re-plating to more or less diluted according to how much
bacterial growth would be found. MSA dilutions used for plating were 10-1, 10-2, and 10-3
due to showing less overall growth than the other media.
Media:
Trypticase Soy Agar, being a general-purpose media, is commonly referred to as
Soybean-Casein Digest Agar USP23 and is a nutritional base with supplements used as a
universal culturing media. This was the first agar to have been chosen due most types of
bacteria being able to readily grow on this media.
Mannitol salt agar (MSA) was used, being both a selective and differential media
containing the carbohydrate mannitol and phenol red indicator. MSA is selective due to a
high 7.5% concentration of sodium chloride (NaCl) which creates an extreme
environment that not many bacterial species can survive (Beaver, 2000). Differential
mannitol fermentation is observed when an acidic byproduct is formed causing the
23
phenol red indicator to turn yellow. Only the genus Staphylococcus is well adapted to
these environmental conditions and can utilize this media for proper growth. MSA is a
differential media with an indicator of phenol red, turning yellow if the Staphylococcus
bacterial species produce organic acids from utilizing the mannitol supplied, resulting in
fermentation. Fermentation noted by bacterial colonies is considered positive for
pathogenic Staphylococcus species presenting the idea that kitchen sponges are capable
of contain pathogenic bacteria (Beaver and Rutter, 2000).
MacConkey agar is a selective and differential culturing media containing bile
salts and crystal violet. MacConkey is selective for gram negative bacteria, inhibiting
gram-positive bacterial growth. Differential aspects are containing neutral red indicator,
being a differential test indicating possible lactose fermentation of the cultured bacteria.
This media was chosen because gram negative bacteria are able to readily grow on this
media, comprising most of sponge flora, and the ability to detect lactose fermentation,
being a notable characteristic of opportunistic pathogenic coliforms (Beaver and Rutter,
2000). Lactose fermentation is most notably associated with coliforms which are known
opportunistic pathogenic gram negative bacterial species able to cause harm to immunecompromised hosts.
Xylose Lysine Deoxycholate (XLD) agar was utilized for its ability to isolate and
differentiate enteric bacilli, being gram negative bacteria commonly found in the
intestines. This agar was chosen due to its selective ability to colonize genus Salmonella
spp. and Shigella spp., with Salmonella being normally associated with kitchen sponges
(Neogen corp., 2008).
24
Initial Test Run:
Upon careful consideration of the media to be used in the culturing of the kitchen
sponge bacteria, a test run was performed in order to obtain preliminary information
about what dilutions to use for each plate and if the media made would properly grow the
plated bacteria. This section was conducted in order to limit the amount of media used in
this research by instead of making six media plates at a time only three were created from
preliminary investigation.
The same procedure was followed for 1 gram of sponge subjected to a 1000-watt
microwave for 30 seconds. Serial dilutions from 10-1 to 10-6 were plated on each specific
media and cultured at 37oC for 48 hours in the incubator. Colony forming units per gram
(CFU/g) were then counted, and if colonies shown were 30-300 CFUs then serial
dilutions most suitable for accurate counts in subsequent sponge analysis were
determined.
Macroscopic and Microscopic Observation:
Each colony was then macroscopically assessed for specific morphological
characteristics and gram stained. After an initial test run, six new sponges were collected
all with the same requirements as the previously collected batch and subjected to analysis
through preparation of serial dilutions. The same process was followed as the previous
six sponges with the addition that each batch would alternate between 30 seconds and one
minute of microwaving in order to test the effects of different microwaving times on the
culturing of bacteria. One minute of exposure was conducted every other week. Colony
25
Forming Units (CFU) on plates were counted and recorded before and after microwaving
and after to determine the effects of microwaving on bacteria.
Bacterial colonies on each plate were morphologically categorized and compared
to the bacteria most commonly found on the preceding plates, and noted as to which
plates they were observed on. This process was conducted for all 50 sponges collected.
16s Ribosomal RNA Sequencing:
DNA isolated from 10 sponges, before and after microwaving, 20 samples total
were processed using a bacterial mini-prep kit from Mo-Bio Laboratories Inc. in
Carlsbad, California using a series of solutions and steps in the isolation of DNA from the
organisms. The series of solutions included Solutions 1 through 4 contain RNase, SDS
detergents, alkaline cell lysis solutions, potassium acetate and salt solution, Ethanol, and
10mM Tris buffer all utilized in proper DNA extraction form the bacteria (Mo-Bio,
2012). Steps were followed for the isolation of the bacterial DNA present in the sponges
and sent to the Functional Biosciences Inc. laboratory in Madison, Wisconsin for 16s
ribosomal RNA PCR and sequencing.
Upon receiving results, analysis of the sequences using Molecular Evolutionary
Genetics Analysis (MEGA) software was performed revealing the bacteria present in the
20 sponges comparing both before and after microwaving. This software conducted
automatic sequence alignments in analysis of the sequence providing the most closely
related species. Upon extraction of the DNA, the MO-BIO kit MEGA software was used
to sequence the DNA isolated and classify the bacteria down to species level for all
bacteria present in 10 before microwave dilutions and 10 after microwave dilutions.
26
RESULTS
Table 1
SPONGES COLLECTED WITH CORRESPONDING TIMES USED BY OWNER
AND METHOD OF DISSINFECTION
Sponge #
n= 24
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Time used
Disinfected?
Method of disinfection
3 weeks
No
None
1 month
No
None
2 weeks
No
None
8 days
No
None
3.5 weeks
Yes
Microwave- Once a week
2 months
No
None
2 weeks
Yes
Microwave- once a week
3 weeks
No
None
3 weeks
No
None
2 weeks
No
None
3 weeks
No
None
4 weeks
No
None
1 month
No
None
3 weeks
No
None
1 month
No
None
1 month
No
None
2 months
No
None
4 months
No
None
1 month
No
None
3 weeks
No
None
1 month
No
None
1 week
No
None
2 weeks
No
None
2 weeks
No
None
Footnote- This data was collected for 24/50 sponges collected
27
8.70%
91.30%
Disinfected Sponges
Sponges not disinfected
Sample size (n)= 24
Percentage of prior disinfected sponges upon collection
Figure 1. Percentage of sponges that were disinfected by students before collection versus
sponges that had not been disinfected prior to collection.
28
Table 2
QUANTITATIVE PLATE COUNTS OF SPONGES BEFORE MICROWAVE
DISINFECTION ON TRYPTICASE SOY AGAR (TSA)
Sponge#
2B
3B
4B
5B
6B
7B
8B
9B
10B
11B
12B
13B
14B
15B
16B
17B
18B
19B
20B
21B
22B
23B
24B
25B
26B
27B
28B
29B
30B
31B
32B
33B
34B
35B
36B
37B
38B
39B
40B
41B
42B
43B
44B
45B
46B
47B
48B
49B
50B
TSA
# Colonies
204
162
45
35
297
Too Few to Count
155
132
Too Many to Count
155
157
73
42
117
55
88
Too Few to Count
66
61
Too Many to Count
60
101
Too Few to Count
39
33
276
Too Many to Count
293
Too Many to Count
126
298
206
238
Too Many to Count
Too Many to Count
95
157
185
239
159
Too Many to Count
123
179
181
53
Too Few to Count
54
269
188
Plate dilution
10^-7
10^-7
10^-7
10^-7
10^-7
10^-5
10^-6
10^-6
10^-5
10^-6
10^-7
10^-6
10^-6
10^-5
10^-5
10^-6
10^-7
10^-6
10^-7
10^-3
10^-7
10^-6
10^-4
10^-7
10^-6
10^-6
10^-7
10^-6
10^-9
10^-7
10^-6
10^-8
10^-6
10^-7
10^-6
10^-7
10^7
10^-6
10^-7
10^-5
10^-7
10^-7
10^-7
10^-7
10^-7
10^-7
10^-7
10^-7
10^-7
29
Total count (CFU/g)
2.04x109
1.62x109
4.5x108
3.5x108
2.97x109
1.55x108
1.32x108
1.55x108
1.57x109
7.3x107
4.2x107
1.17x107
5.5x106
8.8x107
6.60x107
6.10x108
6.00x108
1.01x108
3.90x108
3.30x107
2.76x108
2.93x108
1.26109
2.98x108
2.06x1010
2.38x108
9.50x108
1.57x109
1.85x108
2.39x109
1.59x107
1.23x109
1.79x109
1.81x109
5.30x108
5.40x108
2.69x109
1.88x109
Table 3
QUANTITATIVE PLATE COUNTS OF SPONGES BEFORE MICROWAVE
DISINFECTION ON MANNITOL SALT AGAR (MSA)
MSA
Sponge#
2B
3B
4B
5B
6B
7B
8B
9B
10B
11B
12B
13B
14B
15B
16B
17B
18B
19B
20B
21B
22B
23B
24B
25B
26B
27B
28B
29B
30B
31B
32B
33B
34B
35B
36B
37B
38B
39B
40B
41B
42B
43B
44B
45B
46B
47B
48B
49B
50B
# Colonies
Too Few to Count
Too Few to Count
Too Few to Count
Too Few to Count
Too Few to Count
102
193
Too Few to Count
Too Few to Count
Too Few to Count
143
Too Few to Count
Too Few to Count
Too Few to Count
Too Few to Count
Too Few to Count
Too Few to Count
0
0
0
Too Few to Count
54
0
0
0
157
0
Too Few to Count
0
0
0
0
0
0
0
0
291
0
0
0
Too Few to Count
0
0
0
0
0
0
0
Too Few to Count
Plate
dilution
10^-1
10^-1
10^-1
10^-1
10^-1
10^-2
10^-2
10^-1
10^-1
10^-1
10^-2
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-3
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
30
Total count (CFU/g)
0
0
0
0
0
1.02x104
1.93x104
0
0
0
1.43x104
0
0
0
0
0
0
0
0
0
5.40x102
0
0
0
1.57x103
0
0
0
0
0
0
0
0
0
2.91x103
0
0
0
0
0
0
0
0
0
0
0
Differential mannitol
fermentation
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Table 4
QUANTITATIVE PLATE COUNTS OF SPONGES BEFORE MICROWAVE
DISINFECTION ON MACCONKEY AGAR
MacConkey agar
Sponge#
2B
3B
4B
5B
6B
7B
8B
9B
10B
11B
12B
13B
14B
15B
16B
17B
18B
19B
20B
21B
22B
23B
24B
25B
26B
27B
28B
29B
30B
31B
32B
33B
34B
35B
36B
37B
38B
39B
40B
41B
42B
43B
44B
45B
46B
47B
48B
49B
50B
# Colonies
Too Few to Count
194
138
96
100
Too Few to Count
63
71
77
110
Too Many to Count
290
168
288
56
37
112
174
113
Too Few to Count
64
56
298
234
101
117
206
73
103
Too Many to Count
Too Many to Count
196
Too Many to Count
Too Many to Count
51
255
84
Too Many to Count
143
178
215
Too Many to Count
238
176
300
288
98
297
Plate
dilution
10^-7
10^-8
10^-7
10^-5
10^-8
10^-4
10^-6
10^-4
10^-6
10^-7
10^-5
10^-5
10^-4
10^-5
10^-6
10^-6
10^-6
10^-6
10^-3
10^-7
10^-6
10^-4
10^-6
10^-5
10^-6
10^-7
10^-6
10^-9
10^-7
10^-7
10^-7
10^-6
10^-7
10^-7
10^-7
10^-6
10^-5
10^-9
10^-5
10^-7
10^-7
10^-7
10^-6
10^-6
10^-6
10^-6
10^-7
10^-6
Total count (CFU/g)
1.94x1010
1.38x109
9.6x106
1.00x1010
6.3x107
7.1x105
7.7x107
1.10x109
2.9x107
1.68x106
2.88x107
5.6x107
3.7x107
1.12x108
1.74x108
1.13x105
6.40x107
5.60x105
2.98x108
2.34x107
1.01x108
1.17x109
2.06x106
7.30x1010
1.03x109
1.96x108
5.10x108
2.55x108
8.40x106
1.43x107
1.78x109
2.15x109
2.38x108
1.76x108
3.00x108
2.88x108
9.80x108
2.97x108
31
# Differential Lactose
fermenting colonies
0
1
30
6
31
8
46
1
30
6
57
20
12
76
85
95
19
2
121
11
3
18
26
20
0
20
0
51
20
105
66
200
215
73
40
200
98
297
Table 5
QUANTITATIVE PLATE COUNTS OF SPONGES BEFORE MICROWAVE
DISINFECTION ON XYLOSE LYSINE DEOXYCHOLATE AGAR (XLD)
Sponge#
2B
3B
4B
5B
6B
7B
8B
9B
10B
11B
12B
13B
14B
15B
16B
17B
18B
19B
20B
21B
22B
23B
24B
25B
26B
27B
28B
29B
30B
31B
32B
33B
34B
35B
36B
37B
38B
39B
40B
41B
42B
43B
44B
45B
46B
47B
48B
49B
50B
XLD agar
# Colonies
51
167
63
26
67
Too Few to Count
Too Many to Count
80
52
Too Many to Count
58
89
258
Too Few to Count
61
150
31
Too Many to Count
119
37
Too Many to Count
201
Too Many to Count
248
153
41
Too Many to Count
156
35
39
Too Many to Count
Too Many to Count
Too Many to Count
Too Many to Count
Too Many to Count
Too Many to Count
82
35
Too Many to Count
Too Many to Count
Too Few to Count
Too Many to Count
289
287
Too Many to Count
Too Few to Count
Too Few to Count
Too Few to Count
218
Plate dilution
10^-6
10^-8
10^-7
10^-5
10^-8
10^-4
10^-5
10^-6
10^-5
10^-5
10^-6
10^-5
10^-5
10^-4
10^-5
10^-6
10^-6
10^-6
10^-6
10^-3
10^-6
10^-5
10^-2
10^-6
10^-5
10^-6
10^-6
10^-6
10^-9
10^-6
10^-6
10^-6
10^-5
10^-6
10^-7
10^-6
10^-6
10^-5
10^-8
10^-5
10^-7
10^-7
10^-6
10^-6
10^-6
10^-7
10^-7
10^-7
10^-6
32
Total count (CFU/g)
5.1x107
1.67x1010
6.3x108
2.6x106
6.7x109
8.0x107
5.2x106
5.8x107
8.9x106
2.58x107
6.1x106
1.5x108
3.1x107
1.19x108
3.70x104
2.01x107
2.48x108
1.53x107
4.10x107
1.56x108
3.50x1010
3.90x107
8.20x107
3.50x106
2.89x108
2.87x108
2.18x108
Salmonella colonies
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Positive
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Positive
Positive
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Table 6
AVERAGE COLONY FORMING UNIT PER GRAM (CFU/g) RECORDED FOR
SPONGES BEFORE MICROWAVE EXPOSURE ON EACH MEDIA UTILIZED
Trypticase Soy
Mannitol Salt
MacConkey
Xylose Lysine
Agar (TSA)
Agar (MSA)
Agar
Deoxycholate
Averages
(CFU/g)
Agar (XLD)
n= 38
n= 31
n= 38
n= 27
Total
Sponges
collected
= 49
1.58x109CFU/g 8.14x103CFU/g 3.08x109CFU/g
+/- 3.28x109
+/- 7.68x103
33
+/- 1.22x1010
2.04x109CFU/g
+/- 7.12x109
Log of Average CFU/g on All Media Before
Microwave Exposure
12
10
Lof of Average CFU/g
8
6
4
2
9.095286507
3.910446525
9.482236348
9.322873075
TSA
MSA
MacConkey
XLD
0
Media
Log of Average CFU/g on each media before microwave exposure
Figure 2. Bar graph containing the log of the average colony forming units per gram of
sponge found on each media prior to microwave subjection.
34
Table 7
QUANTITATIVE PLATE COUNT AFTER 30 SECONDS OF MICROWAVE
EXPOSURE ON TRYPTICASE SOY AGAR (TSA)
TSA
Sponge #
2A
3A
4A
5A
6A
13A
14A
15A
19A
20A
21A
22A
23A
24A
31A
32A
33A
34A
35A
36A
37A
43A
44A
45A
# Colonies
Too Many to Count
50
42
46
Too Few to Count
42
184
Too Few to Count
277
68
Too Few to Count
181
138
Too Many to Count
134
158
165
158
58
80
Too Many to Count
81
55
73
Plate
dilution
10^-6
10^-7
10^-6
10^-5
10^-5
10^-6
10^-4
10^-5
10^-6
10^-5
10^-6
10^-6
10^-3
10^-4
10^-5
10^-5
10^-5
10^-6
10^-4
10^-3
10^-6
10^-4
10^-4
Total count
(CFU/g)
5.0x108
4.2x107
4.6x106
4.2x106
1.84x108
2.77x107
6.80x107
1.81x108
1.38x108
1.34x106
1.58x107
1.65x107
1.58x107
5.80x107
8.00x105
8.10x107
5.50x105
7.30x105
46A
Too Many to Count
10^-3
-
-
47A
Too Few to Count
10^-5
-
-
35
% Inhibition
69.14%
90.67%
98.69%
94.25%
-338.10%
58.03%
88.85%
69.83%
-36.63%
99.89%
94.70%
99.92%
93.36%
93.41%
99.97%
99.96%
Table 8
QUANTITATIVE PLATE COUNT AFTER 30 SECONDS OF MICROWAVE
EXPOSURE ON MANNITOL SALT AGAR (MSA)
MSA
Differential
Plate
Total count
mannitol
Sponge #
# Colonies
dilution
(CFU/g)
fermentation
2A
143
10^-3
1.43x105
0
3A
Too Few to Count
10^-1
4A
Too Few to Count
10^-1
5A
Too Few to Count
10^-1
6A
Too Few to Count
10^-1
13A
Too Few to Count
10^-1
0
14A
Too Few to Count
10^-1
0
15A
Too Few to Count
10^-1
0
19A
0
10^-1
0
0
20A
0
10^-1
0
0
21A
0
10^-1
0
0
22A
Too Many to Count
10^-4
23A
Too Many to Count
10^-3
24A
0
10^-1
0
0
31A
0
10^-1
0
0
32A
0
10^-1
0
0
33A
Too Few to Count
10^-1
34A
0
10^-1
0
0
35A
0
10^-1
0
0
36A
0
10^-1
0
0
37A
0
10^-1
0
0
43A
0
10^-1
0
0
44A
0
10^-1
0
0
45A
0
10^-1
0
0
46A
0
10^-1
0
0
47A
0
10^-1
0
0
(-)= Unable to calculate data due to information unattainable
36
%
Inhibition
-
Table 9
QUANTITATIVE PLATE COUNT AFTER 30 SECONDS OF MICROWAVE
EXPOSURE ON MACCONKEY AGAR
MacConkey agar
Differential
lactose
Sponge
Plate
Total count
fermenting
#
# Colonies
dilution
(CFU/g)
colonies
6
2A
76
10^-5
7.6x10
11
3A
Too Many to Count
7
4A
233
10^-5
2.33x10
3
5A
35
10^-5
3.5x105
2
6A
Too Many to Count
13A
161
10^-4
1.61x106
0
14A
192
10^-4
1.92x106
30
15A
Too Many to Count
10^-3
19A
185
10^-5
1.85x107
175
20A
290
10^-5
2.90x107
188
21A
Too Many to Count
10^-6
7
22A
169
10^-5
1.69x10
36
23A
Too Many to Count
10^-6
24A
Too Many to Count
10^-6
31A
Too Many to Count
10^-6
32A
71
10^-5
7.10x106
1
33A
252
10^-4
2.52x106
80
34A
279
10^-5
2.79x107
4
7
35A
103
10^-5
1.03x10
8
36A
Too Many to Count
10^-4
37A
Too Many to Count
10^-6
7
43A
46
10^-6
4.60x10
46
44A
47
10^-4
4.70x105
47
5
45A
37
10^-4
3.70x10
37
46A
182
10^-3
1.82x105
105
47A
81
10^-4
8.1x105
70
(-)= Unable to calculate data due to information unattainable
37
% Inhibition
98.31%
96.35%
93.34%
83.48%
83.33%
85.77%
97.86%
99.84%
99.90%
99.73%
Table 10
QUANTITATIVE PLATE COUNT AFTER 30 SECONDS OF MICROWAVE
EXPOSURE ON XYLOSE LYSINE DEOXYCHOLATE AGAR (XLD)
XLD agar
Sponge #
2A
3A
4A
5A
6A
13A
14A
15A
# Colonies
Too Few to Count
110
27
90
Too Many to Count
51
247
Too Many to Count
Plate
dilution
10^-5
10^-5
10^-5
10^-5
10^-4
10^-5
-
Total count
(CFU/g)
1.10x107
2.7x106
9.0x106
5.1x105
2.47x107
-
Salmonella
Colonies
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
%
Inhibition
99.93%
99.57%
-246.15%
94.27%
4.26%
-
19A
20A
21A
22A
23A
24A
31A
32A
33A
34A
35A
36A
37A
43A
44A
45A
251
278
Too Many to Count
Too Many to Count
Too Many to Count
Too Many to Count
Too Many to Count
48
194
50
54
37
Too Many to Count
127
165
33
10^-5
10^-5
10^-5
10^-4
10^-6
10^-5
10^-4
10^-6
10^-6
10^-4
10^-4
2.51x107
2.78x107
4.80x106
1.94x106
5.00x107
5.40x106
3.70x105
1.27x108
1.65x106
3.30x105
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
76.64%
99.43%
99.89%
46A
Too Few to Count
Negative
5
47A
57
10^-4
5.7x10
Negative
(-)= Unable to calculate data due to information unattainable
38
-
Table 11
AVERAGE COLONY FORMING UNIT PER GRAM (CFU/g) RECORDED FOR
SPONGES 30 SECONDS AFTER MICROWAVE EXPOSURE ON EACH MEDIA
UTILIZED
Trypticase Soy
Mannitol Salt
MacConkey
Xylose Lysine
Agar (TSA)
Agar (MSA)
Agar
Deoxycholate
Agar (XLD)
Averages
n= 18
n= 1
n= 17
n= 16
(CFU/g)
Total
Sample
Size= 26
% Inhibition
7.44x107CFU/g
1.43x105*
1.29x107CFU/g
2.05x107CFU/g
+/- 1.22x108
CFU/g
+/- 1.37x107
+/- 3.51x107
89.33%
-
93.79%
82.00%
*Based upon only one sponge on Mannitol Salt Agar that CFU/g was in respectable
ranges
39
TSA Average CFU/g Before Microwaving
versus 30 Seconds
1.80E+09
1.60E+09
1.40E+09
Average CFU/g
1.20E+09
1.00E+09
8.00E+08
6.00E+08
4.00E+08
2.00E+08
1.58E+09
7.44E+07
Before (n= 38)
30 seconds (n= 18)
0.00E+00
Time Exposed
Trypticase Soy Agar (TSA) Average CFU/g Comparison of Before Exposure to
After 30 Seconds
Figure 3.Comparison of the calculated average CFU/g of before microwave exposure to
after 30 seconds of exposure culture on TSA.
40
MacConkey Average CFU/g Before
Microwaving versus 30 Seconds
3.50E+09
3.00E+09
Average CFU/g
2.50E+09
2.00E+09
1.50E+09
1.00E+09
5.00E+08
3.08E+09
1.29E+07
Before (n= 38)
30 seconds (n= 17)
0.00E+00
Time Exposed
MacConkey Agar Average CFU/g Comparison of Before Exposure to After 30
Seconds
Figure 4. Comparison of the calculated average CFU/g of before microwave exposure to
after 30 seconds of exposure culture on MacConkey agar.
41
XLD Average CFU/g Before Microwaving
versus 30 Seconds
2.50E+09
2.00E+09
Average CFU/g
1.50E+09
1.00E+09
5.00E+08
2.04E+09
2.05E+07
Before (n= 27)
30 seconds (n= 16)
0.00E+00
Time Exposed
Xylose Lysine Deoxycholate agar (XLD) CFU/g Comparison of before Exposure to
after 30 Seconds
Figure 5. Comparison of the log of the calculated average CFU/g of before microwave
exposure to after 30 seconds of exposure culture on XLD.
42
Table 12
QUANTITATIVE PLATE COUNTS AFTER 60 SECONDS OF MICROWAVE
EXPOSURE ON TRYPTICASE SOY AGAR (TSA)
TSA
Sponge #
# Colonies
Plate
dilution
Total count
(CFU/g)
% Inhibition
7A
52
10-4
5.2x105
-
8A
Too Few to Count
10-5
-
-
9A
46
10-5
4.6x106
96.52%
10A
Too Many to Count
10-4
-
-
11A
283
10-4
2.83x106
98.17%
12A
Too Many to Count
10-4
-
-
16A
119
10^-3
5
97.84%
7
1.19x10
17A
102
10^-5
1.02x10
88.41%
18A
Too Few to Count
10^-5
-
-
25A
26A
55
48
10^-5
10^-4
6
98.59%
5
98.55%
5.5x10
4.8x10
7
27A
125
10^-5
1.25x10
95.47%
28A
Too Many to Count
10^-5
-
8
29A
107
10^-6
1.07x10
63.48%
30A
Too Many to Count
10^-5
-
-
38A
Too Many to Count
10^-3
-
-
39A
Too Many to Count
10^-3
-
40A
41A
40
175
10^-5
10^-2
6
4.0x10
99.83%
4
99.89%
5
1.75x10
42A
102
10^-3
1.02x10
-
48A
Too Few to Count
10^-1
-
-
49A
50A
94
272
10^-4
10^-2
43
5
9.4x10
4
2.72x10
99.97%
100.00%
Table 13
QUANTITATIVE PLATE COUNTS AFTER 60 SECONDS OF MICROWAVE
EXPOSURE ON MANNITOL SALT AGAR (MSA)
MSA
Total count
(CFU/g)
0
0
Differential
Mannitol
Fermenters
0
0
Sponge #
7A
8A
# Colonies
Too Few to Count
Too Few to Count
Plate
dilution
10-1
10-1
9A
129
10-1
1.29x103
0
10A
Too Few to Count
10-1
0
0
11A
46
10-2
4.6x103
0
12A
Too Few to Count
10-1
0
0
16A
Too Few to Count
10^-1
-
0
17A
91
10^-1
9.1x102
0
18A
Too Few to Count
10^-1
0
0
25A
Too Few to Count
10^-1
0
0
26A
Too Few to Count
10^-1
0
0
27A
Too Few to Count
10^-1
0
0
28A
Too Few to Count
10^-1
0
0
29A
Too Few to Count
10^-1
0
0
30A
Too Few to Count
10^-1
0
0
38A
Too Few to Count
10^-1
0
0
39A
Too Few to Count
10^-1
0
0
40A
Too Few to Count
10^-1
0
0
41A
Too Few to Count
10^-1
0
0
42A
Too Few to Count
10^-1
0
0
48A
Too Few to Count
10^-1
0
0
49A
Too Few to Count
10^-1
0
0
50A
Too Few to Count
10^-1
0
0
44
%
Inhibition
-
Table 14
QUANTITATIVE PLATE COUNTS AFTER 60 SECONDS OF MICROWAVE
EXPOSURE ON MACCONKEY AGAR
MacConkey agar
Sponge #
7A
8A
9A
10A
11A
# Colonies
68
179
99
109
64
Plate
dilution
10^-4
10^-3
10^-5
10^-3
10^-7
Total count
(CFU/g)
6.8x105
1.79x105
9.9x106
1.09x105
6.4x108
#
Differential
Lactose
fermenters
37
2
0
72
0
12A
16A
17A
18A
25A
26A
27A
28A
29A
30A
38A
39A
40A
41A
42A
48A
56
31
63
180
34
276
285
177
39
219
Too Many to Count
Too Many to Count
232
293
282
Too Few to Count
10^-4
10^-3
10^-5
10^-4
10^-5
10^-3
10^-4
10^-4
10^-6
10^-5
10^-3
10^-3
10^-4
10^-2
10^-2
10^-1
5.6x105
3.1x104
6.3x106
1.80x106
3.4x106
2.76x105
2.85x106
1.77x106
3.9x107
2.19x107
2.32x106
2.93x104
2.82x104
-
26
31
3
50
13
0
2
17
2
45
19
21
3
-
99.95%
99.89%
88.75%
95.14%
98.86%
98.82%
97.18%
-17.93%
99.97%
99.80%
100.00%
-
49A
50A
62
Too Many to Count
10^-4
10^-1
6.2x105
-
17
150
99.94%
-
45
%
Inhibition
-731.17%
Table 15
QUANTITATIVE PLATE COUNTS AFTER 60 SECONDS OF MICROWAVE
EXPOSURE ON XYLOSE LYSINE DEOXYCHOLATE (XLD) AGAR
XLD agar
Sponge #
# Colonies
Plate
dilution
Total count
(CFU/g)
Salmonella
Colonies
%
Inhibition
7A
86
10^-2
8.6x103
Negative
-
8A
Too Many to Count
10^-3
-
Negative
-
9A
37
10^-4
5
3.7x10
Negative
99.54%
5
10A
245
10^-3
2.45x10
Negative
95.29%
11A
153
10^-6
1.53x108
Negative
-
12A
96
10^-3
9.6x104
Negative
99.83%
16A
Too Few to Count
10^-2
-
17A
18A
25A
85
84
74
10^-4
10^-4
10^-4
Negative
-
5
Negative
99.43%
5
Negative
97.29%
5
Negative
99.70%
4
8.5x10
8.4x10
7.4x10
26A
57
10^-3
5.7x10
Negative
99.63%
27A
41
10^-4
4.1x105
Negative
99.00%
28A
93
10^-4
9.3x105
29A
30A
179
173
10^-5
Negative
-
7
Negative
89.10%
6
1.70x10
10^-4
1.73x10
Negative
100.00%
4
38A
30
10^-3
3.0x10
Negative
99.96%
39A
Too Few to Count
10^-3
-
Negative
-
5
40A
90
10^-4
9.0x10
Negative
-
41A
Too Few to Count
10^-4
-
Negative
-
42A
Too Few to Count
10^-2
-
Negative
-
48A
49A
Too Few to Count
49
10^-1
10^-4
4.9x105
Negative
Negative
-
50A
Too Few to Count
10^-2
-
Negative
-
46
Table 16
AVERAGE COLONY FORMING UNIT PER GRAM (CFU/g) RECORDED FOR
SPONGES 60 SECONDS AFTER MICROWAVE EXPOSURE ON EACH MEDIA
UTILIZED
Trypticase Soy
Mannitol Salt
MacConkey
Xylose Lysine
Agar (TSA)
Agar (MSA)
Agar
Deoxycholate
Agar (XLD)
Averages
n= 14
n= 3
n= 19
n= 16
(CFU/g)
Total
Sample
Size= 23
% Inhibition
1.06x107CFU/g 2.27x103CFU/g 2.28x106CFU/g 1.60x106CFU/g
+/- 2.80x107
+/- 2.03x103
+/- 2.71x106
+/- 4.23x106
94.73%
72.11%
98.03%
98.07%
47
TSA Average CFU/g Before Microwaving
Exposure versus After 60 seconds of Exposure
1.80E+09
1.60E+09
1.40E+09
Average CFU/g
1.20E+09
1.00E+09
8.00E+08
6.00E+08
4.00E+08
2.00E+08
1.58E+09
1.06E+07
Before (n= 38)
60 seconds (n= 14)
0.00E+00
Time Exposed
Trypticase Soy agar (TSA) CFU/g Comparison of before Exposure to After 60
Seconds
Figure 6. Comparison of the calculated average CFU/g before and after microwave
exposure at 60 seconds of exposure culture on TSA.
48
MSA Average CFU/g Before Microwaving
Exposure versus After 60 seconds of Exposure
9.00E+03
8.00E+03
7.00E+03
Average CFU/g
6.00E+03
5.00E+03
4.00E+03
3.00E+03
2.00E+03
1.00E+03
8.14E+03
2.27E+03
Before (n= 6)
60 seconds (n=3)
0.00E+00
Time Exposed
Mannitol Salt agar (MSA) CFU/g Comparison of before Exposure to After 60
Seconds
Figure 7. Comparison of the calculated average CFU/g of culture on MSA before
microwave exposure to after 60 seconds of exposure.
49
MacConkey Average CFU/g Before
Microwaving Exposure versus After 60
seconds of Exposure
3.50E+09
3.00E+09
Average CFU/g
2.50E+09
2.00E+09
1.50E+09
1.00E+09
5.00E+08
3.08E+09
2.28E+06
Before (n= 38)
60 seconds (n= 19)
0.00E+00
Time Exposed
MacConkey agar CFU/g Comparison of before Exposure to After 60 Seconds
Figure 8. Comparison of the calculated average CFU/g of before microwave exposure to
after 60 seconds of exposure culture on MacConkey agar.
50
XLD Average CFU/g Before Microwaving
Exposure versus After 60 seconds of Exposure
2.50E+09
2.00E+09
Average CFU/g
1.50E+09
1.00E+09
5.00E+08
2.04E+09
1.60E+06
Before (n= 27)
60 seconds (n= 16)
0.00E+00
Time Exposed
Xylose Lysine Deoxycholate agar (XLD) CFU/g Comparison of before Exposure to
After 60 Seconds
Figure 9. Comparison of the calculated average CFU/g of before microwave exposure to
after 60 seconds of exposure culture on XLD.
51
Table 17
RECORDED AVERAGE CFU/g ON EACH MEDIA IN COMPARISON OF BEFORE,
30 SECONDS, AND 60 SECONDS OF MICROWAVE EXPOSURE
Before
TSA
MSA
30 Seconds
60 Seconds
1.58x109 CFU/g
7.44x107 CFU/g
1.06x107 CFU/g
+/- 3.28x109
+/- 1.22x108
+/- 2.80x107
n= 38
n=18
n= 14
8.14x103CFU/g
1.43x105CFU/g
+/- 7.68x103
+/- 2.03x103
n= 6
MacConkey
XLD
2.27x106CFU/g
n= 1
n= 3
3.08x109 CFU/g
1.29x107 CFU/g
2.28x106CFU/g
+/- 1.22x1010
+/- 1.37x107
+/- 2.71x106
n= 38
n= 17
n= 19
2.04x109 CFU/g
2.05x107 CFU/g
1.60x106CFU/g
+/- 7.12x109
+/- 3.51x107
+/- 4.23x106
n= 27
n= 16
n= 16
52
Average CFU/g Before Microwave and After
30 Seconds on all Media Utilized
12
10
Average CFU/g
8
6
Before
30 seconds
4
2
n value:
0
38
18
TSA
6
38
1
MSA
17
MacConkey Agar
27
16
t-test
value0.041105
XLD
Comparison of before microwave exposure to 30 seconds recorded in the average
CFU/g on each media
Figure 10. Calculated difference between before microwave exposure on all media in
comparison to 30 seconds.
53
Photograph of TSA Colonies before Microwave Exposure
Figure 11. Sponge 13 Dilution plate 10-5 before microwaving on TSA
Photograph of TSA Colonies after 30 Seconds of Microwave Exposure
Figure 12. Sponge 13 Dilution plate 10-5 after 30 seconds of microwaving on TSA
54
Photograph of MacConkey Colonies before Microwave Exposure
Figure 13. Sponge 13 Dilution plate 10-5 before microwaving on MacConkey
Photograph of MacConkey Colonies after 30 Seconds of Microwave Exposure
Figure 14. Sponge 13 Dilution plate 10-5 after 30 seconds of microwaving on MacConkey
55
Average CFU/g Before Microwave and After
60 Seconds on all Media Utilized
Title
12
10
Average CFU/g
8
6
Before
60 seconds
4
2
n0
38
14
TSA
6
3
MSA
38 19
MacConkey Agar
27
XLD
16
t-test value0.03985
Comparison of before microwave exposure to 60 seconds recorded in the average
CFU/g on each media
Figure 15. Calculated difference between before microwave exposure on all media in
comparison to 60 seconds
56
Photograph of XLD Colonies before Microwave Exposure
Figure 16. Sponge 13 Dilution plate 10-5 before microwaving on XLD
Photograph of XLD Colonies after 60 Seconds of Microwave Exposure
Figure 17. Sponge 13 Dilution plate 10-5 after 60 seconds of microwaving on XLD
57
Table 18
COMAPRISON OF AVERAGE CFU/g DECREASE FOR BEFORE MICROWAVE TO
AFTER 30 SECONDS MICROWAVE SUBJECTION
Before
TSA
30 Seconds
1.58x109 CFU/g
+/- 3.28x10
7.44x107 CFU/g
9
+/- 1.22x10
n= 38
MSA
% Decrease
8
89.33%
n=18
8.14x103CFU/g
1.43x105CFU/g
-
+/- 7.68x103
n= 6
MacConkey
XLD
n= 1
3.08x109 CFU/g
1.29x107 CFU/g
+/- 1.22x1010
+/- 1.37x107
n= 38
n= 17
2.04x109 CFU/g
2.05x107 CFU/g
+/- 7.12x109
+/- 3.51x107
n= 27
n= 16
58
93.79%
82.00%
Table 19
COMAPRISON OF AVERAGE CFU/g DECREASE FOR BEFORE MICROWAVE TO
AFTER 60 SECONDS MICROWAVE SUBJECTION
Before
TSA
MSA
MacConkey
XLD
60 Seconds
% Decrease
1.58x109 CFU/g
1.06x107 CFU/g
+/- 3.28x109
+/- 2.80x107
n= 38
n= 14
8.14x103 CFU/g
2.27x106 CFU/g
+/- 7.68x103
+/- 2.03x103
n= 6
n= 3
3.08x109 CFU/g
2.28x106 CFU/g
+/- 1.22x1010
+/- 2.71x106
n= 38
n= 19
2.04x109 CFU/g
1.60x106 CFU/g
+/- 7.12x109
+/- 4.23x106
n= 27
n= 16
59
94.73%
72.11%
98.03%
98.07%
Percent Inhibition of 30 and 60 Seconds
Compared to Before Microwave on TSA
105.00%
100.00%
Percent Decrease
95.00%
90.00%
85.00%
80.00%
89.33%
94.73%
30 Seconds (n= 18)
60 Seconds (n= 14)
75.00%
Time Exposed
Calculated Percent of Induced Inhibition Due to Microwave Exposure
Figure 18. Percent induced inhibition due to microwave exposure at 30 and 60 seconds in
comparison to before microwave subjection on TSA
60
Percent Inhibition of 60 Seconds Compared
to Before Microwave on MSA
78.00%
76.00%
Percent Decrease
74.00%
72.00%
70.00%
68.00%
66.00%
72.11%
64.00%
60 Seconds (n= 3)
Time Exposed
Calculated Percent of Induced Inhibition Due to Microwave Exposure
Figure 19. Percent induced inhibition due to microwave exposure at 60 seconds in
comparison to before microwave subjection on MSA
61
Percent Inhibition of 30 and 60 Seconds
Compared to Before Microwave on
MacConkey
102.00%
100.00%
98.00%
Percent Decrease
96.00%
94.00%
92.00%
90.00%
88.00%
93.79%
98.03%
30 Seconds (n= 17)
60 Seconds (n= 19)
86.00%
Time Exposed
Calculated Percent of Induced Inhibition Due to Microwave Exposure
Figure 20. Percent induced inhibition due to microwave exposure at 30 and 60 seconds in
comparison to before microwave subjection on MacConkey Agar
62
Percent Inhibition of 30 and 60 Seconds
Compared to Before Microwave on XLD
120%
100%
Percent Decrease
80%
60%
40%
20%
82%
98.07%
30 Seconds (n= 16)
60 Seconds (n= 16)
0%
Time Exposed
Calculated Percent of Induced Inhibition Due to Microwave Exposure
Figure 21. Percent induced inhibition due to microwave exposure at 30 and 60 seconds in
comparison to before microwave subjection on XLD.
63
Table 20
AVERAGE OF LACTOSE FERMENTING COLONIES FOUND ON BEFORE
MICROWAVING, AFTER 30 SECONDS, AND AFTER 60 SECONDS PLATES,
WITH CALCULATED PERCENTAGE OF BACTERIA FERMENTING LACTOSE
DECREASE IN COMPARISON TO BEFORE MICROWAVING TOTAL COLONIES
MacConkey Agar
Before (n= 38)
30 Seconds (n=17)
60
Seconds
(n=
20)
Average of lactose
55.50 CFU/g
49.59 CFU/g
25.50 CFU/g
fermenting colonies
+/- 69.76205
+/- 58.41453
+/- 35.38436
10.65%
54.05%
found (CFU/g)
Percent inhibition
comparison to before
microwaving
64
Average Lactose Fermenting Colonies
70
Before to after 30
seconds
t- test= 0.373132
Before to after 60
seconds
t- test= 0.017031
60
Average of Number of Colonies Recorded
50
40
30
20
10
55.5
49.58823529
25.5
Before (n= 38)
30 Seconds (n= 17)
60 Seconds (n= 20)
0
Average of Lactose Fermenting Colonies Isolated from MacConkey Agar
Figure 22. Average number of recorded lactose fermenting colonies on MacConkey agar
plates for before microwaving, after 30 seconds, and after 60 seconds respectively.
65
Table 21
MANNITOL SALT AGAR COMMON COLONIES MACROSCOPICALLY AND
MICROSCOPICALLY DESCRIBED
MSA Common Colonies
Sponges differential bacteria was found present on
Macroscopic
Gram Stain
Observations
and
Before-
30 seconds-
60 Seconds
Not found
Not found
microscopic
Round
Gram
colony
Positive
Mannitol
Cocci
fermenting
2µm
17B
colony
66
Percentage of MSA Plates Positive for
Mannitol Fermenting Colonies
2.00%
Plates Positive for Mannitol
fermention
Plates Negative for Mannitol
fermention
99.98%
Mannitol Fermenting Colonies Isolated on MSA
Figure 23. Graphs of the total percentage of occurrences of mannitol fermenting colonies
for before microwave subjection on MSA media.
67
Table 22
MACCONKEY AGAR COMMON COLONIES MACROSCOPICALLY AND
MICROSCOPICALLY DESCRIBED
MacConkey Common Colonies
Macroscopic
Gram
Observations
Stain
Sponges differential bacteria was
found present on
Before
30 Seconds
60 Seconds
Large, round
Gram
2B, 4B, 5B,
2A, 4A, 5A,
7A, 8A, 9A,
Light purple edges
negative
6B, 7B, 8B,
15A, 19A,
10A, 12A, 16A,
with
rods
9B, 10B, 11B,
20A, 21A,
17A, 18A, 25A,
cream center
2µmx1µ
12B, 13B, 14B,
22A, 23A,
27A-30A
Positive lactose
m
15B, 16B, 18B,
24A, 32A-
19B, 20B, 21B,
37A
fermentation
22B, 23B, 24B,
25B-30B,
31B-37B
68
Percentage of MacConkey Plates Lactose
Fermenting Colonies Were Found on Before
Microwave Subjection
30.61%
Plates Positive for Lactose
Fermenting Colonies
Plates Negative for Lactose
Fermenting Colonies
69.39%
Differential Lactose Fermenting Colony Isolated on MacConkey Agar
Figure 24. Graphs of the total percentage of occurrences for lactose fermenting colonies
for before microwave subjection on MacConkey media.
69
Percentage of MacConkey Plates Lactose
Fermenting Colonies Were Found on After 30
Seconds of Microwave Subjection
38.46%
Plates Positive for Lactose
fermenting colonies
61.54%
Plates Negative for lactose
fermenting colonies
Differential Lactose Fermenting Colony Isolated on MacConkey Agar
Figure 25.Graphs of the total percentage of occurrences for lactose fermenting colonies
after 30 seconds of microwave subjection on MacConkey media.
70
Percentage of MacConkey Plates Lactose
Fermenting Colonies Were Found on After 60
Seconds of Microwave Subjection
43.48%
Plates Positive for Lactose
Fermenting Colonies
56.52%
Plates Negative for Lactose
Fermenting Colonies
Differential Lactose Fermenting Colony Isolated on MacConkey Agar
Figure 26. Graphs of the total percentage of occurrences for lactose fermenting colonies
after 60 seconds of microwave subjection on MacConkey media.
71
Table 23
XYLOSE LYSINE DEOXYCHOLATE AGAR COMMON COLONIES
MACROSCOPICALLY AND MICROSCOPICALLY DESCRIBED
XLD Common Colonies
Sponges selective bacteria was found present on
Macroscopic
Observations
Before
Round
Black dot
middle
Gram Stain
in
Gram
negative rod
26B,
36B
72
30 Seconds
35B,
Not
found
60 Seconds
Not
found
Percent of XLD Agar Plates Positive for
Salmonella Before Microwaving
Salmonella
positive
6%
Salmonella
Negative
94%
Salmonella positive
Salmonella Negative
n= 27
Selective Colony Isolated on XLD Agar
Figure 27. Graphs of the total percentage of occurrences of Salmonella spp. on selective
XLD agar before microwave subjection.
73
DISCUSSION
Data analysis
Sponges are common tools often utilized in the process of cleaning, but these
instruments for cleaning can also be a primary source of contamination, capable of
harboring and spreading harmful microbes, such as pathogenic bacteria, to dishes and
kitchen surfaces. As stated in an article published by the New York Times, not only are
people’s hands are a primary source of bacteria but also dish towels, sinks, refrigerator
door handles, and moist sponges are breeding grounds for bacteria, meaning that there is
the potential of spreading pathogens when using a sponge to wipe a surface (Hesser,
2004). Disinfection of sponges in order to reduce these harmful bacteria is suggested by
experts, but still some people remain unaware of the risks of not disinfecting sponges and
also the benefits, even 30 seconds of microwave exposure can have on the reduction of
harmful microbes, some even related to common foodborne illnesses.
Data collected of the first 24 out of 50 sponges revealed that only 8.7% of the
sponge owners disinfected their sponges through any means prior to collection. Time of
use of these collected sponges ranged from 8 days to as long as 4 months. Studies have
shown in as little as two uses harmful bacteria can be isolated (Hesser, 2004) (Fig 1 and
Table 1). Sponges 5 and 7 were the only sponges collected to have been previously
disinfected through microwave exposure out of the batch, which upon analysis of colony
forming units per gram (CFU/g) were shown to be relatively low in comparison to other
sponges collected due to disinfection methods used to reduce bacterial build up over time
(Table 2).
74
In accordance with this data, our results revealed that the percent of individuals
who disinfect their sponges are significantly lower than individuals who do not disinfect
their sponges as supported by the findings of Layton et al., (2006) which revealed similar
findings. This significant difference could be due to individuals unaware that pathogenic
microbes can be easily spread and can cause various forms of forborne illnesses. Our
study and others show kitchen sponges provide an ideal environment for the spreading of
disease.
The average CFU/g were calculated using the number of colonies found
multiplied by the dilution factor for all colonies found on TSA, MSA, MacConkey, and
XLD agars before microwave subjection with data in created Tables 2 through 5. These
values were taken and the average CFU/g was calculated for the 49 sponges on each
media along with standard deviation in Table 6. Average CFU/g calculated for each
media revealed initially gram negative bacteria colonizing on MacConkey agar were
shown to be the most prevalent with a calculated average of 3.08x109 CFU/g, with gram
negative species of bacteria on XLD close in total number being 2.04x109 CFU/g (Table
6). These data reveal that gram negative species are most prevalent in total bacterial load
in cellulose kitchen sponges before microwave subjection, which is a concern due to the
link to opportunistic pathogenicity by containing lipopolysaccharide. Gram negative
bacterial species were found to be the most prevalent of the total bacteria culture on all
four media as shown by the large average CFU/g calculated (Table 6), in which gram
negative bacterial species seem to more suitable for living in an environment with
moisture and food particles available for optimal proliferation. This idea is supported by
research by Bergquist published in 1979, revealing that CFU/g are not only dependent on
75
the time of the sponge being used but that sponges provide an ideal source of moisture
and nutrients in which gram negative species seem to take advantage of this (Bergquist,
1979).
Mannitol Salt Agar plate counts, being a media selective for bacteria of the genus
Staphylococcus, were recorded in Table 3 for sponges prior to microwave exposure.
MSA was a troublesome media in this research as bacterial cultures did not grow readily.
Staphylococcus spp. are more readily associated with skin than cellulose fibers, making
the transfer of the bacteria from skin to sponge less likely as shown in the data collected
in which most sponges did not show any growth on MSA media. Most plates revealed
zero to too few to colonies cultured to count even on the lowest dilution of 1/10th (Table
3). Microbial attachment is shown to be influenced by the design of the food-contact
surface as stated by Silva et al., (2010) revealing that it is more likely to transfer onto
food than on human skin (Silva et al., 2010).This may explain why our MSA data was
hard to obtain and analyze.
Average CFU/g for before sponges subjected to the microwave were calculated
from Tables 2-4 and recorded in Table 6 for future comparison of before total average
CFU/g to after 30 seconds and 60 seconds of microwave subjection. Table 6 revealed an
average CFU/g on TSA was found to be 1.58x109 CFU/g, 8.14x103 CFU/g on MSA,
3.08x109 CFU/g on MacConkey, and 2.04x109 CFU/g on XLD (Table 6). All of these
average CFU/g calculated and recorded were all relatively similar in that all were a very
large amount of bacteria colonies per gram of cut sponge, with the exception on MSA
where the average CFU/g was notably lower than the rest by thousands (Table 6). Figure
2 shows this data collected of the average CFU/g in which it can be shown that MSA was
76
very low in forming units per gram compared to on the other three media. Figure 2
revealed that the most bacterial colonies per gram of sponge were culture on MacConkey
agar, selective for gram negative species (Figure 2). This total bacterial growth was
followed closely by XLD, also culturing gram negative bacterial species, and TSA
culturing both gram positive and negative species (Figure 2).
The same processes were repeated for sponges subjected to 30 seconds of
microwave radiation with the addition of percent inhibition calculated for each sponge
proper data was supplied. Thirty seconds of microwave subjection on the TSA (Table 7),
MSA (Table 8), MacConkey (Table 9) and XLD (Table 10) revealed a decrease in most
sponges subjected to 30 seconds of microwave exposure. Average calculated CFU/g of
each of these media collected showed TSA plates culturing the largest amount of bacteria
being an average of 7.44x107 CFU/g in comparison to MSA, MacConkey, and XLD
being 1.43x105 CFU/g, 1.29x107 CFU/g, and 2.05x107 CFU/g respectively (Table 11).
Overall inhibition revealed that 30 seconds of microwave exposure decreased bacteria on
TSA by 89.33%, MacConkey by 93.79%, and XLD by 82.00% (Table 11). MSA percent
inhibition was unable to be calculated due to the media not properly culturing bacterial
colonies (Table 8). Percent inhibition was shown to have been negative on a few sponges
such as sponge 14A on TSA, this is due to different pieces of the same sponge being
used for before and after analysis revealing that the after exposure piece initially
contained more bacteria per gram than the before exposure piece (Table 7). Thirty
seconds of microwave exposure revealed similar inhibition of gram negative bacteria on
both MacConkey agar and XLD with this evidence being supported by a study done by
77
Ermolaeva et al., (2009) which stated that in general gram negative bacteria are more
sensitive to microwave radiation than gram positive bacteria (Ermolaeva et al., 2009).
A comparison of bacterial growth on all media was made regarding before
microwave subjection to after 30 seconds and graphed in Figures 3, 4, and 5, with the
exception of MSA which couldn’t be properly analyzed at 30 seconds. Across all media
clear overall decrease can be shown of the average bacteria per gram before subjection to
after 30 seconds of subjection. This data suggests that even at 30 seconds of exposure
bacterial inhibition can be clearly noted.
Twenty three sponges subjected to 60 seconds of microwave radiation were tested
with results listed in Tables 12-15, showing recorded colonies and corresponding dilution
plates for TSA, MSA, MacConkey and XLD agars respectively. Calculated data of
average CFU/g for sponges subjected to 60 seconds of microwave exposure were noted
as being 1.06x107 CFU/g on TSA, 2.27x103 CFU/g on MSA, 2.28x106 CFU/g on
MacConkey agar, and 1.60x106 CFU/g on XLD (Table 16). TSA percent bacterial
inhibition revealed data showing 100% bacterial reduction on sponge 50A to as little as
63.48% on sponge 29A (Table 12). Similar rates of percent inhibition were found on
MacConkey and XLD agars (Table 14 and Table 15). MSA again was difficult to analyze
in regards to percent inhibition for each sponge do to their being no growth on the sponge
either before or after subjection (Table 13).
Graphical analysis of this data set revealed that TSA (Figure 6) showed the most
colonies relating to the average CFU/g, followed by MacConkey (Figure 8), XLD (Figure
9), and then MSA (Figure 7); all media showing overall average bacterial decreases
greater than 30 seconds of inhibition (Table 16). Average percent decrease comparing
78
before microwave subjection to 60 seconds were reported in Table 16 and ranged from
72.11% decrease on MSA to a 98.07% decrease on XLD agar suggesting gram positive
bacteria are less susceptible to microwave exposure than gram negative bacteria (Table
16). Gram positive bacteria possess a cell wall which is able to protect bacteria from
turbid environments, in which it is suspected that this is why gram positive bacteria are
less susceptible to microwave exposure.
In comparison of all the average CFU/g on all media it was shown that TSA,
having an average of 1.58x109 CFU/g, was shown to have decreased to 7.44x107 CFU/g
after 30 seconds and further decreased to 1.06x107 CFU/g after 60 seconds of microwave
exposure (Table 17). Similar results can be seen with MacConkey agar and XLD starting
with 3.08x109 CFU/g and 2.04x109 CFU/g respectively and decreasing to 1.29x107 CFU/g
and 2.05x107 CFU/g, then further decreasing after 60 seconds to 2.28x106 CFU/g and
1.60x106 CFU/g respectively (Table 17). MSA was shown to contain 8.14x103 CFU/g in
the before microwaving sample and shown to have 2.27x103 CFU/g after subjection for
60 seconds showing clear decrease in total bacteria per gram (Table 17). This data
presented in Table 17, and graphically in Figure 10 comparing before to 30 seconds and
Figure 15 comparing before to 60 seconds, shows data supporting the idea that
microwave exposure after 30 and 60 seconds shows a clear decrease in total bacterial
numbers per gram of sponge on all media and that microwave radiation treatment does in
fact have an effect on bacterial disinfection at these intervals. It is shown that in a study
by Shamis et al., 2008, that in concern to E. coli bacteria 50oC was enough to decrease
CFU/g by 31%, while 60oC temperatures were enough to decrease bacteria by 100%,
with common 100 watt microwaves temperature around 71.11oC (Shamis et al., 2008;
79
FSIS, 2012). This idea can give insight why the effects of microwave on bacterial
reduction differ from 30 and 60 seconds in that the microwave must have enough time to
reach optimal temperature in order for optimal reduction.
Through further analysis of the percentage decrease, comparisons of before
microwaving and after 30 seconds were first made as shown in Table 18. Using the
average CFU/g of all media it was calculated that TSA showed a total decrease of
89.33%, on MacConkey plates 93.79%, and on XLD a decrease was shown of 82.00%
(Table 18). Data on MSA percentage decrease was not calculated due to only one value
being present for 30 seconds, which would give inaccurate data of accurate percentage
decrease. Bacteria grown on MacConkey agar were shown to have the greatest
percentage decrease after 30 seconds of microwave radiation exposure followed closely
by bacteria grown on XLD agar (Table 18). In comparison of before microwaving to after
60 seconds of microwaving MacConkey agar was again shown to be the greatest decrease
percentage of total bacteria, showing a decrease of 98.03% (Table 19). This was shown to
be followed closely by XLD, showing a decrease in 98.07%, then TSA showing a
94.73% decrease (Table 19). Percentage decrease of MSA was able to be calculated
showing a decrease of 72.11% of total bacteria after 60 seconds of microwave exposure.
The percentage decrease in comparison before microwave exposure to 30 seconds
and 60 seconds was graphed for each media in Figures 18-21. In analysis of TSA, the 60
seconds of exposure showed a clear larger percent decrease in comparison to before
exposure than 30 seconds of exposure did (Figure 18). MacConkey agar, XLD, and MSA
showed similar results in that 60 seconds of exposure showed a larger percent decrease of
overall bacteria than 30 seconds in comparison to before exposure (Figures 19, 20, and
80
21). In comparison of total bacteria grown before microwave exposure on TSA and
MacConkey agars to after 30 seconds photographs were taken and supplied revealing
clear overall decrease in total bacteria on the plates using the same dilution for before and
after (Figures 11, 12, 13, and 14). In comparison of XLD agar, containing Salmonella
spp., before microwave exposure to after one minute of exposure similar photographs
were taken which revealed dramatic decrease in overall bacteria cultured on the plates
similarly utilizing the same dilution from the same sponge (Figures 16 and 17). Through
the use of this data it can be concluded that microwave treatment does show an overall
increase in bacterial inactivation and additionally in comparison of percentage decrease
between gram positive bacterial species to gram negative species, it can be speculated
that gram negative rod bacteria are more susceptible to microwave radiation than gram
positive species.
The average number of lactose fermenting colonies was recorded for before
microwave subjection and compared to after 30 seconds and after 60 seconds. This data
revealed the average recorded number of lactose fermenting colonies on MacConkey agar
before microwave subjection was calculated as 55.5 CFU/g (Table 20). Additional
calculations revealed that after 30 seconds of microwave subjection the average number
of colony forming units per gram was decreased to 49.69 CFU/g, a 10.65% decrease then
decreased even further after 60 seconds to a low of 25.5 CFU/g of sponge, showing a
54.05% decrease in lactose fermenting opportunistic pathogenic bacteria (Figure 22 and
Table 20). Photographs of lactose fermenting bacteria were taken on MacConkey plates
before microwave exposure and after 30 seconds in comparison of the effects of
microwave induced inhibition on the bacteria revealing a clear decrease in total bacteria
81
after only 30 seconds (Figure 13 and Figure 14). Data clearly shows that microwave
radiation exposure on the sponge effectively decreases lactose fermenting opportunistic
pathogenic coliform bacteria, such as Escherichia coli, Klebsiella spp., and Enterobacter
spp. in as little as 30 seconds, with a decrease in 50% by one minute of exposure (Table
20). Through microwave exposure coliforms, gram negative lactose fermenting bacteria,
often opportunistic pathogenic bacteria can be successfully decreased.
Lactose fermentation was variable, resulting in 0 colonies able to ferment lactose
recorded on 3 sponges to as much as 297 colonies on sponge 50B (Table 4). Through the
analysis of the MacConkey agar lactose indicator on plates collected, it can be strongly
speculated that coliforms are present in 89.29% of the total sponges collected before
microwave subjection (Table 3). Figures were created according to each time exposed
and the percentage of plates positive for lactose fermenting colonies. Before microwave
subjection revealed 69.39% of plates containing lactose fermenting colonies (Figure 24).
This total number was decreased after 30 seconds of exposure to 61.54%, then to 56.52%
of plates positive for colonies after 60 seconds of exposure (Figures 25 and 26). Using
this data it can be speculated that the occurrence of lactose fermenting colonies on plates
is affected by microwave exposure 60 seconds of exposure clearly decreased the
occurrence rate of lactose fermenting opportunistic pathogenic bacteria. Lactose
fermentation is very important as opportunistic pathogenic bacteria such as Escherichia
coli, Genus Klebsiella, and Genus Enterobacter, are known lactose-fermenting bacteria
(Johnson, 1998). Overall total coliform occurrence was decreased and subsequently
opportunistic pathogenic bacteria were discussed.
82
In conjunction with MSA’s selective qualities, the differential qualities of the
media were utilized which tests for mannitol fermentation. This differential quality leads
to the separation of pathogenic and non-pathogenic Staphylococcus species. For before
microwave subjected sponges, it was noted that sponge 17B was shown positive for
mannitol fermentation establishing a link to pathogenicity and suggesting that sponge
contains pathogenic Staphylococcus aureus (Table 3, Table 21, and Figure 3). Mannitol
fermenting colonies were morphologically described through macroscopic characteristics
and gram staining noted in Table 21. Figure 23 revealed that only 2% of the sponges
before microwave subjection contained pathogenic Staphylococcus species, being a very
low percentage (Figure 23). In a study by Chaidez and Gerba, Staphylococcus aureus was
noted on 60% of the cellulose sponges collected and 86% of the loofahs which was
considerably higher than the collected data of the one sponge positive for fermentation,
which calculated data revealed a much lower percentage (Chaidez and Gerba, 2000).
Though only isolated from a small percentage of sponges collected, S. aureus was still
noted in the batch of sponges collected establishing a connection of the sponges to
pathogenicity.
Data regarding CFU/g on Xylose Lysine Deoxycholate agar, which was
particularly used for its ability to culture Salmonella, commonly associated with cross
contamination resulting in foodborne illness, typhoid fever, gastroenteritis, and
paratyphoid fever, was put together in Table 23 (FSIS, 2012 and Davis, 2012). Colony
forming units per gram were calculated on XLD agar with Salmonella being noted
sponges 26B, 35B, and 36B, being characterized by the black dot present in the middle of
the colony due the ability to produce hydrogen sulfide (Table 23; Davis, 2012).
83
Salmonella being present on 6.1% (Figure 27) of the sponge samples collected is a
relatively low percentage, but was still found in this relatively small demographic
surveyed of college students in which 5-7 individuals were using each of these sponges.
In accordance to a study conducted by Chaidez and Gerba, in which it was revealed that
Salmonella spp. was identified in 9.8% of the sponges tested, in which roughly ten
percent of the sponges analyzed contained Salmonella spp. (Chaidez and Gerba, 2000).
Even though only three sponges were positive for Salmonella growth, Salmonellosis
could have been contracted leading to development of diarrhea, fever, and abdominal
cramps around 12 to 72 hours after infection lasting 4 to 7 days in the individuals using
the sponge (CDC, 2012). The idea that Salmonella was found in a small demographic of
numerous people sharing a common sponge could have been problematic in which a
quick means of disinfection could have remedied the situation s. For all types of media
the average CFU/g was found in addition to the average CFU/g being graphed average
and compared.
Data collected from utilization of the 16s ribosomal subunit and MEGA software
for classification of bacteria in samples down to species level were omitted from this
thesis due to unforeseen circumstances. Upon analysis of the 16s subunit variable region
sequences received through Function Biosciences Inc., inaccurate data was obtained.
Inaccurate data was caused in part by very few matches for numerous samples in which
high max identity and low equivalence values were not obtained. Correct identification of
common bacteria found in each sample would not have been possible with the sequence
results received of each sample submitted.
84
Overall Conclusions
Sponges were collected from college students living in a 5-7-person dorm with
one kitchen. It was shown that only 8.7% of the sponges were disinfected through any
means before collection. It can be speculated that this many households do not commonly
disinfect their sponges, allowing bacteria, including opportunistic and pathogenic
bacteria, to build up to a harmful level in sponges. These bacteria may then be spread
around the kitchen, and onto food and dishes. Educating individuals about the potential
hazards of harmful microbes to spread through this vector is an important matter to be
discussed and that there is a quick and effective solution. A microwave, a very common
household appliance, can quickly and effectively solve this problem of bacteria build up.
Overall, these data suggest that microwave radiation exposure is an effective
means to disinfect common cellulose based kitchen sponges. Through research it is
shown that as little as 30 seconds of exposure in a 1000 watt microwave bacteria can
decrease bacteria by 93.79% and 98.03% after 60 seconds of exposure (Tables 18 and
19). The percent inhibition was compared between before exposure to 30 and 60 seconds
after exposure showing a clear increase in percent inhibition compared to before in which
60 seconds showed the largest percent decrease across all media. This method of
disinfection is a very effective and easy way of disinfecting sponges, as almost every
household possesses a microwave already, so no expensive specific cleaners need to be
purchased. Both 30 and 60 seconds of microwave exposure were shown to decrease the
overall average CFU/g on each type of media, with gram negative rods suspected to be
the most affected by the exposure.
85
Gram positive Staphylococcus species seemed to be the least affected utilizing the
data collected by only showing a 72.11% decrease after 60 seconds of microwave
exposure as shown on the MSA plates utilized in this experiment. Relatively no mannitol
fermentation was noted in the sponges collected revealing very little episodes of
Staphylococcus aureus. Additionally microwave exposure showed effective in decreasing
the number of lactose fermenting bacterial colonies from 55.5 CFU/g to 49.69 CFU/g
after 30 seconds, and 25.5 CFU/g after 60 seconds. Data showed a 54.05% decrease in
Enterobacteriaceae opportunistic pathogenic bacteria as shown through the decrease in
Lactose fermenting bacteria on MacConkey agar. Lactose fermenting bacteria, such as
Escherichia coli, Enterobacter spp., and Klebsiella spp., showed a decrease after only 60
seconds of exposure. Through this research presented on MacConkey agar differential
lactose fermenting colonies were isolated on before, after 30 seconds, and after 60
seconds revealing the percentage of plate containing lactose fermenting opportunistic
pathogenic bacteria to be 69.39%, 61.54%, and 56.52% respectively. T-test calculation
revealed a p-value of 0.3731 in comparison of before microwaving to 30 seconds after
microwaving supporting the hypothesis that there is a significant difference between
these two values due to microwave exposure. In comparison on before microwaving to 60
seconds after a p-value of 0.017031 was calculated revealing strong difference between
the two sets of data due to microwave radiation exposure on lactose fermenting bacterial
colonies (Figure 22). Overall decrease in lactose fermenting colonies was noted revealing
microwave exposure has an effect on lactose fermenting colony inhibition in as little as
30 seconds of exposure.
86
Mannitol Salt Agar plates revealed mannitol fermenting colonies on plated
cultured by sponge 17B, revealing positive pathogenic Staphylococcus species (S.
aureus) growth on the sponge. This sponge revealed clear link to pathogenicity.
In regard to XLD, opportunistic bacteria Salmonella was noted on sponges 26B,
35B, and 36B, when plated on XLD agar in which this bacterial species was not present
in subsequent sponges after microwave radiation at 30 or 60 seconds. This bacterial
species was calculated to be found on 6% of the before microwave subjected sponges,
and 0% of the plates after 30 seconds and 60 seconds. Through this data it can be
speculated that microwave radiation exposure is an effective means in reducing
Salmonella present in sponges.
Overall, data reveals an overall decrease in bacteria culture on selective and
differential media, being lactose fermenting colonies, Salmonella species, and mannitol
fermenting colonies on MacConkey, XLD, and MSA agars respectively. Additional
colony
morphologically
described
were
characterized
based
on
macroscopic
characteristics, but could not be concluded to be the same bacteria based on morphology
alone. This information provides insight into the existence of common bacterial colonies
found in each sponge which are readily cultured by the specific agar chosen. Micro- and
macro-scopic characteristics of these common colonies inhabiting kitchen sponges were
recorded for investigation in the future. The data supplied and analyzed in this research
all evidence supports the idea that microwave exposure is an effective method in
decreasing total bacterial count, notably lactose opportunistic pathogenic coliform
bacteria and Salmonella species, both of which are commonly involved in both cross
contamination and foodborne illness cases. However because of the low numbers
87
observed in this research further investigation into microwave exposure induced decrease
of pathogenic Staphylococcus species, additional research must be done in order to
confirm whether microwave radiation does decrease mannitol fermenting colonies.
Due to the circumstances data on 16srRNA analysis was omitted as no
conclusions could be properly processed at this time. Further investigation into this
section is needed in future studies and must be done again.
Through utilization of a common kitchen appliance bacteria growing in a tool
made for cleaning can be dramatically decreased in which cleaning, instead of spreading
around possibly pathogenic microbes, can be truly utilized for a truly cleaner kitchen
space.
Future Studies
Further research into the species of the common colonies characterized on each
media must be pursued in order to properly identify the colonies described, thus
identifying the most common colonies present on sponges. Data collected while running
the original 16s rRNA samples were faulty and gave inaccurate results into the most
common species of bacteria in each sample sent upon analysis. In result, 16s ribosomal
RNA analysis must be redone in order to retrieve data on all species of bacteria found in
before sponges and also sponges subjected to 30 and 60 seconds of microwave exposure
with additional analysis into which bacteria are most susceptible. For more accurate data
of the percentage decrease of exposed sponges the same sponge could be used for before,
30 seconds, and 60 seconds of microwave exposure instead of switching between 30
88
seconds and 60 seconds of exposure every other week. In particular, this would lead to
more accurate results in regards to percentage decrease of total bacteria on sponges
especially the effects of the additional 30 seconds of exposure between 30 and 60
seconds. Further research into the effects of microwave exposure on bacteria, and
common bacteria inhabiting cellulose based kitchen sponges, can be done in the future
using this research as a basis.
Data collected involving microwave radiation exposure on reduction of total
bacterial CFU/g suggests that microwave exposure has a bactericidal effect and can be
successfully used in the process of disinfection of cellulose based kitchen sponges in
order to reduce total bacterial counts per gram of sponge. Microwave exposure was
shown to be successful in decreasing opportunistic pathogenic bacteria through decrease
in mannitol fermenting bacteria, Lactose fermenting bacteria, and Salmonella spp.
Common colonies were noted in each sponge based on selective and differential tests of
each media utilized.
89
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98
APPENDICES
Table 22
TRYPTICASE SOY AGAR COMMON COLONIES MACROSCOPICALLY AND
MICROSCOPICALLY DESCRIBED
TSA Common Colonies
Colon Macroscopic
y
Observations
1
Cream
colored
circle
1mm
shiny
2
cream
colored with
whiter dot
in center
5mm
round
shiny
Gram Stain
Sponges present
Gram positive
cocci
1µm
G- rods, 2um
99
Beforeo 2B, 3B, 4B, 5B, 6B,
8B, 9B, 10B, 11B,
12B, 13B, 14B, 15B,
16B, 17B, 18B, 19B,
20B 21B, 22B, 23B,
24B, 25B-37B, 41B50B
30 secondso 2A, 3A, 4A, 5A, 6A,
13A, 14A, 15A, 19A,
20A, 21A, 22A, 23A,
24A, 31A-37A, 43A47A
60 seconds
o 7A, 8A, 9A, 10A,
11A, 12A, 16A,
17A, 18A, 25A30A, 41A, 42A,
48A-50A
Before
o 2B, 3B, 4B, 5B, 6B,
8B, 9B, 12B, 13B,
14B, 15B, 16B, 17B,
18B, 19B, 21B, 22B,
23B, 24B, 25B-30B,
31B, 33B-37B, 41B50B
30 secondso 2A, 3A, 4A, 5A, 6A,
15A, 20A, 21A, 22A,
23A, 24A, 32A, 34A37A, 43A-47A
60 secondso 7A, 8A, 9A, 11A,
12A, 16A, 17A, 18A,
27A, 30A, 41A, 42A,
48A-50A
3
4
5
6
bright
yellow
mustardy
shiny
round
4mm
G- rods
dull
white
flaky with
jagged
edges
raised
design in
center
circle with
jagged edges
creamish
yellow
dot in center
3-5mm
G- rods,
8um
tannish
brown
circular
wet
4mm
G-rods, 4um
7-
G- rods, 2um
100
Before
o 2B, 3B, 4B, 6B,
15B, 16B, 17B,
22B, 23B, 24B,
30B, 31B-33B,
41B, 42B, 46B
30 secondso 20A, 21A, 22A,
24A, 32A-37A,
47A
60 secondso 11A, 16A, 25A,
41A, 42A
Beforeo 4B, 5B, 9B, 47B
30 secondso 2A, 3A, 45A
60 seconds
o 41A, 42A, 50A
11B,
18B,
25B35B,
23A,
46A,
29A,
Beforeo 7B, 11B, 19B, 22B,
23B, 19B, 22B, 23B,
25B, 26B, 27B, 32B,
33B, 34B,
30 secondso 15A, 32A, 34A, 35A,
36A
60 secondso 7A, 11A, 12A, 17A,
18A, 26A
Beforeo 8B, 9B, 11B, 12B,
13B, 15B, 17B, 19B,
21B, 23B, 25B- 29B,
32B-37B,
30 secondso 15A, 32A-37A
60 secondso 9A, 10A, 11A, 12A,
16A, 18A, 28A-30A,
Table 23
MANNITOL SALT AGAR COMMON COLONIES MACROSCOPICALLY AND
MICROSCOPICALLY DESCRIBED
MSA Common Colonies
Colon Macroscopic
y
Observations
1
0.5mm
white
round
shiny
2
pinkish
.5mm
round
shiny
Gram Stain
G+
cocci
G+
cocci
101
Sponges present
Beforeo 7B, 8B, 9B, 10B, 11B,
12B, 13B, 15B, 16B,
17B, 18B, 22B, 23B,
24B, 27B, 32B
30 secondso 24A
60 seconds
o 11A, 12A
Beforeo 7B, 8B, 9B, 10B, 11B,
12B, 13B, 16B, 21B,
23B, 24B, 25B-30B,
32B, 35B, 36B, 37B
30 secondso 14A, 15A, 22A, 23A,
24A, 32A, 33A
60 seconds
o 8A, 9A, 10A, 11A,
12A, 17A
Table 24
MACCONKEY AGAR COMMON COLONIES MACROSCOPICALLY AND
MICROSCOPICALLY DESCRIBED
MacConkey Common Colonies
Colon Macroscopic
y
Observations
1
purple/greyish
shiny
circle
5mm
2
large, round
light
purple
edges with
cream center
positive
lactose
fermentation
Gram Stain
Grods
Grods
102
Sponges present
Beforeo 2B,
3B,
4B,
5B,
6B,
7B,
9B, 10B,
11B, 12B,
13B, 14B,
15B, 16B,
19B, 20B,
21B, 21B,
22B, 23B,
24B, 25B30B, 31B37B,
30 secondso 2A, 3A, 5A, 13A,
14A, 15A, 19A, 20A,
21A, 22A, 23A, 24A,
31A-37A
60 secondso 7A, 8A, 10A, 11A,
12A, 16A, 17A, 18A,
25A-28A, 30A,
Beforeo 2B, 4B, 5B, 6B, 7B,
8B, 9B, 10B, 11B,
12B, 13B, 14B, 15B,
16B, 18B, 19B, 20B,
21B, 22B, 23B, 24B,
25B-30B, 31B-37B
30 secondso 2A, 4A, 5A, 15A,
19A, 20A, 21A, 22A,
23A, 24A, 32A-37A
60 secondso 7A, 8A, 9A, 10A,
12A, 16A, 17A, 18A,
3
greyish clear
edges with
brownish
black center
2mm
circle
Grods
103
25A, 27A-30A
Beforeo 2B, 5B, 6B, 8B, 12B,
15B, 16B, 19B, 21B,
22B, 23B, 24B, 25B,
26B, 31B-37B
30 secondso 2A, 5A, 13A, 20A,
21A, 22A, 23A, 24A,
32A-36A
60 secondso 7A, 11A, 12A, 17A,
18A, 25A, 26A
Table 25
XYLOSE LYSINE DEOXYCHOLATE AGAR COMMON COLONIES
MACROSCOPICALLY AND MICROSCOPICALLY DESCRIBED
XLD Common Colonies
Colon Macroscopic
y
Observations
1
circle
pink wet edges with
lighter
pink center
shiny
flat
3mm
Gram
Stain
Grods,
11.5µ
m
2
light yellow
same colored dot in
center
looks like a car wheel
raised 5mm
Grods,
0.250.5µ
m
3
yellow/cream
circle
clear
grey
middle
shiny
slightly raised
Grods,
0.250.5µ
m
Sponges present
Beforeo 2B, 3B, 4B, 5B, 6B, 7B, 8B,
9B, 10B, 11B, 12B,13B,
14B, 15B, 17B, 18B, 19B,
20B, 21B 22B, 23B, 24B,
25B-30B, 31B-37B
30 secondso 2A, 3A, 4A, 5A, 6A, 14A
19A, 23A, 24A, 31A-37A
60 secondso 7A, 8A, 9A, 10A, 11A,
12A, 16A, 17A, 18A, 25A30A
Beforeo 2B, 3B, 5B, 6B, 8B, 9B,
11B, 12B, 14B, 16B, 19B,
20B, 21B, 22B, 23B, 24B,
25B, 27B-30B, 32B-35B,
37B
30 secondso 2A, 3A, 4A, 6A, 15A,
22A,23A, 33A, 34A, 36A
60 secondso 7A, 8A, 9A, 11A, 12A, 17A,
25A, 28A-30A
Beforeo 3B, 5B, 8B, 11B, 12B, 16B,
17B, 25B, 26B, 28B-30B,
35B, 37B
30 secondso 3A, 4A, 6A, 29A
60 secondso 8A, 12A, 16A, 17A, 30A
104
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