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Saint Martin’s University
The Interactions between Concentration and Time on
Staphylococcus aureus when using Virex Tb™, pure thyme oil,
and Benefect™ as Germicidal Disinfectants
Michelle Coffey
May 8, 2007
Senior Seminar
Dr. Olney, Dr. Hartman
Abstract
This study was done to compare the effectiveness of Virex Tb™, a commercial cleaner,
to the environmentally friendly cleaners of thyme oil concentrate and Benefect™. The
cleaners were tested against a common bacterium, Staphylococcus aureus. Zone of
inhibition was measured in millimeters after incubating 288 Petri plates per chemical for
48 hours at 37C. Optical density tests were run after sampling floor tiles before and
after cleaning the tiles with one of the three products, 28 before and after samples were
measured for each cleaner. A spectrophotometer was used to measure the amount of
bacteria removed from the floor tiles during cleaning after being incubated for 48 hours at
37C. The amount of bacteria inhibited by the cleaners was greater for the Virex Tb™
then for the thyme oil or the Benefect™ suggesting the thyme oil and Benefect™ are not
good germicidal cleaners. The optical density tests showed that all three cleaners
removed the same amount of bacteria from the floor tiles. Each cleaner had relatively
high absorbency levels meaning each cleaner was useful at removing the bacteria from
the surface of the tiles.
2
Table of Contents
Introduction
4
Methods I
15
Methods II
20
Results
23
Discussion
27
Acknowledgements
31
Literature Cited
32
3
It is estimated that 1/3 of the U.S. population have bacteria known as
Staphylococcus aureus inhabiting their bodies and living within their environment (Davis
et al., 2004). The population abundance of S. aureus has resulted in 5 to 15 % of the
total amount of infections acquired in hospitals (Tortora et al., 2004). Infections
obtained from being in a hospital are called nosocomial infections. Examples of
nosocomial infections are urinary tract infections, lower respiratory infections, skin
infections, and infections of tissue surrounding a surgical site. An estimated 34% of
nosocomial infections are caused by S. aureus (Tortora et al., 2004). The wounds and
depressed immune systems of those who enter the hospital lend themselves as targets to
S. aureus that normally live on the skin’s surface and mucus membranes. The illnesses
caused by S. aureus rarely result in death, but can cause severe discomfort. For instance,
Toxic Shock Syndrome is the result of S. aureus infecting the blood stream. Food
poisoning is associated with the ingestion of S. aureus often found on the surface of meat
products, such as ham and other cured meats. If S. aureus penetrates the surface of a
person’s skin, it can cause a very uncomfortable skin rash and peeling, referred to as
Scalding Skin Syndrome (Stainier et al., 1986).
S. aureus is a facultative anaerobe (able to use oxygen when it is present, but can
also live without it) allowing it to thrive in and on the body (Brown, 2005). This
bacterium survives under high osmotic pressures and low moisture environments,
enabling it to live on the surface of the skin and in the nasal secretions of noses. S.
aureus infections begin when the bacteria either penetrate the epidermis (top layer) of the
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skin or are ingested. Once the bacteria enter the body it produces toxins such as
leukocidin, exfoliative toxin, and enterotoxins resulting in a variety of painful illnesses
(Tortora et al., 2004). Despite aggressive actions to kill S. aureus, it is becoming more
resistant to antibiotics and germicides everyday. Twenty-five to 87 percent of S. aureus
nosocomial infections acquired from infectious are resistant to antibiotics (Tortora et al.,
2004). The primary explanation for the decrease in susceptibility to antibiotics and
cleaners is because of molecular mutations within the bacteria.
In an experiment by Davis et al. (2004), mutations expressed by S. aureus were
studied for their level of resistance to household cleaners. The samples for this study
were a collection of 76 strains of S. aureus from two medical centers. The antimicrobial
susceptibilities of the bacterial strains were tested against common household cleaners
such as Pine Sol™, Orange Clean™, and Simple Green™, which had been stored at
room temperature until use. Resistances for the antibiotics methicillin, vancomycin, and
oxacillin were tested along with the cleaners. In addition to the 76 strains of bacteria,
Davis et al. (2004) also collected 76 strains of methicillin resistant S. aureus (MRSA).
These MRSA strains were analyzed and divided into two categories, MRSA strains with
mutations and those without mutations. Working stocks of the two types of MRSA
strains were prepared in nutrient rich broth and incubated for 24 hours.
The tests of Davis et al. (2004) were carried out using Luria broth agar plates
containing different concentrations of the antibiotics vancomycin and oxacillin. To test
the different antibiotics and cleaners, a gradient plating technique was used. The gradient
plating technique used a double layer of Luria broth agar. The first layer was solidified
as a slant in the bottom of a square dish. The second layer of the dish contained the same
5
Luria broth agar but it contained a specific amount of the cleaner or antibiotic mixed in.
The mixture was poured over the first layer and was allowed to lay flat as it solidified
making a level surface. Once the chemical layer had solidified, the plates were
inoculated with S. aureus and incubated at 37°C for 48 hours. After incubation, Davis et
al. (2004) measured the area of inhibited growth of the bacteria by measuring the
diameter where no bacteria were found on the plates.
Davis et al. (2004) found that known mutant strains of S. aureus increased in
colony size after being exposed to increased vancomycin concentration. S. aureus
strains without mutations showed decreased colony sizes. These results indicate
germicides or antibiotics affect MRSA strains that do not have the mutation, while
MRSA strains with the mutation were not greatly affected by the cleaners or the
antibiotics. With this information in hand, the authors conducted a comparison of the
growth of S. aureus with the ingredients in the germicidal detergents they used. Davis et
al. (2004) discovered cleaners that effectively decreased the colony sizes of the mutant
MRSA strains contained ammonium hydroxide, a powerful germicidal agent. This
discovery is important because it helps provide a method to solving the problem of S.
aureus’ increasing resistances to cleaners. The reliability of cleaners and antibiotics is
decreasing. Therefore, it is not only important to find out why product effectiveness is
declining, but also to find out what will kill S. aureus.
Many studies have attempted to understand what kills S. aureus. Davis et al.
(2004) suggested that knowing the molecular alterations occurring within the bacteria cell
are the key to understanding why S. aureus has increased resistance to germicides and
antibiotics. A group of scientists investigated the antibacterial properties of cationic
6
surfactants (positively charged chemicals exhibiting detergent like properties) on prelaundered 100%-cotton cleaning cloths. Abel et al. (2002) predicted that the cationic
(positively charged) site of the agent would bind with the anionic (negatively charged)
sites of the cell wall of the bacteria. This process would then result in electrostatic
interactions and physical disrupt the bacteria cell. Abel et al. (2002) synthesized
modified carbohydrate surfaces they felt would be able to disturb the cell membrane of
the bacteria allowing electrolytes and nucleic materials to be released from the cell
resulting in cell death.
Abel et al. (2002) conducted their study by first acquiring seven strains of
bacteria, four that were Gram-negative and three were Gram-positive, including S.
aureus. The bacteria were added to carbohydrate-modified surfaces of laminated
tabletops. Eight surface types were created, each having antibacterial properties, with
one modified carbohydrate type on each surface. The bacteria were added to the
modified surfaces and incubated along with three controls on the same plate. Any
bacterial growth visible over the edge of the surface was observed by the naked eye and
documented. After visual observations were recorded, 2 mL of liquid growth medium
was flushed over the surfaces with the bacteria and the solution was collected in test
tubes and incubated for sixteen hours (Abel et al., 2002).
The growth of the bacteria was determined by turbidity testing, where a
spectrophotometer was used to determine the absorbance, or optical density, of the
culture (Brown, 2005). The results of Abel et al.’s (2002) study showed S. aureus were
completely eliminated when exposed to four of the eight antibacterial surfaces. It was
speculated by Abel et al. (2002) that the positive and negative charges of the modified
7
surfaces allowed the cleaner to absorb into the surface of the bacterial cell easier because
the electrical charges of the carbohydrate surfaces combined with the charges of the cell.
The chemical attractions allowed the cleaner to absorb to the bacteria, enabling it to
destroy vital functions taking place within the cell.
After analysis, Abel et al. (2002) found the level of inhibition, or area without
growth, on the four modified surfaces to be great enough that S. aureus was determined
to be susceptible. Even after an increased concentration of the bacteria was placed on the
modified surface, total elimination of the bacteria resulted. However, even though S.
aureus is susceptible to these four surfaces, the other four antibacterial surfaces did not
affect S. aureus providing confirmation that the susceptibility of S. aureus is decreasing.
Also, the bacteria’s susceptibility was only seen on the surfaces that contained the
modified carbohydrates with a chain length of 10-16 carbons in the lipophilic region.
The length of the lipophilic is the most important feature because this region hooks into
the cells surface just far enough that it cannot be expelled initiating cell damage (Abel et
al., 2002). The four surfaces that did not react with the bacteria contained carbon chains
shorter then 10 carbons and were unable to hook into the bacteria properly.
Problems with disinfection have occurred since the mid 1800s when Joseph Lister
first developed techniques for sterilization after medical procedures (Tortora et al., 2004).
Spaulding and Emmons (1958) addressed the problems associated with proper
disinfection. The two scientists suggested that the misconceptions surrounding multipurpose disinfectants are thinking a multi-purpose disinfectant exists. According to the
researchers, one cleaner cannot be used for multiple scenarios. Many people assume a
cleaner that works for floors is also good for cleaning countertops and toilets. But, as
8
Spaulding and Emmons (1958) point out with their research, there is a broad range of
products designed for disinfecting specific areas such as floor cleaning, decontamination
of airways, sterilization of instruments used for surgeries, etc.
There are multiple factors to consider when determining how and what to use for
cleaning a particular surface. According to Spaulding and Emmons (1958) there are three
main factors that influence the success rate of germicidal detergents; concentration,
corrosiveness, and time. Concentration and corrosiveness go together in that the
corrosiveness of a product determines whether or not a strong concentration should be
used. Ideally, the strongest concentration possible should be used when killing
microorganisms because the stronger the solution, the faster it will kill the
microorganism. The third factor of time is the most important and is driven by three
factors. When trying to determine how much time a chemical must be exposed to the
microorganism to properly kill it, the nature of the disinfectant and contaminant must be
taken into consideration along with the overall effectiveness of the chemical as a killing
agent. This is because a porous surface requires different procedures than a smooth
surface. Also, each type of microbe requires special consideration. One product
designed to kill nonsporulating vegetative bacteria will most likely not work on spore
forming microbes, and neither of the products that work on those two would work for
tubercle bacillus.
Spaulding and Emmons (1958) tested several bacterial species. They concluded
that concentration strength coupled with the correct length of time could ensure proper
disinfection. However, these factors vary among types of cleaners, such as chlorines and
germicidal detergents called quats. The authors conclude that different types of
9
germicidal cleaners will be effective against certain bacterial species, such as tubercle
bacillus and nonsporulating vegetative bacteria; but will fail against spore forming types.
Therefore, a multi-purpose cleaner does not exist; as each cleaner is designed for a
specific purpose (Spaulding and Emmons, 1958).
Spaulding and Emmons’ (1958) research began to answer the question: how can
you properly disinfect a surface? Olson et al. (1994) examined this question further by
testing common household cleaners for their efficiency of removing debris, along with
their effectiveness to reduce bacterial levels on those surfaces. Tests were conducted on
kitchen and bathroom laminate tiles covered in chemically manufactured grime, to mimic
soils commonly found in these environments. Once the tiles had been covered in the
fatty acid sebum soil, they were exposed to Serratia marcescens (red pigmented bacteria
thought to contribute to nosocomial infections) and incubated for 24 to 36 hours.
Sponges were used to apply different cleaning agents and were soaked in simulated hard,
warm water before applying the cleaner. The alternative cleaning products tested on the
kitchen tiles were lemon juice, vinegar, baking soda, and Borax™. Along with these
alternative products additional alternative products were used on the bathroom tiles,
mainly household ammonia, and hand washing liquid Ivory™ soap. Both types of tiles
were tested using Spic and Span with Pine™ and Clorox Clean-Up™ for the commercial
cleaners (Olson et al., 1994). After the tests had been completed, a comparison of the
effectiveness for both soil removal and microbial reduction were conducted.
The results of the experiment are divided into two categories, one for microbe
reduction and another for soil removal. For the microbe reduction, the commercial
cleaners had lower bacteria counts than alternative products. However, vinegar showed
10
similar results to the commercial cleaners’ microbe reduction. Water and ammonia did
an average job of removing bacteria from the tiles, while dishwashing liquid and baking
soda resulted in the highest growth out of all of the alternative products. For the soil
removal results a significant difference was found to exist within the alternative cleaner
group. Borax™ had the best soil removal for the alternative group and vinegar had the
lowest amount of soil removal. Baking soda was found to have no statistical significance
from Clorox Clean-Up™. For the kitchen soil removal, ammonia had a high level of soil
removal. The highest level of soil removal was seen from Spic and Span with Pine™
(Olson et al., 1994). The lemon juice results were not analyzed because there were too
few observations. Overall, the commercial cleaners were effective at removing dirt,
grime, and bacteria. But is that always the case?
In a study by Bauer et al. (1995) products similar to those tested by Olson et al.
(1994) were tested for their effectiveness against dirt and bacteria. Borax, vinegar, and
ammonia were tested against Lysol Disinfecting Multi-Purpose Cleaner™. All
detergents were mixed according to the manufacturers’ specifications and were not
altered. The cleansers were tested on sterile slides that had been exposed to bacteria
including S. aureus and Klebsiella pneumoniae. The slides were then subjected to the
different chemicals, ten tests per organism per cleaner. Following these tests, another test
was conducted in which real surfaces were decontaminated with rubbing alcohol prior to
their exposure to S. aureus and Escherichia coli. After exposure to the bacteria, cleaners
were applied and the colonies that grew on the slides were counted. Bauer et al. (1995)
conducted a further test to determine the effectiveness of cleaners on non-food contact
surfaces by observing colonies of S. aureus and K. pneumonia that formed after being
11
exposed to each of the chemicals for five minutes. The results of the tests led to the
conclusion that none of the environmentally friendly or “green” alternative cleaners
effectively reduced the number of colonies formed by the bacteria after disinfection
(Bauer et al., 1995). Borax, vinegar, ammonia, and baking soda are thought to lack the
chemical properties necessary for adequate germicidal removal because of their
molecular structure. In order for a chemical to kill bacteria, it is necessary for the
chemical to penetrate the plasma membrane’s phospholipid bilayer that surrounds the
exterior of the bacterial cell. Once the chemical(s) is inside the bilayer, it chemically
disrupts the production of the bacteria’s survival proteins and causes leaking of
intracellular contents (Tortora et al., 2004). Also, once the chemical seeps further into
the cell membrane where the level of cross-linkages between the glycan strands of the
peptidoglycan layer is lowered, the structural backbone of the cell is weakened (Tortora
et al., 2004). Unfortunately, alternative cleaners such as baking soda, ammonia, and
vinegar cannot successfully maneuver through the bacterial cell membrane. The
cleaners have charges similar to those of the interior of the cell producing a repelling
action and making them ineffective as germicidal detergents (Bauer et al., 1995). The
Lysol Disinfecting Multi-Purpose Cleaner™ showed a 100% reduction in the colony
growth of S. aureus, E. coli and K. pneumonia (Bauer et al., 1995).
If alternative cleaners such as baking soda, ammonia, vinegar, and Borax™ do not
kill bacteria, then maybe there is not a “green” cleaner that can sufficiently kill bacteria.
In an experiment conducted by Rasooli and Abyaneh (2004), two essential oils extracted
from a thyme plant were tested for their effectiveness against the fungi Aspergillus flavus
and Aspergillus parasiticus. Both fungi are spore-forming types most commonly found
12
on molded food. But, unlike other molds that can grow on food, Aspergillus fungi can
cause severe health problems when consumed. In fact, some Aspergillus fungi have been
known to cause cancer (Rasooli and Abyaneh, 2004).
Two types of thyme oil were used in Rasooli and Abyaneh’s experiments. Plants
grown at the National Botanical Garden of Iran were used to acquire the extracts needed
in this experiment. Extracts were collected from two types of thyme plants, Thymus
eriocalyx and Thymus x-porlock. After the oils had been extracted, they were diluted
with methanol. Rasooli and Abyaneh (2004) chose methanol because it lacked antifungal
properties and would ensure little interaction from the solvent. Rasooli and Abyaneh
(2004) used a disk diffusion method for determining the effectiveness of the oils.
Samples of the Aspergillus fungi spores were streaked onto Petri dishes that had been
prepared with agar. After the plates had been streaked, paper disks that had been soaked
in 10 µL of thyme oil were applied to the Petri dishes. The thyme oil was prepared at
dilution levels of 1/2, 1/4, 1/8, 1/16, and 1/32. Ten µL of methanol were added to the
control disks without any thyme oil. The plates were incubated at 30C for 48 to 72
hours, and the zone of inhibition was measured to determine the effectiveness of the oils.
The results of this experiment revealed the growth of the spores was inhibited by both
types of thyme oil (Rasooli and Abyaneh, 2004). However, the level of effectiveness for
the thyme oils decreased as the concentration levels were diluted more than 1/4. Overall,
Rasooli and Abyaneh (2004) determined that essential oil from thyme plants is an
effective germicidal detergent. If further testing on other types of microorganisms shows
similar results, an effective environmentally friendly germicidal cleaner could be widely
used.
13
Many environmentally friendly cleaners do not contain the proper active
chemicals necessary for microbial death. Alternative cleaners tend to lack chemicals that
contain the molecular structures needed to penetrate the bacteria’s plasma membrane.
Therefore, the cellular content cannot be spilled, which is necessary for the bacteria’s
death (Spaulding and Emmons, 1958). However, alternative cleaners have been
discovered to kill other organisms other then bacteria. Rasooli and Abyaneh (2004)
determined thyme oil, an environmentally friendly product, could effectively kill
Aspergillus fungi. But, did this also mean thyme oil have antibacterial properties as well?
In my experiment I tested if thyme oil could effectively inhibit the growth of the bacteria
S. aureus. I hypothesized that when Virex Tb, a commercial cleaner, is compared with
concentrated thyme oil and Benefect™, the zone of inhibition for S. aureus will be the
same. But the zone of inhibition would only be the same if the concentration and length
of exposure time was kept at the manufacturer’s recommended level. I further
hypothesized bacterial removal would be greater for the Virex Tb™ than for the thyme
oil or the Benefect™. This study was conducted in two parts. First, large numbers of
plated S. aureus were exposed to each chemical where the chemical concentrations and
exposure time were altered. Second, laminated floor tiles were tested for microbial
removal for each of the three chemicals. The results were used to determine which
chemical was most effective.
14
Methods I
Pouring plates
To maintain sterile plates the first task in pouring them required creating a sterile
environment. A chemical hood was sterilized with disinfectant and alcohol prior to
pouring the plates. Setting up the plates for pouring required removing them from the
sterile bag they came in and placing them in sets of five. Agar was poured until each
plate was about 70% covered or 10 mL of agar. As each stack of five plates was poured,
they were carefully swirled to ensure the agar covered the entire surface of the plate.
Then, the plates were stacked to allow a slow drying time, which decreased the amount of
condensation that formed on the lid. The plates were allowed to sit undisturbed for
approximately one hour on a flat surface to solidify (Brown, 2005).
Culturing Staphylococcus aureus
The bacterium, Staphylococcus aureus, was cultured in nutrient broth (Smith et
al., 2000). Liquid nutrient broth was made by adding 0.6g of dry nutrient broth to75 mL
of deionized water. Once the broth was mixed thoroughly 25 mL was poured into each of
3 large, clean test tubes. The test tubes were sterilized by autoclaving in a Tuttnauer
2540E autoclave at 121°C for 15 minutes at 17 pounds per square inch of pressure. Once
the autoclave cycle was done, the test tubes were removed and the caps were immediately
tightened to create a seal, and then allowed to cool to room temperature before
inoculation. Once the test tubes had cooled to room temperature one loop of S. aureus,
purchased from Ward’s, was added to test tubes with broth by carefully scraping the loop
across the agar slant containing the bacteria. Then the loop was placed into nutrient
broth, or tryptic soy broth, and swirled vigorously, tapping it along the side of the test
tube and on the bottom to make sure the bacteria came completely off the loop. In
15
between each inoculation, the loop tool was flamed until red and allowed to cool for a
few seconds. After a test tube was inoculated, the threading around the top was flamed to
sterilize it and a cap was placed loosely on top. When all the tubes were inoculated, they
were put into an incubator at 37°C. Upon inspection of the cultures the next day, it was
discovered that only one tube had developed a culture. Since there appeared to be no
growth in the other two tubes, they were discarded by adding bleach to the tubes and
letting them sit for 10 minutes to make sure any bacteria present were dead.
Plating Staphylococcus aureus
The first step in plating S. aureus was sterilizing all of the equipment. Forceps,
paper disks, and pipette tips were placed in sterile autoclave bags and autoclaved at
121°C for 15 minutes at 17 psi. Next, each of the Petri plates were divided into fourths
and labeled. When all the plates had been labeled, 100L of S. aureus culture were
pipetted onto the surface of the agar using a sterilized pipette tip. An ‘L’ bent glass rod
was used to spread the bacteria evenly over the agar surface by lightly holding the rod on
the surface of the agar, while spinning the plate on a turner. Once the bacteria culture
covered the agar evenly, the glass rod was placed into a beaker of isopropyl alcohol and,
immediately prior to spreading the next plate, it was slowly drawn through a Bunsen
burner to sterilize it.
Making specific concentration levels of thyme oil and Virex Tb™
Virex Tb™ provided by South Puget Sound Community College, thyme oil made
by Herb Pharm in Williams, OR and purchased from Radiance in Olympia, WA, and
Benefect™ provided by South Puget Sound Community College were tested for their
effectiveness at inhibiting the growth of S. aureus. The active ingredient in Virex Tb™ is
16
n-Alkyl dimethyl benzyl ammonium chloride and n-Alkyl dimethyl ethylbenzyl
ammonium chloride. The thyme oil contained a 1:5 ratio of the essential oil extracted
from the Thymus vulgaris plant and certified organic grain alcohol. The active ingredient
in Benefect™ is thymol, which is a component of the thyme oil extracted from the
Thymus vulgaris plant. To test each of the chemicals a 50mL beaker was cleaned and
filled with 20 mL of a chemical and no deionized water for the 100% concentration level,
15 mL of a chemical and 5 mL of deionized water for the 75% concentration, or 10 mL
of a chemical and 10mL of deionized water for the 50% concentration level (Rasooli and
Abyaneh, 2004). This procedure was repeated for all three chemicals.
Paper disk application of thyme oil and Virex Tb™
Petri dishes were divided into fourths and labeled after the nutrient agar solidified.
For the first application method, filter paper disks 6 mm in diameter, purchased from the
Cynmar Company, were placed in the center of each quadrant of the plate after they were
sterilized in the autoclave at 121°C for 15 minutes at 17 psi (Charnock, 2006). A pipette
then deposited ~50 µL of the 100% concentrated Virex Tb™ onto the center of the filter
paper disk. It soon became apparent this quantity was too much because the liquid
overflowed the paper disk and throughout the Petri plates rendering them unusable for
this experiment. After this, the amount of chemical pipetted onto the filter paper disks
was reduced to 20µL and this too resulted in an overflow. So, a dipping method was
used to apply the chemical to the filter paper by using forceps to dip the filter paper half
way into the chemical and then the filter paper was placed onto the Petri plate. Twentyfour plates were used for the chemical at each concentration level. Eight plates from each
set of 24 were exposed to one of the chemicals for 5 minutes, 10 minutes, or 15 minutes.
17
After the specific time had elapsed, the paper disks were carefully removed using
sterilized forceps and discarded. Once a set of the Petri dishes had been exposed to the
chemical they were incubated at 37°C for 24 hours (Smith et al., 2000).
After the first 48 hours S. aureus appeared to have no growth on the plates. After
72 hours there was still no growth. After 7 days of being in the incubator the bacteria had
formed large colonies of growth, which was not the intended result indicating a
malfunction had occurred. In an effort to remedy the situation a side test was conducted
before continuing with the zone of inhibition tests.
The test for the zone of inhibition changed from applying the chemical onto the
filter paper to dipping the filter paper directly into the solution halfway so as not to oversaturate the nutrient agar plate. The remainder of the application process remained the
same.
Determining the preference of media by Staphylococcus aureus
Upon the discovery of minuscule growth by S. aureus after the first time
through the procedure, a side study was conducted to determine if S. aureus grew better
in one form of media over another. To do this, 16 test tubes were divided into groups of
four, 3 contained mixtures of nutrient broths with different purchasing years, and one test
tube contained tryptic soy broth. The broths were autoclaved at 121°C for 15 minutes at
17 psi to sterilize them and then each tube was inoculated with the S. aureus purchased
from Ward’s. The tubes were put into the incubator at 37°C for 24 hours. After 24 hours
the tubes were removed from the incubator and examined for growth.
18
It was determined at the time of incubation that the incubator was at a lower
temperature, about 33°C, and so the temperature was adjusted until an internal
thermometer gave a reading of 37°C.
Measuring the Zone of Inhibition
To measure the zone of inhibition on the Petri plates, a millimeter ruler was used
to measure the diameter of the ring formed prior to the application of the chemical. The
diameter from one edge of where the bacteria were growing to the other was measured to
nearest hundredth of a millimeter (Smith et al., 2000).
Statistical Analysis
Once all the data were collected, it was analyzed using two-factor Analysis of
Variance (ANOVA) tests. One of the tests was performed using the factors of chemical
and concentration, and the other ANOVA tested the factors of chemical and exposure
time. If the two-way ANOVA tests showed an interaction follow-up statistical analysis
were done by computing a one-way ANOVA (Walpole et al., 2002). The one-way
ANOVA would look at the three concentration levels of each chemical as the factors for
one test and then the three-exposure times of the chemicals would be examined for the
other test.
19
Methods II
Making the tryptic soy broth for the optical density test
Tryptic soy broth was prepared for the optical density test by combining 0.03g of
tryptic soy broth powder to one mL of deionized water making a final volume of 280 mL
of broth. The broth was pipetted into the test tubes, 5 mL per test tube for 56 test tubes.
The test tubes were loosely capped and autoclaved at 121°C for 15 minutes at 17 psi to
sterilize the broth. The test tubes were cooled to room temperature and the caps were
tightened to create a seal. The test tubes were stored in the refrigerator at 4°C until
needed.
Applying Staphylococcus aureus to the floor tiles
Laminated floor tiles donated by South Puget Sound Community College were
used to test the efficiency of bacterial removal from hard surfaces for each of the three
chemicals along with bleach and water, which were used as positive and negative
controls, respectively. The 12” X 12” tiles were divided into fourths and smeared with S.
aureus using a sterilized glass rod. The glass rod was sequentially sterilized between
“smears” by placing it in isopropyl alcohol and then lightly flaming it. The tiles were
then allowed to sit, undisturbed, for 2.5 hours making sure that excess liquid medium had
evaporated from the surface of the tiles. Once the extra liquid had evaporated, sterile
cotton swabs were used to wipe the center of each quadrant. Each swab was then placed
into small-labeled test tubes that contained 4 mL of sterilized tryptic soy agar. After each
section of the tiles had been swabbed, the tiles were then cleaned with one of the three
chemicals at a 100% concentration level, equal to the manufacturer’s specifications.
20
After all the tiles had been cleaned, each quadrant of the tile was swabbed again, and the
swabs were placed into sterile tryptic soy broth and incubated for 48 hours at 37°C. One
of the test tubes had a sterile cotton swab without any bacteria placed inside of it and the
contents of the test tube served as a blank for zeroing out the spectrophotometer prior to
reading the absorbency for the optical density.
Cleaning the surface
Twenty-eight tiled areas were cleaned with Virex Tb™, thyme oil, or Benefect™.
The cleaning process was conducted using Rite Aid™ sterile, lint free, cotton pads
purchased from Rite-Aid. A small amount of each cleaner, 300µL, were placed on each
sterile pad using a pipette. The pad with the cleaner was then swiped across the tiled
surface area in a zigzag formation moving left to right and from top to bottom. A new
cotton pad was used for each tile section. The cleaned area was then allowed to dry
thoroughly before a sterile cotton swab was used to collect an after sample. These swabs
were then placed in test tubes and incubated for 48 hours at 37°C.
Running the Spectronic 20D+ Spectrophotometer
The Spectronic 20D+ spectrophotometer was set to a wavelength of 600 nm,
which is the wavelength for which S. aureus will absorb light (Abel et al., 2002). The
transmittance was set to 100% and then a small amount of the blank sample, 3.5 mL, was
poured directly from the test tube with the incubated sample into a disposable cuvette
purchased from Wards supply company. The cuvette was inserted into the machine and
the dial was adjusted so that the blank sample had a transmittance reading of zero. After
the machine had been zeroed with the blank, small amounts of the broth from each test
21
tube containing the swabs from the floor tile test were put into cuvettes one at a time. All
measurements were recorded as transmittance and later were converted to absorbance.
Statistical analysis
The optical density values were recorded and converted to absorbency using
equation 1. Absorbance = log (100% / % transmittance). A one-way Analysis of
Variance (ANOVA) test was conducted using Minitab® (2005) to determine if there was
a statistically significant difference between the three cleaners. If a significant difference
was found a Tukey multiple comparison test was performed.
22
Results
The removal of S. aureus from laminated floor tiles showed no difference in the
amount of bacteria removed when Virex Tb™, thyme oil, and Benefect™ were compared
using an ANOVA test. The differences between the optical densities, before and after
cleaning 26 replicate tiles with Virex Tb ™, thyme oil, or Benefect™, were used in the
calculations and the results indicate there was no statistically significant difference
between the levels of bacterial removal among the three chemicals used (F=0.63, df=2,
p=0.535). The means for the optical densities for each of the three cleaners are shown in
Figure 1 confirming none of the chemicals provided superior bacterial removal properties
in this experiment.
2.5
Absorbancy
2
1.5
1
0.5
0
Virex Tb™
Thyme oil
Benefect™
Figure 1. Mean optical density of bacteria after removal of S. aureus from laminated floor tiles; 28 tile
sections were used for each of the three cleaners. The differences between the optical density before and
after cleaning the floor tiles were used to compute the means for each chemical. The vertical bars represent
the standard error about the means.
A two-way ANOVA test for the zone of inhibition of S. aureus on nutrient agar
plates after 48 hours of incubation at 37ºC showed a significant interaction between the
three cleaners, Virex Tb™, thyme oil, and Benefect™, and the three concentration levels,
23
100%, 75%, and 50% (F=22.75, df=4, p=0.0005,). This is shown in Figure 2 where the
average zone of inhibition is graphed, showing Virex Tb™ is more effective at reducing
bacteria then the other two chemicals. Since there was a statistical significant interaction
between the three cleaners and the three concentration levels, a one-way ANOVA was
conducted to examine each factor separately.
Zone of Inhibition (mm)
20
18
16
14
12
10
8
6
4
2
0
Virex
Thyme Oil
100%
75%
Benefect
50%
Figure 2. Mean zone of inhibition for Staphylococcus aureus. Each of the cleaners had 96 replicates, 32
for each of the three concentration levels, 100%, 75%, and 50%. The vertical bars represent the standard
error about the mean for each chemical and concentration level.
The one-way ANOVA for Virex Tb™ at each concentration level showed a
significant difference among the 3 concentration levels (F=34.16, df=2, p=0.0005). A
Tukey test revealed 75% Virex Tb™ was the most effective in inhibiting the bacterial
growth followed by 100% Virex Tb™ and then 50%.
The one-way ANOVA for thyme oil also showed a statistical significance among
the three concentration levels also ( F=39.58, df=2, p=0.0005). A Tukey test showed the
concentration level of 100% was statistically higher than both the 75% and 50%
concentrations. However, the zones of inhibition for the concentration level of 75% and
24
50% of thyme oil were slightly different with a mean of 1.469 for the 50% and 1.000 for
the 75%..
The one-way ANOVA for the Benefect™ showed a statistical significance
between all three-concentration levels (F=8.96, df=2, p=0.0005). A Tukey test computed
with a 98.01% confidence interval showed the concentration level of 100% was
statistically lower then the concentration level of 75% and 50% Benefect. ALos, the
concentration level of 75% had a mean of 1.696 which was close to the mean of the 50%
Benefect™ of 1.673. A graph of the means for each chemical at each of the three
concentrations levels is shown in Figure 2.
A two-way ANOVA test for the zone of inhibition of S. aureus on nutrient agar
plates after 48 hours of incubation at 37ºC showed a statistical significance between the
cleaners and the time they were allowed to remain in contact with the bacteria ( F=3.66,
df=4, p=0.006). Figure 3 shows the mean relationship between the cleaners and the
length of time those cleaners were in contact with bacteria. Virex Tb™ had a larger
mean then thyme oil or Benefect™ and because there was an interaction between the
cleaners a one-way ANOVA was run for each cleaner at the three time frames.
25
Mean zone of inhibition (mm)
16
5 minutes
14
10 minutes
12
15 minutes
10
8
6
4
2
0
Virex Tb™
Benefect™
Thyme Oil
Figure 3. Mean zone of inhibition for Staphylococcus aureus after being exposed to one of three chemicals
for 5 minutes, 10 minutes, and 15 minutes. There were 32 replicates per time frame per cleaner. The
vertical bars represent the standard error about the means for each chemical and time frame .
The one-way ANOVA for Virex Tb™ showed there was no difference between
the lengths of time the chemical was allowed to remain in contact (F=0.99, d.f.=2,
p=0.371). However, the thyme oil did show a significant difference among the three
times (F=8.17, d.f.=2, p=0.0005) and a Tukey test revealed the 15 minute exposure time
was the least effective at inhibiting the bacteria with a confidence level of 98.01%.
Benefect™ showed no difference between the three time frames so they all worked
equally effective (F=1.79, d.f.=2, p=0.169).
26
Discussion
The results of my experiment fail to support my hypothesis that when Virex Tb™,
a commercial cleaner, is compared with thyme oil and Benefect™, the zone of inhibition
for Staphylococcus aureus will be the same for all cleaners if the exposure time and
concentration level for the thyme oil and Benefect™ is at the manufacturer’s
recommended levels of 10 minutes and 100% concentration (Rasooli et al., 2003). The
zone of inhibition for the three cleaners showed some differences when comparing them
to various times and concentration levels. Overall the Virex was the most effective at the
manufacturer’s recommended levels of 100% Virex Tb™ for a 5 minute exposure time.
The thyme oil worked best for the manufacturer’s recommended concentration of 100%
but the exposure time worked best at 10 minutes instead of 5 minutes. The Benefect also
worked best at the manufacturer’s recommended level of 100% but the length of
exposure time showed no difference in the amount of bacteria inhibited. My results
further failed to support my secondary hypothesis that the removal of S. aureus would be
greater for the Virex Tb™ then for thyme oil or the Benefect™ (Olson et al., 1994).
None of the chemicals showed superior bacterial removal from the floor tiles in my
study.
The ineffectiveness of the thyme oil and Benefect™ comes as a surprise to me,
because in previous studies thyme oil was determined to be effective against Aspergillus
flavus and Aspergillus parasiticus fungi (Rasooli et al., 2003) and in studies conducted
by the Environmental Protection Agency Benefect™ was determined effective against S.
aureus, Salmonella choleraesuis, Pseudomonas aeruginosa, Mycobacterium tuberculosis,
HIV-1, and Trichophyton mentagrophytes (Sensible Life Products, 2005). Several
differences between their studies and mine were the concentration levels, the organisms,
27
and the application methods. The concentration levels I used produced a mixture of
results. Virex Tb™ was most effective in reducing bacteria at the 75% level followed by
100% and then 50%. Thyme oil had different results with 100% as the most effective
followed by 50% and 75%. Lastly, Benefect™ had best results when the chemical
concentration level of 100% was used proceeded by 50% and 75%. For further studies of
this nature I recommend using a broader range of concentration levels.
The results of Rasooli et al. (2003) were observed using fungi, which have
different biological properties than bacteria. For instance, the cellular and membranous
structures of fungi are quite different from bacteria. Fungi have cell walls that are
composed of chitin or polysaccharides and bacteria have a cell membrane composed of
peptidoglycans, which are polysaccharides cross-linked with short peptides or proteins
(Brown, 2005). The proteins found within the bacterial cell membrane that serve as a
filter, allowing only certain substances to enter the cell (Boyer, 2006). In contrast, the
cell wall associated with fungi are composed of only polysaccharides, therefore their
filter lacks the protein component bacteria have making fungi susceptible to chemicals in
a different manner from bacteria. Using multiple types of organisms in future studies
would help to better determine the overall effectiveness of a cleaner by providing variety.
The optical density test for the cleaners showed no significant difference, which is
not what I had expected. I thought there would be some difference based on Olson et
al.’s (1994) and Bauer et al.’s (1995) studies where the alternative cleaners yielded lower
effectiveness then the commercial cleaners. The biggest influence producing my
outcomes was the method used for removing the bacteria from the floor tile. Wiping the
tile with a sterile, lint-free, cotton pad may have been too abrasive, removing bacteria
28
regardless of the presence of a cleaner. This is seen when comparing the optical densities
between the two controls, water and bleach. Both water and bleach had the same change
in optical density. Using different types of cleaning tools, like a sponge or towel, could
help distinguish a preferable method for bacterial removal. Other factors that may have
altered the outcome are not allowing enough drying time of the sample bacteria on the
surface of the tile before cleaning. I allowed 3 hours for the culture to dry before
applying any chemical, but if this study were repeated I would change it to 24 hours to be
certain the culture is completely dry. The amount of time the samples were allowed to
incubate before measuring their optical density may have been too long. I measured the
absorbency after 48 hours of incubation. I would recommend changing the incubation
time to checking it after 24 and 48 hours because some organisms maximize their growth
after 24 hours and some peak at 48 hours (Davis et al., 2004). One final aspect I would
change about the methods for my study is the method used for measuring bacterial
presence, removal, and growth. I would keep the zone of inhibition test but I would
change the optical density tests to turbidity tests to measure the amount of organism
removed from a surface. The reason is because the Environmental Protection Agency
uses results from turbidity tests to determine the effectiveness of disinfectants (US EPA,
1982).
If this same experiment were to be conducted again, I would change two main
things. The first would be to include at least one other type of organism, preferably a
fungi since previous studies have already been performed on these organisms (Rasooli
and Abyaneh, 2004). The second thing I would change is the incubation time; I would
not only measure the zone of inhibition at 24 hours, but also at 48 in order to obtain better
29
quality measurements. For the optical density portion of this experiment I would change
the method of applying the chemical, using a variety of cleaning tools and I would use
turbidity to measure the amount of bacteria removed from surfaces.
The results of this study have posed a variety of questions for future research of
this type. There are three main types of questions that are left unanswered and they are;
how does the effectiveness change for various organisms, do concentration levels have an
influence on the effectiveness of a cleaner and if so what is that influence, and lastly how
does incubation time effect the results of a study like this? Also, future studies could
benefit by looking at different types of alternative cleaners to see if there is one that
works effectively.
Our environment is rapidly changing, natural resources are depleting fast. It has
become the purpose of many individuals, particularly those with power, to make changes
and help the environment and those inhabiting it. But at what risk is this happening.
Everyday household chemicals are rapidly being replaced by all natural environmentally
friendly cleaners. The claim is they work equally as well as their dangerous counterparts
but according to my research that is not always true. Thyme oil did not effectively
demonstrate its power to act as a germicide cleaner the way Virex Tb™ does. As a
consequence of this Benefect™ was no more effective either. But, this does not mean
there is not an effective green cleaner that can one take the place of the unfriendly
germicidal detergent. It just had to be found.
30
Acknowledgements
I am happy to thank Dr. Margret Olney for showing me how to apply many
microbiological techniques, Dr. Mary Jo Hartman for helping me with my statistical
analysis, Dr. Gregory Garman for his insight and problem solving strategies, Cheryl
Guglielmo for assisting me with all of my lab needs, Josh Coffey for his support, Deon
Roe for her preparatory help, Katie Cey for her photography ability, Si’i Vulangi for
accompanying me in the lab, and last, but not least, South Puget Sound Community
College for their willingness to supply me with the tools necessary for completing my
research.
31
Literature Cited
Abel, T., Cohen, J. I., Engel, R., Filshtinskaya, M., Melkonian, A., Melkonian, K. 2002.
Preparation and investigation of antibacterial carbohydrate-based surfaces.
Carbohydrate Research. 337: 2495-2505.
Bauer, J.M., Beronio, C.A., Rubino, J.R. 1995. Antibacterial activity of environmentally
“green” alternative products tested in standard antimicrobial tests and a simulated
in-use assay. Journal of Environmental Health. 57: 13-19.
Boyer, R. 2006. Concepts of Biochemistry. Third Edition. John Wiley and Sons, Inc.
pp. 211-222.
Brown, A. 2005. Microbiological Applications. Ninth edition. McGraw Hill, NY. pp.
115-122, 207-208.
Charnock, C. 2006. Are multidose over the counter artificial tears adequately
preserved? J. of Cornea and External Disease. 25: 432-437.
Davis, A.O., O’Leary, J.O., Muthaiyan, A., Langevin, M.J., Delgado, A., Abalos, A.T.,
Fajardo, A.R., Marek, J., Wilkinson, B.J., Gustafson, J.E. 2004. Characterization
of Staphylococcus aureus mutants expressing reduced susceptibility to common
house cleaners. J. of Applied Microbiology. 98: 364-372.
Minitab® Release 14.20. 2005. Minitab
Olson, W., Vesley, D., Bose, M., Dubbel, P., Bauer, T. 1994. Hard surface cleaning
performance of six alternative household cleaners under lab conditions. J. of
Environmental Health. 56: 27-32.
Rasooli, I., Abyaneh, M.R. 2004. Inhibitory effects of thyme oil on growth and aflatoxin
production by Aspergillus parasiticus. Food Control. 15: 479-484.
Sensible Life Products. 2005. [cited 2007 May 7] www.Benfect.com/usa/index.html.
Smith, R., Calviello, C., DerMarderosian, A., Palmer, M. 2000. Evaluation of
antibacterial activity of Belizean plants. Pharmaceutical Biology. 38.
Spaulding, E.H. and Emmons, E.K. 1958. Chemical disinfection. American J. of
Nursing. 58: 1238-1242.
Stainier, R.Y., Ingraham, J.L., Wheelis, M.L., Painter, P. R. 1986. The Microbial World.
Fifth edition. Prentice-Hall, New Jersey. pp. 624-27, 636.
Tortora, G.J., Funke, B.R., Case, C.L. 2004. Microbiology: An Introduction. Eighth
edition. Pearson Education Inc., CA. pp. 322-23, 446-47.
U.S. EPA. Pesticide assessment guidelines subdivisions g: product performance.
Washington, D.C. p.50-96.
Walpole, R., Myers, R., Meyers, S., Ye, K. 2002. Probability & Statistics for Engineers
& Scientist. Seventh edition. Prentice Hall, NJ. pp. 227, 477.
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