Microbiology Lab

Pre-lab Questions:
It is necessary to treat the control with water because bacteria, like other living things, need
water along with food and a suitable environment to survive. Since the effectiveness of
antimicrobial products is being tested, water, which is not an antimicrobial solution, is suitable to
be used in the control to measure the growth rate of bacteria under normal growing conditions
without the interference of antimicrobial solutions.
Some natural products have antimicrobial properties to prevent the food from running out by
getting eaten by bacteria, and to serve as a food safe for consumption by young offspring. For
example, honey is stored food in bee hives for the bees offspring to consume. If it did not have
any antimicrobial properties, bacteria will be able to consume all of it much faster, and there will
be no remaining food for the bees to eat. In addition, the food with bacteria may also bring
sickness or disease to the bees and their young offspring.
In this lab, gram-positive and gram-negative bacteria are being differentiated to determine how
each antimicrobial solution will effect the two types of bacteria with different types of cell walls.
Gram-positive bacterias cell walls are made up of a thick carbohydrate layer with proteins, while
gram-negative bacterias cell walls have a second layer of lipids and carbohydrates, which make
them more resistant to antimicrobial solutions and antibiotics.
Purpose: To determine the effectiveness of store bought versus natural antimicrobial products
on gram-positive and gram-negative bacteria.
Hypothesis: Gram-negative bacteria will be more resistant to the antimicrobial solutions
compared to gram-positive bacteria, and bleach will be the most effective antimicrobial solution,
while garlic powder will be the least effective.
Data: Table 1
The Comparison of the Sizes of Zones of Inhibition of Bacteria with Different Antimicrobial
Solutions
Bacteria Type: Gram-Positive
Plate

Solution

Diameter (mm)

Water

Control

0mm

Bleach

28mm

Store-bought

Plate

Solution

Diameter (mm)

Antibiotic Disk

22mm

Hand Sanitizer

0mm

Water

Control

0mm

Garlic Powder

0mm

Honey

0mm

Cinnamon

0mm

Natural

Discussion:
Comparing with the other groups experimenting on gram-positive bacteria, the zones of
inhibition for the control, water, and for the solutions C, D, E and F on both plates were the same
as our group: all the zones were at 0mm. However, the the zone of inhibition we measured in
our group for the solutions A and B were sightly different compared to the other groups. For
solution A, we measured a zone of inhibition of 28mm; however, two other groups had
measurements of 18mm and 17mm, and another two had insufficient bacterial growth for us to
make a conclusion. For solution B, our group measured a zone of inhibition of 22mm but the
other groups had measurements of 26mm, 20mm, 24mm, and 23mm. Comparing the
measurements for solution A in all groups, there was a significant difference in the size of the
zones of inhibition. Our group had the largest diameter for the zone, while the two other groups
had diameters that were 10mm or 11mm smaller compared to ours. Our zone of inhibition for
solution B, on the other hand, was quite close to the other groups; the measurements from the
plates of the other groups for solution B differentiated from ours by 2mm,1mm or 4mm. These
differences may have been resulted from the slight difference in the supply of food, the amount
of bacteria, the degree of contamination by other bacteria, the amount of oxygen, the amount of
water, and the amount of antimicrobial solution placed in each section of each plate. Hence,
replicates, which mean conducting the experiment several more times, are very important in
science. Due to all the random and systematic errors that come from the tools we use in the
experiment, the way we, the conductors of the experiment, along with the living things that we
are experimenting with react in response to something else, and many other possibilities, a
variety of results can be concluded for the same lab, especially in biology, where the reactions of
the organisms being tested cannot be controlled. Thus, conducting the same lab many times
can help average and decrease the effects of these errors and shorten the uncertainty range,
which represents the range of the different measurements being made. Comparing the results
for the antimicrobial effectiveness of solutions A, B and C on gram-negative bacteria, all the

groups either had insufficient bacterial growth or contamination by other bacteria; hence, a
conclusion could not be made for the effectiveness of the solutions A, B and C on the bacteria.
For solution F, all groups experimenting on gram-negative bacteria had a zone of inhibition of
0mm, which were the same results for gram-positive bacteria. Solution D, on the other hand, did
not have the same effects on gram-negative bacteria as it did on gram-positive bacteria; there
were constant 17mm, 21mm and 17mm zones of inhibition within the groups testing gramnegative bacteria. This was surprising as garlic powder had no antimicrobial effectiveness on all
the groups experimenting with gram-positive bacteria but gram-negative bacteria, which had a
thicker cell membrane and was more resistant compared to gram-positive bacteria, had zones of
inhibitions. This difference may have been caused by some errors in the control variables or
random errors; however, there is also the possibility that garlic powder is more effective when it
reacts to lipids, or is less effective when reacting with proteins because gram-negative bacteria
has a lipid and carbohydrate layer while gram-positive bacteria has a cell membrane of
carbohydrates and proteins. Lastly, the results for gram-negative bacteria interacting with
solution E also had some variation with the results from testing gram-positive bacteria. For
solution E and gram-negative bacteria, there were zones of inhibition of 21mm and 0mm, and
another plate had insufficient bacterial growth to make a conclusion. With gram-positive
bacteria, all groups had no zones of inhibition. Solution E, in this case, may also be similar to
solution D, where there is the possibility that it is more effective reacting with gram-negative
bacteria, increasing its antimicrobial effectiveness. Replicates, in this case, will be useful in
averaging out the errors, and concluding more reliable results about the antimicrobial
effectiveness of solution E and D on gram-negative and gram-positive bacteria.
Bleach is one of the antimicrobial solutions that was being tested in this lab. It is a
chemical called sodium hypochlorite that kills bacteria by attacking proteins, quickly destroying
their delicate shape, and is used as a household cleaner (Ledford). More specifically, as a
hypochlorous acid, bleach (attacks) the amino acids that make up proteins, destroys the cell
membrane, and causes bacterial proteins to lose their structure and form aggregates, which
is a mass of living cells (Ledford; CBC News). This effect caused by bleach has the same
result high temperatures would have on proteins and cells (CBC News). After forming
aggregates, this process will be irreversible; the proteins clumped together usually cannot be
dissolved and will result in the death of the stressed cells (CBC News). However, according to
CBC News, bacteria are not totally defenceless when attacked by bleach; they have evolved a
protein chaperone called heat shock protein Hsp33 which works like an immune system,
jumping into action when the bacterial cell reacts with bleach. This protein helps to protect the
(other) proteins from the aggregation effect and to increase their resistance (CBC News).
Although protein Hsp33 increases bacterial resistance against bleach, most of the time, the
bacteria may still not be sufficient enough to survive because bleach attacks many of the
proteins that are essential for bacterial growth(CBC News). In addition, hypochlorite is also
produced by the immune system as a first line of defence to kill invading bacteria, and a study

by Ursula Jakob from the University of Michigan also suggests the acid may limit the growth of
bacteria in insect guts (CBC News; Ledford).
Conclusion:
The purpose of this lab was to determine the effectiveness of store bought versus natural
antimicrobial products on gram-positive and gram-negative bacteria, and the hypothesis states
that gram-negative bacteria will be more resistant to the antimicrobial solutions compared to
gram-positive bacteria, and bleach will be the most effective antimicrobial solution, while garlic
powder will be the least effective. The data from this lab neither accepts or rejects the
hypothesis as there were insufficient bacterial growth and data, and some inconsistencies in the
zones of inhibition for some of the solutions that were experimented.
Although it is observed that the bleach had the greatest zone of inhibition on the grampositive bacteria in our group, of the total of five groups experimenting on gram-positive bacteria,
two agar plates had insufficient bacterial growth and two other groups had zones of inhibition of
18mm and 17mm for bleach while our group had 28mm. These zones of inhibition tested on the
same type of bacteria had a great difference; thus, it cannot be concluded if bleach was most
effective or if the antibiotic disk with zones of inhibitions of 20mm, 23mm, 24mm and 26mm
was. Replicates would be need to make a more distinct conclusion. For the other results of
gram-positive bacteria, due to the observation of solutions C, D, E and F to all have 0mm of
zones of inhibition in all their agar plates, the solution with the least anti-microbial effectiveness
could not also be concluded. Moreover, there was also insufficient bacterial growth and data to
conclude that gram-negative bacteria is more resistant compared to gram-positive bacteria and
to determine the antimicrobial effectiveness of solution A, B and C on gram-negative bacteria:
solutions A, B and C that were tested with gram-negative bacteria had insufficient bacterial
growth on all the agar plates, and only solution F had zones of inhibition that were the same for
both gram-negative and gram-positive bacteria of 0mm. In addition, contradicting to the
hypothesis, solution D and E, which shown no antimicrobial effectiveness on gram-positive
bacteria, had resulted in constant zones of inhibition of 17mm, 21mm and 17mm on all three
agar plates for solution D and zones of inhibition of 21mm and 0mm, along with an agar plate
with insufficient bacterial growth for solution E. Although gram-negative bacteria has a thin,
single-layered peptidoglycan layer while gram-positive bacterias is thick and multilayered, it has
a outer membrane that contains a high content of lipids and lipoproteins but gram-positive
bacteria do not have an outer membrane; hence, gram-negative bacteria is more resistant to
antimicrobial solution compared to gram-positive, making them resistant to several classes of
antibiotics; even broad-spectrum antibiotics can be ineffective against some bacteria, leaving
healthcare providers no choice but to use older drugs, such as colistin, which can have toxic
side effects ("Gram-negative Bacteria; "Comparative Characteristics of Gram-Positive and
Gram-Negative Bacteria). The results of the gram-negative bacteria interacting with solutions D
and E, garlic powder and honey, compared to the data for gram-positive bacteria, made gram-

negative bacteria appear to have less resistance towards anti-microbial solutions. However,
Mandal stated that the extracts of raw and processed honey showed (zones of inhibition of)
(6.9437.94) mm, against gram-positive bacteria viz., S. aureus, Bacillus subtilis, Bacillus
cereus, as well as gram-negative bacteria like E. coli, P. aeruginosa and S. enterica serovar
Typhi" ; the antimicrobial effectiveness of Tualang honey was also shown effective when it
inhibited the growth of bacterial species that cause gastric infections, such as S. typhi, S.
flexneri and E. coli at the low concentrations. Hence, honey should have anti-microbial
properties against both gram-positive and negative bacteria, not only gram-negative, as
presented in the data for this lab. Since only one zone of inhibition of 21mm was recorded out of
three agar plates, making replicates to test the antimicrobial effectiveness of honey with gramnegative bacteria will be useful to make a conclusion. Garlic, on the other hand, also shown
antimicrobial effectiveness on gram-negative bacteria but not gram-positive; however, the
antibacterial effect of garlic was reported previously for both Gram-positive and Gram-negative
bacteria, including antibiotic-resistant strains; thus, garlic should have also had an effect,
creating a zone of inhibition when it was tested with gram-positive bacteria (Jonkers, D., et al).
An explanation for this result may have been because the antibacterial activity varies with the
species, production method and ripeness of the garlic or due to the ingredients that may have
been added in to the garlic powder when it was processed: as the strongest antibacterial effects
were observed for the raw garlic extract, which was found to have the highest concentration of
allicin, that is responsible for part of the antimicrobial property of garlic (Jonkers, D., et al).
The lab demonstrated the antimicrobial effectiveness of bleach and the antibiotic disk on
gram-positive bacteria but could not determine which antimicrobial solution was most effective,
and which of the six solutions was the least effective. Due to the insufficient bacterial growth for
all three solutions of A, B and C for gram-negative bacteria, the effectiveness of bleach, the
antibiotic disk and the hand sanitizer was also not able to be concluded. Lastly, as there was a
great variation in the two zones of inhibition for solution E of 21mm and 0mm, and the solutions
D and E had antimicrobial effects on gram-negative bacteria but not the gram-positive bacteria,
this lab could not demonstrate that gram-negative bacteria is more resistant to microbial solution
compared to gram-positive bacteria. Furthermore, the data was not able to completely determine
if store-bought antimicrobial products are more effective than natural products. Overall, the lab
did not support nor reject the hypothesis.

Works Cited
CBC News. "U.S. researchers discover how bleach works to kill bacteria." CBC News. CBC
Radio Canada, 13 Nov. 2008. Web. 8 Mar. 2016. <http://www.cbc.ca/news/technology/us-researchers-discover-how-bleach-works-to-kill-bacteria-1.733785>.

"Comparative Characteristics of Gram-Positive and Gram-Negative Bacteria." University of

Maryland College of Computer, Mathematical and Natural Sciences. N.p., n.d. Web. 10
Mar. 2016.
<http://www.life.umd.edu/classroom/bsci424/BSCI223WebSiteFiles/GramPosvsGramNe
g.htm>.
"Gram-negative Bacteria." NIH: National Institute of Allergy and Infectious Diseases. N.p., 30
Apr. 2012. Web. 10 Mar. 2016.
<https://www.niaid.nih.gov/topics/antimicrobialresistance/examples/gramnegative/Pages/
default.aspx>.
Jonkers, D., et al. "Antibacterial effect of garlic and omeprazole on Helicobacter pylori." Oxford
Journals: n. pag. Journal of Antimicrobial Chemotherapy. Web. 10 Mar. 2016.
<http://jac.oxfordjournals.org/content/43/6/837.full#ref-4>.
Ledford, Heidi. "How does bleach bleach?" Nature.com. Nature Publishing Group, 13 Nov.
2008. Web. 8 Mar. 2016.
<http://www.nature.com/news/2008/081113/full/news.2008.1228.html>.
Mandal, Manisha Deb, and Shyamapada Mandal. "Honey: its medicinal property and
antibacterial activity." US National Library of Medicine National Institutes of Health.
N.p., Apr. 2011. Web. 10 Mar. 2016.
<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3609166/#b34>.