Tuesday, February 10, 2009
The InhibitoryAffect on Growth of E. coli and M. luteus due to the ChemicalAgents Scope, Mr.
Clean and Mercury
The domain bacteria is comprised of prokaryotes, which are unicellular organisms that do not
contain membrane bound organelles or nuclei and can function alone or in colonies. Prokaryotes are
essential to all ecosystems and to all life we encounter.As some bacteria are harmful, chemical agents
have been developed to retard the growth of and kill these bacteria. Specific chemical agents often
target bacteria based on their cell wall composition. Gram-positive bacteria have a cell wall comprised
of a thick peptidoglycan layer, whereas Gram-negative bacteria have a lipopolysaccharide outer cell
wall above a thin peptidoglycan layer. This outer wall in Gram-negative bacteria often makes it less
susceptible to some chemical agents (Campbell et al. 2008). In this experiment, the effect of the
chemical agents Scope, Mr. Clean and Mercury on the growth of the bacteria, Escherichia coli and
Micrococcus luteus, is examined. Through examining the zones of inhibition in both species in
response to exposure to the chemical agents, the relative survival of the two species can be analyzed.
E. coli is a Gram-negative bacteria that is present in the human digestive tract, but may also
cause illnesses such as traveler’s diarrhea and urinary tract infections (Biology 108 lab manual, 2008).
M. luteus is a Gram-positive bacteria that is commonly present on human skin and can cause meat to
spoil (Biology 108 lab manual, 2008). The purpose of this experiment is to test the effect of the three
chemical agents on these bacteria, providing insight on their action in bacterial inhibition. This study is
relevant because bacterial infections are a common risk for humans, and in order to stay safe, we must
come up with effective methods in preventing contamination. Through testing the effectiveness of
various chemical agents, the best antiseptic and disinfecting methods can be determined, thus helping
to prevent the spread of infections. If M. luteus and E. Coli are exposed to Mr. Clean, Scope and Mercury, the growth of these
bacterial species will be inhibited in varying amounts. Mercury will likely be the most effective anti-
microbial agent against both the Gram-positive M. luetus and the Gram-negative E. coli because in
high concentrations the Hg will poison both types of bacterial cells. In both bacteria the mercury metal
salt will react with proteins in the cell membrane leading to insoluble heavy metal protein salts, which
will cause the poisoned bacteria to clump together in a heavy mass in which they cannot survive (Rahn,
1945). Mr. Clean’s main active ingredient is NaOH, which is effective in killing Gram-negative
bacteria such as E. coli, but should be less effective in killing Gram-positive bacteria such as M. luteus.
NaOH causes the denaturation of integral membrane proteins and the saponification of membrane
lipids, but the thick peptidoglycan cell wall of Gram-positive bacteria should keep the cells intact,
whereas gram-negative bacteria are more likely to lyse due to their thin peptidoglycan layer (Amoroso
et al. 1994). Mr. Clean will likely be less effective than Mercury in both cases. Scope contains more
than one anti-microbial agent and thus will likely be effective against both bacteria.Alcohol is able to
penetrate the cell wall of bacteria and disrupt hydrogen bonding of cell membrane proteins, causing the
formation of new hydrogen bonds with the alcohol (Campbell et al. 2008).According to the Biology
107 Laboratory Manual (2008), alcohol dissolves the lipopolysaccharide layer of Gram-negative
bacteria, which suggests that E. coli are more susceptible to the alcohol than M. luteus. Sodium
benzoate and benzoic acid are two other chemicals in Scope that are effective in killing Gram +
bacteria (Phillips and Walker, 2007). Thus Scope will affect both E. coli and M. luteus. It will likely be
more effective in inhibiting bacterial growth than Mr. Clean due to its various chemical agents. Scope
will likely be less effective in inhibiting growth than mercury because scope is handled by humans,
suggesting it is not very toxic, whereas mercury is a very poisonous substance that people avoid
handling because of its high toxicity.
Materials and Methods: The Biology 108 lab manual (2008) states that using sterile technique, three PCAplates are
inoculated with M. luteus and three with E. coli. There is a control plate for each bacterial species that
has not been impregnated by a chemical agent. The other plates have one of each disk impregnated
completely by the chemical agent solutions placed in one of three sections on each experimental PCA
plate. There are two experimental plates for each species to incorporate replication, ensuring viable
results.After preparation the cultures are allowed to cultivate and diameters of zones of inhibition are
recorded in mm around the disks of each plate the next week.
It is seen from the results summarized in Table 1 that the three chemical agents tested inhibited
the growth of both types of bacteria in varying amounts. In looking at the control groups for M. luteus
and E. coli, there is no zone of inhibition when no chemical agent has been used. However, when a
chemical agent has been used, there are varying zones of inhibition depending on the species and the
agent. The respective zones of inhibition represent the amount that a specific chemical agent inhibits
bacterial growth, and by measuring the diameter of each zone of inhibition, the growth prevention
caused by each chemical agent can be quantified. It was found that the Mr. Clean treatment resulted in
the smallest zones of inhibition for both species. There was a slightly larger zone of inhibition of M.
luteus (with 7.17mm inhibition) than E. coli (with 6.67mm inhibition) with Mr. Clean. Scope resulted
in the second largest zones of inhibition, inhibiting the growth of M. luteus (with 15.5mm inhibition)
more than E. coli (with 10.5mm inhibition). Mercury resulted in the largest zones of inhibition for both
species with E. coli having a slightly larger zone of inhibition (29.5mm) than M. luteus (26.0mm).
Table 1: The average diameters of zones of inhibition as induced by three chemical agents on 10mm
diameter disks placed in PCAagar plates on the growth of E. coli and M. luteus.
Diameters of Zones of Inhibition (mm)
Mercury Mr. Clean Scope Control Organism
Escherichia Coli 29.5 6.67 10.5 0
Micrococcus luteus 26.0 7.17 15.5 0
As was seen in the control tests, there were no zones of inhibition, demonstrating that the PCA
medium in which the bacteria were grown did not inhibit the growth of either bacterial species. It also
demonstrates that the paper disks, which were impregnated with chemical agents in the experimental
plates, did not inhibit bacterial growth in the absence of chemical agents.
As was predicted in the hypothesis, mercury was the most effective chemical agent in inhibiting
the bacterial growth of both species due to its extreme toxicity to most living organisms. The mercury
was, however, slightly more effective in inhibiting the growth of E. coli than it was in inhibiting M.
luteus. The diameter of the zone of inhibition on E. coli was 29.5mm, suggesting that mercury had a
very strong inhibiting effect on the bacteria. The slightly smaller diameter of 26.0mm for M. luteus still
suggests a very strong inhibiting effect. Mercury is an effective chemical agent because it interacts with
the cell membranes and dissolves into the bacterial cytoplasm, interrupting cell function by forming
heavy, insoluble metal protein salts (Rahn,1945).Apossible reason for E. coli being slightly more
inhibited by the mercury than M. luteus might be because of interaction of the mercury with proteins,
lipoproteins and perhaps lipopolysaccharides and phospholipids of the outer cell wall (Kaiser, 2008). In
Gram-negative bacteria the mercury would immediately come in contact with proteins of the outer
membrane to which it could combine and inhibit the cell faster and more effectively than it would
Gram-positive bacteria. In Gram-positive bacteria, the mercury has to pass through the thick
peptidoglycan layer in order to get to the cell membrane or through the cell wall to begin the process of
interaction with and thus inhibition of the proteins. Also as was predicted, Scope was the second most effective inhibitor of both species of bacteria
due to the large amount of various disinfectants it contains. Scope was significantly more effective in
inhibiting the growth of M. luteus, with a zone of inhibition of 15.5mm, than it was in inhibiting E.
coli, with a zone of inhibition of 10.5mm. Scope contains